!!!!!Study!on!the!role!of!Stx2!gene!in!spermatogenesis!

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2013,!September!Yasuhiro!Fujiwara!

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Graduate!School!of!Natural!Science!and!Technology!!

Okayama!University!

! 1!

Contents

Chapter 1

General Introduction------------------------------------------------------- 3

Chapter 2

Histological and immunohistochemical analysis of the Stx2repro34 mice

Introduction---------------------------------------------------------------------------------- 24

Material and methods---------------------------------------------------------------------- 29

Results----------------------------------------------------------------------------------------- 37

Discussion------------------------------------------------------------------------------------- 42

Figures and tables--------------------------------------------------------------------------- 46

Chapter 3

Expression of STX2 in the seminiferous epithelium

Introduction---------------------------------------------------------------------------------- 60

Material and methods---------------------------------------------------------------------- 62

Results----------------------------------------------------------------------------------------- 65

Discussion------------------------------------------------------------------------------------ 67

Figures and tables-------------------------------------------------------------------------- 70

! 2!

Chapter 4

Aberrant localization of sulfoglycolipids lead to multinucleation of

the germ cells

Introduction---------------------------------------------------------------------------------- 78

Material and methods---------------------------------------------------------------------- 80

Results----------------------------------------------------------------------------------------- 81

Discussion------------------------------------------------------------------------------------ 83

Figures and tables--------------------------------------------------------------------------- 87

Chapter 5

General discussion and Conclusion------------------------------------------------------ 92

Acknowledgement--------------------------------------------------------------------------- 100

References------------------------------------------------------------------------------------ 101

! 3!

Chapter 1

General introduction

! 4!

Spermatogenesis

Spermatogenesis is a process to produce male gamates and a complex biological

process of cellular transformation. This process produces haploid gamates from

diploid spermatogonial stem cells, and the final maturation step in which an

elongated spermatid differentiate into a sperm is called spermiogenesis. During the

spermatogenesis, spermatogonial cells are transformed into spermatozoa over an

extended period of time, about 32 days in mice, within seminiferous epithelium

boundaries of the testis (Fig 1-1). The first cycle of spermatogenesis after the birth is

the synchronized process called the first-wave of spermatogenesis (Fig 1-2). This

precisely timed and highly organized spermatogenic cycle is essential for continuous

sperm production, which is dependent upon numerous factors; both intrinsic (Sertoli

and germ cell) and extrinsic (androgens, retinoic acids), as well as being species-

specific genetic background (1). The primary endocrine hormones are the

gonadotrophins, LH and FSH that are synthesized and secreted by the pituitary gland.

Gonadotropin receptors are expressed only by somatic cells within the testis. The LH

receptors are present on Leydig cells and the FSH receptors on Sertoli cells.

Synthesis of testosterone in the adult Leydig cells is stimulated by LH (2).

Russell, et al. (3) established a 12 staging method of the spermatogenic cycle

in the mouse following the Oakberg's 12-stage classification scheme (4). The

spermatogenic “cycle” is the sum of the stages, and the duration of the cycle is the

period taken for a single germ cell to pass through each of the stages. In rodents, the

first wave of the spermatogenetic process starts a few days after birth, when

gonocytes (prespermatogonia) resume proliferation and become undifferentiated

spermatogonia or spermatogonial stem cells (SSC). Once SSC are engaged in

differentiation process, the cells undergo six successive mitotic divisions, giving rise

! 5!

to meiotic spermatocytes. The spermatocytes go through meiosis which is composed

of two consecutive divisions, with a single round of DNA replication, to give rise to

haploid spermatids. The final step in spermatogenesis consists of the transformation

of spermatids into spermatozoa and this process is specifically called spermiogenesis.

Meiosis

Meiosis is a unique nucleic division to produce haploid nucleus in the germ cell. In

sexual reproduction, in order to inhibit the nucleus from doubling the number of their

chromosomes over generations, reproductive cells experience two meiotic divisions

during gametogenesis. Whereas meiotic division shares many molecular mechanisms

with mitotic division (5), meiotic progression accomplishes several specialized

mechanisms because of two chromosomal distributions without S phase. In many

animals and asexual cells with 2N chromosomes, these cells have each chromosome

containing a pair of two homologous chromosomes originated from their parents but

with subtle difference. On the other hand, in haploid germ cells, each chromosome

contains one homologous chromosome, and the cells differentiate into specialized

germ cells called gamete, such as egg or sperm.

Meiosis is characterized by an extended prophase, followed by two divisions

that produce gametes. In male mammal, a population of spermatogonial stem cells is

present at birth, and meiosis is initiated in maturing cohorts of spermatogonia,

resulting in the continuous production of sperm throughout the reproductive lifespan.

During the prophase of meiosis I, homologous chromosomes pair and synapse, which

is mediated by a meiotic framework called the synaptonemal complex (SC). The

substages of meiosis I prophase is defined by chromosome configurations and

structure (6).

! 6!

Meiotic prophase is classified into four substages, leptotene, zygotene,

pachytene, and diplotene, as shown in Fig 1-3. During leptotene stage, the

homologous chromosomes start to align, although they are not yet fully paired. Axial

element (AE) is a chromosomal scaffold, derived through the cohesin proteins, such

as REC8 and SMC1B, and SC-specific proteins, such as SYCP3 and SYCP2 (7, 8).

The chromatids experience genetically programmed double-strand DNA breaks

induced by SPO11. These DNA breaks are recognized by homologous recombination

repair machinery, such as phosphorylation of H2AX to form gammaH2AX by ataxia

telangiectasia mutated (ATM), which triggers binding of RECA-related proteins, such

as DMC1 and RAD51. These proteins co-localize to electron-dense structures called

recombination nodules (RN) along the developing AE (9). By zygotene stage,

homologous chromosomes start to pair each other and synapsis is initiated. This

initiation of synapsis facilitates the formation of the SC, and the AE begin to “zip”.

Through this process the AE become the lateral elements (LE) of the SC. Pachytene

stage is defined by completion of synapsis, at which point the central element (CE) of

the SC is apparent. CE is composed of proteins such as SYCP1, synaptonemal

complex central element protein 1 (SYCE1) and SYCE2 (10). The pachytene stage is

lengthy and includes maturation of a subset of meiotic recombination sites (<10%)

into crossovers marked by the mismatch repair proteins MutL protein homologue 1

(MLH1) and MLH3, which also co-localize to RN (11, 12). After recombination is

completed, chromosomes undergo desynapsis and condense in the final diplotene

substage. At this stage, the homologues are held together by the recombination sites

(crossovers), which are seen in cytological preparations as chiasmata (6).

! 7!

Mutagenesis

In recent years, several numbers of mouse models that show impaired

spermatogenesis have been made for genetic research. Whole-genome mutagenesis is

a powerful tool for this purpose. N-ethylnitrosourea (ENU) is one of frequently used

alkylating agents, which causes random mutations throughout the genome of all types

of cells, importantly germ cells (13). The Jackson Laboratory created a large number

of mouse infertility models by ENU-induced mutagenesis under the Reproductive

Genomics Program (14). Fig 1-4 shows the mating scheme to create infertile mutant

strains. High mutation load by ENU in treated (G0) males causes high rate of death

due to conditions, such as lymphoma. In order to reduce such conditions, mutated G0

males are bred with wild type females to generate a G1 generation, each of which are

considered to be a line founder during the relatively short period of the time. G1

males are then bred to produce the G2 and backcrossed to produce G3 generation that

will be homozygous for a series of mutations generated in the G0 mouse. Recessive

mutations affecting male infertility (assuming a 100% percentage rate) will be

detected in about 25% of G3 males within an affected line (15). G3 males and females

that fail to reproduce by natural mating to wild-type animals will be used for

secondary phenotype screening to assess gonad and accessory organ histology,

hormone production, gamete production and gamete function in fertilization. The

genetic transmission of the infertility trait in each family is confirmed and each

mutation is genetically mapped to a defined chromosome region, facilitating

identification of candidate genes from DNA sequence and expression database (14).

! 8!

repro34

repro34 is one of infertile mutant strains created by the Reproductive Genomics

Program using ENU at The Jackson Laboratory (14). repro34 males, but not in

females, show infertility due to defective spermatogenesis. Previous study using

linkage analysis and positional candidate approach identified Syntaxin2 (Stx2) gene,

also known as Epimorphin (Epim), on chromosome 5 as the gene responsible for

repro34 (16). A nonsense mutation of the Stx2 gene of repro34, a nucleotide

substitution of C to T in exon 3, led to a premature termination at codon 41. Because

of this non-sense mutation, the most of STX2 domains are expected to be lost in

repro34 (Fig 1-7). This mutation affected specifically male reproductive function

causing defective spermatogenesis with no apparent dysfunction in the other organs in

Stx2repro34. Homozygous females are normal in reproductive function (16).

Syntaxin2/Epimorphin

Epimorphin was initially identified on the surface of mesenchymal cells of mouse

embryonic tissues as the target of a monoclonal antibody that blocked hair follicle

morphogenesis in the dermal epithelium (17). Soon after the identification of

epimorphin, HPC1 (later renamed syntaxin), a neuronal cell-specific membrane

protein, was discovered (18, 19), and it was revealed that this syntaxin/HPC1 protein

was similar in structure to epimorphin but lacked a signal peptide at the N-terminus

(17). Epimorphin is now known to be involved in the morphogenesis and

development of many epithelial organs (20). Epimorphin is also known to mediate

vesicle trafficking (21) and localize throughout many epithelial organs including

intestine, pancreas, mammary gland, lung, gallbladder, liver, skin, kidney, and hair,

! 9!

where epimorphin is secreted from stromal cells (22) in the form of absence of N-

terminal signal peptide (23), playing a role in morphogenesis (Fig 1-8).

Syntaxin2 was known to be identical to nervous system-specific syntaxin1A in

rat, and thought to be important for calcium-regulated secretion during the docking of

synaptic vesicles with the presynaptic plasma membrane (18). Syntaxin2

(Epimorphin) consists of a three-helical N-terminal bundle (3-Hlx), namely SynN

domain, connected by a flexible linker to a C-terminal sequence containing the

SNARE and transmembrane (TM) domains (24), namely t-SNARE domain (Fig 1-6).

Sixty percent of the amino acid sequence of epimorphin/syntaxin2 is identical to

syntaxin/HPC1 (23). However, deletion analysis of epimorphin has revealed that the

morphogenic and vesicle-fusion functions can be separable, in which the C-terminal

SNARE and TM domains were essential for syntaxin-mediated vesicular fusion (24,

25), but they are dispensable for epimorphin-mediated morphogenic activity (23),

thereby N-terminal SynN domain containing three alpha helicles is required for

epimorphin activity (20) (Fig 1-6). In addition, for epimorphin to be functional, it

appeared to change its membrane orientation by unknown mechanism, and by loosing

its TM domain epimorphin would perform its extracelllular function (23) (Fig 1-6). It

has also been shown that membrane topology of proteins lacking N-terminal signal

peptides but containing hydrophobic signal-anchor domain (like epimorphin and

syntaxin family members) largely depends on the three-dimensional configuration of

N-terminal sequence (26, 27). This may indicate that intramolecullar interactions

within the epimorphin sequence allow a subpopulation to orient itself on the outer cell

surface (23). Furthermore, The expression and the subcellular localization of

epimorphin appeared to be highly regulated (23).

! 10!

