elib.tiho-hannover.de · Nils-Holger Zschemisch and Emily Northrup contributed equally to this work. Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch

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Institute for Laboratory Animal Science

Hannover Medical School

Dnd1: From Germ Cells to Teratomas in the Rat

THESIS

Submitted in partial fulfillment of the requirements for the degree

Doctor of Philosophy (PhD)

in

Laboratory Animal Science

at the University of Veterinary Medicine Hannover

by

Emily Northrup

(Nürnberg)

Hannover, Germany 2011

Supervisor: Prof. Dr. H.-J. Hedrich

Advisory committee: Prof. Dr. H.-J. Hedrich

Prof. Dr. U. Martin

Prof. Dr. I. Greiser-Wilke († 2010)

Prof. Dr. L. Haas

1st evaluation: Prof. Dr. H.-J. Hedrich (Hannover Medical School, Institute for

Laboratory Animal Science)

Prof. Dr. L. Haas (University of Veterinary Medicine Hannover,

Institute of Virology)

Prof. Dr. U. Martin (Hannover Medical School, Department of

Cardiothoracic, Transplantation and Vascular Surgery)

2nd evaluation: Prof. Dr. H.-J. Jacobsen (Leibniz Universität Hannover,

Institute for plant genetics)

Date of the oral examination: May 6, 2011

This study was funded by the German Cluster of Excellence REBIRTH

“From Regenerative Biology to Reconstructive Therapy”.

To my family

“It is a bad plan that

admits of no modification”

Publilius Syrus

Table of contents I

Table of contents

Publications ............................................................................................................. IV

List of abbreviations ............................................................................................... VI

1. Introduction ....................................................................................................... 1

2. Literature review ................................................................................................ 2

2.1. The germline ................................................................................................ 2

2.1.1. The germline: linking generations .......................................................... 2

2.1.2. Cell cycle, mitosis and meiosis ............................................................... 2

2.1.3. Germ cells during embryonic development ............................................ 3

2.1.4. Gametogenesis ...................................................................................... 5

2.2. Germ Cell Tumors ........................................................................................ 6

2.2.1. Classification and origin of germ cell tumors .......................................... 6

2.2.2. Teratomas and Teratocarcinomas ......................................................... 7

2.2.3. Incidence of germ cell tumors in humans ............................................... 8

2.2.4. Genetic and environmental factors in human GCTs ............................... 9

2.2.5. Infertility and TGCTs ............................................................................ 10

2.2.6. Teratomas in mice ................................................................................ 11

2.2.7. Teratomas in rats ................................................................................. 12

2.3. The Dead end homolog 1 gene (Dnd1) ...................................................... 13

2.3.1. Dead end (dnd) in anamnia .................................................................. 13

2.3.2. The Ter mutation in the mouse Dead end homolog 1 (Dnd1) .............. 14

2.3.3. Modulating the phenotype of Ter/Ter mice ........................................... 15

2.3.4. Dead end homolog 1 (DND1) in humans ............................................. 16

2.3.5. The RNA binding protein Dnd1 ............................................................ 17

2.3.6. Zebrafish dnd, miRNAs and target genes ............................................ 17

2.3.7. Mammalian Dnd1, miRNAs and target genes ...................................... 18

2.3.8. Interactions of Dnd1 with Apobec ......................................................... 19

2.3.9. Role of Dnd1 in germ cell cycle and gene expression .......................... 20

2.4. Pluripotent germ cells and their in vivo origin ............................................. 21

2.4.1. Primordial germ cell specification in the mouse ................................... 21

2.4.2. Identification of murine PGCs............................................................... 21

II Table of contents

2.4.3. Defining pluripotency ............................................................................ 23

2.4.4. Differentiation potential of PGCs .......................................................... 24

2.4.5. Embryonic stem cells ........................................................................... 24

2.4.6. Embryonic carcinoma cells................................................................... 25

2.4.7. Features of rodent embryonic germ cell derivation and culture ............ 26

2.4.8. The 2i-LIF media enabling culture of EGC and ESC ............................ 26

2.4.9. Epigenetic signature of PGCs and EGCs ............................................. 27

2.4.10. EGCs from different species ................................................................ 28

2.4.11. From ESC to PGCs .............................................................................. 29

3. Goals and objectives ....................................................................................... 30

4. The ter mutation in the rat Dnd1 gene causes gonadal teratomas and

infertility in both genders ............................................................................... 31

4.1. Abstract ...................................................................................................... 32

4.2. Introduction ................................................................................................ 32

4.3. Material and Methods ................................................................................. 35

4.4. Results ....................................................................................................... 39

4.5. Discussion .................................................................................................. 42

4.6. Figures ....................................................................................................... 46

5. Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat

embryonic germ cells...................................................................................... 54

5.1. Abstract ...................................................................................................... 55

5.2. Introduction ................................................................................................ 55

5.3. Material and Methods ................................................................................. 57

5.4. Results ....................................................................................................... 62

5.5. Discussion .................................................................................................. 65

5.6. Figures ....................................................................................................... 69

5.7. Supplementary data ................................................................................... 75

6. Discussion and Conclusions ......................................................................... 79

6.1. The WKY-Dnd1ter/Ztm rat as a model for germ cell tumors ........................ 79

6.1.1. Highly conserved functions of Dnd1 in germ cell development ............ 80

6.1.2. Gonadal tumorigenesis and infertility in rodents ................................... 80

Table of contents III

6.1.3. Implications of the ter rat for human TDS and OGCTs ......................... 81

6.1.4. Gender and environment ..................................................................... 82

6.1.5. Genetic background ............................................................................. 83

6.1.6. DND1 in humans .................................................................................. 83

6.2. Derivation of pluripotent EGCs from the WKY-Dnd1ter/Ztm rat ................... 84

6.2.1. EGCs as a new approach to study rat Dnd1 ........................................ 85

6.2.2. The culture of EGCs ............................................................................. 86

6.2.3. Molecular networks of pluripotency in EGCs ........................................ 88

6.2.4. Germline competence of rat EGCs ...................................................... 89

6.3. Dnd1-related molecular mechanisms at work in germ cells ....................... 90

6.3.1. Interactions of Dnd1 and miRNAs ........................................................ 90

6.3.2. Effects of Dnd1 on EGC derivation and culture .................................... 90

6.3.3. Onset of germ cell loss and transformation .......................................... 92

6.3.4. Dnd1 in mitosis and meiosis................................................................. 92

6.4. Final conclusion and future perspectives ................................................... 93

7. Summary .......................................................................................................... 96

8. Zusammenfassung .......................................................................................... 99

9. References ..................................................................................................... 102

Acknowledgements .............................................................................................. 118

IV Publications

Publications

Research articles:

Northrup E, Zschemisch NH, Eisenblätter R, Glage S, Wedekind D, Cuppen E,

Dorsch M, Hedrich HJ. submitted. The ter mutation in the rat Dnd1 gene

causes gonadal teratomas and infertility in both genders.

Nils-Holger Zschemisch and Emily Northrup contributed equally to this work.

Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch

NH. submitted. Loss of Dnd1 facilitates the cultivation of genital ridge-derived

rat embryonic germ cells.

Held N, Smits BMG, Gockeln R, Schubert S, Nave H, Northrup E, Cuppen E, Hedrich

HJ, Wedekind D. 2011. A Mutation in Myo15 Leads to Usher-Like Symptoms

in LEW-ci2 rats. PlosOne 6(3): e15669.

Presentations at scientific meetings:

Northrup E, Eisenblätter R, Dorsch M, Hedrich HJ, Zschemisch NH; Loss of Dnd1

leads to immortalization of pluripotent rat primordial germ cells in vitro; Rat

Genomics and Models; December 2-5, 2009 in Cold Spring Harbor, New

York, USA

Zschemisch NH, Ji D, Wu Y, Northrup E, Dorsch M, McCoy A, Little L, Hedrich HJ,

Weinstein E, Cui X; Zinc-finger nuclease-mediated disruption of Rag-1 in the

rat; 9th Transgenic Technology Meeting; March 22-24, 2010 in Berlin,

Germany

Transgenic Research, 19:P355, 2010

Northrup E, Eisenblätter R, Dorsch M, Glage S., Rudolph C, Hedrich HJ, Zschemisch

NH; In vitro survival of pluripotent rat embryonic germ cells correlates with

Dnd1 deficiency; Vets for life, 11th Joint Scientific meeting of ESLAV, LAVA

and ComVet AFSTAL; September 26-28, 2010 in Toulouse, France

Publications V

Zschemisch NH, Eisenblätter R, Northrup E, Rudolph C, Dorsch M, Hedrich HJ;

Cultivation and characterization of embryonic stem cells from the WKY/Ztm

rats; The 18th International Workshop on Genetic Systems in the Rat;

November 30-December 3, 2010 in Kyoto, Japan

Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch

NH; Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat

embryonic germ cells; The 18th International Workshop on Genetic Systems in

the Rat; November 30-December 3, 2010 in Kyoto, Japan

VI List of abbreviations

List of abbreviations

2i two inhibitors

+/+ wild type

μg microgram

μl microliter

μM micromolar

A adenine

AP alkaline phosphatase

Az. Aktenzeichen

bFGF basic fibroblast growth

factor

Bmp bone morphogenetic

protein

bp base pair

C cytosine

cDNA copy DNA

CDK cyclin-dependent kinase

cm centimeter

Chr chromosome

CIS carcinoma in situ

c-terminus carboxyl-terminus

CO2 carbon dioxide

d day

d pc day post coitum

DDX4 DEAD box polypeptide 4

(synonyms: Mvh, Vasa,

RVLG)

DNA deoxyribonucleic acid

dnd dead end

Dnd1 dead end homolog 1

EB embryoid body

EC embryonal carcinoma

ECC embryonic carcinoma cell

EGC embryonic germ cell

ESC embryonic stem cell

F filial generation

FCS fetal calf serum

FELASA Federation of European

Laboratory Animal

Science Associations

fig. figure

g gram

G guanine

G0, G1, G2 gap 0, gap 1, gap 2

GCT germ cell tumor

GSK-3 glycogen synthase

kinase-3

h hour

HE hematoxylin-eosin

HSA human chromosome

ICM inner cell mass

Ig immunoglobulin

iPS cell induced pluripotent stem

cell

ITGCN intratubular germ cell

neoplasia

(synonym: CIS)

LIF leukemia inhibitory factor

LOD logarithmic odds ratio

M mitosis (chapter 2 and 6)

M molar (chapter 4 and 5)

List of abbreviations VII

min minute

miRNA/miR microRNA

ml milliliter

mM millimolar

mm millimeter

MMU mouse chromosome

mRNA messenger RNA

Mvh mouse vasa homolog

ng nanogram

nm nanometer

nt nucleotide

OGCT ovarian germ cell tumor

p short chromosome arm

PBS phosphate buffered

saline

PCR polymerase chain

reaction

PGC primordial germ cells

pmol picomolar

q long chromosome arm

RBP RNA binding protein

RRM RNA recognition motif

rEGC rat EGC

RNA ribonucleic acid

RNO rat chromosome

RT reverse transcription

S synthesis

s second

SCF stem cell factor

SD standard deviation

SD Sprague Dawley

(chapter 6)

SNP single-nucleotide

polymorphism

Sry Sex-determining region Y

suppl. supplementary

T thymine

TDS testicular dysgenesis

syndrome

TGCT testicular germ cell tumor

UTR untranslated region

WHO World Health

Organization

YST yolk-sac tumor

Introduction 1

1. Introduction

Teratomas are germ cell tumors (GCTs) consisting of various tissues from all three

germ layers. The malignant version, the teratocarcinoma, also includes

undifferentiated embryonic carcinoma cells (ECCs). It has been confirmed that the

pluripotent ECCs are the actual cancer stem cells capable of differentiating into the

various tumor tissues (Kleinsmith & Pierce, 1964). ECCs are thought to result from

neoplastic transformation of the embryonic precursor cells of the gametes, the so-

called primordial germ cells (PGCs) (Stevens, 1967; Andrews, 1998). Testicular

GCTs are often associated with male infertility and although the molecular

mechanisms remain unknown, it is apparent that malfunctions in testis stem cell

regulation and DNA fidelity can lead to either. Both genetic and environmental factors

influence the incidence of GCTs as well as infertility (Hotaling & Walsh, 2009).

The inactivation of the gene dead end homolog 1 (Dnd1) can lead to reduced fertility

in various vertebrates and gonadal teratocarcinogenesis in mice. The Ter mutation in

the Dnd1 gene of the mouse results in a depletion of PGCs causing sterility in males

and reduced fertility in females. In males some of the PGCs dodge their demise and

are transformed into ECCs leading to a heightened incidence of testicular teratomas

in Ter mice (Noguchi & Noguchi, 1985; Youngren et al., 2005). Ordinarily, teratomas

have a rare occurrence in rodents. Nevertheless, a spontaneous mutation referred to

as ter has been described in the WKY/Ztm rat strain, which led to the formation of

congenital teratomas in the ovaries and testes along with sterility (Länger et al.,

2004). The first objective of this project was to identify the mutation and gather

further information on the phenotype of the mutant rat strain.

PGCs are highly specialized cells that eventually develop into mature oocytes or

sperm. However, at the same time they must maintain the ability exhibited by the

gametes to differentiate into all cell types after fertilization (McLaren, 2003).

Therefore, ontogenetic disorders cannot only lead to a loss of PGC that causes

infertility, but also to the development of teratoma-predecessors, or even to both as

seen in the WKY/Ztm-ter rat. ECC are not the only pluripotent cells derived from

PGCs. Through in vitro culture, PGCs can be reprogrammed into another type of

2 Literature review

pluripotent cells referred to as embryonic germ cells (EGCs). EGCs and to a certain

extent also ECCs possess similar characteristics as embryonic stem cells (ESCs)

(Matsui et al., 1992; Resnick et al., 1992). Culturing wild type and ter-mutant EGCs of

both genders from PGCs of the genital ridge was the second goal of this project. For

one, we wanted the proof of principal that genital ridge-derived EGC culture is

possible in the rat. For another, establishing the EGCs culture creates an ideal and

innovative tool to study teratocarcinogenesis.

2. Literature review

2.1. The germline

2.1.1. The germline: linking generations

In sexually reproducing organisms all the genetic information passed from parents to

their offspring is transmitted through cells of the germline. Early on in embryogenesis,

the first germ cells are specified and can be distinguished from surrounding somatic

cells. This is followed by a series of pre- and postnatal proliferation and differentiation

steps until the first fully differentiated gametes develop during puberty. The fusion of

oocyte and sperm at fertilization leads to a totipotent cell with the ability to create a

new organism by combining genes derived from two parents. The fertilized oocyte is

capable of differentiating into all cell types of both the somatic line and germline, as

well as any cell of the extraembryonic membranes. This brings the germline to a full

circle of specification, differentiation and fertilization that repeats itself over and over

(Seydoux & Braun, 2006; Western, 2009). Thus, the germ cell lineage is frequently

referred to as immortal and its DNA is passed from one generation to the next, while

the somatic cell line simply exists for one lifetime (Jones, 2007; Surani, 2007;

Nayernia, 2008).

2.1.2. Cell cycle, mitosis and meiosis

The cell cycle is an ordered set of events with complex control mechanisms by which

eukaryotic cells reproduce. The four stages are G1 (gap 1), S (synthesis of DNA), G2

Literature review 3

(gap 2: cells prepare for division) and M (mitosis). Mitosis can be subdivided into

prophase, prometaphase, metaphase, anaphase and telophase. It produces two

daughter cells that are identical to the parental cells (Wolgemuth et al., 2002). A cell

can exit the cell cycle at G1 to enter G0, where it maintains its specialized functions

but no longer undergoes cell division (Johnson & Walker, 1999). Gametes are

produced by meiosis, which involves two divisions producing a total of four

genetically different cells with a haploid chromosome set. Recombination of parental

chromosomes during meiosis is responsible for the genetic variety of the daughter

cells (Hochwagen, 2008). In contrast to mitosis, meiosis consists of two successive

meiotic M-phases without an intermediate S-phase (Brunet et al., 1999). Meiosis I

leads to a reduction of chromosomes and is followed by meiosis II in which cells

undergo a division without further chromosome reduction (Hochwagen, 2008).

Analogous to mitosis, each of the meiotic divisions subsist of prophase, metaphase,

anaphase and telophase. The first meiotic prophase distinguishes between the

stages leptotene, zygotene, pachytene, diplotene and diakinesis (Page & Hawley,

2003; Nicholas et al., 2009).

2.1.3. Germ cells during embryonic development

A common signaling and developmental pathway is very probable for mammals

during germ cell development (Donovan & de Miguel, 2003; Aflatoonian & Moore,

2006; De Miguel et al., 2010). Detailed information about the rat is hard to come by,

while their close relative, the mouse, has been extensively studied. In accordance

with the existing data for mouse and rat (Witschi, 1962; McLaren, 2003; Maatouk et

al., 2006; Leitch et al., 2010), it is likely that similar pathways exists for both species,

with the rat being one to three days behind in its embryonic development (Fujinaga et

al., 1992; Hill, 2010). Therefore, the following account and timeline of germ cell

development is that of the mouse, unless specified otherwise.

Embryogenesis starts with fertilization and the formation of the zygote, leading to the

blastocyst around day 3.5 post coitum (d pc) in the mouse and one day later in the

rat (Schmidt & von Kreybig, 1965; Prather & First, 1988; De Miguel et al., 2010). The

first ancestors of the PGCs originate at d6.25pc in the posterior epiblast, in close

proximity to the extraembryonic ectoderm, after Bmp (bone morphogenetic protein)

4 Literature review

signaling (Lawson et al., 1999; Ying et al., 2001; McLaren & Lawson, 2005). During

the course of gastrulation the germ cell precursors move through the posterior

primitive streak and into the extraembryonic region (McLaren, 2003). Final

specification does not occur until d7.25pc in a group of about 40 progenitor cells

present in the extraembryonic mesoderm at the base of the allantois (Saitou et al.,

2002; Chuva de Sousa Lopes et al., 2007). At d8.5pc, the endoderm invaginates to

form the hind gut and the PGCs move along with the endodermal cells. Most of the

PGCs migrate out of the hind gut and into the dorsal body wall by d9.5pc.

PGCs enter the genital ridges from d10-11pc and remain stationary from this time on

(McLaren, 2003). As expected, the rat PGCs enter the genital ridge two days later

between d12pc and 13pc (Kemper & Peters, 1987), while human PGCs enter the

genital ridge around the 6th week of gestation (De Miguel et al., 2010). By the time

the PGCs are located in the genital ridge, they are referred to as late PGCs (De Felici

et al., 2009), gonocytes (McLaren, 2003) or as oogonia and prospermatogonia after

sexual differentiation (Richards et al., 1999; De Felici et al., 2004; Pepling, 2006).

The PGCs undergo several mitotic divisions during migration, with numbers

increasing in the mouse from around 100 at d8.5pc to 1000 at d10.5pc. This is

followed by two or three further rounds of mitosis after arrival in the genital ridge,

reaching a total of 25000 germ cells in each gonad at d13.5pc (Tam & Snow, 1981;

Saiti & Lacham-Kaplan, 2007). PGCs of both genders enter a premeiotic stage and

upregulate meiotic genes by d12.5pc (McLaren, 2003).

Male and female PGCs were indistinguishable up to this point in development and

only now, at d12.5pc, can the first sexual differences be identified (Adams &

McLaren, 2002; Western, 2009; Sabour et al., 2010). Meiosis is initiated by the

expression of the stra8 (stimulated by retinoic acid 8) gene and at d13.5pc female

germ cells start entering meiotic prophase (Koubova et al., 2006; Saiti & Lacham-

Kaplan, 2007), which is marked by germ cell arrest at the G2/M stage of the cell cycle

(Cook & Capel, 2010; Miles et al., 2010). Meiotic entry is blocked in the male by a

retinoid-degrading enzyme (Bowles et al., 2006; Saiti & Lacham-Kaplan, 2007).

Therefore, the male germ cells enter mitotic arrest in G0 between d12.5pc and

Literature review 5

d14.5pc (Adams & McLaren, 2002; Western et al., 2008) and differentiate into

prospermatogonia (McLaren, 1984, 2003; Durcova-Hills & Capel, 2008).

2.1.4. Gametogenesis

Gametogenesis is the process of cell division and differentiation, leading to the

mature haploid gamete.

In males the prospermatogonia in G0 mitotic arrest resume proliferation in the first

week after birth as spermatogonia (Chuma et al., 2005; Phillips et al., 2010). At

puberty the spermatogonia start entering meiosis, and mature sperm is formed

(Donovan, 1998). Spermatogenesis takes place in the seminiferous tubules of the

testis and is completed in about 9 days in the mouse, 13 days in the rat, and 16 days

in humans. The epithelium of the tubules consists of Sertoli cells, which provide

nourishment, different stimuli and protection for the developing sperm. During the

proliferation phase the spermatogonia undergo mitotic divisions to form

spermatocytes, which divide by meiosis and form the haploid spermatids.

Metamorphosis of the spermatids, including structural modifications and acrosome

formation, finally leads to the development of spermatozoa (Hermo et al., 2010). A

subpopulation of spermatogonia, the spermatogonial stem cells, retains the unique

ability of self-renewal as well as the capacity of differentiating to spermatozoa.

The process of gametogenesis is quite different in females. Although there has been

some recent controversy whether neo-oogenesis might exist, it is certain that for the

most part the replication of oocytes ceases before birth (Schnorr, 1996; Notarianni,

2011). The female oogonia only divide by mitosis during embryogenesis. After

entering the meiotic prophase, they differentiate into primary oocytes that arrest in

the diplotene stage of prophase I around the time of birth (McLaren, 2003). This

stage corresponds to the G2 phase of the cell cycle (Goren & Dekel, 1994). At the

end of prophase I, the oocytes and a surrounding layer of epithelial cells generate the

primordial follicles. The meiotic resting phase that then begins is called the dictyotene

and lasts until puberty, when the first oocytes complete meiosis I, resulting in a polar

body and the secondary oocyte. Meiosis II follows suit as it is initiated by the haploid

secondary oocyte. The oocyte is released from the follicle during ovulation (Schnorr,

1996). In most vertebrates meiosis is halted at the metaphase II stage at the time of

http://en.wikipedia.org/wiki/Metaphase_II

6 Literature review

ovulation until fertilization occurs and the second meiotic division is completed

(Verlhac et al., 1994; Eppig et al., 1996).

2.2. Germ Cell Tumors

2.2.1. Classification and origin of germ cell tumors

Tumors classified as germ cell tumors (GCTs) are derived from cells belonging to the

germ lineage (Looijenga & Oosterhuis, 1999). It is a heterogeneous group of

neoplasms with varying histopathological and clinical manifestations (Bahrami et al.,

2007). For the most part analogous tumors can be found in ovary and testes,

however, even though the morphology is similar, incidence and malignity differ

between the genders (Ulbright, 2005). The modern classification system for human

GCTs was first proposed by Oosterhuis and Looijenga in 2005 and is recognized by

the World Health Organization (WHO). The four major types of GCTs diagnosed in

the gonad are as follows: type I (teratomas and yolk-sac tumors of neonates and

infants); type II (seminomas/dysgerminoma and non-seminomas); type III

(spermatocytic seminomas of the testes; typically in older men); type IV (Dermoid

cysts of the ovary) (Oosterhuis & Looijenga, 2005).

Type I GCTs are diagnosed before puberty and the biparental, partially erased

patterns of genomic imprinting suggest that the precursor cells are earlier

PGCs/gonocytes (Stevens, 1967; Oosterhuis & Looijenga, 2005). These tumors lack

typical precursor lesions (carcinoma in situ), and the PGCs are directly transformed

into ECCs (Bahrami et al., 2007; Kristensen et al., 2008). Teratomas are

karyotypically normal (Anderson et al., 2009) while the yolk-sac tumors (YST) may be

aneuploid (Looijenga & Oosterhuis, 1999).

GCTs type II are divided into two histological subgroups with different biological and

clinical features: seminomas/dysgerminoma and non-seminomas (von Eyben, 2004;

Hussain et al., 2008; McIntyre et al., 2008). Some GCTs contain elements of both

forms (McIntyre et al., 2008). Pure seminomas occur in the testes and consist of a

homogeneous cell population that has undergone gonadal differentiation and

resembles PGCs and gonocytes (McIntyre et al., 2008). Dysgerminomas are ovarian

germ cell tumors (OGCT) with an identical morphology as the seminoma (Ulbright,

http://en.wikipedia.org/wiki/Fertilization

Literature review 7

2005). The more malignant non-seminomas are a very heterogeneous group of

tumors that can contain different histological elements including teratoma, embryonal

carcinoma (EC), YST and choriocarcinoma (Lee-Jones, 2003; Oosterhuis &

Looijenga, 2005). Pure forms of non-seminomas are relatively rare and, instead,

mixed GCTs with a combination of components are more frequent (Lee-Jones, 2003;

Chieffi et al., 2009). Genomic imprinting is erased, and the development of the

aneuploid type II GCTs probably involves late PGCs/gonocytes that are blocked from

their physiological differentiation (Oosterhuis et al., 1989; Oosterhuis & Looijenga,

2005; van de Geijn et al., 2009).

All TGCTs type II stem from the precursor lesion carcinoma in situ (CIS) (known as

intratubular germ cell neoplasia, in short ITGCN, according to the WHO) localized in

the seminiferous tubules that progresses towards invasiveness. The CIS gives rise to

either seminoma or ECCs (Andrews, 1998; Leendert et al., 1999; Krausz &

Looijenga, 2008; Kristensen et al., 2008). Cells of the EC are the actual pluripotent

cancer stem cells (Kleinsmith & Pierce, 1964; Wicha et al., 2006) and have the ability

to differentiate into embryonal or adult tissue (teratoma) as well as extraembryonal

tissue (YST and choriocarcinoma) (von Eyben, 2004; van de Geijn et al., 2009).

In contrast to the TGCTs, little is known for certain about the pathogenesis of OGCTs

(Faulkner & Friedlander, 2000). The malignant dysgerminomas are expected to stem

from PGCs before meiosis of the oocytes (Hoei-Hansen et al., 2007), while the

dermoid cysts, also known as type IV benign ovarian teratomas, seem to stem from

germ cells having completed meiosis I (Ulbright, 2005). Postpubertal testis tumors

are often aneuploid and have a consistent chromosomal abnormality of HSA12p

(Oosterhuis & Looijenga, 2005; Ulbright, 2005; Bahrami et al., 2007) also seen in

malignant OGCTs. The histological, genetic, and biological similarities found in

malignant GCTs of ovary and testes suggest a shared pathogenesis (Faulkner &

Friedlander, 2000).

2.2.2. Teratomas and Teratocarcinomas

Teratomas develop from pluripotent ECCs and therefore contain normal derivatives

from all three germ layers: ectoderm, mesoderm and endoderm. They consist of an

assortment of tissues such as cartilage, muscle, epithelium and neuronal tissue and

8 Literature review

can include organoid structures such as teeth or bone fragments (Andrews, 1998;

Donovan, 1998; Bulic-Jakus et al., 2006).

Dermoid cysts of the ovary are usually classified as benign teratomas, albeit the fact

that they only contain derivatives of mesoderm and endoderm (Akgul Ozmen et al.,

2009). In some cases tumors consisting of one germ layer have been defined as

monodermal teratomas (Bahrami et al., 2007). A teratoma is considered immature

when it contains embryonal or other immature structures and mature when it consists

of differentiated, adult-like cells (Bosl & Motzer, 1997). Incomplete somatic

differentiation of the pluripotent cells leads to malignant teratocarcinomas, which

retain clusters of pluripotent ECC alongside the differentiated tissue (Andrews, 1998,

2002; Yu & Thomson, 2008). As teratocarcinomas consist of more than one germ cell

component, they belong to the group of mixed germ cell tumors (Stamatiou et al.,

2009).

Pure, mature human teratomas found in the ovaries and prepubertal testes are

generally benign while teratomas found in the postpubertal testes and immature

teratomas of the adult ovaries are often malignant (Ulbright, 2005). Human teratomas

are extragonadal or gonadal tumors and the main sites are the sacrococcygeal

region, the ovaries and the testes (Harms et al., 2006).

2.2.3. Incidence of germ cell tumors in humans

Type I GCTs include mature and immature teratomas as well as yolk sac tumors and

have a very low incidence of 0.5-2.0 of 100 000 (Nerli et al.). Although even the most

frequent of all TGCTs, type II TGCTs, are rare tumors and comprise only 1% of all

cancers in men, their importance derives from the fact that with 60% it is the most

common malignant tumor in 20-40 year old Caucasian men (Jemal et al., 2004;

Krausz & Looijenga, 2008; Greene et al., 2010). Furthermore, the incidence is on the

rise and has more than doubled over the past 50 years (Huyghe et al., 2003; Jemal

et al., 2004). The reasons for this development are still unclear.

GCTs account for 95% of all testicular tumors, split evenly between seminomas and

non-seminomas with a small fraction being a combination of both (Looijenga &

Oosterhuis, 1999; Hussain et al., 2008). According to Ulbright, pure teratomas

account for 4% of all TGCTs, while embryonal carcinomas make up 10%, and 33%

Literature review 9

are mixed germ cell tumors (Ulbright, 2005). The most frequent mixed GCT are

teratocarcinomas, which are responsible for 25% of all TGCTs (Stamatiou et al.,

2009).

Ovarian Germ Cell Tumors (OGCT) are mainly diagnosed in young women and

account for over 60% of all ovarian neoplasms in children and adolescents and for

15-20% in all age groups (Lee-Jones, 2003). The majority of OGCTs are benign and

the most frequent are the benign teratomas known as dermoid cyst, with an

incidence of 95% (Faulkner & Friedlander, 2000; Ulbright, 2005). Malignant OGCTs

account for the remaining 5% (Faulkner & Friedlander, 2000) and, with an incidence

of only 2%, dysgerminomas are the second most common OGCT (Ulbright, 2005).