In mammalian cells, Syntaxin2 is involved in cytokinesis through two possible

mechanisms. In the first possibility, Syntaxin2 may increase the surface of plasma

membrane during furrowing by mediating the fusion of vesicles with plasma

membrane close to the site of ingteression. On the other hand, in the second

possibility, syntaxin2 may have a direct involvement in the final abscission of the

midbody to completely separate into daughter cells (28).

In the testes, syntaxin2 is known to localize at acrosomal compartment,

playing a critical role in acrosomal reaction (29-31). Furthermore, Akiyama, et al.

(16) and Wang, et al. (32) have previously indicated its involvement in

spermatogenesis. Both Stx2repro34 and Stx2-knockout (KO) show a number of

multinucleated spermatocytes and no spermatid was observed in the seminiferous

tubules. Spermatogenic arrest in these mutants indicates syntaxin2 plays an important

role during spermatogensis, but its function has not yet been revealed.

The intercellular bridges of the germ cells

Mammalian germ cells are connected as syncytium through intercellular bridges that

exist between the spermatogenic cells at the same developing stage (33, 34). The

intercellular bridges are large open channels between clones of spermatogenic germ

cells (35, 36), and stable formation of the intercellular bridges is essential for

spermatogenesis and fertility (37). Disruption of the intercellular bridges prevents

normal formation of syncytium, which arrests spermatogenesis prior to meiotic

prophase, and resulted in male-restricted sterility (37). Although several roles of

intercellular bridges have been hypothesized (38), two hypothesizes are currently

accepted (5). The first hypothesis advocate that “cytoplasmic sharing” of essential

signals, such as transfer of mRNA, proteins, and ribosomes, between interconnected

! 11!

germ cells is required for the synchronous cell divisions (39-41). In the second

hypothesis, the communication between germ cells coordinates some of critical steps

of spermatogenesis, such as the entry of meiosis, and this intercellular communication

is controlled by the relatively rapid transfer of unknown signals or substances through

the intercellular bridges (42, 43). Although many components for intercellular bridges

have been reported, TEX14 is the only essential component (37). In addition, genes

essential for cytokinesis and formation of ring canals are not necessarily required for

ring canals or fertility in female (37, 44).

Formation of intercellular bridges shares many molecular mechanism with

cytokinetic division (5), although most aspects of the signaling process during

cytokinesis remain unknown (45). Proper formation of the intercellular bridge owes a

lot to retaining a midbody, a central part of the bridge which consists of various

molecules. In somatic cytokinesis, recruit of TSG101 and ALIX to the midbody and

their association with CEP55, a component of the midbody, trigger abscission of the

midbody and consequence to complete division of the daughter cells. This hypothesis

is proven by the appearance of multinucleated HeLa cells through siRNA inhibition

of CEP55 (46). In the case of male germ cells, TEX14 is speculated to disturb the

association of TSG101 and ALIX with CEP55 so that the midbody stays stable and

the intercellular bridge are maintained (47) (Fig. 1-8).

Glycolipids

In mammals, two major sulfoglycolipids are produced in the testis; sulfatide and

seminolipids, through catalyzation of alkylacylglycerol (EAG) by common enzymes,

cerebroside sulfotransferase (CST) (48, 49) and ceramide galactosyltransferase (CGT)

(50) one after another. Consequently alkylacylglycerol and ceramide are converted to

! 12!

seminolipids and galactocylsulfatide, respectively (48, 50, 51) (Fig. 1-9). Seminolipid

is synthesized in spermatocytes and maintained in the subsequent germ cell stages

(52). Significance of glycolipids in spermatogenesis has been evidenced with the

arrested spermatogenesis in CST and CGT deficient mice (48, 50). Zhang, et al. (53)

proved that synthesis of glycolipids in germ cells is essential for spermatogenesis,

however the precise function of glycolipids in spermatogenesis has not yet been

elucidated.

Glycans are expressed specifically in the mammalian testis. Seminolipids are

sulfated glycolipids and their distribution is restricted to the testis. Seminolipids

consist of 90% of glycolipids in the testis (54). The sugar chain of seminolipids shows

the same structure as sulfatides that are abundant in myelin sheath, but lipids chain of

seminolipids consists of glycelolipids that binds to ether. The sugar parts of

seminolipids and sulfatides are biosynthesized through CGT and CST. The

homologous deletion of CGT (50) and CST (53) resulted in male-restricted sterility.

In the testis of CGT KO mice, galactosyl-alkyl-acyl-glycerol and seminolipids are

depleted, and spermatogenesis was arrested prior to meiotic division (55, 56). On the

other hand, spermatogenesis of CST KO was arrested at metaphase of meiosis I (48).

These observations indicated that these two testis-specific glycolipids have similar but

distinct roles in mammalian spermatogenesis. Futhermore, transplantation of normal

spermatogonial germ cells into the seminiferous tubules of CST KO mice that

contained glycolipid-depleted somatic cells observed normal spermatozoon (53).

Taken together, glycolipids are required for germ cells themselves. Glycolipids seem

not to be required for the normal function of somatic cells in the seminiferous

epithelium,

! 13!

Several studies suggested that lipids play an important role in regulation of

cytokinesis as a component of lipid-order microdomains, termed lipid rafts (57), and

lipid rafts are thought to be platform of signaling molecules. Seminolipids are

produced in the Golgi membrane of spermatocytes (54) and transported to the plasma

membrane by vesicle transportation (52, 58). Glycolipids including seminolipids are

normally localized to the plasma membrane of spermatocytes or round spermatids,

but not on the spermatogonia (59), and testis-specific glycolipids are also thought to

be a component of lipid rafts on sperm (60). Several studies also suggested that

plasma membrane raft domains are formed in the furrows of dividing cells, indicating

an important role in cytokinesis (57). In addition, seminolipid in sperm is thought to

be a component of lipid rafts and to function for sperm to attach Zona pellucidae (ZP)

to penetrate during capacitation (60).

! 14!

! 15!

! 16!

! 17!

! 18!

! 19!

! 20!

! 21!

! 22!

! 23!

Chapter 2

Multinucleation of the germ cells in the

Stx2repro34 mice

! 24!

Introduction

In the previous study, infertile Stx2repro34 mutants showed spermatogenic failure

during meiosis, and this abnormal spermatogenesis was caused by a mutation on the

Stx2 gene (61). Homologous inactivation of Stx2 also showed similar spermatogenic

failure (62). These observations indicate the essential role of Stx2 in spermatogenesis.

However, the precise function of Stx2 in spermatogenesis is largely unknown.

Therefore, this study focused on further examination of the phenotype of aberrant

spermatogenesis in the Stx2repro34 mutant by histological and cytological analysis.

Firstly, in order to investigate the transition of spermatogenesis in the

Stx2repro34 mutant, the testis sections during the first-wave of spermatogenesis was

examined. When the spermatogenesis is initiated, the spermatogenic cells undergo

synchronized process within seminiferous tubules. During the first-wave of

spermatogenesis, primitive type A spermatogonia are first appeared on the day 6 after

the birth, and meiotic prophase I is initiated on the day 7 to 9 (63). Pachytene

spermatocytes first appeared on from day 12 to 15 (63, 64). Previous study revealed

that the expression of Stx2 start to increase at day 16 after the birth (P16) in the testis

(61), indicating that Stx2 plays an important role after pachytene stage.

Secondly, since the phenotype of spermatogenic defect in the Stx2repro34 was

characterized by remarked high frequency of multinucleation of the germ cells (61).

This study next examined the cytological effects of multinucleation by

immunohistrogical and immunocytrogical analysis using antibodies against several

maker proteins. GammaH2AX is a phosphorylated form of histone variant H2AX on

Serine 139 (65, 66). GammaH2AX marks chromatin domains with DNA damage,

including double strand breaks (DSBs) (66). During meiotic prophase, the

spermatocytes that are successful with repair of DSBs only exhibit restricted

! 25!

expression of γH2AX only in the chromatin of the XY body (sex chromosome) at

pachytene or diplotene stages (65, 67). By observing the pattern of γH2AX labeling in

spermatocytes, the extent of DNA repair machinery can be examined.

Multinucleation of germ cells often appears in the pathological testicular

conditions. One of ceuases for the formation of these abnormal cells is opening the

intercellular bridge, a stable cytoplasmic extension that interconnect a large number

of spermatogenic cells (3). Multinucleation of somatic cells is caused by two

mechanisms; cell-cell fusion and acytokinetic cell division (68). Hence, this study

thirdly examined the formation of the intercellular bridges in the Stx2repro34 by

observing immunohistochemical localization of a component of the intercellular

bridges. Although the intercellular bridges in germ cells have distinct functions and

are different from the interconnections in somatic cell cytokinesis, the molecular

mechanisms to form the intercellular bridges share the large part of those in somatic

cytokinesis (5). The intercellular bridges are large open channels between clones of

spermatogenic germ cells (35, 36), which continuously formed from the stage of

undifferentiated spermatogonia to that of elongated spermatids. Since TEX14 plays an

important role in stable formation of intercellular bridges of the germ cells, this study

observed localization of TEX14 in the multinucleated cells in the Stx2repro34 mutant.

In the Stx2repro34 mutant, multinucleation of germ cells can be observed in

pachynema. During pachytene stage, one of important events, meiotic synapsis, is

completed, which is essential for successful meiosis. Fourthly, in order to reveal the

status of synapsis during meiotic prophase I, surface spread chromosome preparation

was examined by immunocytrogical localization of some marker proteins including

SYCP3, SYCP1, and γH2AX. SYCP1 and SYCP3 are known to form during

prophase I as main components of two side rails of the synaptonemal complex (SC).

! 26!

SYCP1 functions in synaptonemal complex assembly, meiotic recombination, and

XY body formation (69), and SYCP3 is required for the formation of LEs as a main

structural element of the LE (70). Homologue juxtaposition during prophase I can be

divided into three structurally distinct steps, recognition, presynaptic alignment, and

synapsis by the SC (6). In many organisms, these three steps are distinguished by

their dependence on DNA DSBs (71, 72). Since γH2AX foci display the DNA DBSs

(66), fluorescenceimmunohistochemical staining of γH2AX indicates the status of

DNA integrity and chromatic organization.

As previously reported, the spermatogenesis in Stx2repro34 was arrested during

meiotic divisions since disrupted formation of metaphase plate (equatorial plate) and

many degenerating spermatocytes at metaphase were observed (61). In order to

complete a series of meiotic divisions, spermatogenic cells must undergo proper

assemble and segregation of chromosome contents. Abnormal assemble of

chromosome during metaphase resulted in Metaphase-specific cell death (73, 74).

This study fifthly conducted TdT-mediated dUTP nick end labeling (TUENL) assay

that detects DNA fragmentation of these denatured germ cells as an indicator of

programmed cell death, namely apoptosis (75).

In meiosis, a spermatocyte divides into four haploid daughter cells through

two meiotic divisions. During metaphase, homologous chromosomes align in the

middle of the cell before being separated into each of the two daughter cells.

Metaphase accounts for approximately 4% of the cell cycle's duration, and 60% to

70% of them are primary spermatocytes and the remaining is the secondary

spermatocytes (3). Microtubules formed during prophase attached themselves to

kinetochores during metaphase since prometaphase, followed by anaphase. The

centromeres of the chromosomes then are gathered on the metaphase plate in between

! 27!

two centrioles. This alignment is enabled by the counterbalance of the pulling power

generated by the opposing kinetochores (76). During metaphase I, SYCP3 protein co-

localize at the surface of contact region between sister chromatids, so-called

interchromatid domain or centromeres in bivalents (77). Hence, this study, next,

examined the cytological localization of SYCP3 in the spermatocytes at metaphase.

During prometaphase and metaphase, without all the chromosomes being

aligned at the metaphase plate, the cell would not be able to enter anaphase,

controlled by one of the cell cycle checkpoints, called spindle checkpoint (78). In the

case of meiosis, the consequences of error during meiotic division in spermatogenesis

would cause serious problems, such as aneuploid spermatozoa, embryonic lethality,

and developmental abnormalities (79). In mouse oocytes, failure of chromosome

segregation resulted in arrest of meiotic progression at metaphase I (80). In

mammalian cells, mitotic spindles are organized at the time of nuclear envelope

breakdown (NEBD), when two active microtubule-organizing centers located at

opposite poles of he nucleus start to emanate microtubules. A bipolar spindle is

organized when the plus ends of antiparallel microtubule arrays meet at the cell

equator (metaphase plate). During prometaphase, the chromosomes congress toward

the spindle equator where they ultimately attain an equilibrium position at metaphase.

Since a number of multinucleated spermatocytes at metaphase containing multiple

metaphase plates were observed in the Stx2repro34 (61), this study next performed

examination of structurally preserved chromosome preparation to visualize the

construction of spindle body.

Lastly, this study focused on the cellular fate of aberrant germ cells in the

Stx2repro34 mutant, since round spermatid-like germ cells were occasionally observed

! 28!

in the seminiferous epithelium. These abnormal cells expressed spermatid-specific

proteins, including IZUMO, which is required during fertilization (81, 82).

! 29!

Material and methods

Animals

The Stx2repro34 mice used were provided from the mouse mutant resource colony of

the Jackson Laboratory (JAX) (Bar Harbor, Maine, USA). The Stx2repro34 mutation

was induced in a C57BL/6J background and subsequently outcrossed to C3HeB/FeJ

inbred strain (CLEA Japan, Tokyo, Japan). A congenic line for the Stx2repro34 was

created on C3HeB/FeJ. For biological analyses, Stx2repro34 homozygotes and control

wild-type littermates at 10-12th weeks of age or at 1-3rd weeks of age for the sake of

first-wave of spermatogenesis were used. All the experiments using animals were

approved by the regulation of animal protection for the laboratory and animal

husbandry of Okayama University. Mice were kept in the animal facility of the

Okayama University, following the law of animal protection for the laboratory and

animal husbandry of Okayama University. The condition of the breeding-room was

maintained constant throughout the year, under 23-26°C temperature, 30-60%

humidity, and 12 hour light-dark cycle condition. Standard pellet diet (CE-2, CLEA

Japan) was fed to mice. Mice were euthanized by the painless method using carbon

dioxide (CO2) gas.

DNA extraction from tail

Genomic DNA was prepared from cut tails or fragments of ear of mice. These tissue

samples were collected at the weaning day as a part of identification. To break cell

structure and digest DNA-protecting proteins, the tissue samples were treated with 4

µl of ProteinaseK (10 mg/ml) in 150 µl of NP-40 detergent (0.14 M NaCl, 10 mM

EDTA, 100 mM Tris-HCl, 0.5% NP-40) and 150µL of 2X SDS (2% SDS, 20 mM

EDTA, 0.2 M NaCl) at 55°C overnight. Adding 4 µl of RNase A (10mg/ml) (nakarai

! 30!

tesque, Kyoto, Japan) to the mixture to digest RNA, the samples were incubated at

37°C for 2 hours. Protein residual substances and fragmented cellular debris were

separated by adding 300 µl of Phenol/Chloroform and 30 µl of 3 M sodium acetate

(pH 5.2). After gentle inverting for 5 minutes, the mixture was centrifuged at 12,000

rpm for 15 minutes at room temperature (RT). The supernatant was transferred to a

new tube containing 600 µl of anhydrous ethanol, and mixed well. Followed by

centrifuging at 12,000 rpm for 15 minutes at 4°C, the ethanol solution was replaced

with 100 µl of 70% ethanol, and centrifuged at 12,000 rpm for 15 minutes at 4°C. The

ethanol solution was removed to dry, and the collected DNA was dissolved in 50 µl of

TE buffer (10 mM Tris HCL and 1 mM EDTA in milli-Q water, pH 8.0).

Genotyping of Stx2repro34

For genotyping of Stx2repro34, template genomic DNA extracted from tail or ear, as

stated above, was used. The reaction mixture for PRC for each sample contained 0.2

µl of forward (5'-GTGGGATCACGAGTCACTCACT-3') and reverse (5'-

GAGTGAGTTCCACGACAGCCA-3') primers (10 µM), 1 µl of 10X PCR

amplification buffer, 1 µl of dNTP solution, 0.05 µl of Tap Polymerase (5 U/µl)

(Amersham Bioscience, Piscataway, NJ, USA), 1 µl of DNA sample (20 µg/ml) and

6.55 µl of autoclaved milli-Q water. Amplification of nucleic acids was processed by

denaturation, annealing, and polymerization cycles; first denaturation at 94°C for 5

minutes, followed by 40 cycles containing denaturation (94°C for 30 seconds),

annealing (58°C for 30 seconds), and polymerization (72°C for 30 seconds), finished

by the final polymerization step at 72°C for 5minutes. The PCR products were

digested with TaqαI restriction enzyme at 65°C for 16 hours, and thereafter analyzed

by electrophoresis through a 2.5% agarose gel. Wild-type and heterozygous mice

! 31!

showed digestion of amplified DNA fragment, creating 258 and 102 bp fragments,

while the amplified DNA fragment from homozygous mice remained undigested

(Fig1-6B).

Histological analysis by Mayer's HE staining

For histological analysis, mice were euthanized by CO2 gas, and testes and

epididymis were immediately extracted for fixation. The whole testes and epididymis

were fixed in Bouin's solution (picric acid: formaldehyde: acetic acid=15:5:1) for 12-

16 hours at 4°C. The fixed samples were then washed with ethanol from 70% x3,

80%, 90%, 95%, up to 99.5% for 1 hour each, and completely dehydrated in

anhydrous ethanol overnight. The tissue samples were placed in Xylene three times at

RT and paraffin three times at 60°C for 1 hour each, and embedded in paraffin block.

Testes samples were sliced by 5 µm using a microtome (RV-240, Yamato, Japan),

and pasted on slide glasses. After drying the slides on paraffin stretcher overnight, the

slides were de-paraffined in Xylene three times for 5 minutes each, and rehydrated

through ethanol at down graded concentrations from 100% twice, 95%, 90%, 80%, to

70% for 3-5 minutes each. Followed by staining in hematoxylin dye for 5 minutes, the

slides were washed by flowing tap water for about 10 minutes until the tissues were

decolorized to light staining color. The slides were then stained in eosin dye for 1

minute. After quick wash in tap water, the slides were decolorized in 70%, 80%, 90%,

95%, 100% ethanol for less than 30 seconds each to adjust the eosin color, and placed

in Xylene three times for 5 minutes each. The preparation slides were sealed using

Entellan New (Merck, USA). Stages of the seminiferous epithelium cycle were

identified according to established morphological criteria (3).

! 32!

TUNEL assay (Fluorescent)

To distinguish apoptotic cells, TUNEL assay was performed using in situ Cell

Detection Kit (Cat#11684817910, Roche, Basel, Switzerland) according to the

manufacture’s instruction with some modifications. Sectioned testis slides were

treated in the same manner as histological analysis until the deparaffin treatment by

Xylene and hydrophilic treatment by ethanol. 25 µl of Proteinase K (20 µg/ml) in 10

mM Tris-HCl (pH 7.4-8.0) were applied to testes section samples circled by PAP pen

(Daido Sangyo, Tokyo, Japan), and incubated for 20 minutes at 37°C. Followed by

two time washes with PBS, one sample was treated with 25 µl of DNase (1 mg-100

µg/ml) as positive control or 10 minutes at RT. Tissue samples were incubated with

25 µl of TUNEL reaction mixture for 60 minutes at 37°C. After three times of wash,

the preparation slides were sealed with VECTASHIELD Mounting Medium with

DAPI (H-1000, Vector Laboratories. Inc. Burlingame, CA, USA), and observed using

ZEISS AXIO Imager. A1 upright microscope (Carl Zeiss AG, Oberkochen, Germany)

with DFC480 Digital Camera System (Leica Microsystems, Wetzlar, Germany).

TUNEL assay (DAB staining)

For non-fluorescent TUNE assay, DAB staining TUNEL assay was performed using

in situ Cell Detection Kit (Roche, USA) according to the manufacture’s instruction

with some modifications. The procedure was the same as fluorescent TUNEL assay

until the first PBS wash after deparaffin and hydrophilic treatment. To block inherent

peroxidase (POD), 20 µl of 3% H2O2 (nacalai tesque, Japan) in methanol was applied

to the tissue sections for 10 minutes at RT. After three time washes with PBS, the

tissue samples were incubated with 30 µl of Proteinase K (20 µg/ml) for 5 minutes at

RT. Followed by three time washes with PBS, the tissue samples were then reacted

! 33!

with TUNEL reaction mixture (TUNEL enzyme: buffer=1:9) for 20 minutes at 37°C.

The tissue samples were washed ten times with PBS, and incubated with 20 µl of

POD reaction mixture for 20 minutes at 37°C. Washing with PBS once, DAB mixture

(DAB substrate: buffer=1:50-100) was applied to the tissue samples for 2-3 seconds,

and immediately washed well with tap water to stop the reaction. The tissue samples

were conterstained with hematoxylin for 2 minutes, and the color of the dye was

adjusted by dipping through tap water for about 20 minutes. After dehydration

treatment with graded concentration of ethanol and xylene, the slide preparations

were sealed with Entellan new (Merck KGaA, Darmstadt, Germany).

Surface spread preparations and immunostaining

The surface spread preparation of testicular cells was prepared as previously

described with some modifications (83-85). Briefly, cells were collected in DMEM

medium (12100-046, Invitrogen, Carlsbad, CA, USA). After centrifugation at 1,800

rpm for 10 minutes at 4°C, pelleted cells were suspended in 0.5% NaCl solution then

spread on silanized slides. The preparation slides were soaked in 2% PFA containing

0.02% SDS in PBS (pH 7-8 was adjusted with 0.1 M Na2B4O7). After washing with

PBS containing 0.04% DRIWEL (Fuji photo film co., LTD, Tokyo, Japan) then dried,

preparation slides were stored in -80°C until use.

For immunostaining, after blocking with antibody dilution buffer (ADB; PBS

containing 2% BSA and 0.05% Triton-X 100), the slides were incubated with anti-

SYCP1, anti-SYCP3 (86) and anti-gammaH2AX in PBS containing 10% ADB

overnight at 4°C. Secondary antibodies, goat anti-mouse IgG-Alexa Flour 488 (A-

21121, Invitrogen) at 1:200 dilution and goat anti-guinea pig IgG-Alexa Fluor 594

(A11076, Invitrogen, Carlsbad, CA, USA) at 1:200 dilution, or bovine anti-rabbit

! 34!