2.2.4. Genetic and environmental factors in human GCTs

Multiple observations suggest that genetic factors play an important role in the

development of GCTs and could be a contributing factor in over a quarter of all TGCT

patients (Nicholson & Harland, 1995). Familial clusters of OGCTs, TGCTs or both

have been identified (Stettner et al., 1999; Giambartolomei et al., 2009; Greene et al.,

2010; Kratz et al., 2010), and family history is a significant risk factor increasing the

development of TGCT (Greene et al., 2010). Prevalence of TGCTs varies among

different races and geographic locations and is highest in Caucasians, especially in

the residents of Scandinavia, New Zealand and Germany (Bahrami et al., 2007). The

incidence of OGCT in the United States is elevated in Hispanics and Asian-Pacific

Islanders (Smith et al., 2006).

Pre-natal or post-natal environmental factors play a role in TGCT development as

immigrants to Sweden were shown to retain the risk of their native country, while the

incidence seen in their children is comparable to that of native Swedes (Hemminki &

Li, 2002). These factors have not been determined yet, but high maternal estrogen,

organic pollutants in the mother‟s blood or maternal smoking might affect testicular

development and increase chances of TGCTs in sons (Skakkebaek et al., 2007;

Chieffi et al., 2009). The risk is also augmented in some professions or by low birth

weight and small testes (Bosland, 1996; Sharpe, 2006). High maternal body mass

and the use of exogenous hormones during pregnancy raises the risk for OGCTs

(Walker et al., 1988).

10 Literature review

However, low-risk groups such as African Americans maintain their low TGCT risk

even when living in high-incidence areas, showing a case of genetics over

environment (Gajendran et al., 2005). Linkage analysis and simulation analysis

showed that multiple loci and not one major gene are responsible for TGCTs

(Crockford et al., 2006; Zhu et al., 2007a; Krausz & Looijenga, 2008). The overall

mutation rate is low in TGCTs in comparison to other solid cancers. Activating

mutations have been found in the proto-oncogene BRAF, in KRAS-2, c-kit and the

microRNA cluster 371-373 (Voorhoeve et al., 2006; Krausz & Looijenga, 2008).

2.2.5. Infertility and TGCTs

The exact molecular mechanisms remain unknown, but infertility is associated with

testicular cancer, and disturbances in DNA fidelity or stem cell regulation can lead to

either. Organized, high-speed cell divisions are essential in spermatogenesis and

precisely these are responsible for its high sensitivity to interruptions of germ cell

development by different genetic, hormonal and environmental factors (Hotaling &

Walsh, 2009). Infertile men exhibit an increased risk of developing testicular cancer;

furthermore declining fertility might be the first marker for TGCTs (Hotaling & Walsh,

2009; Walsh et al., 2009; Burns et al., 2010).

Skakkebaek and colleagues have suggested that poor semen quality, infertility,

testicular cancer, cryptorchidism and hypospadias are different symptoms belonging

to one entity named the testicular dysgenesis syndrome (TDS) (Skakkebaek et al.,

2001; Skakkebaek et al., 2003; Skakkebaek et al., 2007). Evidence for this is found

in the rising incidence and the similar geographic distributions of the different

symptoms (Hemminki & Li, 2002; Skakkebaek et al., 2007; Hotaling & Walsh, 2009).

The hypothesis is that disruptions in embryonal programming or gonadal

development occur during fetal life, and can result in any of the symptoms belonging

to TDS (Skakkebaek et al., 2001; Walsh et al., 2009; Burns et al., 2010).

The situation in women is a different one as, so far, there is no evidence linking

infertility to the development of GCTs in the ovary.

Literature review 11

2.2.6. Teratomas in mice

To date, all teratomas observed in mice most closely resemble prepubertal GCTs.

GCTs of mice and the infantile testis both arise from PGCs and lack CIS as well as

the karyotypic abnormalities (e.g. in HSA12p) found in adult TGCTs (Walt et al.,

1993; Anderson et al., 2009). Therefore, they are most likely a model for type I

GCTs, which also means that there are no animal models available for type II GCTs

(Looijenga & Oosterhuis, 1999; Oosterhuis & Looijenga, 2005). It has been

suggested that the lack of type II GCTs in rodents is related to their rapid embryonic

development and brief puberty not allowing the neoplastic germ cells to accumulate

further genetic and epigenetic changes (Zhu et al., 2007a). In general, GCTs are

uncommon in rodents (Stevens, 1980). Parthenogenetic oocytes arrested in the

metaphase of meiosis I can lead to ovarian teratomas in the inbred LT/Sv mouse

strain (Eppig et al., 1996). Only mice of the inbred strain 129/Sv develop testicular

teratomas spontaneously at a rate of 1-10% (Stevens & Hummel, 1957; Youngren et

al., 2005). The incidence of teratocarcinomas is considerably increased through the

Ter mutation of the Dnd1 gene in males of this, and only this, strain, while fertility is

reduced in both genders of numerous strains (Noguchi & Noguchi, 1985; Zhu et al.,

2007a). Loss of Dmrt1 causes a high incidence of teratomas in males of the same

129/Sv mouse strain, with only testicular dysgenesis and no tumors found in the

C57BL/6J as background strain (Krentz et al., 2009). The pgct1 locus on

chromosome (Chr) 13 segregates with the male teratoma phenotype of the 129/Sv

strain (Muller et al., 2000). Furthermore, the chromosome substitution strain

129.MOLF-Chr19 in which Chr 19 of 129/Sv+/+ was replaced by its MOLF-derived

homologue shows a high frequency (70-80%) of GCTs in the testis. The PGC-

specific knockout of Pten (phosphatase and tensin homolog) causes teratomas in all

male and even in some female mice and coincides with PGC loss (Kimura et al.,

2003). Another tumor suppressor whose absence leads to TGCTs is p53, which may

play a role in promoting cell cycle arrest in PGCs around d13.5pc (Matin, 2007).

Interruptions in germ cell development can also cause infertility without teratoma

development. This is seen in mice carrying the germ cell-deficient mutation caused

by the deletion of the Fancl gene (Pellas et al., 1991; Agoulnik et al., 2002), as well


as in mice with the homozygous dominant white spotting or steel mutation of the loci

encoding for the c-kit receptor or its ligand the stem cell factor (also known as SCF or

Kitl), respectively. However, teratoma incidence is increased in heterozygous

steel/wild type males (Stevens, 1981; Noguchi & Noguchi, 1985; Donovan & de

Miguel, 2003).

2.2.7. Teratomas in rats

Teratomas rarely occur in rats. A few isolated cases of spontaneous teratogenesis

have been observed in the adrenal gland, kidney, central nervous system and

abdomen of Sprague-Dawley and Wistar rats (Schardein & Fitzgerald, 1977;

Ninomiya, 1983; Itoh et al., 1985). Hereditary teratomas of the ovary and testes have

been found in the Csk:Wistar-Imamichi strain in Japan. The strain was denominated

Tera, and a mutation with a recessive mode of inheritance was thought to cause the

teratomas (Miwa et al., 1987). However, this strain no longer exists and the

underlying mutation was never identified. Länger and colleagues have described a

spontaneous mutation in the inbred WKY/Ztm rat strain leading to teratocarcinomas

of the ovary and testes (Länger et al., 2004). They established a coisogenic mutant

strain by mating the ancestors of the affected animals. Analogous to the Ter mutation

in the mouse and based on the hypothesis of a single recessive ter allele causing the

teratomas, this new animal model for teratomas was denominated WKY/Ztm-ter.

Unlike the Ter mouse, where tumors only arise in males, the ter rat developed

teratomas in both genders with an ongoing high incidence of 25%. However, tumor

progression was influenced by gender, and the females developed mostly bilateral

tumors between day 21 and 63, while the males developed tumors between day 14

and 224, with 50% being unilateral. The partially undifferentiated teratocarcinomas

consisted of derivatives from all three germ layers, approximately 10%

undifferentiated cells and also included immature tissue resembling embryonic

structures (Länger et al., 2004).


2.3. The Dead end homolog 1 gene (Dnd1)

The dead end genes play an important role in survival, migration and development of

primordial germ cells in different species. The dead end homolog 1 genes in mice

(Dnd1), rats (Dnd1) and humans (DND1) are orthologs of the dead end (dnd) gene

expressed in zebrafish and African clawed frog (Youngren et al., 2005).

2.3.1. Dead end (dnd) in anamnia

In some non-mammalian species, such as zebrafish (Danio rerio) and the African

clawed frog (Xenopus laevis), the germ cell lineage is established by the assembly of

germ plasm in the cytoplasm of the oocyte. Cells inheriting the preformed germ

plasm, which is rich in RNAs, RNA-binding proteins and ribosomes, ultimately give

rise to the germ cell lineage (Fox et al., 2007; Richardson & Lehmann, 2010).

Therefore, the germ plasm is one of the unique features of PGCs (Kedde & Agami,

2008), and several germ plasm components involved in the early PGC development

in lower organisms are also expressed in late mammalian PGCs of the genital ridge

(Weidinger et al., 2003). Dead end (dnd) is specifically expressed in the germ plasm

of zebrafish and the African clawed frog throughout embryogenesis (Weidinger et al.,

2003; Horvay et al., 2006). The 3‟untranslated region (UTR) of dnd in zebrafish is

responsible for the germ cell specific expression as 3‟UTR-dnd injected into the 1-cell

stage is degraded in somatic cells and protected in germ cells (Slanchev et al.,

2009).

Knockdown of dnd in zebrafish initially allows normal PGC specification, however, it

then blocks the restriction of PGCs to the deep blastoderm and leads to large

numbers of ectopic PGCs in the outermost cell layer. Furthermore, PGCs no longer

exhibit a motile behavior, and failure to migrate is followed by the demise of PGCs on

the first day of development (Weidinger et al., 2003). PGCs of the African clawed frog

disappear at the tadpole stage of the embryos as a consequence of inhibiting the dnd

translation. Early specification does not appear to be affected, but PGCs do not

migrate dorsally and cease to exist (Horvay et al., 2006).

Consequently, Dnd is required for PGC migration in both species, with no effects

observed in PGC specification or somatic cells. The similar phenotype observed in


anamnia, along with the reciprocal functional redundancy of mouse and zebrafish

Dead end indicates an evolutionary conserved role of dnd in germ cell development

in vertebrates (Kedde et al., 2007; Slanchev et al., 2009).

2.3.2. The Ter mutation in the mouse Dead end homolog 1 (Dnd1)

The spontaneous ter mutation that took place in the 129/Sv strain some 40 years ago

(Stevens, 1973) has been traced to the inactivation of the Dead end homolog 1

(Dnd1) gene on mouse Chr 18. A single base change (cytosine to thymine)

introduces a termination codon in the coding region of Dnd1 and ends up causing

germ cell deficiency in both genders along with testicular teratomas (Asada et al.,

1994; Matin & Nadeau, 2005; Youngren et al., 2005). The teratoma incidence in male

129/Sv-Ter mice was 94% in homozygous Ter/Ter and 17% in heterozygous Ter/+

mice in comparison to the 1.4% in wild type (+/+) mice. Teratocarcinogenesis in

Ter/Ter males was unilateral in 25% of the cases and associated with reduced size

and weight of non-tumorous testis (Noguchi & Noguchi, 1985). No germ cells are

visible in Ter/Ter males after birth as all PGCs either perish or transform into ECC

(Zhu et al., 2007a). Germ cell number is not affected in Ter/+ as there were no signs

of germ cell deficiency in mice presumed to be Ter/+ (Noguchi & Noguchi, 1985), and

the number of PGCs in Ter/+ embryos did not differ from that in +/+ embryos

(Sakurai et al., 1995). Although all homozygous males are sterile, a number of the

females remain fertile. Decreased fertility in Ter/Ter females is linked to smaller

ovaries containing only 10% of the oocytes found in wild type 129/Sv mice (Stevens,

1973; Noguchi & Noguchi, 1985). Transfer of Ter onto other genetic backgrounds

resulted in germ-cell deficient mice with small gonads, but without

teratocarcinogenesis (Noguchi & Noguchi, 1985; Sakurai et al., 1995). Therefore, the

primary effect of the Dnd1 mutation in homozygous mice is the progressive loss of

PGCs in Ter/Ter animals during embryonic development. PGC reduction starts at

d8pc in all congenic strains and can be seen throughout the migratory and

proliferative phase (Sakurai et al., 1995). The migration of the PGCs to the genital

ridge does not seem to be affected by the Dnd1 deficit (Sakurai et al., 1995;

Youngren et al., 2005). Tumor development is presumed to start around embryonic

d12.5pc and the PGCs are transformed into EC cells that are first visible in the gonad


at d14.5pc (Noguchi & Stevens, 1982; Rivers & Hamilton, 1986; Matin, 2007). They

proliferate within the developing seminiferous cord, rupture the basement membrane

to invade the interstitial space and proliferate in the gonads after birth. At about 5

days after birth most EC cells differentiate (Oosterhuis & Looijenga, 2005; Zhu et al.,

2007a) and nearly all tumors can be detected macroscopically by 3-4 weeks of age

(Heaney & Nadeau, 2008).

At d6.75pc Dnd1 is expressed in PGC precursors and somatic neighbors at a specific

frequency, whereas this gene is markedly upregulated only in PGCs after d7.25pc

(Yabuta et al., 2006). Furthermore, Dnd1 expression was detected in the genital

ridges of d11.5pc embryos and it was upregulated in the male gonad between d12.5

and 14.5pc, while it was downregulated in the female gonads during this period

(Youngren et al., 2005). Dnd1 is detectable in embryonic germ cells (EGC) and

embryonic stem cells (ESC) from the mouse (Bhattacharya et al., 2007; Zhu et al.,

2007a), but not in Sertoli cell lines (Youngren et al., 2005) or embryonic Sertoli cells

(Cook et al., 2009). In the adult mouse Dnd1 is expressed in the heart and testis, but

not in other tissues such as brain, spleen, lung, kidney or liver (Youngren et al., 2005;

Bhattacharya et al., 2007).

2.3.3. Modulating the phenotype of Ter/Ter mice

Genetic background and gender are the decisive factors in teratoma development

but only the latter plays a role in germ cell development. Bax-dependent apoptosis is

a physiological pathway to remove extragonadal PGCs in the mouse (Stallock et al.,

2003) and may be one pathway eliminating Ter/Ter germ cells from the fetal testis

prior to neoplastic transformation (Cook et al., 2009). Crossing of Ter/Ter to a Bax-

null background resulted in a partial rescue of germ cells in the embryo (Cook et al.,

2009). Furthermore, teratomas developed in Ter/Ter males on a mixed genetic

background of C57BL/6J and 129/Sv in homozygous (90%) or heterozygous Bax

(44%) mutants (Cook et al., 2009). Complete backcrossing to the C57BL/6J led to

survival of the germ cells without teratocarcinogenesis (Cook et al., 2011). Double-

mutant females on a mixed background did not develop teratomas, but were fertile

and maintained an increased number of oocytes (Cook et al., 2009).


Extrinsic factors, such as the environment provided by somatic cells of the gonads,

play a role in Dnd1-related teratoma formation (Regenass et al., 1982; Matin, 2007).

This is substantiated by the fact that Ter/Ter XX germ cells that develop in a

testicular environment give rise to the same neoplastic clusters as mutant XY germ

cells in a testis (Cook et al., 2009).

Chr X also modulates the teratoma incidence in 129/Sv males, while Chr Y has no

effect. Chr X and Y from the 129/Sv-Ter strain were substituted with those from the

C57BL/6J. Ter/Ter males with a C57BL/6J Chr X were sterile but developed less

teratomas than their counterparts. This is either due to unidentified C57BL/6J tumor

suppressor genes or 129/Sv tumor susceptibility genes on Chr X (Hammond et al.,

2007).

Furthermore, considerable interactions are seen between Ter and six TGCT

susceptibility modifiers, including mutations in p53 and SCF (KitlSl-J) as well as

segments from Chr 19 of the MOLF strain. Trans-generational epistasis affects tumor

development and the presence of one of the six genetic variants in either parent

modifies the tumor rate found in male Ter/+ offspring (Lam et al., 2007).

2.3.4. Dead end homolog 1 (DND1) in humans

Information on DND1 in humans is scarce and its physiological relevance has not

been identified. The gene is shown to be expressed in a range of normal tissues,

including testicular germ cells, Leydig cells, interstitial cells and cells from the

seminiferous tubules (Sijmons et al., 2010). To date, it is known that DND1 maps to

the chromosomal region 5q31.1 and deletions in this region have been found in germ

cell tumor tissue and cell lines (Sijmons et al., 2010). Two studies have been

performed to identify mutations in human DND1 in TGCTs. Linger and colleagues

found a rare heterozygous variant, p. Glu86Ala, with unknown effect in DND1 from 1

of 263 GCTs (Linger et al., 2008). Sijmons and colleagues screened DND1 in 272

men with TGCT and observed only one case of a nucleotide substitution c.657C > G

(p.Asp219Glu) predicted to be non-pathogenic (Sijmons et al., 2010). Therefore,

DND1 mutations are unlikely to contribute to the majority of human TGCTs. DND1 is

upregulated in some cancers, such as adenocarcinoma, leukemia or prostate cancer


(Sijmons et al., 2010). Upregulation of microRNA-24 causes a reduced DND1

expression and is observed in the tongue squamous cell carcinoma (Liu et al., 2010).

2.3.5. The RNA binding protein Dnd1

Dnd1 is an RNA binding protein (RBP) capable of prohibiting the function of several

microRNAs (miRNAs, miR). Crucial processes in normal development as well as

cancer are regulated through miRNAs inhibiting the expression of specific mRNAs, a

mechanism conserved across metazoans. Translation and stability of specific target

mRNAs is disrupted by miRNAs associating with the 3‟ untranslated region (UTRs) of

mRNAs (Valencia-Sanchez et al., 2006; Ketting, 2007).

Most miRNAs are transcribed by the RNA polymerase II as long RNAs that are then

converted to ~22 nt mature miRNAs by the RNase III enzymes Drosha and Dicer.

They are subsequently assembled into ribonucleoprotein complexes and form a

multisubunit complex referred to as miRNA-induced silencing complex (miRISC)

(Kedde et al., 2007; Filipowicz et al., 2008).

Dnd1 can counteract the miRNA-mediated repression of mRNAs by binding to the

uridine-rich regions in the 3‟UTR of target transcripts and protecting them from

miRNAs. Dnd1 contains a conserved RNA binding domain (RBD), also known as

RNA recognition motif (RRM), that it requires to counter miRNA activity (Kedde et al.,

2007; Ketting, 2007; Kedde & Agami, 2008). Dnd1 does not bind directly to the

miRNA and just how it influences miRNAs remains to be uncovered. Two possible

explanations seem plausible: Dnd1 binding of mRNAs could either block miRNA

recognition of target structures or move mRNAs to a location not accessible to

miRNAs (Ketting, 2007).

2.3.6. Zebrafish dnd, miRNAs and target genes

In zebrafish miR-430 is responsible for clearing maternal transcripts from the

developing embryo and causes the repression of nanos1 and TDRD7 in somatic cells

(Mishima et al., 2006). Nanos1 and TRDR7 expression is enabled in germ cells only

as long as dnd is present, as demonstrated by reduced target gene expression after

inhibition of endogenous dnd (Kedde et al., 2007). Furthermore, a mutation in the

3‟UTR of miR-430 led to a no longer germ cell restricted, ubiquitous expression of the


target gene transcripts (Mishima et al., 2006). The c-terminus of zebrafish DND

possesses a Mg2+-dependent ATPase activity, which is essential for the survival of

PGCs. Translation of DND mutant proteins lacking ATPase is accompanied by a

reduced expression of nanos1 and TDRD7, indicating that the ATPase of Dnd is

required for the protection of both target gene mRNAs (Liu & Collodi, 2010).

Functional DND translocates from the germ-cell nucleus to the perinuclear granules,

however, this phenomenon is not observed in proteins mutated in the RRM domain,

as these are restricted to the nucleus. This is in line with the hypothesis that DND

binds RNA in the nucleus and then transfers to the granules, where it protects the

RNA from miRNA-mediated inhibition (Slanchev et al., 2009).

2.3.7. Mammalian Dnd1, miRNAs and target genes

The human miR-93,-302,-372,-373 and-520 have been termed miRNA-373 family

and act as potential oncogenes in human germ cells (Voorhoeve et al., 2006; Kedde

& Agami, 2008). Rapid cellular proliferation as well as enhanced cellular migration,

invasion, and metastasis have been linked to the expression of the miRNA-373

family (Kedde & Agami, 2008).

Absence of p53 or alternatively the presence of miR-372/3 can lead to a neoplastic

transformation of cells (Voorhoeve et al., 2006). p53 is a tumor suppressor protein

that increases levels of p21 and blocks the cell cycle or induces apoptosis (Serrano

et al., 1997; Voorhoeve et al., 2006). High levels of p21 (Cip1) inhibit cyclin

dependent kinases (CDK) and causes cells to arrest in G1 phase. However, miR-

372/3 counteracts the p53-mediated CDK inhibition by inducing resistance to high

p21 levels (Voorhoeve et al., 2006).

Using human cell-culture based assays Kedde and coworkers found that Dnd1

antagonized the miRNA-mediated repression of three genes: LATS2, p27 (Kip1) and

Cx43 (Connexin43) (Kedde et al., 2007). Furthermore, an overexpression of Dnd1

was found to augment p27 and Lats2 expression in mouse cells (Cook et al., 2011).

Gene activity of the tumor suppressor LATS2, a serine threonine kinase, is directly

controlled by human miR-372/3. LATS2 augments p53-mediated apoptosis (Li et al.,

2003), and its inhibition might be responsible for the p21-resistance seen in miR-

372/3 expressing cells (Voorhoeve et al., 2006). The human miR-221 and miR-222


are regulators of p27, a cell cycle inhibitor and tumor suppressor. In certain cancer

cell lines a high activity of miR-221/2 is required to maintain low p27 levels and

continue proliferation (le Sage et al., 2007). Moreover, independent of whether

zebrafish or human Dnd1 were transfected, p27 levels went up in HEK293 cells

expressing miR-221/2 (Kedde et al., 2007). Expression of Cx43 gap junction

channels is required for skeletal myoblast fusion in vitro. The gap junctions are

downregulated after the myoblast fusion by human miR-206 and miR-1, which inhibit

the expression of Cx43 protein during myoblast differentiation despite the presence

of mRNA (Anderson et al., 2006; Kedde et al., 2007).

Through comparisons of the 129 and 129-Chr19MOLF mouse strain, miRNA-107 was

identified as one of the genes involved in germ cell tumor development in 129-

Chr19MOLF mice (Zhu et al., 2007b). This led Kedde and colleagues to suggest a

connection between the GCT susceptibility gene miRNA-107 and the Dnd1 mutation

in the 129 strain (Kedde et al., 2007; Kedde & Agami, 2008).

2.3.8. Interactions of Dnd1 with Apobec

The RRM of Dnd1 exhibits a high homology to the RRM of ACF (APOBEC1

complementation factor) (Youngren et al., 2005; Matin, 2007). ACF is the essential

RNA-binding co-factor of the RNA-editing enzyme APOBEC1 (Zhu et al., 2007a).

This led to speculations whether Dnd1, much like ACF, might interact with APOBEC-

like proteins (Zhu et al., 2007a; Bhattacharya et al., 2008). The APOBEC family is a

group of cytidine deaminases that includes the DNA mutator and antiretroviral factor

APOBEC3 (Conticello, 2008), which is expressed in a variety of tissues including the

testis and also in EGCs in vitro. APOBEC3 binds mRNA to inhibit miRNA-mediated

repression and was found to interact with Dnd1 in mammalian cells and the

embryonic gonads of the mouse. The functional consequences remain unknown, but

together these two proteins might modulate miRNA function. A weak interaction

between Dnd1 and APOBEC1 is thought possible but has not been confirmed

(Bhattacharya et al., 2008).


2.3.9. Role of Dnd1 in germ cell cycle and gene expression

A group of cell cycle inhibitors including p21, p27, Lats2, p53, pRB and Pten are

enriched in mouse cells by overexpressing Dnd1 (Cook et al., 2011). Both p27 and

p21 regulate the G1/G0 mitotic arrest in male germ cells via inhibition of CDKs

(Western et al., 2008). CDKs along with cyclins are the major control switches in the

cell cycle, causing the cell to move from G1 to S or G2 to M (Voorhoeve et al., 2006;

Chu et al., 2008). During the physiological transition from G1 to G0 at d14.5pc p21

and p27 are detectable in germ cells (Western et al., 2008); however, regardless of

strain background, neither is active in mutant Ter/Ter germ cells and these do not

enter G0 mitotic arrest by d17.5pc. Nevertheless, C57BL/6J Ter/Ter germ cells

committed to apoptosis arrest prior to M-phase and the cell cycle arrest is considered

the key event in preventing tumor formation (Cook et al., 2011).

Loss of Dnd1 also results in downregulation of differentiation genes (Mvh, nanos2,

nanos3) followed by an upregulation of meiotic markers (STRA8 and SCP3), which is

typical for females at this stage, and maintenance of pluripotency genes (Nanog,

Sox2, Oct4) (Cook et al., 2011). Under physiological circumstances the male-specific

Nanos2 gene inhibits meiosis by preventing Stra8 expression (Suzuki & Saga, 2008).

The genital ridge at d13.5pc in Bax and Dnd1 double mutants exhibits an

upregulation of the pluripotency marker Nanog and a downregulation of the

differentiation markers Mvh, Nanos2 and Nanos3 in comparison to Bax single

mutants (Cook et al., 2011). Mvh and E-cadherin both label male germ cells after

entry in the genital ridge while Nanog and Sox2 expression declines by the time germ

cells have entered mitotic arrest at d15.5pc (Durcova-Hills & Capel, 2008). In the

129/Sv Ter/Ter mice clusters of Mvh negative and E-cadherin, Sox2 and Nanog

positive cells are found in the gonad in lieu of individual germ cells. The C57BL/6J

Bax and Dnd1 double mutants at d17.5pc do express Mvh and E-cadherin and

downregulate Sox2 and Nanog (Cook et al., 2011).

Summarizing the data suggests that prevention of tumor formation requires

differentiation and cell cycle arrest, and Dnd1 regulates the exit from G1 phase of

mitosis to the quiescent G0 phase.


2.4. Pluripotent germ cells and their in vivo origin

2.4.1. Primordial germ cell specification in the mouse

Cells destined for the germline depend on the repression of somatic pathways and

the induction of germ cell specific programs (Seydoux & Braun, 2006). In the mouse,

the transcriptional repressor Blimp1 (Prdm1) seems to be the critical factor initiating

germ cell specification (Vincent et al., 2005; Durcova-Hills et al., 2008). Blimp1 is first

upregulated at d6.25pc in about six epiblast cells that are then restricted to the germ

cell fate (Ohinata et al., 2005; Chuva de Sousa Lopes & Roelen, 2008). Further

Blimp1-positive PGC precursors emerge between d6.25-7.25pc by induction of

additional epiblast cells and division of the existing Blimp1 population (Chuva de

Sousa Lopes et al., 2007; Chuva de Sousa Lopes & Roelen, 2010). Specification is

not simply a cell-intrinsic process, but depends on extracellular Bmp-mediated

signaling from the extraembryonic ectoderm (Ying et al., 2001; McLaren, 2003). This

has been established through the transplantation of cells to the proximal epiblast that

gave rise to PGCs (Tam & Zhou, 1996) and a chimera experiment proving that Bmp4

is required in the extraembryonic tissue (Lawson et al., 1999). Furthermore, culture of

competent epiblast cells revealed that these acquired germ-cell properties and

expressed Blimp1 after addition of Bmp4 (Ohinata et al., 2009). Germ cells become

restricted to the germline at d7.25pc, meaning that, aside from apoptosis or necrosis,

no more cells can exit or enter the germline. The specification is characterized by

changes in gene expression, cell cycle length and morphology of PGCs (Chuva de

Sousa Lopes & Roelen, 2010).

2.4.2. Identification of murine PGCs

Histologically PGCs can be identified based on their large size, low nucleus to

cytoplasm ratio, clear nuclear borders and granular nuclear chromatin. Moreover,

germ cells can be distinguished from surrounding cells through distinctive patterns of

gene expression (Saiti & Lacham-Kaplan, 2007). Different markers of PGCs have

been extensively researched, however, no single germ cell-specific marker could be

identified that is continuously expressed. Instead, there is a variety of markers


expressed in the different developmental stages (Chuva de Sousa Lopes & Roelen,

2010).

Blimp1 is the first marker of the PGC precursors at d6.25pc and is also expressed in

the 40 PGCs found at d7.25pc (Ohinata et al., 2005; Vincent et al., 2005; Chuva de

Sousa Lopes & Roelen, 2010). The PGCs continue to express Blimp1 until d13pc

and it is also expressed in various other tissues (Chang et al., 2002). Fragilis (Ifitm3)

appears to be expressed in response to Bmp4 signaling (Saitou et al., 2002). It is

detectable throughout the epiblast in d6.0pc embryos until the expression pattern

concentrates on about 150 cells including the PGCs by d7.2pc. Shortly afterwards, at

d8.5pc, Fragilis is downregulated (McLaren, 2003). Stella (Dppa3) is a PGC-specific

marker also detectable in the post natal oocyte, the zygote and the blastocyst (Saiti &

Lacham-Kaplan, 2007). Its expression commences at d7.2pc in cells showing high

Fragilis activity and continues till d15.5pc in males and d13.5pc in females. c-kit is

expressed in PGCs from their initial segregation at d7.5pc up to d13.5pc. Expression

of c-kit reappears in differentiated spermatogonia and spermatocytes until the round

spermatid stage where it is no longer detected (Prabhu et al., 2006; Saiti & Lacham-

Kaplan, 2007). Expression of the Nanos genes in the mouse is restricted to germ

cells. Nanos3 is expressed during PGC migration and expression is continued in the

genital ridge until it disappears prior to d15.5pc in the male and d13.5pc in the female

(Tres et al., 2004; Suzuki et al., 2007). The mouse vasa homologue (Mvh) gene

encodes an ATP-dependant RNA helicase of the DEADbox protein family and is also

known as DEAD box polypeptide 4 (DDX4). It is exclusively expressed in germ cells

after entry into the genital ridge, with its expression continuing in spermatogenic cells

and maturing oocyte stages of adult mice (Toyooka et al., 2000).

Primordial germ cells also maintain or reactivate the expression of typical markers of

pluripotency such as the cell surface markers alkaline phosphatase (AP) and SSEA1.