IgG-FITC (SC-2365, Santa Cruz, CA, USA) at 1:400 dilution in PBS containing 10%

ADB, were used for 1 hour at RT. The slides were then mounted with

VECTASHIELD Mounting Medium (H-1200, Vector Laboratories Inc.) and

observed.

Structurally preserved chromosome preparation

Structurally preserved chromosome preparation was made following the previous

method (87) with some modifications. Testes were dissected out into test tubes

containing 1 ml of ice-cold Ringer solution (155 mM NaCl, 5.6 mM KCl, 2.2 mM

CaCl2, 2.4 mM NaHCO3) for 4 hours, and subsequently minced in the microtubule-

stabilizing buffer (100 mM PIPES (pH 6.8), 1 mM MgSO4, 1 mM EGTA, and 1%

Triton X-100). After centrifuging at 1,500 rpm for 5 minutes, collected suspension

containing germ cells was spread onto slides. The slide preparations were fixed in the

fixative (0.25% glutaraldehyde, 2% formaldehyde, in the microtubule-stabilizing

buffer without detergent) for 30 minutes. For further stabilization of cytoskeletons,

the slides were immersed in methanol at -20°C. The preparation was then treated with

NaBH4 (0.5 mg/ml in PBS), and washed three times in PBS containing 0.02% NaN3

and 0.1% Triton X-100 and once for 5 minutes with the same solution without the

detergent. For immunofluorescence reaction, the cells were reacted with anti-α-

TUBULIN antibody and secondary antibody anti-mouse IgG-Alexa Flour 488 (A-

11008, Invitorogen). After incubation, the slides were sealed with

VECTASHIELDMounting Medium with DAPI, and observed using KEYENCE

BIOREVO BZ-9000.

! 35!

Giemsa-staining air-dried chromosome preparation

Procedure and identification of cell stages were referred to the previously described

methods (88-91) with some modifications. The testes were quickly extracted from

live animals and collected samples were placed in 20 ml of 2.2% sodium citrate in a

laboratory dish. Tunica albuginea was then pealed off, and a cluster of seminiferous

tubules was disentangled using tweezers. These tubules were treated with 1% sodium

citrate solution for 30 minutes at room temperature. The hypotonic solution was then

changed with fixative (Methanol: Acetic acid=3:1) at 4°C for 10 minutes. Followed

by changing the old fixative with new one, the fixed tubules were then transferred to a

new tube containing 50% acetic acid, and waited until germ cells became suspended

due to the tubules being melted. After centrifugal separation at 1500 rpm for 10

minutes, the cell precipitation was resuspended in the same fixative for further use. A

few drops of cell suspension were dropped on a grass slide from the distance above.

The grass slide was placed on the hot plate whose temperature was 37°C to dry. The

chromosomes were stained with 5% Giemsa-solution (Sigma-aldrich, Missouri, USA)

for 5 minutes. The stained slide was gently washed with tap water, and left to dry for

a while. The slide preparation was sealed with Entellan new (Merck KGaA,

Germany).

Immunohistochemistry

Testes from mice and rat were fixed in Bouin's solution or 4% PFA fixative (4%

paraformaldehyde, 0.1 M phosphate buffer, pH 7.4). They were dehydrated then

embedded in paraffin. Sections at 5 µm thickness were cut and deparaffinized. For

immunostaining, deparaffinized sections were autoclaved in 0.1 M Sodium Citrate,

pH6.0, at 120°C for 20 minutes, or the sections were treated with 20 µg/ml

! 36!

ProteinaseK in 10 mM Tris-HCl (pH 7.4) at 37°C for 20 minutes for antigen retrieval,

when necessary. All the sections were incubated in 3% H2O2 in PBS for 15 minutes to

quench the endogenous peroxidase. They were then incubated in PBS containing 10%

BSA for 30 minutes at RT to reduce background. Sections were reacted with primary

antibodies diluted with PBS containing 1% BSA for 3 hours at 37°C. The antibodies

used and each conditions for staining were shown in Table 2-1. Subsequently HRP-

labeled secondary antibodies (Santa Cruz, CA, USA), were reacted at 1:400 dilution

for 1 hour at RT. Antigens were visualized using TSA Plus DNP (HRP) System

(Perkin Elmer, MA, USA) with NEL 747B Dako Liquid DAB+ Substrate Chromogen

System (20 µl/ml) (K3467, DAKO, Glostrup, Denmark). Sections were

counterstained with hematoxylin.

! 37!

Results

Affected spermatogenesis in the Stx2repro34 mutant

In the testis of adult Stx2repro34 mutant males, multinucleation of the germ cells was

observed in pachynema (Fig. 2-1A), diplonema (Fig. 2-1B), spermatocyte at

metaphase (Fig. 2-1C), and round spermatid-like germ cells (Fig. 2-1D). The round

spermatid-like germ cells expressed spermatid-specific protein IZUMO (Fig. 2-9D,E),

an acrosome-localized protein required for fertilization (81). Further histological

analysis observed that many degenerated germ cells were observed in the epididymis

of Stx2repro34 (Fig. 2-1F), while the epididymis of wild-type was filled with mature

spermatozoa (Fig 2-1E).

The postnatal Stx2repro34 mutant males were indistinguishable from the wild-

type males in growth (Fig. 2-2B,C), but the testis weight started to express

differences between two types of males P16 (Fig. 2-2D). The testicular size of

Stx2repro34 mutant is smaller than that of wild-type, although other reproductive organs

of male Stx2repro34 mutant appear to be normal (Fig. 2-2A). Histological examination

of the testes from the mice during first-wave of spermatogenesis revealed that the

abnormal formation of syncytial multinucleated germ cells was also seen in the

seminiferous epithelium of the Stx2repro34 mutant males. At 14, morphology of germ

cells were indistinguishable between wild-type (Fig. 2-3A) and Stx2repro34 mutant

(Fig. 2-3B). In contrast to the progress of meiosis in the wild-type males (Fig. 2-3C),

the Stx2repro34 mutant males at day 16 post birth (P16) showed the multinucleation of

spermatocytes in the seminiferous epithelium (Fig. 2-3D), which presumably

corresponded to mid to late pachytene stage in the normal testis (63). Subsequently, at

P18, multinucleated spermatocytes presumably at diplotene stage as well as at

metaphase were found in Stx2repro34 mutant males (Fig. 2-3F). At P32, the

! 38!

seminiferous tubules of the wild-type testis were filled in with round and elongated

spermatids, whereas the testis of Stx2repro34 mutant males included neither round nor

elongated spermatids, instead, multinucleated germ cells were observed in the inner

layer of the epithelium (Fig. 2-3H). Overall, the phenotypic features in

spermatogenesis of Stx2repro34 mutant males appeared to be the same in the immature

and adult mice. These observations exclude age-dependent regulation of Stx2 in

spermatogenesis.

Multinucleated germ cells

This study confirmed the arrest of spermatogenesis and multinucleation of germ cells

in the adult Stx2repro34 mutant males by immunohistochemistry. Stage-specific

expression patterns of gammaH2AX in the nucleus showed normal progress of

meiosis up to zygotene stage in the mutant. The expression of was restricted to XY

body in the nucleus of the pachynema in the wild-type male (Fig. 2-4C). The same

pattern of expression was observed in the pachytene nucleus in the mutant. In

addition, the positive reaction was observed in the syncytial germ cells (Fig. 2-4D).

The reactions were shown to be a part of the nucleus by staining with LAMIN-B1.

The nucleus of the multinucleated cells displayed positive reaction when

immunostained with a LaminB1 antibody, which indicates the integrity of the nuclear

membrane (Fig. 2-4A, inset).

This study then investigated the formation of the intercellular bridges in the

Stx2repro34 mutants with a TEX14 antibody. The intercellular bridges are large open

channels between clones of spermatogenic cells (35, 36) and TEX14 is one of the

specific proteins in the bridges (37). In the wild-type, the expressions were dotted

around the spermatocytes and spermatids, showing the connection of the germ cells

! 39!

with the bridges (Fig. 2-4E,G). The mutant testis exhibited the same expression in the

spermatocytes. When examined the multinucleated cells, patchy, not dotted

expression of TEX14 around the cells were observed (Fig. 2-4F,H). Although TEX14

shows ring-shape formation in normal mice (Fig. 2-4E,G), the expression of TEX14

was disrupted around many of multinucleated spermatocytes in the Stx2repro34 mutant

males (Fig. 2-4F,H). This abnormal formation of intercellular bridges was observed

during later prophase, which is consistent with the timing of STX2 protein and Stx2

gene expression (61) and multinucleation (Fig. 2-12). Hence, STX2 localized to the

intercellular bridges is in part required for maintenance of the intercellular bridges

especially during later prophase.

Arrest of spermatogenesis at pachytene stage often accompanied with aberrant

chromosome synapsis, therefore we performed immunostaining of spread

chromosome preparations using antibodies against SYCP3, SYCP1, and

gammaH2AX, maker proteins for meiotic synapsis. Labeling pattern of these maker

proteins indicate extent of synapsis. However, the labeling patterns of these proteins

were indistinguishable between wild-type and Stx2repro34 mutants (Fig. 2-5), indicating

that normal synapsis of homologous chromosomes in the Stx2repro34 mutant males.

These results indicated that STX2 is not involved in chromosomal synapsis.

As stated above, several types of multinucleated cells were observed in the

adult Stx2repro34 mutant males (Fig. 2-1A-D). This study, therefore, performed

immunocytochemistry of these syncytial germ cells obtained by means of squash

technique from the seminiferous tubular segment at stage VIII-I to unveil the

structural features. Expression pattern of SYCP3 at centromere appeared to be normal

in multinucleated spermatocytes at metaphase in the Stx2repro34 mutant males,

compared with that of spermatocytes at metaphase in the normal (77), but the number

! 40!

of the positive spots seemed twice or three times as many as those in the normal (Fig.

2-6A,B). Staining of alpha-TUBULIN revealed that multipolar formation of spindle

body in multinucleated metaphase spermatocytes (Fig. 2-6D). The aberrant

chromosomal contents were confirmed by Giemsa stained chromosome preparation

(Fig. 2-6G,H). More than 60% of metaphase spermatocytes were multiploid and

contained more than 20 bivalents or univalents in the Stx2repro34 mutant males, but

about 40% of metaphase spermatocytes contained 20 bivalents like wild type (Fig. 2-

7). Since secondary spermatocytes were barely observed in the Stx2repro34 mutants

(3.66%/N=86 in Stx2repro34, compared with 29.6%/N=145 in wild type), developing

germ cells were not capable of completion of meiotic division due to failure of

chromosome segregation. TUNEL assay further confirmed remarkably higher

frequency of apoptotic cells in the testis of Stx2repro34 mutant males compared with

wild-type (Fig. 2-1E,F), and stage-specific apoptosis at metaphase (Fig. 2-1G-I).

Meiotic arrest in round spermatid-like germ cells in Stx2repro34

In order to determine more precise timing of meiotic arrest in Stx2repro34 mutant, round

spermatid-like abnormal germ cells were labeled with some meiosis specific markers,

SYCP1 and SYCP3. SYCP1, a component of central element (CE), disappears at the

end of prophase I, and expression of SYCP3, a component of lateral element (LE),

can be observed at centromeres until metaphase I (77). In the tubules of wild-type at

XI, diplonema showed SYCP3 expression (Fig. 2-10A,C), and spermatocytes at MI

also showed SYCP3 expression (Fig. 2-6A). In contract, round spermatid-like

abnormal germ cells showed neither SYCP1 nor SYCP3 expression (Fig 2-10D-F).

As shown above, secondary spermatocytes were barely observed in Stx2repro34 mutants

(Fig. 2-6). These observations suggest that the meiotic progression of Stx2repro34

! 41!

mutants is arrested somewhere between anaphase I and prophase II, probably

interphase.

! 42!

Discussion

Previous study showed gene disruption or a nonsence mutation of Stx2 gene caused

defective spermatogenesis, which lead to formation of syncytial multinucleated

spermatocytes (61, 62). This study further investigated the detail of the phenotype of

the Stx2 mutant repro34 to reveal the function of STX2.

Based on histological observation of the testis of both adult and immature

Stx2repro34 homozygotes, multinucleation of the germ cells was initiated during late

pachytene stage. It has been known that multinucleation of the cell can be caused by

two mechanisms: acytokinesis and cell-cell fusion (68), and in the germ cell,

multinucleation is often caused by abnormal opening of the intercellular bridges (3).

This study also revealed aberrant localization of TEX14, an intercellular bridge

specific protein (37), in the multinucleated germ cells, indicating that unstable

formation of the bridge caused opening of these bridges between syncytialy connected

germ cells. Therefore, it is hypothesized that the direct cause of formation of

multinucleated germ cells is unstable formation/maintenance of the intercellular

bridges in the Stx2repro34 mutant. This hypothesis is verified by the observation that the

nuclei within a giant cell are at the same developmental stage. Thus, the loss of STX2

probably affects the open channels of the intercellular bridges at specific stages,

resulting in the formation of abnormal syncytium.

However, the direct cause of multinucleation of germ cells in the Stx2repro34

remains unclear. Several mutant mice showed aberrant spermatogenesis with

multinucleation of the germ cells due to dysfunction of cytokinetic regulation (92),

dietary toxins (93, 94). In addition, the loss of adhesion between spermatids and

Sertoli cells resulted in multinucleation of symplastic giant germ cells that contained

multiple nuclei (95). Disruption of androgen signals also resulted in multinucleated

! 43!

germ cells (96, 97). Since Sertoli cell and Leydig cell play an important role in

differentiation of germ cells by controlling the movement of signal molecules or

maintaining the environmental surrounding of germ cells (98), the loss of STX2 might

affect the microenvironment of the testes. In several kinds of somatic cells, STX2 is

known to play an important role in vesicle fusion during exocytosis (28). Hence,

STX2 may be involved in paracrine communication between somatic cells and germ

cells in the testes, and loss of this function might result in abberant state of either

germ cells or somatic cells, which leaded to multinucleation.

As stated above, there is a possibility that testicular microenvironment is

affected since spermatogenic process is disrupted during later meiotic prophase.

Meiotic progression at pachytene is one of the most important phases for the

structural assemble of homologous chromosomes for recombination, which is

therefore highly regulated by several check point mechanisms during pachytene (99-

101). However, apoptotic cell death was barely observed in spermatocytes during

meiotic prophase (Fig. 2-1G). However, in the spermatocytes of Stx2repro34

homozygotes, in addition, pairing of homologous chromosomes or construction of

synaptonemal complex were apparently normal (Fig 2-5). Hence, multinucleation did

not affect the process of meiotic synapsis, and STX2 appear to have independent

functions from the meiotic synapsis.

In spite of a high frequency of apoptotic cell death in metaphase

spermatocytes due to spermatogenic arrest through spindle checkpoint, histological

analysis revealed the existence of multinucleated round spermatid-like germ cells (Fig

2-9D,E). It was therefore hypothesized that some spermatocytes were capable of

completion of meiotic progression. Immunostaining of the testes of Stx2repro34 with

two of marker proteins for meiotic prophase, SYCP1 and SYCP3, revealed that the

! 44!

nuclei of these round spermatid-like germ cells were at least completed meiotic

prophase (Fig 2-10). Other study examined haploidity of the nuclei in the syncytial

cells by means of reproductive engineering technique, round spermatid injection

(ROSI). Although the study did neither succeed to obtain siblings by ROSI using the

nucleus of the aberrant cells as donors nor confirm haploidity of the nuclei by

chromosome preparations, vestiges of the implantation of the transferred embryo were

detected. This might suggest that few of the nuclei in the syncytial cell completed

meiosis to have haploid genome. However, it was noteworthy that high rate of traces

of implantation was observed, and some recipient mice bred on the day 15 of

pregnancy. This may imply development of fetus to some extent, but abnormally.

Similar results, such as traces of implantations or abnormally developed fetuses, were

reported by microinjection of artificially made trisomy cell previously tested.

Aneuploidy also often leads to failure of normal development in transplanted embryo

(unpublished data). Chromosomal abnormalities due to aneuploidy in male are known

to cause several genetic diseases, such as several types of cancers (102), Alzheimer's

disease (103, 104), Down syndrome (105), or Turner's syndrome (106).

Contrary to the expectation of aneuploidy in the round spermatid-like germ

cell, no chromosome with aneuploidy was observed from the second trial of

microinjection. Since the nucleus of round spermatid-like cell was not a haploid but in

the status of spermatocytes, the two meiotic divisions in the Stx2repro34 were not

probably completed in full state. The chromosome of spermatocytes was rather

arrested during interphase, but morphologically "differentiate" into round spermatid-

like cells expressing some spermatid specific marker proteins including IZUMO (82)

(Fig. 2-9E) and TRA54 (data not shown).

! 45!

As a concluding remark of this chapter, apparent abnormality in spermatogenic

process was initially observed at late pachytene as a formation of multinucleated germ

cells, and the arrest of spermatogenic process in the Stx2repro34 mutant was probably a

secondary outcome owing to disrupted spindle assembly due to the multinucleation of

the germ cells. The multinucleation of the germ cells appeared to be caused by

disrupted intercellular bridges at specific stages in the Stx2repro34 mutant. Therefore, the

function of SXT2 seems to be related to the formation/maintenance of the intercellular

bridge. The later chapters will focus on this respect based on the observations of this

chapter.

! 46!

! 47!

! 48!

! 49!

! 50!

! 51!

! 52!

! 53!

! 54!

! 55!

! 56!

! 57!

! 58!

! 59!

Chapter 3

Expression of STX2 in the Seminiferous

Epithelium

! 60!

Introduction

The STX2 is one of SNARE (soluble N-ethylmaleimide-sensitive factor attachment

protein receptor) members, and many of SNARE family are involved in membrane

trafficking pathways during cytokinesis in a variety of organisms (107). In somatic

cytokinesis, since STX2 is essential for abscission of the midbody in HeLa cells (28),

it was assessed whether the protein localizes to the intercellular bridges of

spermatogenic cells. Further, STX2 is exhibited to localize in acrosomes of sperm to

induce membrane fusion during acrosomal reaction, resulting in release of acrosomal

components during fertilization (29, 30). Precise function of STX2 in the

spermatogenic cells remains to be addressed, but it is apparent that STX2 is one of

key factors for spermatogenesis.

Two Stx2 mutants shoswed an importnace of Stx2 in spermatogenesis (16, 32),

and previous chapter suggested that STX2 is required for maintenance of intercellular

bridges between germ cells. However, precise function of STX2 remains largely

unknown. To reveal the precise function of STX2 in spermatogenesis, this study

examined the expression and subcellular localization of secretory SNARE protein

STX2, and found that STX2 is localized to the Golgi apparatus in developing

spermatocytes and to the intercellular bridges between developing spermatogenic

cells. The SNARE hypothesis postulated that SNARE proteins are categorized into

two broad categories, v-SNAREs in transport vesicles and t-SNAREs in target

membranes, both of which pair specifically for membrane fusion during exocytosis

(108). STX2, t-SNARE protein, is involved in secretory granule fusion in pancreatic

cells (109), platelets (110), and alveolar cells (111), and is localized to the apical

plasma membrane in pancreatic acinar cells (112), and actively cycles between the

plasma membrane and endosomes under cultured cell (113). These data suggest that

! 61!

STX2 is involved in signaling transmission within a cell, but it has not been

elucidated which molecule is the target for the STX2.

! 62!

Material and methods

Animals

For biological analyses, Stx2repro34 homozygotes and control wild-type littermates at

10-12th weeks of age or at 1-3rd weeks of age for the sake of first-wave of

spermatogenesis were used. Rats of a Jcl:Wister closed colony rat at 9-10th weeks of

age were purchased from CLEA Japan (Tokyo, Japan). To obtain protein lysate for

western blotting analysis, immature Jcl:ICR closed colony mice (CLEA, Tokyo,

Japan) were used.

Western blotting

The testes removed from Stx2repro34 homozygous and normal mice were homogenized

in RIPA buffer (150 mM NaCl, 10 mM Tris, pH7.2, 0.1% SDS, 1.0% Triton X-100,

1% Deoxycholate, and 5 mM EDTA). The protein concentration of cell lysate was

measured using the bicinchoninic acid (BCA) protein assay kit (B9643, Sigma,

Missouri, USA), and 50 µg of total protein in each sample were electrophored on 10%

SDS-polyacrylamide gels and transferred onto Immobilon-P Transfer Membranes

(PVDF-Plus) (IPVH15150, Millipore, MA, USA). Immunoblots were incubated with

anti-Epimorphin antibody (BAF2568, R&D Systems, Minneapolis, USA) and anti-

βactin antibody (ab8227, abcam, Cambridge, UK) and subsequently with a

horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG secondary antibodies

(1:20000, Santa Cruz Biotechnology). The membrane was developed using ECL

Advance Western Blotting Detection Kit (RPN2135, Amersham Biosciences,

Buckinghamshire, UK), and observed.

! 63!

Immunohistochemistry

Testes from mice and rat were fixed in Bouin's solution. They were dehydrated and

embedded in paraffin. Sections (5 µm) were deparaffinized and autoclaved in 0.1 M

Sodium Citrate, pH 6.0, at 120°C for 20 minutes, or the sections were treated with 20

µg/ml ProteinaseK in 10 mM Tris-HCl (pH 7.4) at 37°C for 20 minutes for antigen

retrieval. All the sections were incubated in 3% H2O2 in PBS for 15 minutes to

quench the endogenous peroxidase. They were then incubated in PBS containing 10%

BSA for 30 minutes at RT to reduce background. Sections were reacted with primary

antibody against STX2 (114) diluted at 1:100 in PBS containing 1% BSA for 3 hours

at 37°C. Subsequently HRP-labeled secondary antibodies (Santa Cruz, CA, USA),

were reacted at 1:400 dilution for 1 hour at RT. Antigens were visualized using TSA

Plus DNP (HRP) System (Perkin Elmer, MA, USA) with NEL 747B Dako Liquid

DAB+ Substrate Chromogen System (20 µl/ml) (K3467, DAKO). Sections were

counterstained with hematoxylin.

Co-localizations of STX2 and TEX14 were examined using paraffin sections

of the testis fixed in Bouin’s solution. Following the incubation with the first

antibodies (Table 2-1), sections were incubated with second antibodies, donkey anti-

goat IgG-DyLight 488 (705-485-003, Jackson Immuno Research) and donkey anti-

rabbit IgG-Cy3 (711-165-152, Jackson Immuno Reseach). The nuclei were

counterstained with DAPI then mounted using VECTASHIELDMounting Medium

(H-1200, Vector Laboratories Inc.), and observed as described above.