Other pluripotency markers expressed are the transcription factors required for the

pluripotency of the inner cell mass (ICM) of the blastocyst: Oct4, Nanog and Sox2

(De Felici et al., 2009; Chuva de Sousa Lopes & Roelen, 2010). In the embryo Oct4

becomes restricted to the germline at d8pc (McLaren, 2003). It persists throughout

male fetal development, and is maintained in prospermatogonia and undifferentiated


spermatogonia. However, Oct4 expression is repressed in female germ cells at

meiotic prophase I and expression is resumed after birth during the growth phase of

the oocytes (Saiti & Lacham-Kaplan, 2007). Nanog is initially downregulated in the

epiblast and then expressed during germ cell proliferation from d8.0pc. It is lost

during meiotic entry in females and decreased during mitotic arrest in male PGCs

(Yamaguchi et al., 2005). Sox2 is progressively upregulated in PGCs prior to Stella

and is downregulated after sexual differentiation (Yabuta et al., 2006; Western,

2009).

2.4.3. Defining pluripotency

Pluripotency is defined as the potential of a cell to differentiate into cells of the three

germ layers and give rise to any fetal or adult cell type. Totipotent cells have the

additional ability to develop extraembryonic tissue and exist only temporarily from the

zygote to the 4-cell stage. Cells of the ICM of the blastocyst and the succeeding

epiblast cells are considered to be pluripotent embryonic cells (Zwaka & Thomson,

2005; De Miguel et al., 2010).

Immortal cell lines exhibiting pluripotent characteristic and capable of remaining in an

undifferentiated state can be generated in vitro by more than one way (Kerr et al.,

2006). These cells include embryonic stem cells (ESC) from the ICM, as well as

EGCs and ECC from the PGCs. Only recently, the first pluripotent cells have been

derived from the testis of both neonatal mice (Kanatsu-Shinohara et al., 2004) and

adult mice (Ko et al., 2009). Even somatic cells can be induced to pluripotency and

create induced pluripotent stem (iPS) cells by transduction with vectors encoding

factors such as Oct4, Sox2, Klf4 and c-myc (Chuva de Sousa Lopes & Roelen,

2010).

Pluripotency can be analyzed in vitro through the expression of the markers

mentioned in chapter 2.4.2 and the ability to differentiate and form embryoid bodies

(EB) under specific culture conditions (Keller, 1995). Developmental potential of cells

is tested in vivo by the ability to form teratocarcinomas after transplantation to ectopic

sites or by analyzing the fate of genetically marked cells after blastocyst injection

(Donovan, 1998; Kerr et al., 2006). Pluripotent cells are capable of integrating into

the blastocyst and produce chimeras, which are made of two genetically distinct


populations of cells. The hallmark of true pluripotency is the ability of the injected cell

line to give rise to the gametes and to transmit through the germline (Kerr et al.,

2006). Cells in possession of said property can be used to generate genetically

modified animals. This is accomplished by targeting specific genes in the pluripotent

cells using homologous recombination, selecting a clone with the correct insertion

and injecting it into the blastocyst (Capecchi, 1989).

2.4.4. Differentiation potential of PGCs

Although PGCs express pluripotency associated genes, they are in fact differentiated

cells restricted in their developmental potency to becoming either oocyte or sperm.

However, the fusion of oocyte and sperm generates the totipotent fertilized oocyte

and marks the beginning of a complex new organism. Consequently, PGCs are

unipotent cells capable of giving rise to the totipotent germline. (McLaren, 1981;

Donovan, 1998; Kerr et al., 2006). This, along with the ability of PGCs to give rise to

pluripotent EGCs or ECCs, shows that PGCs retain a well-regulated capacity for

developmental pluripotency. Teratomas can be generated experimentally in vivo by

transplantation of pluripotent cells or PGCs to ectopic sites. PGCs from d11.5pc-

d13.5pc embryos generate ECCs after transplantation into the kidney or testis

capsule. The ability of genital ridges to give rise to teratomas declines with

developmental age to a point that no tumors developed from grafts of d14.5pc

gonads (Stevens, 1966). These results infer that the PGC potential changes after

colonization of the gonad anlagen (Stevens, 1967) and that mitotically proliferating

PGCs are responsible for teratoma development (Zhu et al., 2007a). However, unlike

pluripotent cells, PGCs do not differentiate into embryoid bodies and do not

contribute to either germline or soma after injection into a blastocyst (McLaren &

Durcova-Hills, 2001; Durcova-Hills et al., 2006; Kerr et al., 2006).

2.4.5. Embryonic stem cells

ESC are derived from the inner cell mass of the preimplantation blastocyst and were

first established from the 129/Sv mouse strain in 1981 by Evans and Kaufman using

feeder cells, LIF (leukemia inhibitory factor) (Evans & Kaufman, 1981). Mouse ESCs

have allowed the targeted manipulation of the mouse genome and have become the


principal technology used for genetic engineering in the mouse, due to their relatively

high capacity to contribute to germline chimeras (Capecchi, 1989; Glaser et al., 2005;

Yu & Thomson, 2008). Human ESCs have been derived under different culture

conditions and expressing a set of markers different from the mouse (Thomson et al.,

1998). For instance, SSEA1 is expressed in mouse but not human ESCs, while

SSEA3 and SSEA4 are expressed in human but not mouse ESCs (De Miguel et al.,

2010).

All attempts to establish rat ESCs failed until recently, when the first germline-

competent rat ESC were generated by inhibiting the MEK and GSK-3 pathways (Li et

al., 2008). The same inhibitors used for rat ESCs have also enabled deriving mouse

ESCs from different strains (Ying et al., 2008; Nichols et al., 2009). The first report

about a knockout of p53 using rat ESC was given little later (Tong et al., 2010).

2.4.6. Embryonic carcinoma cells

ECCs are the PGC-derived pluripotent stem cells of teratocarcinomas that can also

be kept in culture (Kahan & Ephrussi, 1970; Zwaka & Thomson, 2005). They were

the first pluripotent cells to be successfully maintained in vitro, thereby paving the

way for all others. They closely resemble EGCs and ESCs as well as their precursor

cells, the PGCs, in that they continue to express typical markers such as AP, Oct4

and Nanog (De Miguel et al., 2010). However, unlike ESC or EGC, the ECC lines are

usually genetically unstable and have an aneuploid chromosome set (Kerr et al.,

2006; De Miguel et al., 2010). Many human ECC lines and some murine ECC lines

display a limited developmental potency and are not capable of differentiation

(Andrews, 1998; Kristensen et al., 2008). Some of the mouse ECC lines are capable

of forming chimeras (Brinster, 1974), however, the majority is not, which has been

attributed to their karyotypic abnormalities (Nichols & Smith, 2009). In a couple of

cases the chimeras have morphological abnormalities and develop tumors (Rossant

& McBurney, 1982). Unlike other pluripotent cells in the embryo, the ECC remain

pluripotent in vivo. Therefore, they can be serially transplanted between adult mice

and always produce new teratomas in their transplantation site (Stevens, 1970).


2.4.7. Features of rodent embryonic germ cell derivation and culture

PGCs have the ability to de-differentiate into pluripotent cells showing clonal growth

in vitro and are then termed EGCs (Durcova-Hills et al., 2006). In the mouse the

complete conversion of PGCs to EGCs takes about 10 days (Durcova-Hills et al.,

2006). The first EGC were derived from mouse PGCs on feeder cells with media

containing the cytokines bFGF, SCF and LIF (Matsui et al., 1992; Resnick et al.,

1992). Contribution of these cells to all somatic cell lineages and the germline in

chimeras confirmed that the cultured PGCs gave rise to truly pluripotent cells. Mouse

EGCs can be obtained from different embryonic stages: pre-migratory (8.0-8.5pc),

migratory (9.5pc) and post-migratory EGCs (11.5 and 12.5pc) (Matsui et al., 1992;

Resnick et al., 1992; Labosky et al., 1994; McLaren & Durcova-Hills, 2001). After

many failed attempts, culture of d13.5pc EGCs was achieved only recently, however,

it is not known whether these cells contribute to chimeras (Shim et al., 2008).

It was long thought that derivation of EGC depends on the presence of the above-

mentioned cytokines (Donovan, 1994; Donovan et al., 2001; De Miguel et al., 2010).

However, recently mouse EGCs were derived without SCF and bFGF using feeder

cells and the 2i-LIF media described in the following chapter 2.4.8. (Leitch et al.,

2010). A similar protocol also derived the first EGCs from the rat at d10pc, which is

the equivalent to d8.5pc in the mouse. The rat PGCs were collected in media

containing FCS and bFGF on SCF-producing feeder cells and on the third day of

culture the original media was exchanged for 2i-LIF (Leitch et al., 2010).

EGC and ESC from mouse and rat appear indistinguishable in most aspects such as

morphology and expression of specific markers (Shovlin et al., 2008; Leitch et al.,

2010). However, differences are found between the ICM-derived ESC and PGC-

derived EGC lines in their respective methylation status (De Miguel et al., 2010).

2.4.8. The 2i-LIF media enabling culture of EGC and ESC

As revealed by the name, the 2i-LIF media contains two inhibitors (PD0325901,

CHIR99021) blocking different cellular pathways and the cytokine LIF. Extrinsic

factors thought to induce differentiation or cell commitment such as serum and its

substitutes are not included in the medium (Buehr et al., 2008).


Under physiological circumstances, the fibroblast growth factor FGF4 induces the

mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)

MEK pathway and drives cells into committing to a cell lineage (Voigt & Serikawa,

2009). The inhibitor PD0325901 blocks the MEK pathway by suppressing the

activation of ERK1/ERK2 (Bain et al., 2007) and helps sustain pluripotent cells

(Buehr et al., 2008; Ying et al., 2008; Leitch et al., 2010). The Glycogen synthase

kinase-3 (GSK-3) is a negative modulator of various anabolic pathways and is part of

the Wnt-signaling cascade. Inhibition of GSK-3 by CHIR99021 increases cytoplasmic

and nuclear β–catenin, which in turn activates the Wnt pathway and enhances self-

renewal (Buehr et al., 2008; Bone et al., 2009). LIF is important for maintaining the

self renewal in culture via activation of STAT3 and induction of c-myc (Bone et al.,

2009).

2.4.9. Epigenetic signature of PGCs and EGCs

Unlike cells of the ICM the PGCs are highly specified cells and are subjected to an

array of epigenetic changes during development (Shovlin et al., 2008). The original

biparental pattern of imprinting of the zygote is erased, and paternal or maternal

imprints are established (Oosterhuis & Looijenga, 2005). PGCs do not self-renew for

longer periods of time but follow a series of differentiation events instead (De Miguel

et al., 2010). Between germ cell specification at d7.25pc and sex differentiation at

d12.5pc, the germ cells exchange histone modification, reactivate the silent X-

chromosome in females, remove parental imprints so that the parental genomes

become epigenetically equal and undergo DNA demethylation (Hajkova et al., 2002;

Shovlin et al., 2008; Chuva de Sousa Lopes & Roelen, 2010). Correct sex-specific

somatic imprints are reestablished after birth in females or in later embryonic

development in males (Yamazaki et al., 2003; Chuva de Sousa Lopes & Roelen,

2010; De Miguel et al., 2010).

Methylation status of PGCs is not necessarily reflected in EGC lines, as these have

undergone epigenetic reprogramming of their own (Durcova-Hills et al., 2001; Shovlin

et al., 2008). EGC lines of different developmental stage show a reduced methylation

of many imprinted genes in comparison to ESCs (Shovlin et al., 2008; De Miguel et

al., 2010). A few of the chimeras made with late mouse EGCs exhibit skeletal


abnormalities, and this might be attributed to erasure of differential methylation in

imprinted genes. Epigenetic changes upon arrival in the genital ridge are also

thought to be responsible for the reduced efficiency seen in the derivation of EGCs

from later embryonic stages (Kerr et al., 2006; Durcova-Hills et al., 2008).

Furthermore, early EGC lines show heterogeneous DNA methylation patterns, in

contrast to the uniformity of methylation patterns seen in late EGC and ESC lines

(Shovlin et al., 2008).

Consequently, epigenetic programming displays differences between pluripotent

EGC or ESC lines and unipotent PGC lines. Clarifying the relationship between germ

cells and pluripotent cells could help shed some light on the nature of the pluripotent

state (Surani et al., 2008).

2.4.10. EGCs from different species

EGCs have been derived from a whole array of species; the mouse was simply the

first and rat the most recent. Mouse EGCs and ESCs have a similar propensity to

generate germline chimeras (McLaren & Durcova-Hills, 2001). After aggregation with

eight-cell embryos, the EGCs are shown to contribute to embryonic and to some

extent to extraembryonic lineages (Durcova-Hills et al., 2003). The EGCs from the rat

have led to genetically modified chimeras; however, these were not germline

competent (Leitch et al., 2010). Culture of porcine PGCs of the genital ridge

generated EGCs (Shim et al., 1997) and genetically modified porcine EGCs have

been able to contribute to chimeras shown to transmit through the germline

(Piedrahita et al., 1998). PGCs from chicken have been cultured and also genetically

modified while staying committed to the germline (van de Lavoir et al., 2006). The

culture conditions used to maintain chicken EGCs can also be used to obtain duck

EGCs (Guan et al., 2009). Furthermore, the successful culture of caprine EGCs

contributed to chimeric goats (Jia et al., 2008). Deriving EGCs from the PGCs in the

genital ridge of human embryos has also been possible. Reprogramming PGC to

EGCs is possible using embryos between 5-10 weeks of gestation in humans (Kerr

et al., 2006). These EGCs express the same markers as human ESCs with the

addition of SSEA1 (Shamblott et al., 1998). It has been suggested that the human

EGCs could be used as an ESC-alternative for cell and tissue replacement therapy


(Shamblott et al., 2001), however, this would require improving culture conditions and

a better understanding of human EGCs (Aflatoonian & Moore, 2005).

2.4.11. From ESC to PGCs

ESC can develop into PGCs of various developmental stages in culture, either

spontaneously or by applying differentiating culture conditions such as retinoic acid

or Bmp4-producing cells (Toyooka et al., 2003; Geijsen et al., 2004; Ko & Schöler,

2006). Germ cell markers stella and fragilis are detectable in ESCs even before the

onset of differentiation, even though they are not expressed in the inner cell mass

and early epiblast cells in vivo. In vitro PGCs are identified based on the expression

of stella and fragilis coupled with Mvh (DDX4), a marker initially not expressed in the

studied ESCs. ESC-derived Mvh-positive cells can produce functional sperm in vitro

(Toyooka et al., 2003). Using a germ cell specific Oct4 promoter, ESCs were

differentiated into oogonia that enter meiosis (Hübner et al., 2003). However,

functionality of the putative oocytes and sperm has not been established yet (Ko &

Schöler, 2006). Thus, ESCs already express specific germ cell markers and it is

relatively simple to induce complete differentiation to PGCs. A possible explanation

given by Ko and Schöler is that a subpopulation of ESCs is already committed to the

germline (Ko & Schöler, 2006).

30 Goals and objectives

3. Goals and objectives

In the WKY/Ztm rat the spontaneous ter mutation initiated infertility as well as

congenital teratomas in the rat testes and ovaries. The primary aim of this project

was to gain a better understanding of tumorigenesis and the developmental potential

of germ cells in the WKY/Ztm-ter rat strain.

This strain is the only available rat model found among the small number of animal

models already existing for GCTs. The first objective of the project was to

characterize the genotype and phenotype of the ter rat. A strong genetic component

of susceptibility for GCTs is found in both humans and mice. This made it critical to

identify the mutation causing the neoplastic transformation in the ter rat. For one, this

enables direct comparisons between different species, and for another, this is

essential to help unravel the molecular pathways leading to tumor development. In

general teratocarcinomas of the ovary are much rarer than of the testis and this rat

model has the unique ability of causing both. Therefore, a special focus of the

phenotypic analysis was to identify gender differences and similarities. Another

central point was gathering data that would allow comparing the ter rat with the Ter

mouse.

PGCs can de-differentiate into pluripotent ECCs through tumorigenesis in vivo or

EGCs through specific culture conditions in vitro. EGCs could present a powerful tool

to determine the relevant mechanisms involved in the neoplastic transformation of

PGCs. Therefore, the second goal of the project was to establish a stable culture of

wild type and ter rat EGCs derived from late PGCs of the genital ridge.

Characteristics of these EGCs were examined in various pluripotency tests, and the

expression of specific germ cell and pluripotency markers was analyzed. The

capacity of PGCs to generate EGC lines was assessed along with the proliferation

rate of these cell lines, and compared between the two different genotypes to

determine the effects of ter. Furthermore, it was discerned whether or not the wild

type cell line has the ability to contribute to germline competent chimeras. This is the

final test of pluripotency and essential, should rat EGCs ever be used as a tool for

genetic manipulation in the rat.

Chapter 4 31

4. The ter mutation in the rat Dnd1 gene causes gonadal

teratomas and infertility in both genders

Nils-Holger Zschemisch1, Emily Northrup1, Regina Eisenblätter1, Silke Glage1, Dirk

Wedekind1, Edwin Cuppen2, Martina Dorsch1, Hans-Jürgen Hedrich1

Nils-Holger Zschemisch and Emily Northrup contributed equally to this work

1 Institute for Laboratory Animal Science, Hannover Medical School

Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

2 Hubrecht Institute, KNAW and University Medical Center Utrecht

Uppsalalaan 8, 3584CT Utrecht, the Netherlands

submitted

Acknowledgements:

We thank Isabell Wittur and Cindy Elfers for excellent laboratory assistance.

Funding for this project was provided by the Cluster of Excellence REBIRTH,

Grant number: EXC 62/1.

32 Chapter 4

4.1. Abstract

We detected the formation of congenital tumors in the ovaries and testes of

WKY/Ztm rats. Histological examination revealed derivatives from all three germ

layers, thereby identifying the tumors as teratomas. These teratomas are presumed

to originate from the embryonic precursors of germ cells and the underlying mutation

was referred to as ter. Linkage analysis of 58 (WKY-ter x SPRD-Cu3) F2 rats with 52

polymorphic microsatellite markers associated the ter mutation with RNO18 (LOD =

3.25). Fine mapping with 64 SNP markers polymorphic between WKY and SPRD

rats, narrowed the ter mutation down to RNO 18p11. Sequencing of candidate genes

detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1) that

introduced a premature stop codon and is assumed to cause a truncation of the

Dnd1 protein leading to a loss of function. Genotyping of the recessive ter mutation

revealed a complete penetrance of teratocarcinogenesis in homozygous ter rats of

both genders. Moreover, the loss of the c-terminus of the Dnd1 protein caused

infertility in male and female rats. Morphologically non-tumorous testes of

homozygous ter males were reduced in size and weight. Immunohistochemical and

histological staining linked the gonadal malformation with a lack of spermatogenesis.

Additionally, oocytes were completely absent in the primary follicles of homozygous

ter females, which indicates an ontogenetic loss of germ cells. Our WKY-Dnd1ter/Ztm

rat could serve as an animal model to investigate gonadal teratocarcinogenesis and

the molecular mechanisms of gamete development, as well as the differentiation

potential of primordial germ cells.

4.2. Introduction

Tumors classified as germ cell tumors (GCTs) are derived from cells belonging to the

germ lineage (Looijenga & Oosterhuis, 1999) and are a very heterogeneous group of

neoplasms with varying histopathological and clinical manifestations (Bahrami et al.,

2007). Testicular germ cell tumors (TGCT) are the most common malignant

neoplasms in young Caucasian men. The incidence of TGCTs ranges from 6-11 per

100,000 and has doubled during the past 50 years (Chieffi et al., 2009; Hotaling &

Chapter 4 33

Walsh, 2009). In young women, 20-25 % of all ovarian neoplasms are ovarian germ

cell tumors (OGCT). The majority of OGCTs are benign ovarian teratomas (dermoid

cysts), and only 5% are characterized as malignant OGCTs (Faulkner & Friedlander,

2000; Giambartolomei et al., 2009). Genetic factors play an important role in the

development of GCTs, although multiple loci and not one major mutation seem to be

involved. To date, the molecular principles underlying OGCT development remain

poorly understood. However, in the testes silencing of p53, microRNA-372/373

activity, as well as mutations in the c-KIT, KRAS-2, and BRAF genes can contribute

to the neoplastic transformation of germ cell precursors (Crockford et al., 2006;

Krausz & Looijenga, 2008). Various male reproductive disorders, including infertility

and TGCTs, are thought to result from similar disruptions during fetal development

and have been grouped together in the so-called testicular dysgenesis syndrome

(TDS) (Skakkebaek et al., 2001).

Teratocarcinomas are malignant GCTs consisting of derivatives from all three germ

layers and undifferentiated embryonic carcinoma cells (ECCs). The pluripotent ECCs

are the stem cells of GCTs that are capable of differentiating into the various tumor

tissues (Kleinsmith & Pierce, 1964) and originate from a neoplastic transformation of

germ cells during embryonic development (Stevens, 1967; Vilar et al., 2006). In

general, teratomas can be classified into prepubertal type I GCTs and the more

common postpubertal type II GCTs. Type I GCTs have a biparental partially erased

pattern of genomic imprinting suggesting that the precursor cells are earlier

primordial germ cells (PGCs) or gonocytes that are directly transformed into ECCs.

Genomic imprinting is erased in the aneuploid type II GCTs and probably involves

late PGCs/gonocytes that are transformed into precursor lesions (CIS/ITGCN) before

being transformed into ECCs (Oosterhuis & Looijenga, 2005; Kristensen et al., 2008;

van de Geijn et al., 2009).

Spontaneous formation of testicular teratomas was described in the 129/Sv mouse

strain in the early 1950ies (Stevens, 1959, 1973). TGCT development was detected

in 1% of male 129/Sv mice, while an additional mutation in a modifier gene referred

to as Ter increased the incidence of TGCT to 17 % in Ter/+ and to 94 % in Ter/Ter

males (Stevens, 1984; Noguchi & Noguchi, 1985; Asada et al., 1994).

34 Chapter 4

Teratocarcinogenesis in the male 129/Sv-Ter mice was associated with infertility in

male and decreased fertility in Ter/Ter female mice (Noguchi & Noguchi, 1985),

which has been attributed to the embryonic loss of PGCs (Rivers & Hamilton, 1986;

Sakurai et al., 1995). Linkage analysis by Asada and colleagues located the modifier

gene carrying the Ter mutation on mouse chromosome 18. The Ter mutation was

identified as a C to T substitution in the Dnd1 gene introducing a premature stop

codon and leading to functional inactivation (Asada et al., 1994; Youngren et al.,

2005).

Weidinger and co-workers showed that the downregulation of dnd in zebrafish

inhibited the embryonic migration of PGCs into the developing gonads and led to the

ontogenic loss of the PGCs (Weidinger et al., 2003). In Xenopus laevis embryos, the

ablation of dnd also blocked PGC migration and initiated the death of PGCs, thereby

showing the conserved functions of the dead end gene in anamnia (Horvay et al.,

2006).

Dnd1 is an evolutionary conserved RNA-binding protein (RBP) that competes with

microRNAs for the binding of target gene mRNAs, as shown in human and mouse

cells. Dnd1 inhibits the miRNA-mediated repression of the tumor suppressor genes

p27 and Lats2, the germ cell specific genes Nanos1 and Tdrd7 as well as

Connexin43 (Kedde et al., 2007; Ketting, 2007). Moreover, Dnd1 is postulated to

form complexes with APOBEC3, a multifunctional protein also involved in the

inhibition of miRNA-mediated mRNA repression (Bhattacharya et al., 2008).

In 2004 Länger and co-workers reported a spontaneous recessive mutation in

WKY/Ztm rats referred to as ter that initiated congenital teratomas in the rat testes

and ovaries (Länger et al., 2004). Here we report the identification of ter as a point

mutation in the rat Dnd1 gene, which leads to a premature stop codon. Unlike the

mouse, Dnd1 is essential for survival and inhibition of the neoplastic transformation

of germ cells in all rats of the WKY-Dnd1ter/Ztm strain. While the mouse Dnd1 was

characterized as a modifier gene that simply modulates teratoma formation in males,

our data suggests that rat Dnd1 acts as a tumor suppressor gene in both genders.

Altogether, the inactivation of Dnd induces the loss or reduction of fertility in

vertebrates and is accompanied by gonadal teratocarcinogenesis in rodents.

Chapter 4 35

4.3. Material and Methods

Animals

All animals were bred and maintained at the Institute for Laboratory Animal Science,

Hannover Medical School, Germany (subline code: Ztm: http://www.mh-

hannover.de/einrichtungen/tierlabor). The experiments were in accordance with the

German Animal Welfare Legislation (Tierschutzgesetz 2006).

Husbandry

The WKY/Dnd1ter/Ztm (further abbreviated as WKY-ter) colony is maintained as a

segregating inbred strain by mating littermates or parents known to carry the

mutation. Microbiological status was monitored according to FELASA

recommendations (Nicklas et al., 2002) and the WKY/Ztm rats were positive for

parvovirus and apathogenic protozoa. Rats were kept in groups of three animals

under a 14:10 light-dark cycle and 55 5% humidity. They received an autoclaved

commercial pelleted diet (Altromin 1314) (protein 22%, fat 5%, raw fiber 4.5%, ash

7%, utilizing energy 3.1 kcal/g) and water ad libitum. The commercial softwood

granulate bedding was sterilized (Lignocel, Altromin; Lage, Germany).

Linkage analysis

We created a F1 generation by mating heterozygous male WKY-ter rats with female

SPRD-Cu3 rats to identify teratocarcinoma susceptibility loci. SPRD-Cu3 was

chosen, because it is easily distinguished from the WKY-ter strain by microsatellites.

The F1 generation was used to generate a [WKY-ter x SPRD-Cu3] F2 generation

(n=58). All offspring was monitored daily for tumors by inspection and palpation of the

scrotum and the abdominal cavity.

DNA samples

Genomic DNA was extracted from the tissues using the NucleoSpin™ Tissue kit

(Macherey-Nagel, Dueren, Germany) according to the manufacturer‟s instructions.

http://www.mh-hannover.de/einrichtungen/tierlabor


36 Chapter 4

Microsatellite analyses

All oligonucleotides used in this study were synthesized by MWG Biotech AG

(Ebersberg, Germany). The microsatellite markers were selected using rat genome

maps published by the Whitehead Institute, the Rat Genome Database and Ratmap.

All primers were tested against a panel of the progenitor strains and the F1

generation to determine the polymorphic nature of the microsatellite markers.

Approximately 52 microsatellite markers proved to be polymorphic between WKY-ter

and SPRD-Cu3 and were therefore used in a genome wide screen of the F2

progeny. A complete list of all used microsatellites can be requested from the

authors.

The PCR reaction was performed according to the manufacturer‟s instructions

(Peqlab Biotechnologie, Erlangen, Germany) using 100 ng DNA template per well in

a 96-well plate (MultiRigid Ultra Plates™, Roth, Karlsruhe, Germany) and amplified in

a PTC-200 thermocycler (Biozym, Hess. Oldendorf, Germany). The amplification

consisted of the following steps: 4 min at 94 °C; 35 cycles: 15 s at 94 °C, 1 min at

55°C, 2 min at 72 °C; 7 min final extension at 72°C. PCR products were analyzed by

electrophoresis in 3 % Nusieve™ agarose gels (Biozym, Hess. Oldendorf, Germany).

Gels were stained using Gelstar™ (Cambrex, Apen, Germany) and documented by

UV light illumination at 312 nm.

Sequencing

Resequencing of all four exons was managed using LIMSTILL, LIMS for Induced

Mutations by Sequencing and TILLing (Victor Guryev, E.C., unpublished). This web-

based publicly accessible information system (http://limstill.niob.knaw.nl) was used to

generate the Dnd1 project and visualize the gene structure based on the Ensembl file

ENSRNOG00000016894. The primer design application within LIMSTILL is Primer3-

based, and parameters are set to design primers with an optimal melting temperature

of 58°C.

PCR was done using a touchdown thermocycling program (92C for 60 s; 12 cycles

of 92C for 20 s, 65C for 20 s with a decrement of 0.4C per cycle, 72C for 30 s;

followed by 20 cycles of 92C for 20 s, 58C for 20 s and 72C for 30 s and 72C for

Chapter 4 37

180 s; GeneAmp9700, Applied Biosystems). PCR reaction mixes contained 5 µl

genomic DNA, 0.2 µM forward primer and 0.2 µM reverse primer, 200 µM of each

dNTP, 25mM Tricine, 7.0% Glycerol (w/v), 1.6% DMSO (w/v), 2 mM MgCl2, 85 mM

Ammonium acetate pH 8.7 and 0.2 U Taq Polymerase in a total volume of 10 µl.

PCR products were diluted with 20 µl H2O, and 1 µl was used as template for the

sequencing reactions. Sequencing reactions, containing 0.25 µl BigDYE (v1.1;

Applied Biosystems, Foster City, CA), 3.75 µl 2.5x dilution buffer (Applied

Biosystems) and 0.4 µM gene specific primer in a total volume of 10 µl, were

performed using cycling conditions as recommended by the manufacturer.

Sequencing products were purified by ethanol precipitation in the presence of 40 mM

sodium-acetate and analyzed on a 96-capillary 3730XL DNA analyzer (Applied

Biosystems). Sequences were analyzed for polymorphisms using polyphred software

(Nickerson et al., 1997). Primers for PCR amplification and sequencing were

designed using the Ensembl genome database (http://www.ensembl.org) and a

customized interface to Primer3.

Genotyping

For the identification of the ter allele PCRs were performed with the primer pair

(terFor2: 5‟-GTCTGGTCTTAAGTGCTTGG-„3; terRev2: 5‟-TCACTGCTTCACCACAG

AAC-3‟) amplifying a 560 bp sequence in a total volume of 25 µl containing 12.5 µl

HotStarTaqMatermix Kit (Qiagen, Hilden, Germany), 10 pmol of each primer and 1 µl

template DNA. After initial 15 min at 95°C followed by 35 cycles (95°C for 30 s, 54°C

for 30 s, 72°C for 30 s), the PCR products were digested adding 0,5 µl KpnI-HF, 3 µl

NEB4 buffer and 0.3 µl BSA (100x) amplification (NEB, Ipswich, MA). Cleavage

fragments from PCR products of wild type Dnd1 (180 bp and 380 bp) and unchanged

560 bp ter allele products were separated by electrophoresis in a 1.5 % agarose gel

in 1x TBE buffer.