Squash preparation and immunocytochemistry

Squash preparation of germ cells was prepared based on the previously described

method with some modifications (115). Briefly, mouse testis was removed and

! 64!

decapsulated in PBS. Seminiferous tubules were segmented into 1-2 mm length, and

both side ends were cut to identify the stages of spermatogenesis by staining with

hematoxylin. The middle part of the tubular segments was applied for

immunocytochemistry, when the both side were identified at the same stage. The

segments at approximately 0.5 mm length were placed in 15 µl of 100 mM sucrose on

a glass slide. The cells were flown out by squashing with a coverslip. The slide was

quickly frozen in liquid nitrogen for 20 seconds, and the coverslip was removed using

a scalpel. The preparation slides were fixed with 4% PFA in PBS for 15 minutes at

RT, followed by treating with 0.1% Triton X-100 in PBS for 10 minutes at RT. After

washing twice in PBS and blocking with 5% skim milk in PBS, the preparation slides

were incubated with primary antibodies in 5% skim milk in PBS to examine co-

localizations of STX2 and TEX14. The second antibodies used were donkey anti-

rabbit IgG-Cy3 (711-165-152, Jackson Immuno Research) and donkey anti-goat IgG-

DyLight488 (711-485-033, Jackson Immuno Research. The nuclei were

counterstained with DAPI then mounted using VECTASHIELDMounting Medium

(H-1200, Vector Laboratories Inc.), and observed using BIOREVO BZ-9000

(KEYENCE).

! 65!

Results

Expression and subcellular localizations of t-SNARE STX2: the Golgi apparatus

of spermatocytes and the intercellular bridges

This study examined the localization of STX2 in the mouse and rat testis. In the rat

testis, the cytoplasmic localization of STX2 was observed in the pachytene

spermatocyte at restricted stage of IX-X. On the other hand, localizations in the Golgi

apparatus in the pachytene and diplotene spermatocyte were seen at the stage VIII-

XIII. STX2 localizations were also detected in the pro-acrosome and acrosome

regions of the round and elongating spermatids in all the stages (Fig. 3-1A). Dividing

spermatocytes did not express STX2 in the rat testis section (Fig. 3-1B). The mouse

testis showed the results quite similar to those observed in the rat testis, but the signal

was not as clear as in the rat (Fig. 3-2C,D). It was revealed that STX2 was localized

in the Golgi apparatus, which confirmed with co-localization of Golgi maker, TRA54

in the immunofluorescence of the mouse testis (Fig. 3-2A,B). The Leydig cell and the

Sertoli cells did not firmly express STX2 in both the rat and mouse testes.

Since STX2 is shown to be an element of the midbody in the cytoplasmic

bridge (28), this study assessed whether the protein was localized in the intercellular

bridge of the germ cells. In the mouse testis, STX2 was present in the intercellular

bridges between both spermatocytes and spermatids (Fig. 3-3, 3-4). Localization of

TEX14, a marker protein of the bridge, was completely merged with the expression of

STX2 (Fig. 3-3A). The intercellular bridge increases its diameter as the germ cells

differentiate; 1-1.3 µm in spermatogonia, 1.4-1.7 µm spermatocytes, and 1.8 µm< in

spermatids (36). This study observed the tendency of an increase in the diameter of

the midbody ring. This study did not observe any consistent or definitively positive

reaction for STX2 in the testicular interstitial tissue. In immunostained germ cells

! 66!

from stage II-IV seminiferous tubule segments, STX2 was localized to bridges

between intermediate spermatogonia and between early pachytene-stage

spermatocytes (Fig. 3-3B). However, signal intensity for STX2 was not as strong in

these cells as it was in late pachytene spermatocytes and spermatids. To optimize

visualization of STX2 in bridges between spermatogonia and spermatocytes at early

stages of the meiotic prophase, this study utilized squash preparations of germ cells.

Examination of germ cells released from stage IX-X tubule segments revealed that

STX2 was localized to the perinuclear region (Fig. 3-4G). In mature spermatozoa,

STX2 is localized to the acrosomal region (30). In round and elongating spermatids,

STX2 was also localized to the pro-acrosome and acrosome regions, respectively

(Fig. 3-4H,I).

Western blotting confirmed that the expression of STX2 (37kD) throughout

the first-wave of spermatogenesis and was increased at P16 (Fig. 3-5). The increased

expression was consistent with the expression of Stx2 mRNA in the previous report

(61). There was no STX2 expression in the testes of adult Stx2repro34 mutants (Fig. 3-

5).

! 67!

Discussion

In reproductive cells, STX2 is known to co-localize with acrin 1, a 90kD acrosomal

protein, and functions in acrosomal reaction during fertilization (30). In somatic cell

cytokinetic division, STX2 and endobrevin colocalize laterally to the midbody matrix,

which is essential for abscission of the midbody in Hela cells. RNAi inhibition of

these proteins resulted in binucleated cells without any defects in nuclear envelope

and nuclear divisions (28). STX2 is target-SNARE, localized to the plasma

membrane, for vesicle-associated v-SNARE, such as endobrevin, localized to

signaling vesicle in somatic cells (28). This study revealed that STX2 is localized to

the Golgi complex of spermatocytes during later prophase. Hence, it was

hypothesized that molecules produced in the Golgi complex are transported to the

place needed via vesicular mechanism (52, 58) by STX2 as an important role for a

signaling receptor. Like mammalian Stx2repro34 mutants, previous reports also showed

that a Golgi-localized SNARE Syntaxin5 (Stx5) is required for ER-Golgi traffic in

yeast and Drosophila, and the loss of Stx5 lead to blockade of the ER-Golgi traffic for

secretory proteins, and failure of germ cells to complete cytokinesis which resulted in

the formation of multinucleated germ cells (116). The loss of genes expressing other

Golgi proteins that are required for vesicle trafficking also leads to multinucleation of

germ cells in Drosophila (117).

In the wild-type testis, STX2 was distinctively detected in the Golgi apparatus

of the spermatocyte and acrosomal vesicle/acrosome in the spermatid. Testicular

localization of STX2 was previously reported by Wang, et al. (32) and Roqueta-

Rivera, et al. (118), however, the results were not in accordance each other. Wang, et

al. (32) described Leydig cell-specific localization, whereas, Roqueta-Rivera, et al.

(118) reported acrosome specific localization in the spermatid and perinuclear

! 68!

localizations in the spermatocytes, speculating that the presence of STX2 in the

endoplasmic reticulum of spermatocytes. This study observed the localization of

STX2 commonly in mouse and rat testes using an antibody against the cytoplasmic

domains of rat STX2 (114). Although this study could not explain the discrepancy

among the studies, the presence of STX2 in the Golgi apparatus and in the acrosome

of the spermatid seems certain because the results are in accordance with the studies

that revealed localizations and functions of STX2 in acrosomes of the spermatozoa to

function in the membrane fusion in fertilization (29, 30).

This study also revealed localization of STX2 to the intercellular bridges of

the germ cells. STX2 is localized to the intercellular bridges of germ cells throughout

the spermatogenic process, while Golgi-localized STX2 is only found in the

spermatocytes during later prophase. These two distinct localizations of STX2

indicate Stx2 gene plays different roles in the different reproductive stages (Fig. 3-6).

A large part of molecular mechanism of the bridges in the germ cells share with that

of somatic cytokinesis (5). In somatic cell division, failed cytokinesis triggers

multinucleation. Binucleations were also observed when STX2 and endobrevin, a

ubiquitously expressed v-SNARE protein, were functionally impaired (28), indicating

that STX2 takes part in midbody abscission in somatic cell cytokinesis. Since almost

nothing is understood about the mechanism of enlargement of the cleavage furrow

following the failed abscission, it is also unknown whether the common mechanism

lies in the formation of multinucleated cells in the somatic and germ cell STX2

deficiency. The midbody in the intercellular bridge of the germ cells is kept by

TEX14, a germ cell intercellular bridge specific protein (47). The TEX14 deficient

testis exhibited lack of the intercellular bridge and frequent apoptosis of the germ

cells (37). Thus, these phenotypic differences indicate that direct molecular

! 69!

interaction between TEX14 and STX2 is less probable. The observation that TEX14

was abnormally labelled to the large region of the membrane of the multinucleated

cell in the Stx2repro34 mutant possibly reflects the structure of the midbody remained,

although its completeness could be lost. Therefore, STX2 rather seems to be required

for maintenance of the integrity of the intercellular bridges in germ cell, and STX2 is

not likely to function to promote midbody abscissions in the intercellular bridge of

germ cells, different from the role of STX2 in the somatic cell cytokinesis. STX2

seems to have opposite features in somatic cells and germcells. This might be because

STX2 has a different role from the one in somatic cell or because STX2 has different

target moelcule(s) as a member of SNAREs mechanism.

! 70!

! 71!

! 72!

! 73!

! 74!

! 75!

! 76!

! 77!

Chapter 4

Aberrant Localization of

Sulfoglycolipids Lead to

Multinucleation of the Germ Cells

! 78!

Introduction

Both Stx2 mutants (repro34 homozygous and knockout mice) exhibited arrested

spermatogenesis during meiotic divisions with the formation of multinucleated

spermatocytes, suggesting an essential role of the Stx2 during meiosis (61, 62). With

respect to the appearance of multinucleated cells in the affected spermatogenesis,

seminolipid deficient mice were also reported to show a similar phenotype to those

observed in the Stx2 mutants (multinucleation) (50, 53), which led us to speculate that

STX2-deficient mice might be impaired in sulfoglycolipid localization. Seminolipid is

sulfated glycolipid and is predominantly distributed in the testis. Seminolipid consists

of 90% of sulfoglycolipids in the testis (54), and their biosynthesis is responded by

ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). The

loss of CGT (50) and CST (48) resulted in complete deficiency of seminolipid in the

testis and disruption of spermatogenesis. Moreover, Zhang, et al. (53) evidenced that

aberrant spermatogenesis in the CST deficiency was caused by impaired seminolipid

in germ cell components and that impaired seminolipid in the somatic components

could sustain spermatogenesis.

Multinucleation of the germ cells was common feature of the two kinds of

seminolipid deficiency (48, 50, 53). Multinucleated germ cells often appear in the

pathological testicular condition possibly due to opening of the intercellular bridges

(3), and this abnormal multinucleation is generally caused by two mechanisms; cell-

cell fusion or acytokinetic cell division (68). Although the function of the intercellular

bridges in the germ cells is distinct from that of somatic cell cytokinesis, the

molecular mechanism of formation of the intercellular bridges shares the large part of

that of somatic cytokinesis (5). Recent studies suggested that lipids play an important

role in regulation of cytokinesis as a component of lipid-order microdomains, termed

! 79!

lipid rafts (57). Seminolipid in sperm is a component of lipid rafts and functions for

sperm to attach Zona pellucidae (ZP) to penetrate during capacitation (60). These

biological and biochemical information directed to speculate that a common

molecular mechanism lies in the multinucleation of the germ cells in the STX2 and

seminolipid deficiencies, especially in breakage or instability of the intercellular

bridges.

In Chapter 2 and Chapter 3, it was suggested that t-SNARE STX2 is required

for maintenance of the intercellular bridges during spermatogenesis. To reveal the

regulation of testicular sulfoglycolipids through STX2 and the function of

sulfoglycolipids in spermatogenic process, this chapter shows testicular and

cytological localization of sulfoglycolipids in the germ cells of wild-type and the

Stx2repro34 mutant.

! 80!

Material and methods

Immunohistochemistry of sulfoglycolipids

Immunostaining of sulfoglycolipids was undertaken following the previously

described method (119). Briefly, testes samples were fixed in 4% PFA after

immobilization. Following substitution with sucrose in HBSS (137 mM NaCl, 5 mM

KCl, 0.44 mM KH2PO4, 5.6 mM Glucose, 0.34 mM Na2HPO4 anhydrous, 0.81 mM

MgSO4, 1.26 mM CaCl2, and 4.17 mM NaHCO3) with an increase gradient from 12

to 18%, tissue samples were embedded in OCT compound, and frozen. Frozen

sections (7 µm) were incubated with DI8 antibody (119) in PBS(-) solution containing

5% skim milk 4°C overnight. Following the first antibody incubation, the sections

were incubated with a secondary antibody, goat anti-mouse IgG-Alexa Flour 488

(A-21121, Invitrogen, CA, USA) at 1:200 dilution with the same buffer as the

primary antibody for 1 hour at RT. The nuclei were counterstained with DAPI, then

mounted using VECTASHIELDMounting Medium (H-1200, Vector Laboratories

Inc., Burlingame, CA, USA), and observed using KEYENCE BIOREVO BZ-9000

(KEYENCE, Osaka, Japan).

Squash preparation and immunocytochemistry

Squash preparation of germ cells was prepared and immunostained using anti-TEX14

and DI8 antibodies (Table 2-1) in the same manner as chapter 3. The secondary

antibodies for TEX14 and DI8 antibodies were goat anti-mouse IgG-Alexa Flour

488 (A-11001, Invitrogen) and donkey anti-goat IgG-Alexa Flour 568 (A-11057,

Invitrogen), respectively.

! 81!

Results

Affected intracellular localization of sulfoglycolipids in the Stx2repro34 mutant

germ cells

Considering the morphological similarity of affected spermatogenesis in the

sulfoglycolipids deficiency, it was speculated the relevance of these molecules with

STX2 in regulations of spermatogenesis. In order to address to this idea, this study

examined localization of sulfoglycolipids in the wild-type and the Stx2repro34 mutant

testes using the DI8 mouse monoclonal antibody against sulfoglycolipids, including

both sulfatide and seminolipids, that contain the terminal 3-O-sulfated galactose

structure (119).

In the wild-type testis, spermatogonia exhibited very little immunoreactivity

(Fig. 4-1A), while spermatocytes at preleptotene, leptotene/zygotene and early

pachytene stages exhibited a strong and characteristic immunoreaction of fine

granular distribution at peripheral plasma membrane (Fig. 4-1A-C). In late pachytene

and diplotene spermatocytes and spermatids, labeling in plasma membrane was quite

weak and entirely cytoplasmic (Fig. 4-1C). In Stx2repro34 mutant testes, spermatogenic

cells did not exhibit robust localization of sulfoglycolipids in peripheral plasma

membrane (Fig. 4-1D-F). In addition, an abnormal and patchy intercellular

localization was frequently observed at the periphery of mutant pachytene

spermatocytes or cytoplasm of Sertoli cells in stages I-IV of the seminiferous

epithelium (Fig. 4-1D). Cytoplasmic accumulation of sulfoglycolipids in mutant

spermatocytes at late pachytene and diplotene stages (Fig. 4-1E,F) was significantly

greater than that of that of wild-type (Fig. 4-1C).

! 82!

Localization of sulfoglycolipids to the intercellular bridges

Immunostaining of the wild-type germ cells obtained by squash preparation

also evidenced that sulfoglycolipids were localized to the plasma membrane of

spermatocytes (Fig. 4-2B) and spermatids. In addition, spermatocytes and spermatids

at any stages, but not spermatogonia, also exhibited ring-shaped sulfoglycolipid

localization, which was perfectly co-localized with TEX14 (Fig. 4-2A,B). This

reactivity was weak in preleptotene to early pachytene stages and stronger in

developing germ cell at later and post-pachytene stages. This confirms that

sulfoglycolipids are localized to intercellular bridges in specific developmental stages

of spermatogenesis (Fig. 4-3). Together, these observations suggest aberrant

localization and dispersal of sulfoglycolipids in mutant germ cells. Interestingly,

sulfoglycolipids were observed in the intercellular bridges of Stx2repro34 mutant germ

cells (Fig. 4-2C,D). The diameter of the intercellular bridges between mutant

spermatocytes was greater (Fig. 4-2C) than that of wild type (Fig. 4-2A,B), an

observation consistent with opening of the intercellular bridges in Stx2repro34 mutant

spermatocytes.

! 83!

Discussion

In this chapter, immunohistochemical staining of the testis of the Stx2repro34 mutant

revealed that sulfoglycolipids are aberrantly localized in the seminiferous epithelium.

Although this study did not perform experiments to show normal catalytic activity to

produce sulfoglycolipids, it is speculated that sulfoglycolipids are normally produced

but not properly localized in the Stx2repro34 mutant germ cells. In the wild-type testis,

STX2 was co-localized with the Golgi marker protein (Chapter 3), and Roqueta-

Rivera, et al. (118) reported perinuclear localizations of STX2 in the spermatocytes.

These observations suggested that STX2 is localized in the endoplasmic reticulum of

spermatocytes, most likely Golgi apparatus. Sulfoglycolipids are produced at the

outer membrane of the Golgi apparatus in pachytene spermatocytes and are present in

the plasma membrane of germ cells (52, 54, 58). This study also showed the

localization of sulfoglycolipids to the plasma membrane in the wild-type

spermatocyte. In contrast, in the Stx2repro34 mutant spermatocyte, sulfoglycolipids

were not markedly detected in the plasma membrane, and were abnormally

accumulated in the cytoplasm of later pachynema and diplonema. This may imply that

sulfoglycolipids were not guided to the plasma membrane or the intercellular bridges

and they accumulated in the cytoplasm. As a result, distinct expression of

sulfoglycolipids was not observed on the plasma membrane of spermatocyte during

later prophase in the normal testis (Fig. 4-4). Therefore, the results could implicate

that STX2 is required for transportation or distribution of sulfoglycolipids from the

Golgi complex to the site needed, especially to the cellular membrane in stage-

specific manner.

It was suggested that seminolipid is a component of lipid rafts in the germ

cells along with other lipid families (53). Lipid rafts are membrane domains which

! 84!

could function in membrane trafficking and/or remodeling. Considering the

accordance of subcellular localization of STX2 and sulfoglycolipids in the pachytene

spermatocyte, the same phenotype (multinucleation) in impairment of each molecule

might suggest that these molecules cooperate to prevent abscission of the midbody in

the intercellular bridge. Lipid rafts hypothesis postulates that sphingolipids and sterols

assemble to form a raft platform which contributes to membrane signaling and

trafficking (59). Since several studies suggested that plasma membrane raft domains

are formed in the furrows of dividing cells, indicating that lipid raft domains play an

important role in cytokinesis (120). The observation of this study suggested that the

loss of STX2 caused formation of multinucleated cells in Stx2repro34 mutant males. In

addition, this study showed the cytological localization of sulfoglycolipids to the

intercellular bridges. Hence, since the molecular mechanism of formation of

intercellular bridges in germ cells shares a large part of that in mitotic cytokinesis (5),

perhaps sulfoglycolipids play an important role in maintenance of intercellular

bridges in germ cells.

Interestingly, common morphological characteristics, abnormal fusion of

spermatogenic cells, in the testes of Stx2repro34 mutant males, CST (50) and CGT KOs

(53), was observed only at the late pachytene stage. This suggests that both

sulfoglycolipids and STX2 are required for maintenance of intercellular bridges. In

addition, MX, alpha-mannosidase IIx, is an enzyme to form intermediate asparagine-

linked carbohydrates (N-glycans), which plays an importnat role in germ cel-Sertoli

cell adhesion, and MX deficient mice also showed some multinucleated germ cells in

the seminiferous epithelium of the testis (121). It is, therefore, hypothesized that the

abnormal fusion of spermatocytes in Stx2repro34 and glycolipid-deficient mutants was

due to defective formation of intercellular bridges, since mammalian germ cells are

! 85!

connected as syncytium through intercellular bridges that exist between the

spermatogenic cells at the same developing stage (33, 34), and stable formation of the

intercellular bridges is essential for spermatogenesis and fertility (37). Disruption of

the intercellular bridges prevents normal formation of syncytium, which arrests

spermatogenesis prior to pachytene, and resulted in male-restricted sterility (37).

Moreover, glycolipid-deficient mutants also showed multinucleated spermatocytes

and male-restricted sterility, which resembles the phenotpe of repro34 (50, 53). In

addition, genes essential for cytokinesis and formation of male ring canals are not

necessarily required for female ring canals or fertility (37, 44).

During spermatogenesis, germ cells experience dynamic morphological

changes; late pachytene spermatocytes are larger than those in the previous stages (3),

while the diameter of intercellular bridges of spermatocytes remains the same (122). It

was postulated that both sulfoglycolipids and STX2 regulate stage-specific membrane

dynamics, and the observations on aberrant intracellular localization of

sulfoglycolipids in Stx2repro34 mutant testes are consistent with this hypothesis.

With respect to these phenotypes of several mutants, STX2 and

sulfoglycolipids appear to be required for maintenance of the intercellular bridges of

the germ cells, and the loss of either STX2 or sulfoglycolipids induced defect of the

intercellular bridges. Present data might indicate accurate localization of

sulfoglycolipids could be a key factor for spermatogenesis and STX2 could be

involved in the mechanisms that convey and settle them into the bridges between

pachynema.

The distinct STX2 localization to the Golgi apparatus we observed also could

implicate STX2 as interacting with target sulfoglycolipids, whose biosynthesis in the

Golgi membranes also involves CGT catalysis (54). Sulfoglycolipids form the

! 86!

platform of lipid raft domains that contribute to membrane signaling and trafficking

(59) and function in capacitation of the mouse and pig spermatozoa (60, 123). Mice

lacking complex gangliosides, other candidate components of lipid rafts, due to

disruption of ganglioside GM2/GD2 synthase also exhibited multinucleation of the

spermatids (124, 125) as do STX2-deficient mice. Taken together, these studies lead

to the idea that sulfoglycolipids may contribute to raft domains within germ-cell

plasma membrane and intercellular bridges. Further studies should identify molecules

interacting with STX2 and mediating transport of sulfoglycolipids and analyze

membrane domain function in maintaining the constricted intercellular bridges that

are a key characteristic of spermatogenesis.

! 87!

! 88!

! 89!

! 90!

! 91!

Chapter 5

General discussion and conclusion

! 92!

Conclusion

Expression and Subcellular localization of t-SNARE STX2 in the Golgi

Apparatus and Intercellular Bridges

This study evidenced the expression and subcellular localization of secretory SNARE

protein STX2 in both the Golgi apparatus and the intercellular bridges of

spermatocytes at meiotic prophase and of spermatids. Furthermore, this study

revealed that co-localization of sulfoglycolipids and STX2 to the intercellular bridges.

t-SNARE protein STX2 is one of two SNARE protein categories, and along with

reciprocal v-SNAREs in transport vesicles, STX2 function specifically for membrane

fusion during exocytosis (108). Although several studies suggested the functions of

STX2 in diverse signaling transmission in the cell, none of the target molecules in the

events have not been previously identified. Together with the facts of co-localization

of TEX14 and sulfoglycolipids to the intercellular bridges of the germ cells (Fig. 4-2),

disrupted distribution of glycolipids in the seminiferous epithelium of Stx2repro34 mice

(Fig. 4-1) and the resultant multinucleation of the germ cells (Fig. 2-1),

sulfoglycolipids are target molecular candidates for t-SNARE STX2. This

transportation system possibly functions in the maintenance of the intercellular bridge

in spermatogenesis. In the wild-type testis, STX2 was specifically detected in the

Golgi apparatus of the spermatocyte and acrosomal vesicle/acrosome in the

spermatid. This study observed the localization of STX2 commonly in mouse and rat

testis using an antibody against the cytoplasmic domains of rat STX2 (Fig. 1-5) (114).

Further, two distinct localizations of STX2 to the intercellular bridge and to the Golgi

apparatus indicate that the gene plays different roles in the different reproductive

stages.

! 93!

Abberant Localization of Sulfoglycolipids in Stx2repro34 Mutant Germ Cells

Immunochemical observations of sulfoglycolipids present in the cytoplasm

and dotted in the plasma membrane of the wild-type spermatocyte (Fig. 4-1, 4-2)

showed good coinsistency with the previous reports (52, 54, 58). Moreover, this study

revealed that sulfoglycolipids specifically localized to the intercellular bridge of these

cells in the squash preparation. In mammals, two major sulfoglycolipids, sulfatide and

galactosylsulfatide, are produced in the testis through catalyzation of

alkylacylglycerol and ceramide with cerebroside sulfotransferase (CST) (48, 49) and

ceramide galactosyltransferase (CGT) (50) one after another, consequently

alkylacylglycerol and ceramide are converted to seminolipids and sulfatide,

respectively (Fig. 1-9) (48, 50, 51). Significance of sulfoglycolipids in

spermatogenesis has been demonstrated with male specific infertility in CST and

CGT deficient mice (48, 50, 53). However, the precise roles of these molecules in

spermatogenesis are still to be clarified. The Stx2repro34 mutants exhibited defective

spermatogenesis with appearance of syncytial multinucleated germ cells during

meiotic divisions (61). This phenotype is quite similar to the glycolipid-deficient

mutants described above (50, 53). Here this study showed that sulfoglycolipids were

localized to the plasma membrane of normal pachynema but accumulated in the

cytoplasm of the mutant spermatocytes at late pachytene and diplotene stages (Fig. 4-

1). These results indicate that glycolipids could be a key factor for maintenance of the

intercellular bridges.

Multinicleated Germ Cell in Stx2repro34 mutants

Meiotic processes appear to proceed normally until the multinucleation

occurs. Immunocytochemical staining of chromosome preparation confirmed normal

chromosome synapsis (Fig. 2-5) and chiasma formation in the mutant testis (Fig. 2-6).

! 94!

Histological examination showed several types of the nuclei, including those of late

pachytene, diplotene and metaphase spermatocytes in the mutant syncytial germ cells

(Fig. 2-1). The multinucleated cells with the nuclei at metaphase suggested ongoing

meiosis after the defect of the intercellular bridge. Although this study did neither

succeed to obtain siblings by ROSI using the nucleus of the aberrant cells as donors

nor confirm haploidity of the nuclei by chromosome preparations, vestiges of the

implantation of the transferred embryo were detected (Fig. 2-10). This at least

suggested that some of the nuclei in the syncytial cell had haploid genome and that

multiploidy of the donor nuclei caused the arrest of embryo development through the

spindle checkpoint.

Absence of STX2 in the Golgi Apparatus and Intercellular Bridges Affect

Integrity of Intercellular Bridges

In somatic cell division, failed cytokinesis triggers multinucleation.

Completion of cytokinesis owes a lot to the abscission of the midbody, a central part

of the bridge that consists of various molecules. Recruitment of TSG101 and ALIX to

the midbody to interact with CEP55, a component of the midbody, triggers abscission

then consequently completes the division of the daughter cells. Once the abscission of

the midbody is failed, the mitotic cell results in formation of binucleated cells, as

observed when the CEP55-ALIX/TSG101 association was disrupted (46, 126). Also,

binucleations were observed when STX2 and endobrevin, a ubiquitously expressed v-

SNARE protein, were functionally impaired (28). Almost nothing is understood about

the mechanism in which the enlargement of the cleavage furrow following the

impaired abscission lies. It is also unknown whether the common molecular system

lies in the multinucleation in the STX2 deficiency of the Stx2repro34 mutant. The

midbody in the intercellular bridge of the germ cells is kept by TEX14, a germ cell

! 95!

intercellular bridge specific protein, which interacts with CEP55 instead of TSG101

and ALIX to disturb the midbody abscission (47). The TEX14 deficient testis

exhibited lack of the intercellular bridge and frequent apoptosis in the germ cells (37),

whereas the STX2 deficient Stx2repro34 mutant testis showed TEX14 expression and

normal formation of the intercellular bridges. Thus, these phenotypic differences

indicate that direct molecular interaction between TEX14 and STX2 is less probable.

STX2 rather seems to be required for maintenance of the integrity of the intercellular

bridges. Furthermore, since the structure of the midbody appeared to be remained in

the Stx2repro34, STX2 is not likely to function to promote midbody abscissions in the

intercellular bridge of the germ cells unlike the case of the somatic cell cytokinesis.

This controversy might come from the roles of the target molecule that the SNAREs

mechanism transports.

STX2 Is Implicated in Intracellular Transportation of Sulfoglycolipids

This study evidenced localizations of STX2 in the intercellular bridge of

spermatogonia, spermatocytes and spermatids (Fig. 3-3), however influences of the

STX2 deficiency in spermatogenesis appeared in the late pachytene stage with an

aberrant intercellular localization of sulfoglycolipids. From these results and the

common abnormal morphology (multinucleation) in the sulfoglycolipids deficiency, it

seems certain that both STX2 and sulfoglycolipids function in cooperation to maintain

the intercellular bridge. Interestingly, the mice lacking complex gangliosides by the

disruption of ganglioside GM2/GD2 synthase showed multinucleation of the

spermatids (124, 125). Gangliosides and choresterols are thought to form the lipid raft

platform which contributes to membrane signaling and trafficking (59), therefore the

lipid raft possibly plays roles to maintain the intercellular bridge of the spermatids.

On the other hand, sulfoglycolipids are shown to form the platform of the raft domain

! 96!

which functions in capacitation of the mouse and pig spermatozoa (60, 127). These

studies would lead to the idea that sulfoglycolipids lubricate the raft domain in the

spermatocytes, which are prerequisite for the intercellular bridge to stay stable.

Therefore, it is favored to speculate that STX2 transports sulfoglycolipids to the raft

with the SNARE membrane fusion mechanism. Further studies on the membrane

domain function for maintaining the furrowing of the intercellular bridge and

identification of the molecules which mediate STX2 transportation of sulfoglycolipids

to the membrane of the bridge are necessary.

Possible Function of STX2 in Sertoli cell

Xu, et al. (128) hypothesized that residual sulfoglycolipids that are observed

from the cytoplasm of elongated spermatids are “recycled” within the Sertoli cell to

convert them into a precursor for de novo synthesis of sulfoglycolipids through

arylsulfatase A (ARSA). In the Sertoli cells of the ARSA deficient mouse,

sulfoglycolipids remained its native status (sulfolipid) and abnormally accumulated in

the cytoplasm of the Sertoli cells (128). In the testis of the Stx2repro34 mutant,

abnormal granules of sulfoglycolipids were frequently observed presumably in the

cytoplasm of Sertoli cells (Fig. 4-1), which is similar to the phenotype of ARSA

deficient-mutant (128). Previous study showed active cycles of STX2 between the

plasma membrane and endosomes in cultured cell (113), and the localization of STX2

in cytoplasm of the Sertoli cell in vitro (129). Taken together, this study postulated

that t-SNARE STX2 is also involved in dynamic transportation of “recycled”

sulfoglycolipids in the Sertoli cells with stage specific-manner (Fig. 5-1). Future

studies need to be conducted to prove this hypothesis.

! 97!

Functions of Glycan in Reproduction

In the reproductive system, biological features of glycans have recently

attracted increasing attention. Although no human reproductive disorder related to

abnormality of glycans was previously reported, several studies have shown the

importance of carbohydrate moiety of zona pellucida (ZP) (130, 131) and endometrial

epithelial tissues (132, 133) in fertilization and implantation, respectively. During

capacitation, seminolipid in sperm is required for sperm to attach to ZP during

capacitation (60) and carbohydrate moiety of sulfoglycolipids on sperm plays an

important role in species-specificity of fertilization (134, 135). This study proposes

the indispensable role of sulfoglycolipids in male gametogenesis, specifically in

maintenance of stable intercellular bridges of germ cells. The loss of stable formation

of intercellular bridges resulted in sterility due to spermatogenic arrest prior to

meiosis (37). However, in Stx2repro34 mutant males, in contrast, the progression of

spermatogenesis and the formation of intercellular bridges were apparently affected

after mid to late pachytene. These observations suggested that cellular development

requires stage-specific mechanisms that regulate the maintenance of the intercellular

bridges at certain stages of spermatogenesis, and the results of this study suggested

that sulfoglycolipids could be one of key factors.

Conclusion

As a concluding remark of this study, STX2 is important for two distinctive

functions; exocytotic transportation of sulfoglycolipids from the Golgi complex and

subcellular guide to the intercellular bridges. However, since molecular mechanisms

of STX2 and functional roles of sulfoglycolipids during spermatogenesis remain

largely unknown, further studies must be conducted.

! 98!

The goal of whole genome, ramdom ENU mutagenesis is to identify novel

genes and functions. Infertile mouse models can provide useful findings of genes

essential for reproduction. Understanding the biology of reproduction is essential for

clinical application not only for human infertility but also diverse contraceptive

approaches. Contribution of this study is that repro34 mutant mice emphasized

importnace of intercellular bridges and sulfoglycolipids in spermatogenesis, and this

study showed a possible correlation between these two unrelated factors, although

precise functions of each factor are largely unknown. Therefore, how STX2 is

involved in these needs to be answered. This study using an ENU mutant took

advantage of foreward genetic approach. Hence, repro34 was found out to be an

importnat model mouse for reproduction, and findings of this study contribute to

reveal future directions that can be exlpoited in understandng of spermatogenesis.

! 99!

! 100!

Acknowledgement

I am grateful to Dr. Tetsuo Kunieda for his support of this study throughout

my academic study in Okayama University. I thank Dr. Junko Noguchi at National

Institute of Agrobiological Sciences, Drs. Atsuo Ogura, Kimiko Inoue and Ms.

Narumi Ogonuki at RIEKN Bioresource center, Dr. Mary Ann Handel at The Jackson

Laboratory for their experimental support and useful comments on my research. I

thank Drs. Takehito Tsuji, Takuro Oikawa, Katsunori Satoh, Hiroaki Funahashi and

members of the Animal Genetics Laboratory in Okayama University for their

comments and discussion on this study. I also thank Mr. Neil Allara for his

grammatical correction of early manuscript.

I would like to express my gratitude to my family and relatives for their

understanding and support.

Lastly, I am grateful to my wife Chizuru and my son Yuta for wholehearted

support.

! 101!

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