Tumors and tissues

Ten males and ten females from WKY as well as from WKY-Dnd1ter with +/+, ter/+

and ter/ter genotype were sacrificed at 3, 6 and 9 weeks of age by cervical

dislocation after CO2 anesthesia. Testes and ovaries were prepared, inspected,

38 Chapter 4

weighed and fixed for histology. In addition, GCTs were harvested from 46 male and

38 female Dnd1-deficient rats at the pre-final stage of cancer, which were

characterized by a significant increase of body weight accompanied by abdominal

swelling, inactivity, reduced food intake and piloerection/rough coat. Tumors were

excised, examined macroscopically, weighed and prepared for histology.

Histology and Immunohistochemistry

Tissues were fixed in 4% formaldehyde solution or in Bouin‟s solution (Sigma-Aldrich,

St.Louis, MO) for 3-5 days. Paraffin embedded tissues were cut in 3 µm slices,

transferred to SuperFrost slides (Menzel, Braunschweig, Germany) and stained with

hematoxylin-eosin following standard procedures.

For immunohistochemical staining, slides from formaldehyde-fixed, paraffin-

embedded tissues were heated in 10 mM citric buffer (pH 6) for 5 min at 125°C in a

pressure cooker for antigen retrieval. Samples were stained with a 1:100 dilution

(stock 200µg IgG/ml PBS) of an anti c-kit antibody (sc-168; Santa Cruz

Biotechnology Inc, Santa Cruz, CA) for 1 hour. For secondary antibody reaction and

HRP staining, the Zytochem Plus HRP Polymer System (Zytomed Systems, Berlin,

Germany) was used according to the manufacturer‟s instructions.

Statistical analyses

Linkage analyses were performed with the JoinMap V 2.0 program (Agricultural

Research Department, Wageningen, Netherlands). The LOD scores of the

teratocarcinoma susceptibility region were calculated using the R/qtl program

provided by Dr. K. Browman (Department of Biostatistics, Johns Hopkins University,

Baltimore, MD) (Xu & Atchley, 1996; Broman et al., 2003). E/M algorithms estimated

susceptibility regions in a binary model using the teratocarcinoma of the animals as a

trait.

A permutation test was performed based on our current genotypic and phenotypic

data to calculate an individual threshold value for significance (LOD score > 2.3)

independent from the theoretical model of Lander and Kruglyak (Churchill & Doerge,

1994; Lander & Kruglyak, 1995). Tumor data analysis and Kaplan-Meyer survival

analysis were performed and statistically verified using One-way Anova and

Chapter 4 39

Bonferroni Multiple Comparison Test by the PRISM 5 for Mac OSX Software

(GraphPad Software Inc., La Jolla, CA).

4.4. Results

We traced the ter mutation in the WKY/Ztm rat strain to a point mutation in the Dnd1

gene and observed teratocarcinomas and infertility in all homozygous rats of both

genders.

Teratocarcinomas in the WKY strain

We established a coisogenic segregating inbred WKY strain that carries a mutation

referred to as ter leading to germ cell tumors (GCT) in both testes and ovaries (Fig.

4.1A). Histological examination of these tumors revealed different tissues that

originated from all three germ layers, thereby characterizing the GCTs as teratomas.

Besides mesodermal tissues such as cartilage, skeletal and cardiac muscle we also

identified glandular structures arising from the endodermal germ layer. Furthermore,

immature neuronal tissue, neural tube-like formations and squamous epithelia

originating from the ectoderm were present in teratomas of both ovaries and testes

(Fig. 4.1B). Tumor development in the ovaries was often accompanied by ascites

and cystic alterations in the early stages of tumorigenesis (Fig. 4.1C, left).

Identification of the ter mutation

Linkage analysis of 58 (WKY-ter x SPRD-Cu3) F2 rats using 52 polymorphic

microsatellite markers indicated an association of RNO18 (D18Rat61, LOD = 3.25)

with tumor development (Fig. 4.2A). A panel of 64 SNP markers, which are

polymorphic between WKY and SPRD-Cu3 was selected. Genomic loci were

amplified by PCR from 58 F2 animals of the WKY-ter x SPRD-Cu3 cross and

genotyped by dideoxy re-sequencing. Strong linkage to a single locus in the middle

of chromosome 18 was observed (data not shown). Based on synteny with the

recently cloned mouse Ter mutation within MMU18qB2, the homologous rat Dnd1

gene in RNO18p11 was re-sequenced (Fig. 4.2B).

40 Chapter 4

A G to A mutation was identified in exon 4 at position 1975, which introduces a

premature stop codon thought to result in a 62 amino acid truncation at the c-

terminus of the Dnd1 protein (Fig. 4.2C) and most likely a complete loss-of-function

of the encoded protein. The ter point mutation also disrupts the recognition site of the

restriction endonuclease KpnI. A 560 bp PCR amplification product of wild type Dnd1

was cleaved by KpnI into a 380 bp and a 180 bp fragment, while no cleavage was

detected in homozygous ter/ter animals, which confirms the absence of the KpnI

restriction site (Fig. 4.3A).

Using this genotyping approach we were able to distinguish between rats carrying

only one ter allele (ter/+), and animals carrying two ter-alleles (ter/ter), and wild type

(+/+) animals. The ter/+ animals showed normal fertility in both genders and were

used to establish the coisogenic WKY-Dnd1ter strain. Litters from breeding

heterozygous rats were of the same size (7-10 pups) as litters from wild type matings

of WKY-Dnd1ter or WKY. Offspring from ter/+ parents showed normal Mendelian

ratios with 22.8% +/+, 53.7 % ter/+ and 24.5% ter/ter genotypes.

Age dependent GCT development

Teratomas in WKY-Dnd1ter became clinically apparent during adolescence. OGCT in

female ter/ter rats were detected between 21 and 66 days after birth with a median

survival of 34 days, and only 7.8 % of the ter/ter females survived day 43 (Fig. 4.3B).

Of the male ter/ter rats 51% had to be sacrificed by day 58 because of

tumorigenesis, and the rapid increase of tumor progression led to the sacrifice of an

additional 39% of ter/ter males by day 70. However, 10% of the ter/ter males

exhibited a delayed TGCT formation and survived up to 196 days, before exhibiting

visual teratomas. Altogether, the median survival was 58 days in males.

In contrast to the 100% tumor incidence in ter/ter rats, tumorigenesis was entirely

absent in the gonads of heterozygous animals. Furthermore, there was no

spontaneous teratocarcinogenesis detectable in wild type rats from WKY-Dnd1ter or

WKY strains (Fig. 4.3B).

Tumor size and localization were examined in male and female rats at three, six and

nine weeks of age to determine tumor progression. The mean TGCT weight

increases in ter/ter male rats from 2.5 g (SD±2.32) at 3 weeks to 3.8 g (SD±2.96) and

Chapter 4 41

4.7 g (SD±3.76) at 6 and 9 weeks (Fig. 4.3B). At 3 and 6 weeks of age, 40% of the

homozygous ter rats had unilateral TGCTs, while both testes were affected in 20%

and in 40% both testes were macroscopically degenerated without signs of tumor

formation. At 9 weeks of age bilateral teratomas increased to 68%, while 18% had

unilateral tumors and 14% degenerated testes.

At 3 weeks of age the OGCTs in female ter/ter rats were smaller (0.54 g±0.38) than

the TGCTs. Rapid growth of the OGCTs was detected from 3 to 6 and 9 weeks of

age up to mean weights of 4.68 g (SD±4.58) and 5.11g (SD±4.86), with large

variations in tumor size (Fig. 4.3C). Only 4 females survived up to 9 weeks of age

and 3 of these females had bilateral OGCTs. Only 1 of the surviving 4 females at 9

weeks of age exhibited unilateral teratocarcinogenesis, however, the non-tumorous

ovary was cystic (Fig. 4.1C). This was the only case of a female ter/ter rat developing

a teratoma in one ovary and not in both. Ovary degeneration in ter/ter females

without tumor formation was never found due to an early onset of tumor

development. The rapid tumor progression was correlated to the fast drop of survival

between 3 and 6 weeks of age compared to male ter/ter rats (Fig. 4.3C).

Testicular malformation in ter/ter males

We defined the degenerated testis as lower in weight and size than the wild type

testis (Fig. 4.1C, right). The mean weight of wild type testes increased from 0.14 g

(SD±0.02) at 3 weeks to 0.83 g (SD±0.07) at 6 weeks and to 1.14 g (SD±0.07) at 9

weeks in WKY males, reflecting sexual maturation. In wild type WKY-Dnd1ter males

the mean testes weight was 0.09 g (SD±0.02) at 3 weeks, 0.65 g (SD±0.05) at 6

weeks and 1.05g (SD±0.14) at 9 weeks of age. There was no significant difference to

heterozygous WKY-Dnd1ter males with 0.1 g (SD±0.03), 0.7 g (SD±0.04) and 1.12 g

(SD±0.14) of testes weight at 3, 6 and 9 weeks of age. In ter/ter males the

malformed, non-tumorous testes were reduced in size with 0.06 g (SD±0.01), 0.32 g

(SD±0.13) and 0.81 g (SD±0.26) from 3 to 9 weeks of age (Fig. 4.4A).

Histological evaluation revealed normal testicular development and maturation in wild

type WKY-Dnd1ter rats from 3 to 9 weeks. In ter/ter testes the failure of germ cell

development was demonstrated by HE staining, which showed a reduced diameter of

the tubuli seminiferi, a loss of germ cells and a lack of spermatogenesis at 3 and 6

42 Chapter 4

weeks of age. At 9 weeks of age, the degenerated ter/ter testes showed an onset of

neoplastic transformation in the seminiferous tubules (Fig. 4.4B).

To confirm the loss of the sperm precursor cells we performed immunohistological

staining with an anti c-kit antibody as a specific marker for stem and germ cells. As

expected, at 3 weeks of age wild type testes showed widely undifferentiated germ

cell precursors in the seminiferous tubules, while at 9 weeks of age the complete

differentiation process from spermatogonia to sperm could be observed. However, in

the testes of both 3- and 6-week-old ter/ter rats only a few c-kit positive cells were

detected in the seminiferous tubule epithelium, and this suggests that primordial

germ cells were lost during embryonic development. Remaining germ cells often

appeared to be aggregated to foci of neoplastic cells (Fig. 4.5).

Ovarian malformation in ter/ter females

At 3 weeks of age, we found no significant differences in ovary size between females

from the WKY or WKY-Dnd1ter strain (0.005-0.01 g). Three weeks later the ovaries

grew to 0.032 g (SD±0.009) in WKY while the ovaries remained significantly smaller

in WKY-Dnd1ter wild type (+/+) with 0.02 g (SD±0.006) and 0.015 g (SD±0.007) in

heterozygous (ter/+) females. At 9 weeks of age the ovaries from WKY/Ztm females

grew to 0.058 g (SD±0.011), in WKY-Dnd1ter/Ztm wild type (+/+) to 0.034 g

(SD±0.007), and to 0.04 g (SD±0.007) in heterozygous (ter/+) females confirming

sexual maturation (Fig. 4.6A). All ter/ter females had developed tumors at 6 and 9

weeks of age (Fig. 4.3C). Histological examination of early OGCTs in female ter/ter

rats revealed that the non-neoplastic primary follicles lack oocytes, indicating the loss

of primordial germ cells (Fig. 4.6B).

4.5. Discussion

Teratocarcinogenesis in the rat: a novel animal model for GCTs

Teratomas are rare tumors in rats. Only a very few cases of spontaneous

teratocarcinogenesis in adrenal gland, kidney, central nervous system or abdomen

have been reported (Schardein & Fitzgerald, 1977; Ninomiya, 1983; Itoh et al., 1985).

Chapter 4 43

Aside from the extra gonadal teratomas, the formation of testicular GCT has been

described in testes of a SPRD IGS rat (Sawaki et al., 2000). Hereditary teratomas

have also been found in testes and ovaries of the Tera strain that was developed

from the Csk:Wistar-Imamichi rat (Miwa et al., 1987); however, the underlying

mutation was never identified. We detected a congenital, recessive point mutation

referred to as ter in the rat Dnd1 gene on chromosome 18p11 that induces

teratocarcinogenesis and infertility in all animals of both genders. The mutant rat

strain was denominated WKY-Dnd1ter/Ztm and it is a valuable new animal model for

research on germ cell development, TDS, TGCTs and OGCTs.

The 129/Sv-Ter mouse is most likely a model for prepubertal type I TGCTs, whereas

there are no animal models available for type II TGCTs (Looijenga & Oosterhuis,

1999; Oosterhuis & Looijenga, 2005). It remains to be established whether the

teratomas of the ter rat show a closer resemblance to type I or type II GCT. In

analogy to the mouse, and because the majority of rat teratomas occurred before or

during puberty, it seems more plausible that the ter rat is a model for type I GCTs.

Species and gender differences in Dnd1-related teratocarcinogenesis

In the WKY-Dnd1ter/Ztm rat, we identified a point mutation at position 1975 of the

Dnd1 gene that substituted G for A and introduced a premature stop codon, which is

presumed to cause a truncation of 62 amino acids of the Dnd1 protein at the c-

terminus. A similar point mutation has been found in the mouse Dnd1 gene with a C

to T exchange in exon 2 on chromosome 18 that also generates a premature stop

codon thought to result in a c-terminal truncation of the Dnd1 protein (Noguchi and

Noguchi, 1985; Asada et al, 1996; Youngren et al, 2005). A rescue of Dnd1 in mutant

Ter mice showed that the loss of functional Dnd1 is responsible for the phenotype

observed in the mouse. Dominant negative effects were excluded in the mouse, as

tumors and germ cell deficient testes no longer expressed regular or truncated Dnd1

(Youngren et al., 2005). The close similarities found between Dnd1 mutant mouse

and rat in the mutation and the phenotype make it highly probable that a functional

inactivation of Dnd1 also causes germ cell loss and teratomas in the ter rat.

Despite the similarities in the rodent Dnd1 mutations, differences between the

species mouse and rat were apparent in tumor incidence and severity of fertility

44 Chapter 4

disorders. In the mouse, Dnd1 has been characterized as a modifier gene amplifying

the incidence of spontaneous teratocarcinogenesis in heterozygous and homozygous

Ter males. Other modifier genes (KitlSl-J, Trp53nul, Ay, M19, M19-A2, M19-C2) interact

with mouse Dnd1 and induce a 2-3 fold increase of TGCT incidence in heterozygous

Ter/+ males (Lam et al, 2007). Unlike the co-dominant Ter mutation in the mouse, the

recessive ter mutation in rat Dnd1 induced gonadal teratoma formation in 100% of

the homozygous animals, while heterozygous and wild type animals completely

lacked teratocarcinogenesis. Therefore, we postulate that Dnd1 is an essential factor

involved in teratocarcinogenesis and acts as a tumor suppressor gene and not as a

tumor modifier in germ cells of the WKY/Ztm rat strain.

Furthermore, the malignant transformation of rat germ cells initiates not only

testicular, but also ovarian teratocarcinogenesis. OGCTs developed in all

homozygous Dnd1-deficient female rats, whereas no tumor development has been

observed in female mice. Cook and colleagues demonstrated that the testicular

environment is a crucial factor in GCT development of mice, by showing that female

Dnd1Ter/Ter germ cells neoplastically transform in the testes (Cook et al., 2009). The

somatic cells of the testes secrete paracrine factors such as SCF, retinoic acid,

FGF2, LIF, EGF and GDFN as well as androgenic hormones, and these might play a

role in providing a tumor-promoting environment in the mouse (Krentz et al., 2009).

The gender in the WKY-Dnd1ter/Ztm rats had no effect on tumor incidence, which

was 100%, and only modified the tumor progression in homozygous animals.

Surprisingly, on the average tumors became clinically apparent earlier in females

where both ovaries were affected, while tumors developed slower in males with

about half of the tumors being unilateral. This could indicate that, in the rat, the

surroundings provided by the ovary might contain tumor-promoting factors or,

alternatively, the testis might include inhibitory factors.

Dnd1 ablation causes PGC loss and infertility

In response to the repression or ablation of dnd in Xenopus laevis and zebrafish

embryos, PGCs failed to migrate into the developing gonads (Weidinger et al., 2003;

Horvay et al., 2006). A recent publication showed that the c-terminus of the zebrafish

Dnd protein possesses an ATPase domain, which is essential for PGC formation and

Chapter 4 45

survival (Liu & Collodi, 2010). The presumed c-terminal truncation of Dnd1 in the

mouse and rat was also linked to the ontogenetic loss of germ cells; however, it

remains to be established which parts of the c-terminus are crucial for a functioning

Dnd1 protein in rodents and whether an ATPase domain is involved.

Loss of PGCs started on embryonic day 8.5 post coitum in Dnd1 deficient mice of

both genders (Sakurai et al., 1995). Apoptosis is at least in part responsible for the

germ cell loss, as additional inactivation of the pro-apoptotic Bax gene in Dnd1Ter/Ter

mice rescued up to 50% of the PGCs in both genders (Cook et al, 2009). Deficit of

the PGCs caused abnormally small testes and male sterility in homozygous 129/Sv-

Ter mice, while in the homozygous females a few germ cells survived and even

matured to oocytes in ovaries of reduced size (Noguchi and Noguchi, 1985).

In contrast to the mouse, gametogenesis was absent in both degenerated testes and

in non-neoplastic ovarian follicles of ter/ter rats, and this may be the result of a

complete ontogenic loss of normal PGCs. The WKY-Dnd1ter/Ztm rat strain exhibited

infertility, small degenerated testis and TGCTs in males. The older the male ter/ter

rats were, the lower the rate of degenerated testes and the higher the rate of bilateral

tumors. Hence, it is highly likely that the infertile and degenerated testes exhibited by

ter/ter males are the predecessors of cancer. Every one of the ter females was

infertile and all but one developed bilateral OGCTs without ever exhibiting any cases

of degenerated ovaries, and this might be due to the early onset of cancer (under 66

days of age) in females compared to males (under 196 days of age).

Conclusion

The WKY-Dnd1ter/Ztm rat provides a promising new model to study Dnd1-dependent

GCT development in gonads of both genders and investigate the molecular

mechanisms of GCT development in testes and ovaries. This model offers the

opportunity to establish new therapeutic and diagnostic approaches for GCTs in men

and women.

46 Chapter 4

4.6. Figures

Figure 4.1

Chapter 4 47

Fig. 4.1: Teratomas in 6 week old ter/ter rats of the strain WKY-Dnd1ter/Ztm: (A) Bilateral

GCTs in female rat (black arrows: ovarian GCTs) and unilateral GCT in male rat (white

arrow: non-neoplastic testes; red arrow: left TGCT. (B) Upper left: ectodermal tissues in

OGCT of a 5-week-old female; neural tube formation (black star) and squamous epithelium

(black arrow). Upper right: mesodermal and endodermal tissues in an OGCT of a 9-week-old

female; cartilage (white star), glandular structures (white arrow) and skeletal muscle (black

arrow). Lower left: heart muscle-like tissue from a contractile ovarian teratoma. Lower right:

immature neuronal tissue from a TGCT of a 6-week-old male. (C) Rat gonads from 3 weeks

old siblings. Left: non-tumorous ter/ter testes are significantly smaller than wild type. Right:

normal wild type ovaries compared to an OGCT and an abnormal, cystic ovary (confirmed by

histological section and HE staining).

Figure 4.2

48 Chapter 4

Fig. 4.2: Identification of the ter mutation in the rat Dnd1 gene: (A) Genome-wide mapping of

teratoma susceptibility loci of the WKY-ter rat. 58 (WKY-ter x SPRD-Cu3) F2 animals were

typed using genomic DNA and the complete panel of polymorphic microsatellite markers as

described in Material and Methods. The incidence of teratomas of the F2 population was

15%. The LOD scores of the teratoma susceptibility region were calculated using the R/qtl

program. A permutation test assumed a LOD score > 2.3 to be associated with the teratoma

development. (B) Resequencing of all 4 exons of Dnd1 was managed using LIMSTILL, LIMS

for Induced Mutations by Sequencing and TILLing (Victor Guryev, E.C., unpublished). This

web-based publicly accessible information system (http://limstill.niob.knaw.nl) was used to

generate the Dnd1 project and visualize the gene structure based on Ensembl file

ENSRNOG00000016894. (C) The 2625 bp coding sequence of the Dnd1 gene is separated

in 4 exons. The RNA Recognition Motif (RRM) essential for nucleic acid binding of Dnd1 was

located in exon 3. The ter point mutation was identified as G to A substitution in exon 4 at

position 1975 introducing a premature stop codon.

Chapter 4 49

Figure 4.3

Fig. 4.3: (A) The ter mutation disrupts a KpnI restriction site used for genotyping of the ter

allele performing PCR amplification and restriction digest. KpnI digest (black arrows) cleaved

the PCR product of 560bp into 380bp and 180bp fragments, N negative control. (B) Kaplan-

Meyer survival analysis of male and female WKY-Dnd1ter/Ztm rats carrying heterozygous and

homozygous ter alleles compared to wild type animals. (C) Sizes of TGCTs and OGCTs after

3, 6 and 9 weeks of age in homozygous WKY-Dnd1ter/Ztm rats.

50 Chapter 4

Figure 4.4

Chapter 4 51

Fig. 4.4: non-tumorous testes. (A) Box plot analysis of the size of non-tumorous testes in

WKY/Ztm and WKY-Dnd1ter/Ztm with wild type (+/+), heterozygous (ter/+) and homozygous

(ter/ter) genotype at 3, 6 and 9 weeks of age. ter/ter testes are degenerated and significantly

smaller than +/+ and ter/+ testes (B) HE staining of paraffin sections from wild type and

homozygous ter/ter testes of WKY-Dnd1ter/Ztm rats from 3 to 9 weeks of age (: p <

0,001).

Figure 4.5

Fig. 4.5: Immunohistochemical anti c-kit staining of paraffin section from wild type and

homozygous ter/ter testes of WKY-Dnd1ter/Ztm rats at 3 and 6 weeks of age (black arrows:

clusters of surviving c-kit positive germ cell).

52 Chapter 4

Figure 4.6

Fig. 4.6: non-tumorous ovaries. (A) Box plot analysis of non-tumorous ovaries from

WKY/Ztm and WKY-Dnd1ter/Ztm with wild type (+/+), heterozygous (ter/+) and homozygous

(ter/ter) genotype (: p < 0.01, : p < 0.001). (B) HE staining of paraffin section from

wild type (+/+, upper panel) and homozygous ovaries (ter/ter, lower panel) of WKY-

Dnd1ter/Ztm rats at 6 weeks of age. While in the wild type ovaries different stages of follicle

maturation (upper panel, left) and primary follicles with a central oocyte (upper panel, right)

were evident, in the ter/ter ovaries only a very few follicle-like structures without germ cells

(lower panel, right, black arrows) remained.

53

54 Chapter 5

5. Loss of Dnd1 facilitates the cultivation of genital ridge-

derived rat embryonic germ cells

Emily Northrup1, Regina Eisenblätter1, Silke Glage1, Cornelia Rudolph2, Martina

Dorsch1, Brigitte Schlegelberger2, Hans-Jürgen Hedrich1, Nils-Holger Zschemisch1

1 Institute for Laboratory Animal Science

Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

2 Institute of Cell and Molecular Pathology


submitted

Acknowledgements:

We thank Isabell Wittur and Cindy Elfers for excellent laboratory assistance.

Funding for this project was provided by the Cluster of Excellence REBIRTH,

Grant number: EXC 62/1.

Chapter 5 55

5.1. Abstract

Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the

embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A

homozygous mutation (ter) leading to the ablation of the dead-end homolog 1 gene

(Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic

transformation and loss of PGCs. We mated heterozygous ter/+ WKY-Dnd1ter/Ztm

rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo

at day 14.5 post coitum (d pc). Genotyping revealed that ten EGC lines were Dnd1

deficient, while only one wild type cell line had survived in culture. This suggests that

the inactivation of the putative tumor suppressor gene Dnd1 facilitates the

immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts

resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs

offer an innovative approach for studies on germ cell development and germ cell

tumor development as well as a new tool for genetic manipulations in rats.

5.2. Introduction

The gametes of adult vertebrates, oocytes and sperm, develop from their embryonic

precursors, the primordial germ cells (PGCs), and are responsible for the

transmission of genetic information from one generation to the next. During

ontogenesis, the PGCs migrate through various tissues before finally reaching the

genital ridge. PGCs themselves are highly specialized cells restricted in their

developmental potency (McLaren, 1981). Pluripotent cells referred to as embryonic

germ cells (EGCs), can be obtained from PGCs in vitro. Culture of PGCs leads to this

not well-understood process of epigenetic reprogramming, which transforms the

unipotent PGCs to pluripotent EGCs (McLaren, 2003). The EGCs proliferate

indefinitely and, unlike PGCs (McLaren & Durcova-Hills, 2001), can result in germ-

line competent chimeras upon blastocyst injection (Labosky et al., 1994; Stewart et

al., 1994). EGCs have been made from PGC in the mouse before and during

migration, as well as after entry into the genital ridge (Durcova-Hills et al., 2001;

McLaren & Durcova-Hills, 2001). Only recently, the first EGC were obtained from pre-

56 Chapter 5

migratory PGCs of the rat. Culture conditions similar to those for rat ESC, using the

two inhibitors (2i) and LIF on feeder cells, could procure these rat EGCs (Leitch et al.,

2010).

Loss of Dnd1 can lead to the formation of germ cell tumors and infertility. Dnd1 is an

RNA-binding protein that competes with miRNAs for the binding of specific target

gene mRNAs. In doing so, Dnd1 inhibits the miRNA-mediated repression and

enables the expression of certain target genes even though miRNAs are present.

The persistence of mRNAs from the tumor suppressor genes Lats2 and p27 as well

as the genes Nanos1, Tdrd7 and Connexin43 in germ cells depends on expression

of Dnd1 (Kedde et al., 2007; Ketting, 2007). Dnd1 has also been shown to interact

with the multifunctional protein APOBEC3 (Bhattacharya et al., 2008). The Ter

mutation in the 129/Sv mouse strain was traced back to a point mutation leading to a

premature stop codon in Dnd1 (Asada et al., 1994; Youngren et al., 2005). This

mutation causes decreased fertility in Ter/Ter females and infertility in Ter/Ter males

and increases the teratoma incidence in the testis to 17% in Ter/+ and 94% in

Ter/Ter males, compared to the 1% in wild type 129/Sv (Stevens, 1984; Noguchi &

Noguchi, 1985). Loss of PGCs during ontogenesis was first detectable in Ter/Ter

mouse embryos of both genders at day 8.5 post coitum (d8.5pc) (Sakurai et al.,

1995).

A spontaneous mutation with a recessive mode of inheritance leading to teratomas in

ovaries and testis also occurred in the WKY/Ztm rat strain and was named ter

(Länger et al., 2004). This ter mutation was identified as a point mutation in exon 4 of

the Dnd1 gene that introduces a premature stop codon. Resulting loss or truncation

of the Dnd1 protein in the homozygous ter/ter rats leads to infertility due to the

ontogenetic death of germ cells, and initiates gonadal teratoma formation in all

animals through neoplastic transformation of surviving germ cells. The onset of

teratocarcinogenesis and the teratoma progression were influenced by gender. While

tumors were detected between 21 and 66 days of age in female ter/ter rats with a

mean survival of 34 days, the tumorigenesis was delayed in Dnd1-deficient males

where 61% survived longer than day 70 and 10% longer than day 196 with a mean

Chapter 5 57

survival of 58 days. Furthermore, both ovaries were affected in the females whereas

50% of the males had unilateral tumors in the testis (chapter 4).

Thus, Dnd1 fulfils different roles in PGC migration, development and survival,

depending on the species. So far the molecular mechanisms involved in the rat

remain entirely unknown. We cultured rat embryonic germ cells (EGC) in vitro derived

from embryonic PGCs of the genital ridge as a tool to investigate the mechanisms of

gender-dependent germ cell differentiation and germ cell tumor development.

5.3. Material and Methods

Animals

All animals were bred and maintained at the Institute for Laboratory Animal Science,


(subline code: Ztm: http://www.mh-hannover.de/einrichtungen/tierlabor). The

experiments were in accordance with the German Animal Welfare Legislation

(Tierschutzgesetz 2006), approved by the local Institutional Animal Care and

Research Advisory Committee and permitted by the Animal Welfare Service of the

Lower Saxony State office for Consumer Protection and Food Safety (Az.10/0226;

Az.10/0209).

Strain origin

The rat strain WKY-Dnd1ter/Ztm traces back to a spontaneous mutation that occurred

in the Institute for Laboratory Animal Science, Hannover Medical School in the inbred

generation F73 of the WKY/Ztm rat strain(Länger et al., 2004). The ter mutation leads

to the development of teratomas and infertility with an incidence of 100% in

homozygous animals, while no effects are seen in heterozygous animals. WKY-

Dnd1ter/Ztm colony is maintained as a segregating, coisogenic inbred strain.

Husbandry

Animals were maintained under standardized conditions in the following environment:

temperature of 22 ± 2°C, relative humidity of approximately 55%, and artificial light


58 Chapter 5

for 14h. The commercial softwood granulate bedding was sterilized (Lignocel,

Altromin; Lage, Germany). They received an autoclaved commercial pelleted diet

(Altromin 1314) (protein 22%, fat 5%, raw fiber 4.5%, ash 7%, utilizing energy 3.1

kcal/g) and water ad libitum.

The rats were kept under conventional housing conditions, as pairs or in sibling

groups. Mice were housed under specified pathogen free (SPF) conditions,

separated by gender and in individually ventilated cages according to ETS123.

Microbiological status was monitored according to FELASA recommendations

(Nicklas et al., 2002). The mice were free of the listed microorganisms. The DA.1M

rats were positive for parvovirus, helicobacter hepaticus and apathogenic protozoa

and the WKY/Ztm rats were positive for parvovirus and apathogenic protozoa.

EGC Derivation

WKY-Dnd1ter/Ztm rats with a ter/+ genotype were mated and the pregnant females

were sacrificed at day 14.5 post coitum. Embryos were collected and EGCs were

isolated as previously described by Cooke and colleagues. with minor modifications

(Cooke et al., 1993): The genital ridge containing the PGC was dissected free from

the embryo without the mesonephros and a single-cell suspension was obtained by

mechanical disruption and digestion with accutase.

EGC Culture

EGC were maintained on -irradiated TRF-O3 cells as feeder in N2B27 media

supplemented with 2i-LIF (Buehr et al., 2008; Li et al., 2008). 2i-LIF includes the

MEK-inhibitor PD032591 (1µM; Axon, Groningen, The Netherlands), the GSK-3

inhibitor CHIR99021 (3µM; Axon) and rat LIF (1000 U/ml; Millipore, Billerica, MA).

The TRF-O3 cells stem from a teratoma that developed in the ovaries of a female

ter/ter WKY-Dnd1ter/Ztm rat and are maintained in DMEM media with FCS. Between

7-10 days of primary culture all visible EGC colonies were picked and expanded. The

EGC colonies were identified based on their morphology and the fact that they were

easy to detach from the feeder cells. To maintain the cells, medium was changed

every other day and cells were split with accutase every 3-4 days. Cells were

Chapter 5 59

cryopreserved in 2i-LIF medium supplemented with 10% DMSO and survived

multiple freeze-thaw cycles.

Immunocytofluorescence and Alkaline Phosphatase Staining

Immunostaining was performed by fixation of the cells in 4% paraformaldehyde for

2h. Cells were then made permeable using 0.05% Triton-X-100 (Sigma) together and

1% SDS in PBS for 10min. Primary antibodies were: rabbit IgG anti Oct-4 (Millipore,

1:50), rabbit IgG anti DDX4/MVH (Abcam, 1:50), rat IgM anti SSEA3 (Millipore,

1:100), goat IgG anti Nanog (Everest, 1:100), mouse IgM anti SSEA1 (Santa Cruz,

1:100). The following Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA)

were used: donkey anti-rabbit IgG (1:800), donkey anti-goat IgG (1:800), goat anti-rat

IgM (1:400), goat anti-mouse IgM (1:800).

AP staining was performed with the Stem Tag™ alkaline phosphatase staining kit

(Cell Biolabs, San Diego, CA) according to the manufacturer‟s instructions.

Genotype and Gender Determination

DNA from rat tissue or EGCs was extracted using TENS buffer (50mM Tris-HCl,

100mM EDTA, 100mM NaCl, 1% SDS and 0,27 mg/ml proteinase K) followed by

isopropanol precipitation. For the identification of the ter allele, PCR and restriction

digest were performed as described (chapter 4). Cleavage fragments from PCR

products of wild type Dnd1 (180 bp and 380 bp) and unchanged 560 bp ter allele

products were separated by electrophoresis in a 1.5 % agarose gel in 1x TBE buffer.

The gender of the EGCs was determined by PCR amplification of the Y-

chromosome-specific Sry gene using the HotStarTaqMastermix Kit (Qiagen, Hilden,

Germany) with the primers 5‟-CTGAAGACCCTACACAGAGA-3‟ and 5‟-

GCTTTTCTGGTTCTTGGAGG-3‟ at an annealing temperature of 52°C. The 210 bp

product in males was identified by electrophoresis in a 2% agarose gel in 1 x TBE

buffer.

Cloning

Dnd1 from the wild type EGC line and one ter/ter EGC line were cloned into the

vector pSCA-amp/kan using the StrataClone PCR cloning kit (Agilent Technologies,

60 Chapter 5

Waldbronn, Germany) according to the manufacturer‟s recommendations. For

cloning, 0.5µl cDNA were amplified using Phusion® Hot Start High-Fidelity DNA

Polymerase (NEB, Ipswich, MA). An A-overhang was ensured by adding 1µl of Taq

Polymerase (Qiagen, Hilden, Germany) to the samples and incubating for an

additional 10 min at 72°C after amplification. The forward primer 5‟-

CCCGGGCATGCAGTCCAAACGGGAGTG- 3‟ was used with the reverse primer 5‟-

CCCGGGGGCCGGCCCTGCTTCACCACAGAACC-3‟ for wild type Dnd1, while the

5‟-CCCGGGGGCCGGCCGAAGCGATGCCAGCCGGC-3‟ reverse primer was used

for the mutated Dnd1. The additional XmaI and FseI restriction sites were added to

the forward and the reverse primers for further cloning efforts. The primers were

synthesized by Eurofins MWG Operon (Ebersberg, Germany). Sequencing of Dnd1

cDNA was performed by Seqlab (Göttingen, Germany) using the primers T3 and T7.

Cytogenetic Analysis

Metaphase chromosomes were prepared by treating the cells with colcemid at a final

concentration of 0.035µg/ml for 5.5 hours, incubated in 0.075M KCl for 20 min at

37°C and fixed in a freshly prepared mixture of methanol:acetic acid (3:1) at room

temperature. Cell suspension was dropped onto glass slides in a climate chamber

(Polymer, Kassel, Germany) at 22°C and 48% humidity and stained with Giemsa.

Images were acquired using a Zeiss Axio Imager.M1 microscope equipped with the

BandView EXPO Software (ASI; Applied Spectral Imaging, Ltd., Migdal HaEmek,

Israel). Altogether, 20-25 metaphases per case were selected for chromosome

counting.

Cell Proliferation Assay

Cell proliferation reagent WST-1 (Roche, Mannheim, Germany) was used according

to the manufacturer‟s instructions. 1x104 cells were seeded onto a 96-well plate and

after 48h of culture 10µl of the cell proliferation reagent WST-1 were added per well

followed by incubation for 3.5h.

Chapter 5 61

Embryonic Stem Cells (ESC)

ESCs were derived from the inner cell mass of +/+ WKY-Dnd1ter/Ztm rat embryos

d4.5 pc as described previously and maintained under the same culture conditions as

the EGCs (Buehr et al., 2008; Li et al., 2008).

RNA Extraction and Reverse Transcription

RNA was extracted from cell culture and tissue using the RNeasy Mini Kit (Qiagen)

according to the manufacturer‟s instructions. cDNA was synthesized from 1µg of

RNA using the Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany) with

Oligo(dt)18 primers (Fermentas, St. Leon Rot, Deutschland) following the suppliers

recommendations.

Gene Expression Analysis

Expression analysis was performed with the HotStarTaqMastermix Kit (Qiagen)

according to the supplier‟s instructions using 0.1µl of cDNA.

The primers for c-Myc, Klf4, Nanog, Oct4, Stella, Rex1, Sox2, Blimp1, DDX4/MVH

and GAPDH have been described previously (Komiya & Tanigawa, 1995;

Zschemisch et al., 2006; Buehr et al., 2008). Other primers used were: Nanos3

forward 5‟-CTTCTGTCTACTGCTACACC-3‟ and reverse 5‟-

CTTCCTGCCACTTTTGGA-3‟; Fragilis forward 5‟-AGAACTCTCCATCCTCTACC-3‟

and reverse 5‟-CAGAGTAGGCATAGGCAATG-3‟; c-kit forward 5‟-

TCAAGGAAGGTTTCCGAA-3‟ and reverse 5‟-AGGAGAGGCTGTGTGGAAGA-3‟.

Teratoma Induction

EGC were harvested and resuspended in PBS to form a slightly clumpy suspension.

Cells were drawn into a 1ml syringe, kept at 4°C and warmed to room temperature

prior to injection. Teratomas were then induced by subcutaneous injection of the

5x106 EGCs in 150µl of PBS cells into the ventro-lateral region of NOD.CB17-

Prkdcscid/J mice. Teratomas were removed when they had reached a maximal

diameter of 1-1.5cm or weight loss of over 20% was evident in the recipients.

Formalin-fixed and paraffin-embedded teratomas were cut into 4µm slices before

62 Chapter 5

staining with HE. Microphotographs were taken using a Zeiss AxioCam MRc camera

and analyzed histologically for structures of all three germ layers.

Blastocyst Injection and Embryo Transfer

Blastocyst injection was performed as described with some modifications (Hogan et

al., 1994). DA.1M blastocysts were injected at d5pc after mating. 10 embryos were

transferred to each uterine horn of a pseudopregnant rat (Hedrich, 1990). Chimeric

rats were identified by coat color. Germline transmission was tested by mating of

chimeras with WKY/Ztm rats.

5.4. Results

Derivation of embryonic germ cells

Our goal was to culture and characterize pluripotent rat EGCs from the WKY-

Dnd1ter/Ztm rat strain. We isolated PGCs from embryos at day 14.5 post coitum after

breeding of Dnd1ter/+ rats. The genital ridges were dissected from the embryo,

mechanically disrupted and digested with accutase (Fig. 5.1A). Resulting cells were

seeded in 2i medium containing recombinant rat LIF on radiation-inactivated rat

tumor fibroblasts as feeder cells. After 7-10 days of culture clonal growth of colonies,

consisting of small and spherical cells referred to as embryonic germ cells (EGC),

was observed along with differentiated cells. To establish the EGC lines, the colonies

from 60 embryos were picked and expanded leading to 11 different cell lines. All cells

lines were propagated in an undifferentiated state for more than 40 passages by

dissociation with the enzyme accutase (Fig. 5.1B). Morphology did not differ

significantly between passages and is also comparable to that of the rat EGCs from

d10pc depicted in the publication by Leitch et al. (2010).

Characterization of the embryonic germ cell lines

Due to the fact that heterozygous ter/+ WKY-Dnd1ter/Ztm rats were mated, it was

essential to determine the genotype of the cell lines as well as the gender. The

results for the 11 EGC lines showed that 3 male and 7 female cell lines were Dnd1

Chapter 5 63

deficient while only one, the female cell line denominated rEGC2, carried the

homozygous wild type Dnd1 (Fig. 5.2A). These genotyping results were verified by

cloning Dnd1 from the +/+ cell line and a ter/ter cell line into the vector pSCA-

amp/kan and subsequent sequencing. The point mutation in which a G-base was

substituted for a T-base could be confirmed in the ter/ter cell line, whereas no

abnormalities were seen in Dnd1 of the +/+ cell line (data not shown). In accordance

with the mating of heterozygous rats, 25% +/+, 50% ter/+ and 25% ter/ter cell lines

would be expected if Dnd1 played no role in EGC survival. However, there were no

ter/+ and only one +/+ cell line with the rest of the cell lines being ter/ter.

Furthermore, no +/+ EGCs could be established from the mating of +/+ WKY-

Dnd1ter/Ztm, as all surviving cells ended up differentiating in culture. These results

suggest that loss of Dnd1 facilitates EGC survival in vitro. Aside from the Sry-PCR,

the gender of the EGCs could be determined through the morphology of cell

colonies. While the female EGCs produced mostly round colonies with smooth

edges, male EGC were prone to a more irregular growth with an angular shape of the

colonies (Fig. 5.2B).

Cytogenetic analysis revealed that four of the eight analyzed rEGC lines (rEGC 1, 2,

4, 8) retained euploid chromosome complements with 42 chromosomes in 67-80% of

the analyzed metaphase at passage 10. A slight increase of numerical aberrations

was detectable in these cell lines over the next passages, with only 60-70% of the

metaphases being euploid at passage 20. The other four cell lines (rEGC 3, 5, 6, 7)

showed mostly aneuploid chromosome complements and included tetraploid and

complex aberrant metaphases at passage 10 and 20. The wild type rEGC2 line was

among the cell lines with a euploid chromosome complement and was also analyzed

at passage 30 showing numerical aberrations in less than 40% of the metaphase

spreads (Fig. 5.3A). Since there were gender-differences in tumor progression in

vivo, we decided to evaluate the proliferation rate of the different EGC lines. A WST-

1 assay was performed with cell lines rEGC1-8 and the inactivated TRF-O3 feeder

cells as control. Proliferation rate varied between the cell lines; however, this seemed

to be independent of gender, genotype or chromosome number. The genetically

instable cell lines showed both the two highest (rEGC5: absorbance 1.655 SD±0.080;

64 Chapter 5

rEGC7: absorbance 1.684 SD±0.169) and the lowest (rEGC3: absorbance 1.316

SD±0.084) proliferation rates (Fig. 5.3B).

Proof of Pluripotency

EGCs, much like ESC, are pluripotent cells and as such express specific markers.

Pluripotency of EGCs could be demonstrated by strong activity of alkaline

phosphatase (AP) in various passages, as late as passage number 25 (Fig. 5.2B).

Furthermore, immunostainings demonstrated the expression of the pluripotency

markers Nanog, Oct4, SSEA1 and SSEA3 in all of the 8 examined cell lines. EGCs

were characterized as PGC-derived by detection of DDX4/MHV in

immunofluorescent stainings (Fig. 5.4). DDX4/MVH, also known as RVLG (rat vasa-

like gene), is the vasa-homologue in the rat and used as a specific marker for germ

cells (Komiya & Tanigawa, 1995; Toyooka et al., 2000). Pluripotency and germ cell

associated markers expressed in the ICM or in PGCs can differ from those

expressed in their in vitro counterparts ESCs and EGCs. Therefore, expression

analysis was performed by RT-PCR with cDNA from EGCs, ESCs and tissue

containing PGCs. The pluripotency-associated genes Nanog, Oct4, Sox2, Rex1, c-

kit, c-myc and Stella were detected in the ESCs and EGCs as well as in the genital

ridge containing the PGCs. The levels of Nanog were higher in the ESCs than in the

EGCs and very low in the genital ridge. There was a strong signal for Klf4 in the

feeder cells; therefore it cannot be excluded that the signal in the ESCs and EGCs

could be due to a slight contamination with feeder cells. Nanos3, which is expressed

during hind gut migration of the PGCs and essential for PGC survival in the mouse

(Tsuda et al., 2003), was found in the genital ridge and a very weak expression in

EGCs and ESCs. The results for Blimp1 mirror those found previously in mouse and

rat at an earlier embryonic stage (Durcova-Hills et al., 2008; Leitch et al., 2010):

Blimp1 could only be identified in tissue from the embryo and was no longer present

in the EGC lines. DDX4/MVH was expressed in the genital ridge and in the EGC and

unexpectedly also in the ESCs. When comparing the genital ridges it was noted that

the DDX4/MVH and Sox2 expression was lower in ter/ter than in +/+ embryos. To put

it briefly, the RT-PCR showed that the EGCs expressed all of the expected

pluripotency and germ cell markers (Fig. 5.5). To allow the use of EGCs as a tool for

Chapter 5 65

further research it was also vital to show their pluripotency through the ability to

develop into the different tissue types. The differentiation potential was demonstrated

through subcutaneous injection of rat EGCs into immunodeficient NOD.CB17-

Prkdcscid/J mice, which resulted in tumor formation. Histological analysis revealed

derivatives from all three embryonic germ layers thereby showing the classical

characteristics of teratomas. Cartilage, neuronal tissue, skeletal muscle tissue,

mucosal epithelium and squamous epithelium could be found among the identified

tissues. Genotype and gender of the cell line played no role in the ability to induce

teratomas as these were obtained from every single injected cell line (Fig. 5.6A).

True pluripotency of cells is only given when cells are capable of integrating into the

developing embryo and are transmitted through the germline. Wild type rEGC2 cells

from different passages injected into blastocysts were capable of integrating into the

embryo. Chimeras were identified through coat coloring. The EGCs stem from an

albino rat and the injected blastocysts from the agouti colored rat strain DA.1M.

Blastocysts were injected with cells from passage 5 before being transferred to a

pseudopregnant female. Of the 5 pups born in the litter, 1 female and 3 males were

chimeric. The chimeras showed a normal development and demonstrated no defects

or malfunctions up to 8 months of age. Furthermore, the female chimera was capable

of transmitting through the germline with 36% (14 of 38 pups) of the offspring

carrying the cell line genome (Fig. 5.6B).

5.5. Discussion

We were able to culture EGCs from post-migratory PGCs of the genital ridge using

d14.5 embryos from the rat strain WKY-Dnd1ter/Ztm. The loss of Dnd1 clearly helped

render the isolation of EGC lines possible. Propagated EGCs were pluripotent and

half of the isolated cell lines retained a stable chromosome number. The wild type

cell line, rEGC2, contributed to the first germline competent chimera made with rat

EGCs.

EGCs have been derived from the genital ridge of other species such as mice, pigs,

chicken and goats (Matsui et al., 1992; Resnick et al., 1992; Shim et al., 1997; Jia et

66 Chapter 5

al., 2008). However, the only rat EGC derived so far are early pre-migratory EGCs

from Sprague Dawley rat embryos at d10pc (Leitch et al., 2010). Here we isolated

late EGCs from the genital ridge. Our protocol for EGC-derivation differs from that of

Leitch et al. (2010) in the embryonic stage (d14.5pc compared to d10pc), in the

feeder cell line (TRF-O3 instead of stem cell producing Sl4-m220 and MEF) and

additionally our EGC colonies emerged earlier in culture (at passage zero and not

passage one).

Despite the fact that we cultured wild type as well as ter/ter PGCs, with the exception

of +/+ rEGC2, only the latter could be propagated in an undifferentiated state. The

mutation of the putative tumor suppressor gene Dnd1 supports or simply accelerates

the immortalization of cultured PGCs from d14.5pc to EGCs. This reveals that the

fate of the PGCs is influenced as early as d14.5pc by the loss of Dnd1. Something

similar can be seen in mice where the tumor suppressor phosphatase and tensin

homologue protein (PTEN) was knocked out, as the PGC-restricted PTEN knockouts

resulted in a high incidence of testicular tumors and the mutant mPGCs formed

EGCs more efficiently than wild type PGCs (Kimura et al., 2003). Moreover,

hyperactivation of the serine/threonine kinase AKT and its suppression of p53

activation also augmented the efficiency of EGC establishment (Kimura et al., 2008).

Dnd1 is known to protect certain mRNAs, including those of tumor suppressor genes

such as p27 and Lats2, from repression by miRNAs (Kedde et al., 2007). The

immortalization of PGCs could be facilitated through the inactivation of tumor

suppressor genes, a well-known process in other (tumor) cell lines. An example

would be the inactivation of p53, a tumor suppressor gene, which enhances

spontaneous cellular immortalization and initiates the tumorigenic transformation of

cells in vivo while playing a key role in the immortalization of murine embryonic

fibroblasts (MEFs) in vitro (vom Brocke et al., 2006; Fridman & Tainsky, 2008).

Gonadal teratocarcinomas originate from PGCs and consist of differentiated tissues

from all three germ layers along with undifferentiated, pluripotent cells (Stevens,

1967). In ter/ter rats all teratomas were restricted to the gonads, meaning that all

tumor precursor cells reach the genital ridge. The growth rate of the teratomas was

gender-specific. First gender-dependent differences such as varying gene expression

Chapter 5 67

profiles can be seen after sex differentiation which starts at d12.5pc in the mouse

embryo (McLaren, 2003; Sabour et al., 2010). The embryonic stage of a mouse at

d12.5pc is comparable to d14pc in the rat (Hill, 2010). These facts made late rat

EGCs from the genital ridge our preferred tool for further research on teratoma

development in connection with Dnd1.

Although seven of the cell lines stem from ter/ter WKY-Dnd1ter/Ztm the number of

chromosomes remained remarkably stable in three of them. In fact, the chromosomal

stability was comparable to that found in rEGC2 or ESC (unpublished data) of +/+

animals from the same rat strain.

The pluripotency of our EGCs could be shown by AP-staining, immunostainings, RT-

PCR, and teratoma development after injection of EGCs into immunodeficient mice.

High alkaline phosphatase activity is characteristic for all pluripotent cells.

Immunocytology detected further markers of pluripotency (Oct4, SSEA1, SSEA3 and

Nanog) along with the germ cell maker vasa (DDX4/MVH). Derivatives of all three

germ layers were identified in the tumors that developed in the NOD.CB17-Prkdcscid/J

mice and confirmed the pluripotent potential of the cells.

Comparative expression analysis by RT-PCR for different pluripotency and germ cell

markers in EGC and/or ESC lines have been performed before in humans, mouse

and rat (Toyooka et al., 2000; Clark et al., 2004; Buehr et al., 2008; Conrad et al.,

2008; Durcova-Hills et al., 2008; Leitch et al., 2010). In the following we compare our

results to those of others. Most markers (Nanog, Oct4, Sox2, Rex1, c-kit, c-myc,

Stella) were expressed in EGCs, ESCs and the genital ridges of rats, which is in

accordance with the cited literature (Buehr et al., 2008; Durcova-Hills et al., 2008;

Leitch et al., 2010). Unexpectedly, the PCR amplification of germ cell markers

showed that all ESC and EGC lines expressed DDX4/MVH. In the mouse,

DDX4/MVH is a specific marker from the late migration to the post-meiotic stage only

expressed in germ cells after entry into the genital ridge. It is not expressed in ESC,

EGCs from migrating PGCs or in PGCs before entering the genital ridge (Toyooka et

al., 2000). The results for undifferentiated human ESCs are varied: Clarke et al.

(Clark et al., 2004) found no expression of VASA while Conrad et al.(Conrad et al.,

2008) detected high levels of VASA in their ESCs. This difference could therefore be

68 Chapter 5

either species-specific or a result of different culture conditions, as it is known that

ESCs can differentiate to germ cells in vitro (Toyooka et al., 2003; Clark et al., 2004).

The fact that we did not find DDX4/MVH expression in the distal part of the embryo at

d10.5pc (supplementary data), which contains the PGCs, leads us to lean towards

the latter. The visibly reduced expression of DDX4/MVH and Sox2 expression found

in ter/ter genital ridges when compared to +/+ genital ridges could be explained

through an early loss of PGCs in this tissue. This would be analogous to the

observations made in the Ter mouse where fewer PGCs survive and enter the genital

ridge of the Ter/Ter than of the +/+ embryo (Sakurai et al., 1994). Furthermore, a

slight expression of Klf4 was seen in the embryo at d10.5 pc, whereas Klf4 was not

detected in PGCs at a comparable embryonic stage in the mouse (Durcova-Hills et

al., 2008) or in the posterior fragment of a rat at d10pc (Leitch et al., 2010).

The +/+ EGC line was capable of chimera contribution and also germline

transmission. This is the first time that germline competence could be shown for

EGCs from the rat, thereby proving that the conversion of once unipotent PGCs to

truly pluripotent EGCs is possible in the rat. Leitch and colleagues (2010) showed

that genetically manipulated EGCs can result in chimeric rats; however, the offspring

did not carry the transgene. The fact that germline transmission could not be shown

in those rat EGCs could be either due to the genetic manipulation or maybe only the

increased number of passages. EGCs have even been used as an alternative to

ESC for the successful germline-competent genetic manipulation in other species

such as pig (Piedrahita et al., 1998) or chicken (van de Lavoir et al., 2006) and could

present an alternative tool to ESCs for gene targeting in the rat.

Conclusion

Here we cultured the first pluripotent and germline competent EGCs from late rat

embryos. The genital ridge-derived rat EGCs offer an innovative approach for studies

on germ cell differentiation and germ cell tumor formation in both genders.

Furthermore, our data indicates that the loss of the putative tumor suppressor gene

Dnd1 enhances the cultivation of late rat EGCs and supports EGC immortalization.

The culture of EGCs gives us a new opportunity to research the interactions between

Dnd1 and its target genes.

Chapter 5 69

5.6. Figures

Figure 5.1

Fig. 5.1: (A) Embryo at d14.5pc and the extracted genital ridges (white arrow) attached to

the mesonephros. The genital ridges contain the PGCs and are cultured. (B) Genital ridge-

derived EGC colonies at passage 0 (P0) and passage 10 (P10)

70 Chapter 5

Figure 5.2

Fig. 5.2: Genotype and gender. (A) Genotyping: Dnd1 was amplified by PCR followed by a

restriction digest with Kpn1. The wild type allele was cut, whereas the ter allele lost the KpnI

recognition site and remains uncleaved (rEGC2: +/+; rEGC1, 3-11: ter/ter). The gender

detected by PCR amplification of the Y chromosome specific Sry gene (rEGC 4, 7, 8: male;

rEGC1-3, 5, 6, 9-11: female). (B) Alkaline phosphatase (AP) staining. Morphological

differences visible between the AP positive colonies of a male and a female EGC line.

Chapter 5 71

Figure 5.3

Fig. 5.3: Differences in-between EGC lines. (A) Metaphase spreads of rEGCs. Euploid

chromsosome complement of rEGC2 showing 42 chromosomes in passage 10 and 20.

EGC3 with structural and numerical aberrations in passage 10. Aneuploid chromosome

complement of rEGC7 with a reunion figure (arrow) in passage 20. (B) WST-1 assay to

determine the proliferation rates of rEGC1-8 and inactivated TRF-03 as control.

72 Chapter 5

Figure 5.4

Fig. 5.4: Staining of rat EGC for germ cell and pluripotency markers. The

immunocytofluorescence was positive for SSEA3, SSEA1, Nanog and Oct4, as well as the

germ cell marker DDX4/MVH.

Chapter 5 73

Figure 5.5

Fig. 5.5: Expression analysis: RT-PCR of pluripotency markers using cDNAs from late rat

EGC lines (rEGC1-8), rat ESC from WKY-Dnd1ter/Ztm, and the genital ridges of d14.5pc

embryos containing PGCs. Controls: TRF-O3: feeder cells cultured in 2i medium. C: negative

control.

74 Chapter 5

Figure 5.6

Fig. 5.6: Differentiation potential of EGCs (A-D) Injection of EGCs into immunodeficient

NOD.CB17-Prkdcscid/J mice led to formation of teratomas with derivatives of all three germ

layers. (A) Mucosal epithelium (representing Endoderm). (B) Neuronal tissue (representing

Ectoderm). (C) Cartilage (representing Mesoderm). (D) Skeletal muscle tissue (representing

Mesoderm). (E-F) Chimeras and germline transmission: (E) Chimeric siblings. (F) Chimeric

mother with albino germline pup and littermates.

Chapter 5 75

5.7. Supplementary data

Recently, we mated heterozygous ter/+ rats from the WKY-Dnd1ter/Ztm and cultivated

pre-migratory PGCs from rat embryos at d10.5pc (Suppl. Fig. 1). PGCs were

collected as described by Leitch and colleagues (2010). The culture conditions used

were identical to those described for the embryos d14.5pc, aside from the fact that

the EGC colonies were detached from the feeder between 9-12 days of culture and

not 7-10 days. Altogether PGCs from 21 different embryos were cultured and each

embryo gave rise to EGCs. Genotyping of the EGC lines revealed that ter/ter, wild

type and heterozygous cell lines from both genders had survived in vitro (Suppl. Fig.

2). This shows that inactivation of Dnd1 does not play a role in deriving pluripotent

cells from early PGC in vitro. Pluripotency was depicted in all cell lines by staining for

alkaline phosphatase (Suppl. Fig. 1B). Expression analysis was performed by RT-

PCR with four randomly chosen wild type and ter/ter cell lines from d10.5pc. The

posterior fragment of the embryo, which contains the PGCs, was used as a control.

The pluripotency and germ cell markers Nanog, Oct4, Sox2, Klf4, Rex1, c-kit and

Stella were detected in the EGCs and the embryo. Expression of DDX4/MVH was

only exhibited by EGCs, and not by the PGCs in the embryo d10.5pc (Suppl. Fig. 3).

76 Chapter 5

Supplementary Figures

Supplementary Figure 5.1

Suppl. Fig. 5.1: (A) Embryo at d10.5pc. The pre-migratory PGCs are located in the distal

end of the embryo. (B) Early WKY-EGC colonies stained with AP.


Suppl. Fig. 5.2: Genotyping and gender determination of early WKY-EGC lines from 21

embryos. 8 EGC lines are male and 13 are female. 9 EGC lines are ter/+, 4 are +/+, and 8

are ter/ter.

Chapter 5 77


Suppl. Fig. 5.3: Expression analysis: RT-PCR of pluripotency markers using cDNAs from

four early WKY-EGC lines (21, 22, 25, 37), posterior fragment of d10.5pc embryos containing

pre-migratory PGCs (dE), and ESCs from the WKY-Dnd1ter/Ztm rat strain; C: negative

control.

78

Discussion and Conclusions 79

6. Discussion and Conclusions

Incidence of infertility and TGCTs are on the rise and the key to understanding these

diseases lies in discerning the factors and genetics regulating germ cell

differentiation to mature gametes and de-differentiation to pluripotent cells (Huyghe

et al., 2003; Hotaling & Walsh, 2009). A new phase in the intensive research on

genetic and molecular mechanisms involved in germ cell differentiation was

introduced by generating germ cells from embryonic stem cells and the isolation of

pluripotent spermatogonial stem cells from germ cells as a therapeutic tool

(Mardanpour et al., 2008; Guan et al., 2009; Ko et al., 2009). The goal of this study

was to characterize and establish two models that can be used for further research

on germ cell development. In general, this is the WKY-Dnd1ter/Ztm rat strain and, in

particular, the cultured EGCs of said strain. Dnd1 is a relevant gene in germ cell

development of the rat and this is elucidated in the manuscripts containing the two

parts of this project:

Chapter 4: The ter mutation in the rat Dnd1 gene causes gonadal teratomas and

infertility in both genders

Chapter 5: Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat

embryonic germ cells

This chapter summarizes the principal results of the two aforementioned chapters

and provides a combined discussion based on the literature review (chapter 2) while

giving an outlook on the future perspectives for germ cell and teratoma research.

6.1. The WKY-Dnd1ter/Ztm rat as a model for germ cell tumors

Linkage analysis, fine mapping and subsequent sequencing of candidate genes

traced the WKY/Ztm-ter rat to a point mutation in exon 4 of the Dnd1 gene on

RNO18. Accordingly, the strain was denominated WKY-Dnd1ter/Ztm, and it is a

valuable animal model for research on GCTs in both genders (chapter 4). Studies on

80 Discussion and Conclusions

germ cell development have confirmed a central role of Dnd1 in the regulation of

PGC migration, survival and tumor suppression in different vertebrates (Ketting,

2007).

6.1.1. Highly conserved functions of Dnd1 in germ cell development

The Dnd1 mutations on Chr 18 in mice (Youngren et al., 2005) and rats occurred

spontaneously with a single base change introducing a premature stop codon, which

probably causes a c-terminal truncation of the Dnd1 protein. The manifestation of

these highly related genetic modifications in impairment of gametogenesis along with

neoplastic transformation of PGCs to ECC emphasizes a central and conserved

function of Dnd1, in particular of the c-terminal end of the protein. The c-terminus of

the zebrafish Dnd protein possesses an ATPase domain, which is essential for PGC

formation and survival (Liu & Collodi, 2010); however, it remains to be established

which parts of the c-terminus are crucial for a functioning Dnd1 protein in rodents and

whether an ATPase domain is involved. Downregulation of dnd in the germ plasm of

zebrafish and African clawed frog inhibits PGC migration and results in their

disappearance (Weidinger et al., 2003; Horvay et al., 2006). Mammalian PGCs lack

migration disorders, while tumorigenesis is not observed in anamnia, thereby

illustrating the varying functions of Dnd1. The ontogenetic death of PGCs in response

to the functional inactivation of the dead-end gene, however, is a common

phenomenon in all species, including the rat. Consequently, dead end is a highly

conserved and essential gene regulating the survival of PGCs in both anamnia and

amniota.

6.1.2. Gonadal tumorigenesis and infertility in rodents

Genotyping of WKY-Dnd1ter/Ztm rats revealed that the homozygous Dnd1 mutation

resulted in complete penetrance with 100% of the ter/ter animals of either gender

developing teratomas, whereas no teratomas were found in their wild type or

heterozygous counterparts. This deviates from the phenotype observed in the

129/Sv-Ter mouse model for GCTs. In general mice of the 129/Sv strain develop

spontaneous teratomas on a regular basis without a mutation in Dnd1, which is

something unheard of in rats of the WKY strain. Loss of functional Dnd1 in male


129/Sv mice is responsible for increasing the TGCT incidence from 1% in wild type to

17% in heterozygous and 94% in homozygous males. At the same time, none of the

mutant females develop any teratomas (Noguchi & Noguchi, 1985). Consequently,

the Dnd1 mutation followed a recessive mode of inheritance in male and female rats,

which is distinct from the co-dominant Ter-allele involved in teratocarcinogenesis of

male mice.

Rats with a homozygous mutation in the Dnd1 gene were infertile due to a loss of

germ cells, while the heterozygous animals possessed normal fertility and litter sizes.

Similarly, all male Ter/Ter mice are also sterile and Ter/+ mice do not show any signs

of germ cell deficiency. The fertility of female Ter/Ter mice is only reduced as some

germ cells are capable of survival and progress to mature oocytes (Noguchi &

Noguchi, 1985; Sakurai et al., 1995).

Teratoma development in the rat testis was either bilateral or unilateral. The

underlying defects initiated proliferation of neoplastic cells in one or both testicles

while the non-tumorous testicle showed a complete depletion of germ cells and loss

of the typical structure of the tubuli seminiferi without GCT. Every one of the ter

females was infertile and developed OGCTs without ever exhibiting any cases of

degenerated ovaries. This could be related to the relatively short time period of about

42 days (at 21-66 days of age) during which tumors were observed in all females, not

being long enough to allow complete germ cell deterioration in the entire ovary prior

to neoplastic proliferation. The fact that non-neoplastic primary follicles found in early

OGCTs did lack oocytes supported this presumption. In comparison, tumor

progression varied strongly between male individuals, and tumors were diagnosed

over a time period of 182 days (at 14-196 days of age). The wide range of time

probably allowed one testis to develop teratomas, while the other degenerated.

6.1.3. Implications of the ter rat for human TDS and OGCTs

Human disorders of germ cell development leading to the assorted symptoms of TDS

are common and are believed to stem from ontogenetic disruptions (Skakkebaek et

al., 2001; Walsh et al., 2009). The WKY-Dnd1ter/Ztm rat strain is a model for TDS as

the Dnd1 mutation could result in infertility, small degenerated testis and TGCTs in

males. The older the male ter/ter rats were, the lower the rate of degenerated testes


and the higher the rate of bilateral tumors. Hence, it is highly likely that the infertile

and degenerated testes exhibited by ter/ter males are the predecessors of cancer.

This supported the view of Burns and colleagues (2010) that infertile men are at an

increased risk of developing testicular cancer.

In contrast to the Ter mouse, the WKY-Dnd1ter/Ztm rat was also a model for OGCTs.

It has been proposed that TGCTs and OGCTs share a common pathogenic

mechanism as, for the most part, both ovaries and testis are capable of developing

identical types of GCTs (Faulkner & Friedlander, 2000). Furthermore, clusters of

OGCTs and TGCTs have been discovered in one and the same family

(Giambartolomei et al., 2009) and chromosomal abnormalities of HSA 12p are found

in GCTs of either gender in humans (Faulkner & Friedlander, 2000). The phenotype

of the WKY-Dnd1ter/Ztm supported the idea of a common pathogenesis, as a shared

mutation in a genetic locus resulted in GCTs with equal frequency in both genders. It

is probable that closely related or even identical mechanisms were responsible for

the neoplastic transformation of germ cells in both genders of the WKY-Dnd1ter/Ztm

rat. This might also be the case in GCTs of some men and women.

6.1.4. Gender and environment

Gender and environmental influences affect the frequency of germ cell tumors in

humans (Skakkebaek et al., 2007; Greene et al., 2010), and this is to some extent

reflected in the gender dissimilarities observed in the rodent models. Deviating

factors present in the gonad as well as intrinsic factors of the germ cells can both be

held responsible for the gender differences observed in mammalian

teratocarcinogenesis. In humans, factors secreted by the male sertoli cells, as well as

estrogen and follicle-stimulating hormone (FSH), have long been strongly believed to

play a role in TGCT development (Sharpe, 2006). Furthermore, estrogen-like

compounds and endocrine disrupters are both discussed as possible causes for the

rising incidence of the TDS (McGlynn et al., 2008). A hormonal aetiology has also

been proposed for human OGCTs (Walker et al., 1988). The surroundings provided

by the gonad seem to be required in teratocarcinogenesis of both Dnd1–mutant

mouse (Matin, 2007) and rat, since neither species developed teratomas in ectopic

sites. This is substantiated by the fact that the restriction of teratomas to male Ter


mice is linked to the testicular environment, wherein female germ cells are as

capable as male germ cells of generating neoplastic clusters (Cook et al., 2009).

Differences between male and female rats existed, but were limited to tumor

progression. On the average tumors were diagnosed earlier in female than in male

rats, which is obviously the opposite of testicular surroundings enabling teratoma

growth in the mouse. Nevertheless, regardless of whether in the mouse or in the rat,

gender-specific features alter the course of germ cell development towards

teratocarcinogenesis in Dnd1-mutants. Which factors are responsible for inhibiting or

promoting tumorigenesis in the Dnd1 rodent models remains to be determined, but

the WKY-Dnd1ter/Ztm rat shows us that female factors have to be taken into account

as much as those of males. Using both the mouse and the rat as models for GCT will

help in identifying the different paracrine, autocrine or hormonal signals involved in

tumorigenesis. This could help comprehend the pathogenesis of human GCTs and

identify the source of the growing incidence.

6.1.5. Genetic background

The genetic component in susceptibility to TGCTs is as strong as it is complex. This

is applicable for humans with their high familial index of TGCTs and despite all efforts

no known susceptibility genes (Heaney & Nadeau, 2008), and is also true for the Ter

mouse and the ter rat. In the Ter/Ter mouse tumors only develop in the 129/Sv strain

and modifier genes (SlJ, Trp53null, Ay, CSS M19, M19-A2 and M19-C2) modulate the

teratoma incidence by interacting with the mutated Dnd1 (Lam et al., 2007).

Replacing the X-chromosome from the 129/Sv-ter mouse through that of the

C57BL/6 mouse was enough to shift the Ter/Ter phenotype from teratoma formation

to infertility (Hammond et al., 2007). In the rat backcrossing of the ter mutation to the

DA strain also yielded a change in phenotype, which proved that genetic factors

other than Dnd1 are associated with tumorigenesis in the rat (results not shown).

6.1.6. DND1 in humans

The role of DND1 in humans remains uncertain, but screening of over 500 men with

type II TGCTs made it fairly obvious that a DND1 mutation is an unlikely cause in the

majority of cases (Linger et al., 2008; Sijmons et al., 2010). Nevertheless, it is still


conceivable that DND1 plays a role in teratocarcinogenesis through mechanisms

more complicated than those investigated in the two previous studies. This could

include the presence of duplications and large deletions in the gene (Sijmons et al.,

2010), as well as imprinting mechanisms, miRNA suppression or epigenetic

silencing. Testisin, MGMT, SCGB3A1, RASSF1A, HIC1 and PRSS21 are examples

for putative tumor suppressor genes that are inactivated through imprinting or

epigenetic silencing in TGCTs (Honorio et al., 2003; Kempkensteffen et al., 2006;

Lind et al., 2007). Furthermore, elucidating the pathways of Dnd1 in the mouse and

rat could help identify new processes and modifier genes that are involved in human

GCT development since the pathways of DND1 and GCTs do cross. For instance,

DND1 inhibits the miR-373 mediated LATS2 repression (Kedde et al., 2007), and the

same miR-373 is believed to act as oncogene in TGCTs (Voorhoeve et al., 2006).

Additionally, increasing the knowledge on DND1 is of importance since it could play a

role in other cancers such as the tongue squamous cell carcinoma associated with a

DND1 downregulation (Liu et al., 2010). Information on the role of DND1 in

prepubertal GCTs is lacking. The 129/Sv-ter mouse lacks CIS or characteristic

karyotypic abnormalities, and teratomas are detected soon after birth, which makes it

a model for human prepubertal type I TGCTs. Hence, it is quite possible that human

DND1 mutations could be responsible for prepubertal TGCT (Zhu et al., 2007a;

Anderson et al., 2009). The new-found role of Dnd1 in rat OGCTs signifies that the

involvement of DND1 in human OGCTs should be evaluated in future studies. Dead-

end is a highly conserved protein associated with reduced fertility and infertility

throughout different species, strains and genders. Therefore, it is feasible that

disruption of DND1 could cause infertility or reduced fertility in adult human beings.

6.2. Derivation of pluripotent EGCs from the WKY-Dnd1ter/Ztm rat

PGCs of various species can be reprogrammed into pluripotent EGCs through the

application of specific culture conditions. We were able to culture the first pluripotent

EGCs from post-migratory PGCs of the genital ridge using the rat strain WKY-

Dnd1ter/Ztm. Mating of heterozygous WKY-Dnd1ter/Ztm rats and culture of PGCs from

60 embryos resulted in clonal proliferation of ten Dnd1-deficient ter/ter EGC lines of


both genders, while only one female wild type EGC line and no heterozygous cell

lines survived without differentiating in vitro. Therefore, Dnd1 played a role in

reprogramming cultured PGCs from d14.5pc to EGCs (chapter 5). EGCs were also

cultured from early rat embryos at d10.5pc, however, no Dnd1-dependent effects

were observed (chapter 5, supplementary data).

6.2.1. EGCs as a new approach to study rat Dnd1

The new rat EGCs can be used as a tool to determine the relevant mechanisms

involved in the neoplastic transformation of ter/ter PGCs. Previously, mouse EGCs

were employed to help clarify the interactions of Dnd1 with the APOBEC family

(Bhattacharya et al., 2008). Use of PGCs in research is restricted by their low number

and limited survival in the embryo, while their in vitro counterparts have the

advantage of being immortal and growing indefinitely. PGCs are the precursor cells

of both EGCs and ECCs and in the mouse these two pluripotent cell lines express

the same markers and differ only in their genetic stability. The tight link between

EGCs and ECCs was ascertained by the fact that mutations in Pten or p53 trigger

teratomas while also enhancing EGC derivation (Kimura et al., 2003; Kimura et al.,

2008). The advantage of culturing EGCs instead of ECCs as a tool to research Dnd1-

related effects is that the former enables comparisons between the wild type and

ter/ter genotype. Additionally, the EGCs have had less time than the instable ECCs

to accumulate genetic aberrations on top of the Dnd1 mutation. In general, ECCs are

aneuploid, while EGCs are usually euploid and maintain a normal set of

chromosomes (De Miguel et al., 2010). Almost half of the tested ter/ter EGC lines

and the +/+ EGC line used here remained euploid in culture for multiple passages.

In this project we cultured EGCs from d14.5pc WKY-Dnd1ter/Ztm embryos, despite

the fact that reprogramming of PGCs is more difficult and less efficient in late

embryonic stages (McLaren, 2003; Kerr et al., 2006). Both the neoplastic

transformation and sexual differentiation of PGCs starts at d12.5pc in the mouse,

which is the equivalent embryonic stage to the rat at d14.5pc. Furthermore, all tumor

precursor cells reach the genital ridge in Dnd1 mutants, as seen by the fact that no

ectopic GCTs developed. Altogether, the genital ridge-derived EGCs were more

likely than their earlier counterparts to display differences between wild type and


ter/ter EGCs; making them more prone to successfully identify the differences

causing ter/ter PGCs to develop into teratomas and +/+ to develop into gametes in

the adult animal. This hypothesis proved to be true as seen by the influence of

mutated Dnd1 on the efficiency of deriving pluripotent cells from late, and only late,

PGCs. Early EGCs from d10.5pc were also cultured and Dnd1 did not exert such

strong effects in primary culture. In fact, no significant differences were seen between

embryos of different genotypes in their aptitude to generate EGCs. Teratoma

progression in the rat was gender-specific, and the late EGCs were more likely to

display germ cell intrinsic differences between the sexes. However, the growth rate

did not differ significantly between female and male ter/ter EGC lines. Consequently,

the differences in tumor progression of the rat are either induced by the somatic

surroundings of the germ cell or possibly the result of sexual differentiation of

PGCs/ECCs in later stages.

6.2.2. The culture of EGCs

Though EGCs of mouse and other species have been successfully cultured for some

time now, the rat proved to be more elusive, and rat EGC culture was achieved only

recently. The only rat EGCs to date stem from pre-migratory PGCs of Sprague

Dawley (SD) rat embryos at d10pc (early SD-EGC) (Leitch et al., 2010). Slightly

modified culture conditions were used to generate our WKY-Dnd1ter/Ztm EGCs from

d14.5pc embryo (late WKY-EGC). The specification of PGCs in the embryo is

accompanied by an upregulation of pluripotency markers such as Nanog, Oct4 and

Sox2. Nevertheless, PGCs differ significantly from EGCs as they are unipotent cells

restricted in their developmental potency to the gametes and incapable of integrating

into the blastocyst (McLaren & Durcova-Hills, 2001; Durcova-Hills et al., 2008). The

complete conversion of mouse PGCs to EGCs takes about 10 days, even though

some large EGC-like colonies can be observed after 6 days in culture (Durcova-Hills

et al., 2006). Similar to this, most of our rat EGC colonies from the genital ridge of

WKY-Dnd1ter/Ztm appeared between 7-10 days. This differs from findings of Leitch

and colleagues, who passaged their cells after 14 days of primary culture and

described the emergence of the first rat EGC colonies 7-15d after passaging, thereby

adding up to a total of 21-29 days. It is possible that loss of Dnd1 accelerated the


formation of ter/ter EGC colonies, and that further +/+ EGC colonies might have

formed during a longer period of culture. However, that does not explain the

presence of the one EGC line from a d14.5pc +/+ embryo and the fact that d10.5pc

+/+ colonies also formed without having to be passaged first. Another reason might

be the culture conditions applied, which in the case of the early SD-EGCs where

based on the culture of mouse EGCs. The cells were cultured in media containing

FCS and bFGF and were plated on SCF-producing feeder cells. The media was

changed on day 3 and replaced with serum free N2B27 media supplemented with 2i-

LIF (Leitch et al., 2010). In contrast, our late WKY-EGCs were cultured using the

same conditions applied to rat ESCs. They were cultured directly in 2i-LIF media and

did not come into contact with either serum, or bFGF and SCF for that matter. Serum

induces differentiation or death in ESCs of the rat (Li et al., 2008), and a similar effect

might be inhibiting or prolonging the derivation of pluripotent early SD-EGCs from

PGCs.

To this date the culture conditions for rat EGCs have not been optimized, and almost

certainly do not achieve the utmost efficiency in deriving EGCs. Even though we

showed that 2i-LIF media is sufficient to generate EGCs, it remains to be determined

whether bFGF or SCF might enhance derivation or growth. In the mouse this is the

case, as neither SCF nor bFGF are required when using the 2i-LIF media, yet bFGF

still augments the number of EGCs developing from PGCs. As feeder cells are

eliminated during initial exposure to the inhibitors and removed in subsequent

changes of media, the number of media changes during primary culture might play

an important role in EGC derivation. Replacing media every other day as described

for both the early SD and our late WKY-EGCs might be excessive and unwarranted.

A greater sensitivity of rat EGCs than mouse EGCs to feeder cell elimination was

proposed by Leitch and colleagues (2010) to be one cause for the species

differences between mouse and rat in EGC derivation.

Prior to 2i-LIF media, long-term culturing of any pluripotent cells of the rat and many

mouse strains was not possible. Until now, human EGCs have been cultured in

media containing bFGF, LIF and forscolin (Shamblott et al., 1998). However, these

culture conditions are in need of improvement, as these human EGCs appear to


have a limited long-term proliferative potential and do not contribute to teratomas (Yu

& Thomson, 2008). Therefore it would be interesting to see whether applying 2i-LIF

media might be a superior method for culturing human EGCs. This might not only

hold true for humans, but might also open new doors for deriving EGCs from other

species.

6.2.3. Molecular networks of pluripotency in EGCs

Comparisons of pluripotency and germ cell markers demonstrate almost identical

expression patterns between mouse or rat PGCs and EGCs (De Miguel et al., 2010).

Yet there are minor differences, and Blimp1 is the key gene expressed in PGCs but

not in EGCs or ECCs (Ancelin et al., 2006; Durcova-Hills et al., 2008; Leitch et al.,

2010). This result could be confirmed for all of the established genital ridge-derived

EGC lines. It is hypothesized that Blimp1 is crucial for repressing PGCs from

acquiring a genuinely pluripotent phenotype. High levels of the Blimp1 target genes

Dhx38 (after 8 days of culture), c-myc (after 7 days of culture) and Klf4 (after 3 days

of culture) are expressed in EGC while neither one is transcribed in the mouse PGCs

at d8.5pc (Durcova-Hills et al., 2008). Nevertheless, gene activity of Klf4 and c-myc

was found in the genital ridges of the WKY-Dnd1ter/Ztm rat. In analogy to the mouse

(Durcova-Hills et al., 2008) the c-myc expression probably results from the

surrounding somatic cells of the gonad, however, the source of the Klf4 expression

remains unknown. In general, the expression of homologs of the vasa gene (VASA,

Mvh, DDX4/MVH) is restricted to germ cells. The Mvh protein is only detectable in

mouse PGCs after entry in the genital ridge at d11.5 pc, and it is lost after 3 days of

culturing in EGCs (Durcova-Hills et al., 2006). The situation observed in our culture is

different, as the expression of the rat DDX4/MVH was retained by the rat EGCs and

even acquired by rat ESCs. This could result from differences between the two

species mouse and rat or be a direct result of our culture conditions. We found no

expression of DDX4/MVH in the early embryo containing the PGCs and suspect that

our culture conditions are responsible for its expression. In the mouse Mvh-activity

can be induced in EGCs, but not in ESCs, through co-culture with gonadal somatic

cells (Toyooka et al., 2000) and the differentiation of human or mouse ESC to vasa-

positive PGCs can occur spontaneously or in specialized culture conditions (Ko &


Schöler, 2006). Consequently, the teratoma fibroblasts (TRF-O3) used as feeder

cells may produce factors inducing ESCs and EGCs to express DDX4/MVH. To this

date, no other study has investigated the expression of vasa in pluripotent cells in 2i-

LIF media. Hence another possibility is that 2i-LIF media induces pathways activating

DDX4MVH expression. However, whatever the reason, the gene activity of

DDX4/MVH does not compromise the pluripotency of said EGCs or ESCs.

6.2.4. Germline competence of rat EGCs

Pluripotent cells have the extraordinary ability to give rise to all cells of the body and

this includes those of the germline. The pluripotency of our +/+ and ter/ter EGC lines

could be shown by alkaline phosphatase staining, immunostainings and RT-PCR for

pluripotency markers, as well as teratoma development after injection of EGCs into

immunodeficient mice. Furthermore, the +/+ EGC line produced chimeric offspring

after injection into the blastocyst. Mating of a female chimeric animal with an albino

rat resulted in 40% albino germline pups, thus proving that the EGC line contributed

to germ cells. This is the first time that germline competence could be shown for rat

EGCs and proved that the 2i-LIF conditions suffice to generate truly rat pluripotent

EGCs. Leitch and colleagues show that their genetically manipulated EGCs can

result in chimeric rats; however, these lack the ability of producing germline offspring.

The fact that germline transmission is not seen in those chimeras made with early

SD-EGCs could result from minor difference such as culture conditions, the genetic

manipulation or the higher passage number of the injected EGCs.

Germline transmission opens new possibilities for EGCs of the rat as they could

present an alternative tool to ESCs for gene targeting. EGCs have even been used

for the successful genetic manipulations in other species such as pig or chicken

(Piedrahita et al., 1998; van de Lavoir et al., 2006). The capacity of mouse ESC and

EGC in generating chimeras is identical (McLaren & Durcova-Hills, 2001). The

frequency of chimeras after injection of the early rat EGCs is higher than that

previously published for ESCs. This led Leitch and co-workers to speculate on

whether they might present a better tool than ESCs for genetic manipulations in the

rat (Leitch et al., 2010). Our late WKY-EGC line was highly capable of generating

chimeras, which would be in agreement with this. Further data is necessary to prove


or refute this hypothesis, as factors such as rat strain, passage number and

karyotype of the specific cell line all influence the ability to contribute to chimeras.

6.3. Dnd1-related molecular mechanisms at work in germ cells

Nothing was known about the functions of Dnd1 in the rat prior to this study. A

spontaneous mutation causing teratomas gave rise to this project and ended up

demonstrating the importance of Dnd1 in germ cell development. Loss of Dnd1

enhanced the ability of germ cell precursors to form pluripotent germ cells in vivo

(ECC) and in vitro (EGC), as described in the previous two chapters 4 and 5.

Although the effects of the Dnd1 mutation in the rat are described here, the involved

molecular pathways remain in the dark for now. Nevertheless, the newfound

knowledge on Dnd1 in the rat in addition to the molecular data gained in other

species provides ample room for speculations and conclusions.

6.3.1. Interactions of Dnd1 and miRNAs

Dnd1 interacts with miRNAs of mice, zebrafish and humans and permits the

expression of specific target genes. The extent to which Dnd1 modifies the activity of

what genes remains to be established (Kedde & Agami, 2008). In zebrafish Dnd

modulates Nanos1 and Trdr7 expression through miR-430. Human cell culture

showed that DND1 allows the transcription of Lats2 through miR-372/373 inhibition,

Cx43 through miR-1/206 inhibition and p27 through miR221/222 inhibition (Kedde et

al., 2007). In the mouse Dnd1 is thought to allow upregulation of cell cycle inhibitors

in mitotically arresting germ cells even though inhibiting miRNAs such as the miR-17-

92 and the miR 290-295 are present (Hayashi et al., 2008; Cook et al., 2011). Due to

the ter-mutant rat and the culture of its EGCs, it becomes possible to elucidate the

role of Dnd1 with hither to unspecified target genes and miRNAs.

6.3.2. Effects of Dnd1 on EGC derivation and culture

The zebrafish miR-430 family, the mouse miR-290 cluster (miR-291-3p, miR-294,

and miR-295), and the human miR-371/2/3 and miR-302 clusters are homologous to

one another and highly expressed in the early embryo (Wang & Blelloch, 2009). In


humans, miR-372 maintains p21 at a low level and is probably in part responsible for

the accelerated G1/S transition of dividing ESCs (Qi et al., 2009). The mouse miR-

290 cluster is known to be highly expressed in PGCs and pluripotent cells, including

in EGCs. Its presence has been associated with the expression of pluripotency

markers and maintaing a pluripotent state (Hayashi et al., 2008; Zovoilis et al., 2008).

Similar to human miR-372, miR-290 controls cell cycle progression by allowing a

rapid G1/S transition in ESC through suppression of multiple inhibitors such as p21,

p27, pRB and Lats2 (Wang & Blelloch, 2009). Incidentally, p21, p27 and Lats2 are

the cell cycle inhibitors linked to Dnd1 expression, and absence of Dnd1 causes loss

of p21 and p27 in the mouse embryo during mitotic arrest (Cook et al., 2011).

Therefore, the enhanced EGC derivation of the rat in absence of Dnd1 could possibly

be related to a miRNA-mediated absence of specific cell cycle inhibitors. However, it

was deemed improbable to be the main cause after a proliferation assay was

performed with the +/+ and ter/ter cell lines and showed no significant Dnd1-

dependent differences in growth rates. The results of the proliferation assay indicated

that Dnd1 affects the process of immortalization of PGCs rather than their in vitro

growth rate as EGCs.

The precise pathways causing PGCs to transform into pluripotent EGCs or ECCs are

unknown, but similar mechanisms seem to be involved. Hyperactivation of Akt

signaling in PGCs increases the efficiency with which EGCs are established. The

tumor suppressor Pten is a negative regulator of the PI3K dependent Akt activation

(Kimura et al., 2003). Distinct mutations in Pten or p53 induce gonadal teratomas in

mice while also augmenting the number of EGCs derived from PGCs of the genital

ridge in the mouse (Kimura et al., 2003; Kimura et al., 2008). The same was also true

for the Dnd1 mutation of the rat, where the efficiency of deriving late WKY-EGCs was

increased in the mutant animals sure to develop teratomas. Effects of the Ter

mutation on EGC-derivation in the mouse are likely, but unknown. In view of the

similar effects on PGCs, it is conceivable that Pten, p53 and Dnd1 could be involved

in similar pathways; a notion which is supported by the fact that mouse Dnd1 is

capable of binding transcripts of the negative cell cycle regulators p53 and Pten

(Cook et al., 2011). Furthermore, the Dnd1 and the p53 mutation both require similar


factors for the induction of GCTs in vivo, as teratomas primarily develop in the 129/Sv

strain and not on a mixed C57BL/6 x 129/Sv genetic background (Donehower et al.,

1995).

6.3.3. Onset of germ cell loss and transformation

Dnd1 is upregulated in PGCs after specification at d7.25pc and the first effects in the

Ter-mutant mice are seen shortly afterwards when germ cell deficiency starts at

d8.0pc. At what point germ cells are lost in the rat remains to be determined,

however, a situation similar to that of the mouse is not unlikely. Germ cells are

completely depleted in immature males at three weeks. An embryonic onset of germ

cell loss is insinuated by the lower expression of germ cell-specific markers, such as

DDX4/MVH and Sox2, in ter/ter than in +/+ rat genital ridges.

The morphological changes found in PGCs of ter/ter mice in the genital ridge

pinpoints the beginning of teratocarcinogenesis in Ter/Ter mice to d12.5pc (Matin,

2007). Intrinsic changes of ter/ter rat PGCs were exhibited at a similar embryonic

stage: the loss of Dnd1 was found to enhance derivation of pluripotent EGCs at

d14.5pc. The ter mutation played no substantial role in generating pluripotent cells as

such, since deriving EGCs from embryos at d10.5pc showed no significant Dnd1-

related effects (chapter 5, supplementary data). These results reveal that the loss of

Dnd1 had influenced the fate of the PGCs by d14.5pc, and the development leading

to teratocarcinomas instead of gametes commenced.

6.3.4. Dnd1 in mitosis and meiosis

In the mouse, tumor development depends on male factors present in the genital

ridge, and the initiation at d12.5pc (Matin, 2007) coincides with the time healthy male

germ cells start entering G0 mitotic arrest (Western et al., 2008). Ter/Ter germ cells

fail to enter G0, which has led to the suggestion that Dnd1 regulates mitotic arrest in

male germ cells (Cook et al., 2011). The contrasting situation in the rat, with

teratocarcinogenesis in both sexes, expands the role previously conferred to Dnd1.

In the rat it cannot be reduced to the inability of germ cells to enter mitotic arrest in

males, as this does not offer an explanation for teratocarcinogenesis in females. The

high level of the Dnd1 target gene p27 found in mitotically arresting germ cells


between d13.5pc and 15.5pc is not exhibited by male mutant mice or by female germ

cells between d12.5-14.5pc (Western et al., 2008). Should the male rat mirror the

situation in the male mouse, this would signify that Dnd1 target genes and

tumorigenesis differs between female and male rats. An alternative explanation could

be that the downregulation of specific cell cycle inhibitors might be a secondary effect

instead of the cause of tumor development.

Between d12.5pc and 14.5pc, Dnd1 is downregulated in XX and upregulated in XY

germ cells. Similar to the rostro-caudal wave of meiotic entry, the progressive

downregulation of Dnd1 ensues from anterior to posterior in the female gonad

(Youngren et al., 2005). Whether rat (or mouse) Dnd1 played a major role during

meiosis is unknown, however, it was deemed improbable. After all, Dnd1 is

downregulated in the mouse during meiosis (Youngren et al., 2005), and the rat

ter/ter germ cells are on the road to pluripotency before meiotic entry as seen by their

ability to generate EGCs at d14.5pc. Furthermore, the absence of Dnd1 in the mouse

actually results in an upregulation of meiotic markers in male germ cells (Cook et al.,

2011). It is more likely that Dnd1 is required for the suppression of

teratocarcinogenesis before the meiotic entry of germ cells. This, together with the

fact that female rats were affected by teratomas, also makes improbable the idea

harbored by Youngren and colleagues (2005), that tumor resistance or susceptibility

might be conferred through the gender-dependent patterns of Dnd1 activity. To what

extent differences and similarities exist in the tumor development of male and female

ter/ter rat needs to be established and might provide new insights into

teratocarcinogenesis.

6.4. Final conclusion and future perspectives

Two models capable of giving new information in the area of rat germ cell

development are described in this study: the WKY-Dnd1ter/Ztm rat and the EGCs

derived from it.

Both models clearly emphasize the importance of Dnd1 in germ cell development.

Loss of Dnd1 is responsible for an increased tendency of germ cells to form


pluripotent cells in vivo and in vitro. Furthermore, its absence results in infertility due

to the inability of non-neoplastic germ cells to survive. The other principal

achievement of this study is the establishment of the first germline competent EGC

line of the rat. This proves that the conversion of previously unipotent PGCs to truly

pluripotent EGCs is possible in the rat by applying the described culture conditions.

Further research in the WKY-Dnd1ter/Ztm rat strain includes continuing the analysis of

mRNA expression and protein translation of both the truncated and wild type Dnd1.

Moreover, a project is in progress to generate a rescue with an inducible expression

of wild type Dnd1 in the ter/ter rat. The revision of the ter/ter phenotype would be the

final proof that the ter mutation leads to the loss of a functional Dnd1. Additionally,

the inducible Dnd1-expression would allow investigating the role of Dnd1 during

different developmental stages and in prospective target locations other than the

gonad, such as the heart.

In the mouse the Ter phenotype differs between strains. Backcrossing of the mutated

rat Dnd1 onto another strain, in this case the DA rat, has resulted in a modulation of

the phenotype (results not shown). The next step is the comparison of different

strains with varying phenotypes to help identify other aspects involved in

teratocarcinogenesis. Backcrossings of the mutated Dnd1 to different rat strains such

as LEW and BN remain to be established. Having the ter mutation in different rat

strains could help identify modifier genes regulating the ter phenotype and define the

precise functions of Dnd1 in the rat.

At what point in time the teratocarcinogenesis or the germ cell loss commences

remains unknown and this could potentially be clarified through immunohistological

staining of germ cells or ECC in the embryo and early gonads. Ascertaining the

absence or presence of ECCs in the degenerated testes or a unilateral orchiectomy

in ter/ter males, followed by long term tumor check-ups, could help identify whether

the non-tumorous testis found in some male rats stays degenerated and does not

form teratomas. Identifying genomic imprinting and chromosomal abnormalities in the

ECCs would help identify the embryonic stage in which teratoma precursors develop


and allow more precise comparisons between the rat animal model and the different

types of TGCTs in humans.

The environment allowing growth of TGCTs includes specific autocrine, paracrine

growth factors and hormones. The effects of different factors on the efficiency of

EGC formation and growth could be tested in vitro. As TGCT and EGC derivation are

linked, this could help identify the factors responsible for tumor growth and the

gender dependent differences visible in the WKY-Dnd1ter/Ztm rat. The expression

levels of Dnd1 and its target genes need to be identified in the EGCs and compared

between +/+ and ter/ter cell lines. Microarrays could be another method with which to

identify differences between +/+ and ter/ter cell lines. Also of interest would be the

determination whether culture of earlier EGCs or ESCs exhibit any Dnd1-dependent

effects. Preliminary data from early WKY-EGCs (d10.5pc) showed no significant

differences in EGC derivation between +/+, ter/ter and ter/+ animals; however,

growth rate and genetic stability remain to be evaluated. Additionally, the ability of

early +/+ EGCs to contribute to germline chimeras needs to be assessed.

A whole new field of opportunities has been opened in rat research through the

breakthrough in culturing pluripotent and germline competent cells from the rat. The

capacity of pluripotent cells to generate knockouts or to replace tissue has been long

studied in the mouse; which pluripotent rat cells are the most resourceful remains to

be determined. For now, a comparison of multiple ESC and EGC lines, cultured and

injected using the same conditions and rat strains, would be required to find out

which has the higher potential of resulting in germline chimeras.

The basic functions of Dnd1 in the rat have now been identified. However, much

more remains to be accomplished, particularly in terms of germ cell development and

pluripotency. This project might simply mark the beginning of significant future

research.

96 Summary

7. Summary

Emily Northrup

“Dnd1: From Germ Cells to Teratomas in the Rat”

Human germ cell tumors (GCT) can be observed in all age groups, ranging from

infancy to adulthood, and are frequently diagnosed at a young age.

Teratocarcinomas are GCTs consisting of derivatives from all three germ layers and

undifferentiated embryonic carcinoma cells (ECCs). A disruption during embryonic

development transforms unipotent primordial germ cells (PGC)/gonocytes into

pluripotent ECCs. Other reproductive problems including infertility have been

associated with testicular GCTs. For unknown reasons the incidence of male GCTs

and infertility are on the rise in humans, making it increasingly important to elucidate

the mechanisms triggering the disease.

The key to understanding GCTs and infertility lies in discerning the factors and

genetics regulating germ cell differentiation to mature gametes or de-differentiation to

pluripotent cells. The ter mutation in the WKY/Ztm rat strain causes infertility along

with ovarian and testicular teratomas. Genetics and phenotype of the WKY/Ztm-ter

rat were investigated in the first part of this thesis. Linkage analysis, fine mapping

and sequencing of candidate genes traced the ter mutation of the rat to a point

mutation introducing a premature stop codon in the dead-end homolog 1 gene

(Dnd1). Genotyping revealed a recessive mode of inheritance with complete

penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both

genders, and no teratomas in their wild type or heterozygous counterparts. This

differs strongly from the co-dominant Ter mutation in the Dnd1 gene of the 129/Sv

mouse, which induces testicular teratoma and infertility but never ovarian teratomas.

Gonadal teratocarcinogenesis indicates that Dnd1 acts as a tumor suppressor gene

in mouse and rat. Teratoma progression in the rat was gender dependant and on the

average tumors developed earlier in females than males. Morphologically non-

tumorous testes of homozygous ter rats were reduced in size and weight.

Immunohistochemical along with histological stainings associated the gonadal

Summary 97

malformation with the absence of spermatogenesis. Additionally, oocytes were

lacking in non-neoplastic follicles of homozygous ter females, thereby showing that

Dnd1 is required for survival of non-neoplastic rat germ cells. The advantage of the

ter rat over the Ter mouse model is the ability of the former to allow studies on

ovarian GCTs and enable comparative studies on GCT development between

genders. In general, the WKY-Dnd1ter/Ztm rat is a novel animal model for further

studies on gonadal teratocarcinogenesis and the molecular mechanisms involved in

germ cell differentiation or de-differentiation. A special focus lies on the tumor

precursor cells, the PGCs, and identifying the factors inducing or accelerating their

neoplastic transformation to ECCs.

Although the developmental potency of PGCs is restricted to the germ lineage, ECCs

are not the only pluripotent cells derived from PGCs. Through in vitro culture,

unipotent PGCs can be converted into pluripotent embryonic germ cells (EGCs).

EGCs from the WKY-Dnd1ter/Ztm rat present an innovative approach allowing in vitro

studies on Dnd1 in germ cell differentiation and tumorigenesis. The successful

culture of EGCs from the genital ridge of the WKY-Dnd1ter/Ztm rat was established in

the second part of this thesis. Heterozygous ter/+ rats were mated and the PGCs

from embryos at day 14.5 post coitum (d pc) were cultivated in media containing

inhibitors of differentiating pathways (2i-LIF). The generated cell lines were

genotyped, and almost all were found to stem from Dnd1-deficient ter/ter embryos.

EGCs were also cultured from early rat embryos at d10.5pc, however, no Dnd1-

dependent effects were observed. Taken together, these facts demonstrate that the

inactivation of Dnd1 facilitates the immortalization of late, but not of early, PGC in

vitro. This reveals that the loss of Dnd1 has altered the fate of PGCs by d14.5pc, and

the development resulting in teratocarcinomas instead of gametes has commenced.

The EGC lines could be maintained in a pluripotent state as demonstrated by

expression of pluripotency markers and teratoma formation in immunodeficient mice.

Injection of the wild type cell line into blastocysts resulted in germline competent

chimeras, making this the first time that germline competence could be shown for a

rat EGC line.

98 Summary

In conclusion, this thesis demonstrates the importance of Dnd1 in rat germ cell

development, with its loss resulting in de-differentiation of germ cells to pluripotent

cells in vivo and in vitro. A further achievement is establishing the culture of genital

ridge-derived rat EGCs. Both, the characterized WKY-Dnd1ter/Ztm rat strain and the

established culture of EGCs from the genital ridge, open new possibilities for

research in conjunction with the Dnd1 gene and the mechanisms involved in germ

cell development.

Zusammenfassung 99

8. Zusammenfassung

Emily Northrup

„Dnd1: Von Keimzellen zu Teratomen in der Ratte“

Humane Keimzelltumoren (GCT) kommen vom Kleinkind zum Erwachsenen in allen

Altersgruppen vor und werden häufig in einem frühen Lebensabschnitt diagnostiziert.

Teratokarzinome, eine Form von GCT, weisen die Besonderheit auf, Gewebe aller

drei Keimblätter zu bilden und undifferenzierte Embryonale Karzinomzellen (ECC) zu

enthalten. Pluripotente ECC entstehen durch neoplastische Transformation aus

unipotenten Primordialen Keimzellen (PGC) bzw Gonozyten. Weitere

Reproduktionsstörungen, wie z.B Infertilität, wurden bereits mit GCT der Hoden in

Verbindung gebracht. Aus noch unbekannten Gründen nimmt die Inzidenz von

männlichen GCT und Infertilität zu, wodurch die Identifizierung der

krankheitsauslösenden Mechanismen zunehmend an Bedeutung gewinnt.

Um GCT und Infertilität zu ergründen, ist es erforderlich die Faktoren und

genetischen Hintergründe zu identifizieren, welche für die Differenzierung von

Keimzellen zu reifen Gameten oder De-Differenzierung zu pluripotenten Zellen

verantwortlich sind. Die ter Mutation in dem WKY/Ztm Rattenstamm verursacht

Infertilität sowie Keimzelltumoren von Ovar und Hoden. Genetik und Phänotyp der

WKY/Ztm-ter Ratte wurden in dem ersten Teil dieser Dissertation untersucht. Die ter

Mutation konnte anhand von Kopplungsanalysen, Feinkartierung und

Sequenzanalysen von Kandidatengenen auf eine Punktmutation zurückgeführt

werden, welche ein vorzeitiges Stopcodon in dem dead-end homolog 1 Gen (Dnd1)

verursacht. Genotypisierungen zeigten, dass der genetische Defekt rezessiv vererbt

wird mit kompletter Penetranz von Teratokarzinogenese und Infertilität in

homozygoten ter Ratten beider Geschlechter, wobei keine Teratome in wildtypischen

oder heterozygoten Tieren vorkommen. Dies unterscheidet sich stark von der

codominanten Ter Mutation in dem Dnd1 Gen der 129/Sv Maus, welche Infertilität

und Hodenteratome, jedoch niemals ovariale Teratome, zur Folge hat. Die

Teratokarzinogenese legt nahe, dass es sich bei Dnd1 um ein Tumorsuppressorgen

100 Zusammenfassung

handelt. Die Tumorprogression in der Ratte war zudem geschlechtsspezifisch

unterschiedlich. Im Durchschnitt entwickelten sich die Teratome früher in weiblichen

Ratten als in männlichen. Die Tatsache, dass morphologisch nicht tumoröse Hoden

von homozygoten ter Ratten kleiner und leichter waren, konnte anhand von

histologischen und immunhistochemischen Nachweisverfahren mit fehlender

Spermatogenese in Zusammenhang gebracht werden. Da außerdem die nicht-

neoplastischen Follikel von homozygoten ter Weibchen frei von Oogonien waren,

liegt der Schluss nahe, dass Dnd1 als Survivalfaktor die Erhaltung von Keimzellen in

der Ratte sichert. Der Vorteil der Ter Ratte gegenüber der ter Maus als Tiermodell

liegt darin, dass diese auch Studien an Ovarteratomen ermöglicht, sowie einen

Vergleich zwischen den Geschlechtern erlaubt. Generell ist die WKY-Dnd1ter/Ztm

Ratte als neues Tiermodell anzusehen, insbesondere für weitere Studien zur

gonadalen Teratokarzinogenese und zur Identifikation von molekularen

Mechanismen, die im Rahmen von Keimzelldifferenzierung und De-Differenzierung

eine Rolle spielen. Von zentraler Bedeutung sind hierbei die Tumorvorläuferzellen,

die PGCs, und das Erkennen von Faktoren, die die neoplastische Transformation der

PGCs zu ECCs induzieren oder beschleunigen.

Ungeachtet der Tatsache, dass sich das Entwicklungspotential von PGCs auf die

Keimbahn beschränkt, sind ECC nicht die einzigen pluripotenten Zellen, die von PGC

abstammen. Durch in vitro Kultur können unipotente PGC zu pluripotenten

embryonalen Keimzellen (EGC) reprogrammiert werden. EGC von der WKY-

Dnd1ter/Ztm Ratte können als innovativer Ansatz dienen, um in vitro Studien zur Rolle

von Dnd1 in Keimzelldifferenzierung und Tumorigenese zu ermöglichen. Die

erfolgreiche Kultur von EGCs aus der Keimleiste der WKY-Dnd1ter/Ztm Ratte wurde

im zweiten Teil dieser Dissertation etabliert. Heterozygote ter/+ Ratten wurden

verpaart und die PGCs von Embryonen an Tag 14.5 post coitum (d pc) wurden in

Medium kultiviert, das Inhibitoren enthielt, welche eine Ausdifferenzierung verhindern

(2i-LIF). Die generierten Zelllinien wurden genotypisiert. Bis auf eine Zelllinie

stammten alle von Dnd1-defizienten Embryonen ab. Dnd1 hatte dagegen keinen

Einfluss auf die Kultivierung von prä-migratorischen EGC aus einem früheren

Embryonalstadium. Dies bedeutet, dass die Inaktivierung von Dnd1 die

Zusammenfassung 101

Immortalisierung von späten, nicht jedoch von frühen PGC in vitro beeinflusst hat.

Somit hat der Verlust von Dnd1 in PGC an d14.5pc vermutlich bereits die

Entwicklungsrichtung in der Gametendifferenzierung zu Teratokarzinomen geändert.

Die EGC-Linien konnten in Kultur Ihren pluripotenten Status aufrechterhalten, was

anhand der Expression von Pluripotenzmarkern und der Bildung von Teratomen in

immundefizienten Mäusen gezeigt wurde. Keimbahngängige Chimäre konnten durch

Injektion der Wildtyp Zelllinie in Blastozysten generiert werden. Somit wurde

erstmalig Keimbahngängigkeit für eine Ratten EGC-Linie gezeigt.

Zusammenfassend konnte durch diese Arbeit die Bedeutung von Dnd1 in der

KeimzelIentwicklung der Ratte aufgezeigt sowie veranschaulicht werden, dass der

Verlust die De-Differenzierung von Keimzellen zu pluripotenten Zellen in vivo und in

vitro zur Folge hat. Eine weitere Errungenschaft ist die Etablierung der Kultur von

Keimzellen aus der Keimleiste der Ratte. Sowohl der charakterisierte WKY-

Dnd1ter/Ztm Rattenstamm als auch die etablierte Kultur von EGCs aus der Keimleiste

schafften neue Möglichkeiten für die weitere Erforschung des Dnd1 Gens und der

Mechanismen, die an der Keimzellentwicklung beteiligt sind.

102 References

9. References

Adams IR, McLaren A. 2002. Sexually dimorphic development of mouse primordial

germ cells: switching from oogenesis to spermatogenesis. Development (Cambridge, England) 129:1155-1164.

Aflatoonian B, Moore H. 2005. Human primordial germ cells and embryonic germ cells, and their use in cell therapy. Current opinion in biotechnology 16:530-535.

Aflatoonian B, Moore H. 2006. Germ cells from mouse and human embryonic stem cells. Reproduction (Cambridge, England) 132:699-707.

Agoulnik AI, Lu B, Zhu Q, Truong C, Ty MT, Arango N, Chada KK, Bishop CE. 2002. A novel gene, Pog, is necessary for primordial germ cell proliferation in the mouse and underlies the germ cell deficient mutation, gcd. Human molecular genetics 11:3047-3053.

Akgul Ozmen C, Nazaroglu H, Yildirim M, Akay H, Bayrak A, Sentürk S. 2009. A rare case of dermoid cyst originating from the submandibular gland. The internet journal of radiology.

Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, Kouzarides T, Surani MA. 2006. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nature cell biology 8:623-630.

Anderson C, Catoe H, Werner R. 2006. MIR-206 regulates connexin43 expression during skeletal muscle development. Nucleic acids research 34:5863-5871.

Anderson PD, Lam MY, Poirier C, Bishop CE, Nadeau JH. 2009. The role of the mouse y chromosome on susceptibility to testicular germ cell tumors. Cancer research 69:3614-3618.

Andrews PW. 1998. Teratocarcinomas and human embryology: pluripotent human EC cell lines. Review article. Apmis 106:158-167; discussion 167-158.

Andrews PW. 2002. From teratocarcinomas to embryonic stem cells. Philosophical transactions of the Royal Society of London 357:405-417.

Asada Y, Varnum DS, Frankel WN, Nadeau JH. 1994. A mutation in the Ter gene causing increased susceptibility to testicular teratomas maps to mouse chromosome 18. Nature genetics 6:363-368.

Bahrami A, Ro JY, Ayala AG. 2007. An overview of testicular germ cell tumors. Archives of pathology & laboratory medicine 131:1267-1280.

Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P. 2007. The selectivity of protein kinase inhibitors: a further update. The Biochemical journal 408:297-315.

Bhattacharya C, Aggarwal S, Kumar M, Ali A, Matin A. 2008. Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1). PloS one 3:e2315.

Bhattacharya C, Aggarwal S, Zhu R, Kumar M, Zhao M, Meistrich ML, Matin A. 2007. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability. Biochemical and biophysical research communications 355:194-199.

References 103

Bone HK, Damiano T, Bartlett S, Perry A, Letchford J, Ripoll YS, Nelson AS, Welham MJ. 2009. Involvement of GSK-3 in regulation of murine embryonic stem cell self-renewal revealed by a series of bisindolylmaleimides. Chemistry & biology 16:15-27.

Bosl GJ, Motzer RJ. 1997. Testicular germ-cell cancer. The New England journal of medicine 337:242-253.

Bosland MC. 1996. Hormonal factors in carcinogenesis of the prostate and testis in humans and in animal models. Progress in clinical and biological research 394:309-352.

Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P. 2006. Retinoid signaling determines germ cell fate in mice. Science (New York, NY 312:596-600.

Brinster RL. 1974. The effect of cells transferred into the mouse blastocyst on subsequent development. The Journal of experimental medicine 140:1049-1056.

Broman KW, Wu H, Sen S, Churchill GA. 2003. R/qtl: QTL mapping in experimental crosses. Bioinformatics (Oxford, England) 19:889-890.

Brunet S, Maria AS, Guillaud P, Dujardin D, Kubiak JZ, Maro B. 1999. Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase. The Journal of cell biology 146:1-12.

Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A. 2008. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135:1287-1298.

Bulic-Jakus F, Ulamec M, Vlahovic M, Sincic N, Katusic A, Juric-Lekc G, Serman L, Kruslin B, Belicza M. 2006. Of mice and men: teratomas and teratocarcinomas. Collegium antropologicum 30:921-924.

Burns WR, Sabanegh E, Dada R, Rein B, Agarwal A. 2010. Is male infertility a forerunner to cancer? Int Braz J Urol 36:527-536.

Capecchi MR. 1989. Altering the genome by homologous recombination. Science (New York, NY 244:1288-1292.

Chang DH, Cattoretti G, Calame KL. 2002. The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development. Mechanisms of development 117:305-309.

Chieffi P, Franco R, Portella G. 2009. Molecular and cell biology of testicular germ cell tumors. International review of cell and molecular biology 278:277-308.

Chu IM, Hengst L, Slingerland JM. 2008. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 8:253-267.

Chuma S, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Hosokawa M, Nakatsuji N, Ogura A, Shinohara T. 2005. Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development (Cambridge, England) 132:117-122.

Churchill GA, Doerge RW. 1994. Empirical threshold values for quantitative trait mapping. Genetics 138:963-971.

104 References

Chuva de Sousa Lopes SM, Hayashi K, Surani MA. 2007. Proximal visceral endoderm and extraembryonic ectoderm regulate the formation of primordial germ cell precursors. BMC developmental biology 7:140.

Chuva de Sousa Lopes SM, Roelen BA. 2008. Primordial germ cell specification: the importance of being 'blimped'. Histology and histopathology 23:1553-1561.

Chuva de Sousa Lopes SM, Roelen BA. 2010. On the formation of germ cells: The good, the bad and the ugly. Differentiation; research in biological diversity 79:131-140.

Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, Pera RA. 2004. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Human molecular genetics 13:727-739.

Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Buhring HJ, Mattheus U, Mack A, Wagner HJ, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert KD, Stenzl A, Skutella T. 2008. Generation of pluripotent stem cells from adult human testis. Nature 456:344-349.

Conticello SG. 2008. The AID/APOBEC family of nucleic acid mutators. Genome biology 9:229.

Cook MS, Capel B. 2010. Shifting gears and putting on the brakes: Female germ cells transition into meiosis. Cell cycle (Georgetown, Tex 9:447-448.

Cook MS, Coveney D, Batchvarov I, Nadeau JH, Capel B. 2009. BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1(Ter/Ter) mice. Developmental biology 328:377-383.

Cook MS, Munger SC, Nadeau JH, Capel B. 2011. Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background. Development (Cambridge, England).

Cooke JE, Godin I, Ffrench-Constant C, Heasman J, Wylie CC. 1993. Culture and manipulation of primordial germ cells. Methods in enzymology 225:37-58.

Crockford GP, Linger R, Hockley S, Dudakia D, Johnson L, Huddart R, Tucker K, Friedlander M, Phillips KA, Hogg D, Jewett MA, Lohynska R, Daugaard G, Richard S, Chompret A, Bonaiti-Pellie C, Heidenreich A, Albers P, Olah E, Geczi L, Bodrogi I, Ormiston WJ, Daly PA, Guilford P, Fossa SD, Heimdal K, Tjulandin SA, Liubchenko L, Stoll H, Weber W, Forman D, Oliver T, Einhorn L, McMaster M, Kramer J, Greene MH, Weber BL, Nathanson KL, Cortessis V, Easton DF, Bishop DT, Stratton MR, Rapley EA. 2006. Genome-wide linkage screen for testicular germ cell tumour susceptibility loci. Human molecular genetics 15:443-451.

De Felici M, Farini D, Dolci S. 2009. In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells. Curr Stem Cell Res Ther 4:87-97.

De Felici M, Scaldaferri ML, Lobascio M, Iona S, Nazzicone V, Klinger FG, Farini D. 2004. Experimental approaches to the study of primordial germ cell lineage and proliferation. Human reproduction update 10:197-206.

De Miguel MP, Fuentes-Julian S, Alcaina Y. 2010. Pluripotent stem cells: origin, maintenance and induction. Stem cell reviews 6:633-649.

Donehower LA, Harvey M, Vogel H, McArthur MJ, Montgomery CA, Jr., Park SH, Thompson T, Ford RJ, Bradley A. 1995. Effects of genetic background on tumorigenesis in p53-deficient mice. Molecular carcinogenesis 14:16-22.

References 105

Donovan PJ. 1994. Growth factor regulation of mouse primordial germ cell development. Current topics in developmental biology 29:189-225.

Donovan PJ. 1998. The germ cell--the mother of all stem cells. The International journal of developmental biology 42:1043-1050.

Donovan PJ, de Miguel MP. 2003. Turning germ cells into stem cells. Curr Opin Genet Dev 13:463-471.

Donovan PJ, De Miguel MP, Hirano MP, Parsons MS, Lincoln AJ. 2001. Germ cell biology--from generation to generation. The International journal of developmental biology 45:523-531.

Durcova-Hills G, Adams IR, Barton SC, Surani MA, McLaren A. 2006. The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem cells (Dayton, Ohio) 24:1441-1449.

Durcova-Hills G, Ainscough J, McLaren A. 2001. Pluripotential stem cells derived from migrating primordial germ cells. Differentiation; research in biological diversity 68:220-226.

Durcova-Hills G, Capel B. 2008. Development of germ cells in the mouse. Current topics in developmental biology 83:185-212.

Durcova-Hills G, Tang F, Doody G, Tooze R, Surani MA. 2008. Reprogramming primordial germ cells into pluripotent stem cells. PloS one 3:e3531.

Durcova-Hills G, Wianny F, Merriman J, Zernicka-Goetz M, McLaren A. 2003. Developmental fate of embryonic germ cells (EGCs), in vivo and in vitro. Differentiation; research in biological diversity 71:135-141.

Eppig JJ, Wigglesworth K, Varnum DS, Nadeau JH. 1996. Genetic regulation of traits essential for spontaneous ovarian teratocarcinogenesis in strain LT/Sv mice: aberrant meiotic cell cycle, oocyte activation, and parthenogenetic development. Cancer research 56:5047-5054.

Evans MJ, Kaufman MH. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156.

Faulkner SW, Friedlander ML. 2000. Molecular genetic analysis of malignant ovarian germ cell tumors. Gynecologic oncology 77:283-288.

Filipowicz W, Bhattacharyya SN, Sonenberg N. 2008. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102-114.

Fox MS, Clark AT, El Majdoubi M, Vigne JL, Urano J, Hostetler CE, Griswold MD, Weiner RI, Reijo Pera RA. 2007. Intermolecular interactions of homologs of germ plasm components in mammalian germ cells. Developmental biology 301:417-431.

Fridman AL, Tainsky MA. 2008. Critical pathways in cellular senescence and immortalization revealed by gene expression profiling. Oncogene 27:5975-5987.

Fujinaga M, Brown NA, Baden JM. 1992. Comparison of staging systems for the gastrulation and early neurulation period in rodents: a proposed new system. Teratology 46:183-190.

Gajendran VK, Nguyen M, Ellison LM. 2005. Testicular cancer patterns in African-American men. Urology 66:602-605.

106 References

Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. 2004. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427:148-154.

Giambartolomei C, Mueller CM, Greene MH, Korde LA. 2009. A mini-review of familial ovarian germ cell tumors: an additional manifestation of the familial testicular germ cell tumor syndrome. Cancer epidemiology 33:31-36.

Glaser S, Anastassiadis K, Stewart AF. 2005. Current issues in mouse genome engineering. Nature genetics 37:1187-1193.

Goren S, Dekel N. 1994. Maintenance of meiotic arrest by a phosphorylated p34cdc2 is independent of cyclic adenosine 3',5'-monophosphate. Biology of reproduction 51:956-962.

Greene MH, Kratz CP, Mai PL, Mueller C, Peters JA, Bratslavsky G, Ling A, Choyke PM, Premkumar A, Bracci J, Watkins RJ, McMaster ML, Korde LA. 2010. Familial testicular germ cell tumors in adults: 2010 summary of genetic risk factors and clinical phenotype. Endocrine-related cancer 17:R109-121.

Guan W, Wang Y, Hou L, Chen L, Li X, Yue W, Ma Y. 2009. Derivation and characteristics of pluripotent embryonic germ cells in duck. Poultry science 89:312-317.

Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, Reik W, Walter J, Surani MA. 2002. Epigenetic reprogramming in mouse primordial germ cells. Mechanisms of development 117:15-23.

Hammond S, Zhu R, Youngren KK, Lam J, Anderson P, Matin A. 2007. Chromosome X modulates incidence of testicular germ cell tumors in Ter mice. Mamm Genome 18:832-838.

Harms D, Zahn S, Gobel U, Schneider DT. 2006. Pathology and molecular biology of teratomas in childhood and adolescence. Klinische Padiatrie 218:296-302.

Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, Tang F, Hajkova P, Lao K, O'Carroll D, Das PP, Tarakhovsky A, Miska EA, Surani MA. 2008. MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis. PloS one 3:e1738.

Heaney JD, Nadeau JH. 2008. Testicular germ cell tumors in mice: new ways to study a genetically complex trait. Methods in molecular biology (Clifton, NJ 450:211-231.

Hedrich H. 1990. Genetic monitoring of inbred strains of rats, pp 282-284. Mutant genes and polymorphic loci of the laboratory rat. Stuttgart, New York: Gustav Fischer Verlag.

Hemminki K, Li X. 2002. Cancer risks in Nordic immigrants and their offspring in Sweden. Eur J Cancer 38:2428-2434.

Hermo L, Pelletier RM, Cyr DG, Smith CE. 2010. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes. Microscopy research and technique 73:241-278.

Hill M. 2010. Rat Development. UNSW Embryology: http://embryology.med.unsw.edu.au/OtherEmb/Rat.htm.

Hochwagen A. 2008. Meiosis. Curr Biol 18:R641-R645. Hoei-Hansen CE, Kraggerud SM, Abeler VM, Kaern J, Rajpert-De Meyts E, Lothe

RA. 2007. Ovarian dysgerminomas are characterised by frequent KIT

http://embryology.med.unsw.edu.au/OtherEmb/Rat.htm

References 107

mutations and abundant expression of pluripotency markers. Molecular cancer 6:12.

Hogan B, Beddington R, Constantini F, Lacy E. 1994. Manipulating the Mouse Embryo: A Laborarory Manual. New York: Cold Spring Harbor Press.

Honorio S, Agathanggelou A, Wernert N, Rothe M, Maher ER, Latif F. 2003. Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours. Oncogene 22:461-466.

Horvay K, Claussen M, Katzer M, Landgrebe J, Pieler T. 2006. Xenopus Dead end mRNA is a localized maternal determinant that serves a conserved function in germ cell development. Developmental biology 291:1-11.

Hotaling JM, Walsh TJ. 2009. Male infertility: a risk factor for testicular cancer. Nat Rev Urol 6:550-556.

Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF, 3rd, Boiani M, Schöler HR. 2003. Derivation of oocytes from mouse embryonic stem cells. Science (New York, NY 300:1251-1256.

Hussain SA, Ma YT, Palmer DH, Hutton P, Cullen MH. 2008. Biology of testicular germ cell tumors. Expert review of anticancer therapy 8:1659-1673.

Huyghe E, Matsuda T, Thonneau P. 2003. Increasing incidence of testicular cancer worldwide: a review. The Journal of urology 170:5-11.

Itoh T, Shirota K, Kagiyama N. 1985. Spontaneous teratoma in a Sprague-Dawley rat. Jikken dobutsu 34:207-209.

Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ. 2004. Cancer statistics, 2004. CA: a cancer journal for clinicians 54:8-29.

Jia W, Yang W, Lei A, Gao Z, Yang C, Hua J, Huang W, Ma X, Wang H, Dou Z. 2008. A caprine chimera produced by injection of embryonic germ cells into a blastocyst. Theriogenology 69:340-348.

Johnson DG, Walker CL. 1999. Cyclins and cell cycle checkpoints. Annual review of pharmacology and toxicology 39:295-312.

Jones DL. 2007. Aging and the germ line: where mortality and immortality meet. Stem cell reviews 3:192-200.

Kahan BW, Ephrussi B. 1970. Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. Journal of the National Cancer Institute 44:1015-1036.

Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T. 2004. Generation of pluripotent stem cells from neonatal mouse testis. Cell 119:1001-1012.

Kedde M, Agami R. 2008. Interplay between microRNAs and RNA-binding proteins determines developmental processes. Cell cycle (Georgetown, Tex 7:899-903.

Kedde M, Strasser MJ, Boldajipour B, Oude Vrielink JA, Slanchev K, le Sage C, Nagel R, Voorhoeve PM, van Duijse J, Orom UA, Lund AH, Perrakis A, Raz E, Agami R. 2007. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell 131:1273-1286.

Keller GM. 1995. In vitro differentiation of embryonic stem cells. Current opinion in cell biology 7:862-869.

108 References

Kemper CH, Peters PW. 1987. Migration and proliferation of primordial germ cells in the rat. Teratology 36:117-124.

Kempkensteffen C, Christoph F, Weikert S, Krause H, Kollermann J, Schostak M, Miller K, Schrader M. 2006. Epigenetic silencing of the putative tumor suppressor gene testisin in testicular germ cell tumors. Journal of cancer research and clinical oncology 132:765-770.

Kerr CL, Gearhart JD, Elliott AM, Donovan PJ. 2006. Embryonic germ cells: when germ cells become stem cells. Seminars in reproductive medicine 24:304-313.

Ketting RF. 2007. A dead end for microRNAs. Cell 131:1226-1227. Kimura T, Suzuki A, Fujita Y, Yomogida K, Lomeli H, Asada N, Ikeuchi M, Nagy A,

Mak TW, Nakano T. 2003. Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production. Development (Cambridge, England) 130:1691-1700.

Kimura T, Tomooka M, Yamano N, Murayama K, Matoba S, Umehara H, Kanai Y, Nakano T. 2008. AKT signaling promotes derivation of embryonic germ cells from primordial germ cells. Development (Cambridge, England) 135:869-879.

Kleinsmith LJ, Pierce GB, Jr. 1964. Multipotentiality of Single Embryonal Carcinoma Cells. Cancer research 24:1544-1551.

Ko K, Schöler HR. 2006. Embryonic stem cells as a potential source of gametes. Seminars in reproductive medicine 24:322-329.

Ko K, Tapia N, Wu G, Kim JB, Bravo MJ, Sasse P, Glaser T, Ruau D, Han DW, Greber B, Hausdorfer K, Sebastiano V, Stehling M, Fleischmann BK, Brustle O, Zenke M, Schöler HR. 2009. Induction of pluripotency in adult unipotent germline stem cells. Cell stem cell 5:87-96.

Komiya T, Tanigawa Y. 1995. Cloning of a gene of the DEAD box protein family which is specifically expressed in germ cells in rats. Biochemical and biophysical research communications 207:405-410.

Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. 2006. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proceedings of the National Academy of Sciences of the United States of America 103:2474-2479.

Kratz CP, Mai PL, Greene MH. 2010. Familial testicular germ cell tumours. Best practice & research 24:503-513.

Krausz C, Looijenga LH. 2008. Genetic aspects of testicular germ cell tumors. Cell cycle (Georgetown, Tex 7:3519-3524.

Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, Zhu R, Matin A, Sarver AL, Parker KL, Griswold MD, Looijenga LH, Bardwell VJ, Zarkower D. 2009. The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proceedings of the National Academy of Sciences of the United States of America 106:22323-22328.

Kristensen DM, Sonne SB, Ottesen AM, Perrett RM, Nielsen JE, Almstrup K, Skakkebaek NE, Leffers H, Meyts ER. 2008. Origin of pluripotent germ cell tumours: the role of microenvironment during embryonic development. Molecular and cellular endocrinology 288:111-118.

Labosky PA, Barlow DP, Hogan BL. 1994. Embryonic germ cell lines and their derivation from mouse primordial germ cells. Ciba Foundation symposium 182:157-168; discussion 168-178.

References 109

Lam MY, Heaney JD, Youngren KK, Kawasoe JH, Nadeau JH. 2007. Trans-generational epistasis between Dnd1Ter and other modifier genes controls susceptibility to testicular germ cell tumors. Human molecular genetics 16:2233-2240.

Lander E, Kruglyak L. 1995. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nature genetics 11:241-247.

Länger B, Dorsch M, Gartner K, Wedekind D, Kamino K, Hedrich HJ. 2004. WKY/Ztm-ter: a new rat inbred strain on the WKY/Ztm genetic background with congenital teratomas. Laboratory animals 38:425-431.

Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL. 1999. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes & development 13:424-436.

le Sage C, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A, Anile C, Maira G, Mercatelli N, Ciafre SA, Farace MG, Agami R. 2007. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. The EMBO journal 26:3699-3708.

Lee-Jones L. 2003. Ovary: Germ cell tumors. Atlas Genet Cytogen Oncol Haematol.: http://AtlasGeneticsOncology.org/Tumors/OvarianGermCellID5067.html.

Leendert HJ, Looijenga LH, Oosterhuis JW. 1999. Pathogenesis of testicular germ cell tumours. Reviews of reproduction 4:90-100.

Leitch HG, Blair K, Mansfield W, Ayetey H, Humphreys P, Nichols J, Surani MA, Smith A. 2010. Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state. Development (Cambridge, England) 137:2279-2287.

Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL. 2008. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135:1299-1310.

Li Y, Pei J, Xia H, Ke H, Wang H, Tao W. 2003. Lats2, a putative tumor suppressor, inhibits G1/S transition. Oncogene 22:4398-4405.

Lind GE, Skotheim RI, Lothe RA. 2007. The epigenome of testicular germ cell tumors. Apmis 115:1147-1160.

Linger R, Dudakia D, Huddart R, Tucker K, Friedlander M, Phillips KA, Hogg D, Jewett MA, Lohynska R, Daugaard G, Richard S, Chompret A, Stoppa-Lyonnet D, Bonaiti-Pellie C, Heidenreich A, Albers P, Olah E, Geczi L, Bodrogi I, Daly PA, Guilford P, Fossa SD, Heimdal K, Tjulandin SA, Liubchenko L, Stoll H, Weber W, Einhorn L, McMaster M, Korde L, Greene MH, Nathanson KL, Cortessis V, Easton DF, Bishop DT, Stratton MR, Rapley EA. 2008. Analysis of the DND1 gene in men with sporadic and familial testicular germ cell tumors. Genes, chromosomes & cancer 47:247-252.

Liu W, Collodi P. 2010. Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development. Faseb J 24:2641-2650.

Liu X, Wang A, Heidbreder CE, Jiang L, Yu J, Kolokythas A, Huang L, Dai Y, Zhou X. 2010. MicroRNA-24 targeting RNA-binding protein DND1 in tongue squamous cell carcinoma. FEBS letters 584:4115-4120.

Looijenga LH, Oosterhuis JW. 1999. Pathogenesis of testicular germ cell tumours. Reviews of reproduction 4:90-100.

http://atlasgeneticsoncology.org/Tumors/OvarianGermCellID5067.html

110 References

Maatouk DM, Kellam LD, Mann MR, Lei H, Li E, Bartolomei MS, Resnick JL. 2006. DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages. Development (Cambridge, England) 133:3411-3418.

Mardanpour P, Guan K, Nolte J, Lee JH, Hasenfuss G, Engel W, Nayernia K. 2008. Potency of germ cells and its relevance for regenerative medicine. Journal of anatomy 213:26-29.

Matin A. 2007. What leads from dead-end? Cell Mol Life Sci 64:1317-1322. Matin A, Nadeau JH. 2005. Search for testicular cancer gene hits dead-end. Cell

cycle (Georgetown, Tex 4:1136-1138. Matsui Y, Zsebo K, Hogan BL. 1992. Derivation of pluripotential embryonic stem cells

from murine primordial germ cells in culture. Cell 70:841-847. McGlynn KA, Quraishi SM, Graubard BI, Weber JP, Rubertone MV, Erickson RL.

2008. Persistent organochlorine pesticides and risk of testicular germ cell tumors. Journal of the National Cancer Institute 100:663-671.

McIntyre A, Gilbert D, Goddard N, Looijenga L, Shipley J. 2008. Genes, chromosomes and the development of testicular germ cell tumors of adolescents and adults. Genes, chromosomes & cancer 47:547-557.

McLaren A. 1981. Germ Cells and Soma: A new look at an old problem. Yale University Press, New Haven, CT.

McLaren A. 1984. Meiosis and differentiation of mouse germ cells. Symposia of the Society for Experimental Biology 38:7-23.

McLaren A. 2003. Primordial germ cells in the mouse. Developmental biology 262:1-15.

McLaren A, Durcova-Hills G. 2001. Germ cells and pluripotent stem cells in the mouse. Reproduction, fertility, and development 13:661-664.

McLaren A, Lawson KA. 2005. How is the mouse germ-cell lineage established? Differentiation; research in biological diversity 73:435-437.

Miles DC, van den Bergen JA, Sinclair AH, Western PS. 2010. Regulation of the female mouse germ cell cycle during entry into meiosis. Cell cycle (Georgetown, Tex 9:408-418.

Mishima Y, Giraldez AJ, Takeda Y, Fujiwara T, Sakamoto H, Schier AF, Inoue K. 2006. Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr Biol 16:2135-2142.

Miwa M, Kojima M, Ohtani T, Tsuji K. 1987. [A recessive mutant causing testicular/ovarian teratoma in rats (Tera strain)]. Jikken dobutsu 36:205-208.

Muller AJ, Teresky AK, Levine AJ. 2000. A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13. Proceedings of the National Academy of Sciences of the United States of America 97:8421-8426.

Nayernia K. 2008. Germ cells, origin of somatic cells? Cell Research 18:26. Nerli RB, Ajay G, Shivangouda P, Pravin P, Reddy M, Pujar VC. Prepubertal

testicular tumors: our 10 years experience. Indian journal of cancer 47:292-295.

Nicholas CR, Chavez SL, Baker VL, Reijo Pera RA. 2009. Instructing an embryonic stem cell-derived oocyte fate: lessons from endogenous oogenesis. Endocrine reviews 30:264-283.

References 111

Nichols J, Jones K, Phillips JM, Newland SA, Roode M, Mansfield W, Smith A, Cooke A. 2009. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nature medicine 15:814-818.

Nichols J, Smith A. 2009. Naive and primed pluripotent states. Cell stem cell 4:487-492.

Nicholson PW, Harland SJ. 1995. Inheritance and testicular cancer. British journal of cancer 71:421-426.

Nickerson DA, Tobe VO, Taylor SL. 1997. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic acids research 25:2745-2751.

Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA, Fumanelli M, Illgen-Wilcke B. 2002. Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Laboratory animals 36:20-42.

Ninomiya H. 1983. Spontaneous teratoma in a Wistar rat. Jikken dobutsu 32:145-149.

Noguchi T, Noguchi M. 1985. A recessive mutation (ter) causing germ cell deficiency and a high incidence of congenital testicular teratomas in 129/Sv-ter mice. Journal of the National Cancer Institute 75:385-392.

Noguchi T, Stevens LC. 1982. Primordial germ cell proliferation in fetal testes in mouse strains with high and low incidences of congenital testicular teratomas. Journal of the National Cancer Institute 69:907-913.

Notarianni E. 2011. Reinterpretation of evidence advanced for neo-oogenesis in mammals, in terms of a finite oocyte reserve. Journal of ovarian research 4:1.

Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T, Saitou M. 2009. A signaling principle for the specification of the germ cell lineage in mice. Cell 137:571-584.

Ohinata Y, Payer B, O'Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, Saitou M, Surani MA. 2005. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436:207-213.

Oosterhuis JW, Castedo SM, de Jong B, Cornelisse CJ, Dam A, Sleijfer DT, Schraffordt Koops H. 1989. Ploidy of primary germ cell tumors of the testis. Pathogenetic and clinical relevance. Laboratory investigation; a journal of technical methods and pathology 60:14-21.

Oosterhuis JW, Looijenga LH. 2005. Testicular germ-cell tumours in a broader perspective. Nature reviews 5:210-222.

Page SL, Hawley RS. 2003. Chromosome choreography: the meiotic ballet. Science (New York, NY 301:785-789.

Pellas TC, Ramachandran B, Duncan M, Pan SS, Marone M, Chada K. 1991. Germ-cell deficient (gcd), an insertional mutation manifested as infertility in transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 88:8787-8791.

Pepling ME. 2006. From primordial germ cell to primordial follicle: mammalian female germ cell development. Genesis 44:622-632.

Phillips BT, Gassei K, Orwig KE. 2010. Spermatogonial stem cell regulation and spermatogenesis. Philosophical transactions of the Royal Society of London 365:1663-1678.

112 References

Piedrahita JA, Moore K, Oetama B, Lee CK, Scales N, Ramsoondar J, Bazer FW, Ott T. 1998. Generation of transgenic porcine chimeras using primordial germ cell-derived colonies. Biology of reproduction 58:1321-1329.

Prabhu SM, Meistrich ML, McLaughlin EA, Roman SD, Warne S, Mendis S, Itman C, Loveland KL. 2006. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction (Cambridge, England) 131:489-499.

Prather RS, First NL. 1988. A review of early mouse embryogenesis and its applications to domestic species. Journal of animal science 66:2626-2635.

Qi J, Yu JY, Shcherbata HR, Mathieu J, Wang AJ, Seal S, Zhou W, Stadler BM, Bourgin D, Wang L, Nelson A, Ware C, Raymond C, Lim LP, Magnus J, Ivanovska I, Diaz R, Ball A, Cleary MA, Ruohola-Baker H. 2009. microRNAs regulate human embryonic stem cell division. Cell cycle (Georgetown, Tex 8:3729-3741.

Regenass U, Friedrich TD, Stevens LC. 1982. Experimental induction of testicular teratomas in dissociated-reaggregated chimaeric gonads. Journal of embryology and experimental morphology 72:153-167.

Resnick JL, Bixler LS, Cheng L, Donovan PJ. 1992. Long-term proliferation of mouse primordial germ cells in culture. Nature 359:550-551.

Richards AJ, Enders GC, Resnick JL. 1999. Differentiation of murine premigratory primordial germ cells in culture. Biology of reproduction 61:1146-1151.

Richardson BE, Lehmann R. 2010. Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nature reviews 11:37-49.

Rivers EN, Hamilton DW. 1986. Morphologic analysis of spontaneous teratocarcinogenesis in developing testes of strain 129/Sv-ter mice. The American journal of pathology 124:263-280.

Rossant J, McBurney MW. 1982. The developmental potential of a euploid male teratocarcinoma cell line after blastocyst injection. Journal of embryology and experimental morphology 70:99-112.

Sabour D, Arauzo-Bravo MJ, Hubner K, Ko K, Greber B, Gentile L, Stehling M, Scholer HR. 2010. Identification of genes specific to mouse primordial germ cells through dynamic global gene expression. Human molecular genetics.

Saiti D, Lacham-Kaplan O. 2007. Mouse Germ Cell Development in-vivo and in-vitro. Biomarker insights 2:241-252.

Saitou M, Barton SC, Surani MA. 2002. A molecular programme for the specification of germ cell fate in mice. Nature 418:293-300.

Sakurai T, Iguchi T, Morikawa K, Noguchi M. 1995. The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation. Development, Growth & Differentiation 37: 293-302.

Sakurai T, Katoh H, Moriwaki K, Noguchi T, Noguchi M. 1994. The ter primordial germ cell deficiency mutation maps near Grl-1 on mouse chromosome 18. Mamm Genome 5:333-336.

Sawaki M, Shinoda K, Hoshuyama S, Kato F, Yamasaki K. 2000. Combination of a teratoma and embryonal carcinoma of the testis in SD IGS rats: a report of two cases. Toxicologic pathology 28:832-835.

Schardein JL, Fitzgerald JE. 1977. Teratoma in a Wistar rat. Laboratory animal science 27:114.

References 113

Schmidt W, von Kreybig T. 1965. Die vorgeburtliche Entwicklung der Gonaden der Ratte. Zeitschrift für Anatomie und Entwicklungsgeschichte 124:588-600.

Schnorr B. 1996. Embryologie der Haustiere, 3. überarb Auflage. Stuttgart: Ferdinand Enke Verlag.

Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. 1997. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88:593-602.

Seydoux G, Braun RE. 2006. Pathway to totipotency: lessons from germ cells. Cell 127:891-904.

Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y, Cheng L, Gearhart JD. 2001. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proceedings of the National Academy of Sciences of the United States of America 98:113-118.

Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD. 1998. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proceedings of the National Academy of Sciences of the United States of America 95:13726-13731.

Sharpe RM. 2006. Perinatal determinants of adult testis size and function. The Journal of clinical endocrinology and metabolism 91:2503-2505.

Shim H, Gutierrez-Adan A, Chen LR, BonDurant RH, Behboodi E, Anderson GB. 1997. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biology of reproduction 57:1089-1095.

Shim SW, Han DW, Yang JH, Lee BY, Kim SB, Shim H, Lee HT. 2008. Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing. Molecules and cells 25:358-367.

Shovlin TC, Durcova-Hills G, Surani A, McLaren A. 2008. Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells. Developmental biology 313:674-681.

Sijmons RH, Vos YJ, Herkert JC, Bos KK, Holzik MF, Hoekstra-Weebers JE, Hofstra RM, Hoekstra HJ. 2010. Screening for germline DND1 mutations in testicular cancer patients. Familial cancer 9:439-442.

Skakkebaek NE, Holm M, Hoei-Hansen C, Jorgensen N, Rajpert-De Meyts E. 2003. Association between testicular dysgenesis syndrome (TDS) and testicular neoplasia: evidence from 20 adult patients with signs of maldevelopment of the testis. Apmis 111:1-9; discussion 9-11.

Skakkebaek NE, Rajpert-De Meyts E, Jorgensen N, Main KM, Leffers H, Andersson AM, Juul A, Jensen TK, Toppari J. 2007. Testicular cancer trends as 'whistle blowers' of testicular developmental problems in populations. International journal of andrology 30:198-204; discussion 204-195.

Skakkebaek NE, Rajpert-De Meyts E, Main KM. 2001. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Human reproduction (Oxford, England) 16:972-978.

Slanchev K, Stebler J, Goudarzi M, Cojocaru V, Weidinger G, Raz E. 2009. Control of Dead end localization and activity--implications for the function of the

114 References

protein in antagonizing miRNA function. Mechanisms of development 126:270-277.

Smith HO, Berwick M, Verschraegen CF, Wiggins C, Lansing L, Muller CY, Qualls CR. 2006. Incidence and survival rates for female malignant germ cell tumors. Obstetrics and gynecology 107:1075-1085.

Stallock J, Molyneaux K, Schaible K, Knudson CM, Wylie C. 2003. The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo. Development (Cambridge, England) 130:6589-6597.

Stamatiou K, Papadopoulos P, Perlepes G, Galariotis N, Olympitis M, Moschouris H, Vasilakaki T. 2009. Mixed germ cell tumor of the testicle with ravdomuosarcomatous component: a case report. Cases journal 2:9299.

Stettner AR, Hartenbach EM, Schink JC, Huddart R, Becker J, Pauli R, Long R, Laxova R. 1999. Familial ovarian germ cell cancer: report and review. American journal of medical genetics 84:43-46.

Stevens LC. 1959. Embryology of testicular teratomas in strain 129 mice. Journal of the National Cancer Institute 23:1249-1295.

Stevens LC. 1966. Development of resistance to teratocarcinogenesis by primordial germ cells in mice. Journal of the National Cancer Institute 37:859-867.

Stevens LC. 1967. Origin of testicular teratomas from primordial germ cells in mice. Journal of the National Cancer Institute 38:549-552.

Stevens LC. 1970. The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Developmental biology 21:364-382.

Stevens LC. 1973. A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. Journal of the National Cancer Institute 50:235-242.

Stevens LC. 1980. Teratocarcinogenesis and spontaneous parthenogenesis in mice. Results and problems in cell differentiation 11:265-274.

Stevens LC. 1981. Genetic influences on teratocarcinogenesis and parthenogenesis. Progress in clinical and biological research 45:93-104.

Stevens LC. 1984. Spontaneous and experimentally induced testicular teratomas in mice. Cell differentiation 15:69-74.

Stevens LC, Hummel KP. 1957. A description of spontaneous congenital testicular teratomas in strain 129 mice. Journal of the National Cancer Institute 18:719-747.

Stewart CL, Gadi I, Bhatt H. 1994. Stem cells from primordial germ cells can reenter the germ line. Developmental biology 161:626-628.

Surani MA. 2007. Germ cells: the eternal link between generations. Comptes rendus biologies 330:474-478.

Surani MA, Durcova-Hills G, Hajkova P, Hayashi K, Tee WW. 2008. Germ line, stem cells, and epigenetic reprogramming. Cold Spring Harbor symposia on quantitative biology 73:9-15.

Suzuki A, Saga Y. 2008. Nanos2 suppresses meiosis and promotes male germ cell differentiation. Genes & development 22:430-435.

References 115

Suzuki A, Tsuda M, Saga Y. 2007. Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development. Development (Cambridge, England) 134:77-83.

Tam PP, Snow MH. 1981. Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. Journal of embryology and experimental morphology 64:133-147.

Tam PP, Zhou SX. 1996. The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Developmental biology 178:124-132.

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. 1998. Embryonic stem cell lines derived from human blastocysts. Science (New York, NY 282:1145-1147.

Tong C, Li P, Wu NL, Yan Y, Ying QL. 2010. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467:211-213.

Toyooka Y, Tsunekawa N, Akasu R, Noce T. 2003. Embryonic stem cells can form germ cells in vitro. Proceedings of the National Academy of Sciences of the United States of America 100:11457-11462.

Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T. 2000. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mechanisms of development 93:139-149.

Tres LL, Rosselot C, Kierszenbaum AL. 2004. Primordial germ cells: what does it take to be alive? Molecular reproduction and development 68:1-4.

Tsuda M, Sasaoka Y, Kiso M, Abe K, Haraguchi S, Kobayashi S, Saga Y. 2003. Conserved role of nanos proteins in germ cell development. Science (New York, NY 301:1239-1241.

Ulbright TM. 2005. Germ cell tumors of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol 18 Suppl 2:S61-79.

Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. 2006. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes & development 20:515-524.

van de Geijn GJ, Hersmus R, Looijenga LH. 2009. Recent developments in testicular germ cell tumor research. Birth Defects Res C Embryo Today 87:96-113.

van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ. 2006. Germline transmission of genetically modified primordial germ cells. Nature 441:766-769.

Verlhac MH, Kubiak JZ, Clarke HJ, Maro B. 1994. Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development (Cambridge, England) 120:1017-1025.

Vilar E, Calvo E, Tabernero J. 2006. Molecular biology of testicular germ cell tumors. Clin Transl Oncol 8:846-850.

Vincent SD, Dunn NR, Sciammas R, Shapiro-Shalef M, Davis MM, Calame K, Bikoff EK, Robertson EJ. 2005. The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development (Cambridge, England) 132:1315-1325.

116 References

Voigt B, Serikawa T. 2009. Pluripotent stem cells and other technologies will eventually open the door for straightforward gene targeting in the rat. Disease models & mechanisms 2:341-343.

vom Brocke J, Schmeiser HH, Reinbold M, Hollstein M. 2006. MEF immortalization to investigate the ins and outs of mutagenesis. Carcinogenesis 27:2141-2147.

von Eyben FE. 2004. Chromosomes, genes, and development of testicular germ cell tumors. Cancer genetics and cytogenetics 151:93-138.

Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R. 2006. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124:1169-1181.

Walker AH, Ross RK, Haile RW, Henderson BE. 1988. Hormonal factors and risk of ovarian germ cell cancer in young women. British journal of cancer 57:418-422.

Walsh TJ, Croughan MS, Schembri M, Chan JM, Turek PJ. 2009. Increased risk of testicular germ cell cancer among infertile men. Archives of internal medicine 169:351-356.

Walt H, Oosterhuis JW, Stevens LC. 1993. Experimental testicular germ cell tumorigenesis in mouse strains with and without spontaneous tumours differs from development of germ cell tumours of the adult human testis. International journal of andrology 16:267-271.

Wang Y, Blelloch R. 2009. Cell cycle regulation by MicroRNAs in embryonic stem cells. Cancer research 69:4093-4096.

Weidinger G, Stebler J, Slanchev K, Dumstrei K, Wise C, Lovell-Badge R, Thisse C, Thisse B, Raz E. 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13:1429-1434.

Western P. 2009. Foetal germ cells: striking the balance between pluripotency and differentiation. The International journal of developmental biology 53:393-409.

Western PS, Miles DC, van den Bergen JA, Burton M, Sinclair AH. 2008. Dynamic regulation of mitotic arrest in fetal male germ cells. Stem cells (Dayton, Ohio) 26:339-347.

Wicha MS, Liu S, Dontu G. 2006. Cancer stem cells: an old idea--a paradigm shift. Cancer research 66:1883-1890; discussion 1895-1886.

Witschi E. 1962. Development: Rat. In: Growth Including Reproduction and Morphological Development. Altman PL, and Dittmer, D. S. , ed. Washington DC: Federation of American Societies for Experimental Biology.

Wolgemuth DJ, Laurion E, Lele KM. 2002. Regulation of the mitotic and meiotic cell cycles in the male germ line. Recent progress in hormone research 57:75-101.

Xu S, Atchley WR. 1996. Mapping quantitative trait loci for complex binary diseases using line crosses. Genetics 143:1417-1424.

Yabuta Y, Kurimoto K, Ohinata Y, Seki Y, Saitou M. 2006. Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling. Biology of reproduction 75:705-716.

Yamaguchi S, Kimura H, Tada M, Nakatsuji N, Tada T. 2005. Nanog expression in mouse germ cell development. Gene Expr Patterns 5:639-646.

References 117

Yamazaki Y, Mann MR, Lee SS, Marh J, McCarrey JR, Yanagimachi R, Bartolomei MS. 2003. Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning. Proceedings of the National Academy of Sciences of the United States of America 100:12207-12212.

Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. 2008. The ground state of embryonic stem cell self-renewal. Nature 453:519-523.

Ying Y, Qi X, Zhao GQ. 2001. Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proceedings of the National Academy of Sciences of the United States of America 98:7858-7862.

Youngren KK, Coveney D, Peng X, Bhattacharya C, Schmidt LS, Nickerson ML, Lamb BT, Deng JM, Behringer RR, Capel B, Rubin EM, Nadeau JH, Matin A. 2005. The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours. Nature 435:360-364.

Yu J, Thomson JA. 2008. Pluripotent stem cell lines. Genes & development 22:1987-1997.

Zhu R, Bhattacharya C, Matin A. 2007a. The role of dead-end in germ-cell tumor development. Annals of the New York Academy of Sciences 1120:181-186.

Zhu R, Ji Y, Xiao L, Matin A. 2007b. Testicular germ cell tumor susceptibility genes from the consomic 129.MOLF-Chr19 mouse strain. Mamm Genome 18:584-595.

Zovoilis A, Nolte J, Drusenheimer N, Zechner U, Hada H, Guan K, Hasenfuss G, Nayernia K, Engel W. 2008. Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles. Molecular human reproduction 14:521-529.

Zschemisch NH, Liedtke C, Dierssen U, Nevzorova YA, Wustefeld T, Borlak J, Manns MP, Trautwein C. 2006. Expression of a cyclin E1 isoform in mice is correlated with the quiescent cell cycle status of hepatocytes in vivo. Hepatology (Baltimore, Md 44:164-173.

Zwaka TP, Thomson JA. 2005. A germ cell origin of embryonic stem cells? Development (Cambridge, England) 132:227-233.

118 References

Acknowledgements

The completion of this thesis would not have been possible without the support and

guidance of many people.

First, I would like to express my gratitude to my thesis supervisor, Prof. Hedrich, for

giving me the opportunity and the freedom to develop my experiments in his

laboratory. I am especially thankful for his discovery that Christmas, his numerous

contributions, the scientific advice, and his full support.

I am grateful to Dr. Martina Dorsch for her helpfulness, her suggestions and her

encouragements, as well as the constructive criticism throughout the last years.

A special and sincere thank you to Dr. Nils-Holger Zschemisch, with all his

enthusiasm, optimism and inspirations. His patience, sound advice and good

teaching were essential for this thesis, from beginning to end.

Furthermore, I thank my other supervisors Prof. Haas, Prof. Martin and Prof. Greiser-

Wilke († 2010) for their interest and guidance throughout this work. I am grateful to

Prof. Greiser-Wilke for the encouragement and advice at the beginning of my studies.

I owe my deepest gratitude to the staff of the Institute for Laboratory Animal Science,

without whom I could not have accomplished this thesis. A special thank you goes to

Matthias Meyer and Elena Wiebe for their technical support, as well as to Dr. Dirk

Wedekind for helpful suggestion in many situations. Furthermore, I would like to

thank Isabell Wittur, Cindy Elfers and especially Regina Eisenblätter, who contributed

to this project with all their knowledge, their patience, their time and their assistance

in varying circumstances.

I am indebted to Dr. Cornelia Rudolph for instructions on cytogenetic analysis,

evaluation of the metaphase spreads and valuable discussions.

Acknowledgements 119

I would like to thank all my friends for the emotional support, comradery,

entertainment, and caring they provided. Silke Glage, Lydia Janus and Gwen

Büchler: your help was indispensible, the regular food intake was essential, and it

would have been a lonely lab without you.

For many hours of happiness I thank Wilma Feuerstein, who made sure I got out of

the lab and exercised on a daily basis.

My special thanks to Tobias Tiedtke for his love, his encouragement, and for being

by my side in good and bad times. He has the common sense that I tend to lack and

the ability to make me laugh in any given situation.

I am grateful to my siblings Jenica and Lukas, who make life so much more fun, and

though both are far away, they are always there when needed.

Lastly, and most importantly, I wish to thank my parents, Sibylle Northrup and Scott

Northrup. They bore me, raised me, supported me, and taught me with unconditional

love. They are responsible for everything I achieved and are the only parents I know

that are continuously encouraging their children to work less, spend more and live life

to its fullest!

Documents

elib.tiho-hannover.de · Nils-Holger Zschemisch and Emily Northrup contributed equally to this work. Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch