Title Acrosomal Integrity, and the In Vitro Inducibility …repository.lib.gifu-u.ac.jp/bitstream/20.500.12099/77969/...Title Studies on the Relationship among Standard Parameters,

Title

Studies on the Relationship among Standard Parameters,Acrosomal Integrity, and the In Vitro Inducibility ofHyperactivation and the Acrosome Reaction in Frozen-thawedBull Spermatozoa( 本文(Fulltext) )

Author(s) Reza Rajabi-Toustani

Report No.(DoctoralDegree) 博士(獣医学) 甲第538号

Issue Date 2019-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/77969

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Studies on the Relationship among Standard Parameters, Acrosomal Integrity, and the In Vitro Inducibility of

Hyperactivation and the Acrosome Reaction in Frozen-thawed Bull Spermatozoa

(

)

2018

The United Graduate School of Veterinary Sciences, Gifu University

(Gifu University)

Reza Rajabi-Toustani

II

Studies on the Relationship among Standard Parameters, Acrosomal Integrity, and the In Vitro Inducibility of

Hyperactivation and the Acrosome Reaction in Frozen-thawed Bull Spermatozoa

(

)

Reza Rajabi-Toustani

i

Contents

Abbreviations ..................................................................................................................... iv

Chapter 1 ............................................................................................................................. 1

General introduction ........................................................................................................... 1

1. 1. Artificial insemination and bull subfertility ........................................................................... 2

1. 2. Acrosome condition .................................................................................................................. 3

1. 3. Capacitation and the acrosome reaction ................................................................................. 5

1. 4. Hyperactivation ......................................................................................................................... 6

1. 5. Objectives of this study ............................................................................................................. 8

Chapter 2 ............................................................................................................................. 9

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa 9

2. 1. Introduction ............................................................................................................... 10

2. 2. Materials and Methods.............................................................................................. 12

2. 2. 1. Reagents and media ............................................................................................................ 12

2. 2. 2. Spermatozoa ........................................................................................................................ 12

2. 2. 3. Washing and fixing of spermatozoa .................................................................................. 12

2. 2. 4. Permeabilization and staining of spermatozoa with FITC-PNA .................................... 13

2. 2. 4. 1. Permeabilization and staining of spermatozoa on smear (On-smear method) ..... 13

2. 2. 4. 2. Permeabilization and staining in suspension (In-suspension method) ................... 13

2. 2. 5. Examination of sperm acrosomes ...................................................................................... 14

2. 2. 6. Experimental design ........................................................................................................... 15

2. 2. 6. 1. Experiment 1: Effect of the staining method (On-smear and In-suspension), different antifades and 24-h storage on the acrosomal staining patterns (Method × Antifade × Storage) ..................................................................................................................................... 15

2. 2. 6. 2. Experiment 2: Effect of different levels of Triton X-100 on the staining patterns of acrosomes using the In-suspension method (Triton X-100) .................................................... 15

2. 2. 6. 3. Experiment 3: Effect of extended times (30 min, 6, 24 , 48 h) for fixation with 3 % PFA on the staining patterns of acrosomes by the In-suspension method (Prolonged fixation) ........................................................................................................................................ 15

2. 2. 6. 4. Experiment 4: Effect of shortened fixation times (10, 20 and 30 min) with different concentrations of PFA on the staining patterns of acrosomes using the In-suspension method (Shortened fixation) ................................................................................... 16

2. 2. 7. Statistical analyses .............................................................................................................. 16

2. 3. Results ........................................................................................................................ 17

2. 3. 1. Experiment 1. – Method × Antifade × Storage ................................................................. 17

2. 3. 2. Effects of the staining condition and the antifade agent (Table 2. 1) – Method × Antifade ............................................................................................................................................ 17

ii

2. 3. 3. Effects of the antifade agent and sample storage for 24 hr using the On-smear method (Table 2. 2) – Antifade × Storage (On-smear) .............................................................................. 17

2. 3. 4. Effects of the antifade agent and sample storage for 24 hr by the In-suspension method (Table 2. 3) – Antifade × Storage (In-suspension) ........................................................................ 18

2. 3. 5. Experiment 2 (Fig. 2. 2) – Triton X-100 ............................................................................ 18

2. 3. 6. Experiment 3 (Fig. 2. 3) – Prolonged fixation ................................................................... 19

2. 3. 7. Experiment 3 (Fig. 2. 4) – Shortened fixation ................................................................... 19

2. 4. Discussion .................................................................................................................. 21

Chapter 3 ........................................................................................................................... 32

Differential responsiveness of cryopreserved Japanese Black bull spermatozoa to calcium and calciumionophore A23187 and its relation to spermtraits and acrosomal integrity ............................................................................................................................. 32

3. 1. Introduction ............................................................................................................... 33


3. 2. 1. An animal use ethics statement .......................................................................................... 36

3. 2. 2. Chemicals and reagents ...................................................................................................... 36

3. 2. 3. Spermatozoa ........................................................................................................................ 36

3. 2. 4. Media .................................................................................................................................... 36

3. 2. 5. Standard semen analysis and of frozen-thawed spermatozoa ........................................ 37

3. 2. 6. Staining of acrosomes of spermatozoa in frozen-thawed semen with FITC-PNA ........ 38

3. 2. 7. Induction of the acrosome reaction with Ca2+ and Ca2+ ionophore A23187 .................. 38


3. 3. Results ........................................................................................................................ 41

3. 3. 1. Categorization of response to Ca2+/A23187 ...................................................................... 41

3. 3. 2. AR response group and conventional parameters ........................................................... 41

3. 3. 3. Acrosomal integrity and conventional parameters .......................................................... 41

3. 3. 4. Acrosomal integrity and AR response group ................................................................... 42

3. 3. 5. Age ........................................................................................................................................ 42

3. 4. Discussion .................................................................................................................. 43

3. 4. 1. Responsiveness of spermatozoa to Ca2+ and A23187 ....................................................... 43

3. 4. 2. Comparison of responsiveness to Ca2+ and A23187 with conventional parameters ..... 45

3. 4. 3. Comparison of acrosomal integrity (FITC-PNA staining) with the conventional parameters ....................................................................................................................................... 46

3. 4. 4. Comparison of AR responsiveness with acrosomal integrity by FITC-PNA ................ 46

3. 4. 5. Bull age and sperm traits.................................................................................................... 47

Chapter 4 ........................................................................................................................... 57

iii

Relationship of standard analyses with hyperactivation, AR and acrosome condition with FITC-PNA in frozen-thawed bull spermatozoa ....................................................... 57

4. 1. Introduction ............................................................................................................... 58


4. 2. 1. Chemicals and reagents ...................................................................................................... 60

4. 2. 2. Media .................................................................................................................................... 60

4. 2. 3. Spermatozoa ........................................................................................................................ 60

4. 2. 4. Standard semen analysis, acrosomal integrity condition with FITC-PNA, induction and assessment of AR with Ca2+ and Ca2+ ionophore A23187 and acrosome double staining procedure ......................................................................................................................................... 61

4. 2. 5. Incubation of spermatozoa with cBiMPS ......................................................................... 61


4. 3. Results ........................................................................................................................ 63

4. 4. Discussion .................................................................................................................. 65

Chapter 5 ........................................................................................................................... 72

General Conclusion ........................................................................................................... 72

Acknowledgements ............................................................................................................ 79

Chapter 6 ........................................................................................................................... 74

Summary ........................................................................................................................... 74

References.......................................................................................................................... 81

iv

Abbreviations

μg Microgram

μl Microlitre

AI Artificial insemination

ANOVA Analysis of variance

AR Acrosome reaction

BO Brackett and Oliphant

BSA Bovine serum albumin

Ca2+ Calcium ion

cAMP Cyclic adenosine monophosphate

cBiMPS sp-5,6-dichloro-1-β-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate

DABCO 1,4-diazabicyclo [2,2,2] octane

DMSO Dimethyl sulfoxide

FITC-PNA Fluorescein isothiocyanate-conjugated peanut agglutinin

FITC-PSA Fluorescein isothiocyanate-conjugated pisum sativum agglutinin

g Gravity

Gly Glycine

hr Hour

IU International unit

M Molar

mg Milligram

min Minute

ml Milliliter

mm Millimeter

mM Millimolar

v

PBS Phosphate buffered saline

PFA Paraformaldehyde

PKA Protein kinase A

PKG Protein kinase G

PVA Polyvinyl alcohol

RT Room temperature

SEM Standard error of the mean

SP Seminal plasma

ZP Zona pellucida

1

Chapter 1

General introduction

2

1. 1. Artificial insemination and bull subfertility

Artificial insemination (AI) is one of the most important tools in cattle breeding programs.

High bull fertility has an important effect on the reproductive efficiency in cattle and thus on

the genetic improvement. Extensive knowledge of factors affecting sperm production and

semen quality is therefore substantial (Fuerst-Waltl et al., 2006).

AI is indispensable for wide use of spermatozoa collected from high-performance sires, and

it has enabled us to produce a large number of high-performance offspring of these cattle.

Moreover, it has also allowed us to limit the number of sires and to reduce the high costs of

feeding and transportation of sires. Currently, bovine reproduction is almost always via AI

techniques using frozen-thawed spermatozoa in Japan (Kishida et al., 2015). However,

conception rates in AI programs for cattle are gradually decreasing in Japan and other countries

(Barbat et al., 2010; Dochi et al., 2010). AI subfertility in cattle is related to both female and

male reproductive dysfunctions. In AI centers, the reproductive performance of the bull is

conventionally predicted by routine standard semen analyses such as total motility, progressive

motility, viability, and normal morphology. However, spermatozoa with high performance of

standard semen parameters resulted in considerably lower conception rates.

The final steps of mammalian oogenesis and spermatogenesis prepare eggs and sperm,

respectively, for fertilization. During ovulation, fully grown oocytes from antral (Graafian)

follicles undergo “meiotic maturation,” a process that transforms fully grown oocytes into

unfertilized eggs prepared to interact with sperm. Similarly, following deposition into and

migration up the female reproductive tract, sperm undergo “capacitation,” a process that

enables sperm to bind to eggs and to undergo the acrosome reaction (AR) (Darszon et al., 1996;

Visconti et al., 1998).

Capacitation probably involves removal of inhibitory factors from sperm accompanied by

membrane protein and lipid rearrangements and/or modifications. Apparently, some alterations

3

are mediated, at least in part, by cAMP-dependent protein tyrosine phosphorylation, as well as

by changes in pH and Ca2+ concentrations. Meiotic maturation of oocytes and capacitation of

sperm propel gametes down a path that leads either to formation of a viable zygote or to

degeneration of the cells.

During fertilization, some physiological procedures (as capacitation, acrosomal reaction,

fusion of sperm and ovum) require active and intact membrane and it is impractical to have

fertilization by sperm with physically inactive membrane (Jeyendran et al., 1984). Due to the

enormous importance of sperm membrane in fertilization, remarkable consideration is given to

the semen analyses primarily determine sperm characteristics such as count, motility, forward

progression, morphology, and agglutination (Jeyendran et al., 1984). During the reproductive

processes, a number of biochemical events are associated with the male gametes and this is not

only essential for the maintenance of sperm motility but also for the initiation of the acrosomal

exocytosis and also other events which are related to fertilization. Therefore, assessment of

these functional aspects of spermatozoa could be a useful addition to the semen analysis as the

success rate of AI is dependent on the quality of semen.

1. 2. Acrosome condition

The mammalian sperm acrosome is a unique structure that is composed of the triple

membranes (plasma, outer acrosomal and inner acrosomal membranes). The AR of mammalian

spermatozoa involves fusion and progressive vesiculation between the outer acrosomal

membrane and the overlying plasma membrane. The subsequent release of acrosomal enzymes

is a prerequisite for spermatozoa to penetrate the zona pellucida (ZP) and to fuse with the egg

plasma membrane (Díaz-Pérez et al., 1988). Since the A is an essential event for fertilization,

information about the acrosomal status is important for semen quality control in AI programs

and in fertilization studies. Acrosomal status can be determined in living, motile sperm of only

4

a few mammalian species such as guinea pigs and hamsters but for other species, many light

microscopic methods have been developed, including colored stains for bright-field

microscopy, and probes for fluorescence microscopy (reviewed by Cross and Meizel, 1989).

The ideal assay for determining the percentage of acrosome reacted sperm should be accurate,

consistent, rapid, applicable to small numbers of cells, innocuous to sperm function, useable in

all biological fluids and environments in which sperm are found, and capable of distinguishing

false from normal AR (Cross and Meizel, 1989).

Methods of examining the sperm acrosome are of interest because its structural

characteristics are an important criterion for assessment of the fertility of ejaculates at fertility

clinics and for AI of domestic animals. The development of deep-freezing techniques for

spermatozoa has increased the importance of studying the acrosome because of its sensitivity

to cryogenic effects (Chacarov and Mollova, 1976).

There are two classes of fluorescent probes of acrosomal status: one is the probe that detects

intracellular acrosome-associated material (and consequently require that the cell be

permeabilized before labeling); this includes lectins and antibodies to intracellular acrosomal

antigens. The other class includes probes that can be used on living, nonpermeabilized cells

such as chlortetracycline and antibodies to externally exposed antigens (Cross and Meizel,

1989). The morphological and functional changes of mammalian spermatozoa are detectable

by various sperm staining methods. The acrosomal status of spermatozoa can be determined

by staining with Fluorescein isothiocyanate-conjugated pisum sativum agglutinin (FITC-PSA)

(Mendoza et al., 1992) and Fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-

PNA) (Cross and Watson, 1994).

The presence or absence of acrosome can be assessed by several methods, including electron

and conventional microscopy. Electron microscopy delivers detailed information on the

acrosomal status but requires an expensive and specialized equipment, as well as a skillful,

5

trained staff. Several techniques have also been described to visualize the acrosome by

conventional microscopy. In different laboratory species, the acrosome can be observed by

phase contrast microscopy. The triple stain technique (Talbot and Chacon, 1981) and

Coomassie Blue labelling (Larson and Miller, 1999) have also been widely used. Combinations

of classical stains are, however, extremely time consuming and are difficult to run in parallel

with other immunohistochemical methods. In contrast, fluorescence techniques allow

simultaneous acrosome detection and labelling of spermatozoan structures (Maier et al., 2003).

The use of fluorescent labelled plant lectins (Aviles et al., 1997) or antibodies raised against

acrosomal proteins (Yamashita et al., 2007) are procedures frequently described for the

evaluation of the acrosomal status. However, many variations in the experimental protocols are

encountered in the scientific literature, and species-related differences in the suitability of each

technique have been reported (Larson and Miller, 1999). In particular, assessment of the

acrosome of rodent sperm is considered as being especially difficult due to the acrosome

thinness and the sperm head morphology.

1. 3. Capacitation and the acrosome reaction

When first released from the male reproductive tract, mammalian sperm are nonfertilizing,

but acquire functional competence after a species-dependent time in the female reproductive

tract (Austin, 1951; Chang, 1951). The earliest event in life is the meeting of the sperm cell

with the egg. Enormous numbers of sperm cells are deposited in the female genital tract, but

only one sperm cell will successfully fertilize the egg. The fusion of sperm and egg leads to the

recombination of fathers and mothers genetical information resulting in a new individual.

Sperm-egg interaction and the subsequent fertilization are highly regulated processes (Flesch

and Gadella, 2000). Mammalian fertilization involves capacitation of the spermatozoa and AR,

the latter process being a major event for sperm penetration through the ZP and subsequent

6

fusion with the egg membrane (Guraya, 2000). The mechanisms involved in the spermatozoa

AR are still under debate. This irreversible physiological process relates to endogenous,

calcium-dependent, molecular events occurring at the level of the acrosomal cap membrane. In

addition, the ZP is considered to be the physiological initiator of the AR in mammalian sperm

(Guraya, 2000).

The AR may be interpreted as an exocytotic event involving membrane fusion between the

outer acrosome and the overlying plasma membranes, resulting in transmembrane pores and

release of acrosome content. In mammals, the AR allows sperm to adhere to the extracellular

oocyte coat, or ZP (Cherr et al., 1986; Florman and Storey, 1982; Myles et al., 1987). In natural

conditions, the reaction occurs in the proximity of the egg in response to specific egg associated

stimuli. Premature AR is associated with a decreased ability to fertilize eggs either in vitro or

in vivo (Fleming and Yanagimachi, 1982).

In vitro, several agonists, such as soluble ZP proteins, follicular fluid, steroid hormones or

calcium ionophores can initiate the AR. The in vitro induction of AR has been demonstrated

to exhibit a significant correlation with fertility in different species (Whitfield and Parkinson,

1992, 1995; Pampiglione et al., 1993). Besides evaluation of semen quality, assessment of the

acrosome status is also used in andrology research for development of male contraception (Suri,

2005) and to detect the gonadotoxic effects of food or drugs (Kumi-Diaka and Townsend,

2003). Hence, the precise assessment and quantification of the acrosomal loss is of high clinical

significance when evaluating sperm fertilizing capacity or damage (Ortloff et al., 2006).

1. 4. Hyperactivation

There is strong evidence that hyperactivation is required for penetrating the ZP. Hamster

sperm were incubated under capacitating conditions until they hyperactivated and then were

added to oocytes. After the sperm bound to the ZP, Ca2+ channel blockers were added to inhibit

7

hyperactivation. Although the sperm remained motile and underwent AR, most failed to

penetrate the ZP (Stauss et al., 1995). Years after those experiments, mice that were null

mutants for CatSper (cation channels of sperm) proteins were developed. The sperm from the

nulls were progressively motile and could undergo AR; however, they could not hyperactivate

and failed to penetrate the ZP of oocytes in vitro. If the ZP were removed, the sperm were able

to fertilize normally (Ren et al., 2001; Quill et al., 2003).

In most species, mature spermatozoa are held immotile within the cauda epididymis until

they are released, whereupon they quickly begin to swim. This process is known as activation

of motility. Activated sperm generate nearly symmetrical flagellar beats, which propel them in

nearly linear trajectories (Suarez and Dai, 1992; Mortimer and Swan, 1995a; Ho et al., 2002).

When sperm become hyperactivated, the amplitude of the flagellar bend increases, usually only

on one side of the flagellum. This produces a beat pattern that is highly asymmetrical, often

causing hyperactivated sperm to swim in circles on glass slides. Extremely asymmetrical bends

produce figure-of-eight movement patterns. During asymmetrical flagellar beating, steady

rolling of the head can result in helical tracks, which have been described for hyperactivated

human sperm (Morales et al., 1988). In some species, particularly in mice, hyperactivated

sperm trace erratic paths due to intermittent production of deep bends. One could argue that

this represents switching back and forth between the activated and hyperactivated state, but

only instantaneous assessment of signaling, which has not been done, can address the issue.

Conventional analyses of spermatozoa measure essentially the ability of spermatozoa to

reach fertilization site (progressive motility mainly). Because sometimes the conventional

parameters fail in detecting subfertile bulls, novel methods are required to successfully detect

subfertility. For that purpose, the fertilizing ability has been attempted, and among sperm

functions, acrosomal exocytosis (Murase et al., 2001b) and hyperactivation (Murase et al.,

2010) have been employed as novel test to detect subfertility.

8

1. 5. Objectives of this study

Bulls with high genetic performances such as milk production and meat quality are selected

and kept for collection of semen to prepare frozen semen. However, if the select bulls are

subfertile, infertile or sterile, high genetic quality of the animal will be useless and cause huge

economic loss, and therefore precise diagnosis and, if possible, therapy of reproductive

disorders are essential for cattle reproduction as well as for other species.

Regarding to Japanese Black bull subfertility, it was observed that some bulls used for AI

show subfertility even with outcomes from standard semen analyses being within the normal

range. Thus, standard semen analysis such as sperm concentration, motility, viability, and

morphology may be carried out routinely in the preparation of bull frozen semen for AI, but

the parameters do not necessarily correlate with conception rates resulting from AI.

The objectives of the study were as follows:

1. To re-evaluate the protocols for staining frozen-thawed Japanese Black bull spermatozoa

with FITC-PNA under gentler conditions. Determine the effective antifades during staining

(on-smear or in-suspension) and the storage of stained spermatozoa with antifade mountant for

24 hr; Determine the best concentration of Triton X-100 to permeabilize spermatozoa; and

determine the best time and concentration of (paraformaldehyde) PFA to fix spermatozoa.

2. To reveal time-course changes in the AR triggered by Ca2+ and A23187 (Ca2+/A23187)

in different bull sperm samples and the relationship among the conventional sperm traits and

the two new parameters of acrosomal integrity assessed by FITC-PNA staining and sperm

responsiveness to Ca2+/A23187 in order to obtain basic information for a development of more

accurate assessment methods to detect bull subfertility.

3. Comparison among hyperactivation induced by cBiMPS (a cAMP analog resistant to

hydrolysis by phosphodiesterase and a potent activator of PKA), standard semen analyses,

acrosome condition by staining with FITC-PNA and AR triggered by Ca2+/A23187.

9

Chapter 2

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed

Japanese Black bull spermatozoa

10

2. 1. Introduction

AI is an essential technique for producing offspring in the cattle industry, and bull semen

used for AI is routinely analyzed to predict fertility. However, there have been subfertile bulls

that exhibited normal standard semen parameters (Kishida et al., 2015; Murase et al., 2010,

2001b). Thus, standard semen analyses may fail to accurately estimates fertility, and novel

methods to predict bull fertility are in demand. Among these methods, acrosomal integrity is

one of the most powerful estimates of the fertilization ability of spermatozoa, because for

successful fertilization in AI, spermatozoa must have intact acrosomes when inseminated into

the female reproductive tract and must react in a timely manner when they reach the site of

fertilization.

The acrosome can be observed by using phase contrast microscopy (Almadaly et al., 2012),

the triple stain technique (Talbot and Chacon, 1981) or Coomassie blue labeling (Larson and

Miller, 1999). More recently, the fluorescein isothiocyanate-conjugated peanut agglutinin

(FITC-PNA), which specifically binds to the sugar Galactosyl ß-1,3 N-acetylgalactosamine in

acrosomal membranes (Mortimer et al., 1987), has been used as a probe to test acrosomal

integrity by Kishida et al. (2015), Almadaly et al. ( 2012) and Harayama et al. (2010). FITC-

conjugated pisum sativum agglutinin (FITC-PSA) has also been utilized for acrosmal staining

(Mendoza et al., 1992). In particular, the report by Harayama et al. (2010) showed that

categorization according to the staining pattern from FITC-PNA could estimate bull fertility;

thus, this method can be useful as a test adjacent to standard semen analyses. This study aimed

to improve staining conditions in the case where stained acrosomes are categorized according

to Harayama et al. (2010).

In our previous report, for staining with FITC-PNA, spermatozoa were fixed with 4% PFA

for 30 min, permeablized with 1% Triton X-100 for 5 min, stained with FITC-PNA and

mounted with 1,4-diazabicyclo [2,2,2] octane (DABCO) for examination according to our own

11

categorization (Almadaly et al., 2012). When frozen-thawed Japanese Black bull spermatozoa

were stained with FITC-PSA, the labelling was not confined to the acrosomal region with a

non-specific labeling of the head and flagellum while labelling with FITC-PNA was specific

to the acrosomal region (Almadaly et al., 2012). Therefore, FITC-PNA is more suitable for

assessing acrosomal status in bull spermatozoa. Moreover, the fluorescent dye FITC is useful

to label sperm acrosomes as conjugate to PNA but the fluorescence generally fades gradually

while stained samples are stored or are being examined under a microscope. Thus retardation

of fading by applying an antifade agent is necessary to assess acrosomal status accurately.

Because PNA binds molecules present inside the sperm plasma membrane, it is necessary

to permeabilize spermatozoa before they are stained for acrosomal integrity with PNA. For

frozen-thawed bull spermatozoa, treatment with 1% Triton X-100 for 5 min has been used to

stain acrosomes with FITC-PNA (Almadaly et al., 2012; Harayama et al., 2010). However,

because Triton X-100 is also used to extract proteins from cells (Gennuso et al., 2004; Hipfner

et al., 1994; Rajagopal et al., 2002), reducing the concentration might minimize the acrosomal

damage caused by excessive treatment.

The aim of this study was to re-evaluate the protocols for staining frozen-thawed Japanese

Black bull spermatozoa with FITC-PNA under gentler conditions to minimize artifactual

damage by using the categorization criteria developed by Harayama et al. (2010) instead of our

previous criteria (Almadaly et al., 2012). To that end, the following were evaluated (1) the

effects of antifades, the sperm environment during staining (on smear or in suspension) and the

storage of stained spermatozoa with antifade mountant for 24 hr; (2) the effects of different

concentrations of Triton X-100 (0, 0.1, 0.5, 1, and 2%) used to permeabilize spermatozoa; and

(3) the effect of time for fixation (10, 20, and 30 min) with different concentrations of PFA (1,

2, and 3%) on the staining patterns of the acrosome.

12

2. 2. Materials and Methods

2. 2. 1. Reagents and media

All chemicals used in this study were purchased from SigmaAldrich (Sigma-Aldrich,

Steinheim, Germany), unless otherwise stated. Saline medium and sucrose medium were used

for washing spermatozoa (Roldan et al., 1994; Roldan and Harrison, 1989).

Next, 12.5% (w/v) PFA in 0.5 M Tris, adjusted to pH 7.4 at 20 °C, was prepared according

to Almadaly et al. (2012) and kept frozen at -30 °C until use.

FITC-PNA stock (444 μg/ml of H2O) was prepared and diluted with Phosphate buffered saline

(PBS; 1060 μl) to 20 μg/ml according to Almadaly et al. (2012).

2. 2. 2. Spermatozoa

Frozen straws of Japanese Black bull semen were generously donated from Hida Beef Cattle

Research Department, Gifu Prefectural Livestock Research Institute, Japan. Cryopreserved

spermatozoa of six Japanese Black bulls were used in this study. This study was approved by

the Committee for Animal Research and Welfare of Gifu University, number: 17125. The use

of cryopreserved semen was approved by the Animal Ethics Committee of the institute.

2. 2. 3. Washing and fixing of spermatozoa

Spermatozoa were washed as described previously (Almadaly et al., 2012) with a slight

modification. Briefly frozen-thawed spermatozoa were resuspended in saline medium by

centrifugation and later washed through 4 ml instead of 7.5 ml sucrose medium.

Washed spermatozoa were diluted with PBS (−) to adjust the final sperm concentration to

100 × 106 spermatozoa/ml after fixation. Spermatozoa (230, 210, and 190 μl) were fixed in

vials for 10, 20, and 30 min at room temperature (RT) by adding (20, 40, and 60 μl) of 12.5%

13

PFA/0.5 M Tris buffer according to the experimental design for final concentrations of 1, 2, or

3%, respectively.

2. 2. 4. Permeabilization and staining of spermatozoa with FITC-PNA

2. 2. 4. 1. Permeabilization and staining of spermatozoa on smear (On-smear method)

The fixed spermatozoa were smeared, permeabilized, and stained as described previously

(smear method by Almadaly et al., 2012). The stained spermatozoa were then covered with 16

μl of 0.22 M DABCO (Sigma-Aldrich, Steinheim, Germany) dissolved in glycerol–PBS

mixture (9: 1), SlowFade® Diamond Antifade Mountant (Thermo Fisher, West Sacramento,

CA, U. S. A. ; Slowfade®) or ProLong® Diamond Antifade Mountant (Thermo Fisher, U. S.

A. ; Prolong®) and a cover slip (24 mm × 50 mm). The procedure was designated the 'On-

smear method'. The 24-hr stained samples were stored at RT in a dark box.

2. 2. 4. 2. Permeabilization and staining in suspension (In-suspension method)

Fixed spermatozoa (250 μl, 100 × 106 spermatozoa/ml) were centrifuged at 1000 × g for 1

min at RT, and the supernatant was removed. Two hundred microliters of bovine serum

albumin (BSA)/Glycine (Gly)-PBS was added, and the spermatozoa were centrifuged again.

This rinse was repeated for a total of 2 times. The pelleted spermatozoa were permeabilized by

adding 200 μl of 0 - 1% Triton X-100 according to experimental design for 5 min at RT and

centrifuged at 1000 × g for 1 min. The supernatant was discarded, 200 μl of BSA/Gly-PBS was

added, and the spermatozoa were centrifuged at 1000 × g for 1 min. This washing procedure

was repeated for a total of 2 times (Tabuchi et al., 2008). The sperm pellets, after being washed

2 times with BSA/Gly-PBS, were mixed in a vial with 200 μl of FITC-PNA (20 μg/ml) at RT

for 30 min. Spermatozoa were washed as the previous step, and then sperm pellets were

resuspended in 250 μl PBS. A portion (8 μl) was taken and mixed with an equal volume of

14

DABCO, SlowFade®, or ProLong®. Two microliters of the mixture was spotted onto a glass

slide, covered with a cover slip (18 mm × 18 mm), and gently pressed between sheets of tissue

paper to remove excess fluid. The procedure was designated the 'In-suspension method'. The

24-hr stained samples were stored at RT in a dark box.

2. 2. 5. Examination of sperm acrosomes

Stained spermatozoa were examined under a phase contrast microscope at a magnification of

1000X using fluorescence illumination (mirror unit U-MWB2: dichroic mirror DM500,

excitation filter, BP460–490 and emission filter BA520IF; Olympus, Tokyo, Japan). The

staining patterns of spermatozoa were first examined by the fluorescence optics, and then

localization was confirmed by phase contrast optics. Two hundred spermatozoa were examined

on each slide, and the staining patterns of the acrosomes were classified into seven categories

following Harayama et al. (2010): Pattern I, normal acrosome; Pattern II, slightly disordered

acrosome; Pattern III, severely disordered acrosome with highly bright fluorescence; Pattern

IV, acrosome with less fluorescence in the whole part; Pattern V, severely deformed acrosome

with less fluorescence in the anterior region; Pattern VI, acrosome with fluorescence only along

its outline; and Pattern VII, acrosome with almost no fluorescence. The percentage of

spermatozoa showing each pattern was obtained, and Patterns I and II were pooled and judged

as intact acrosomes. Higher percentage of intact acrosomes (percent Patterns I + II) and lower

percentage of the other patterns (percent Patterns III to VII) indicated better staining conditions

(Fig. 2. 1).

15

2. 2. 6. Experimental design

2. 2. 6. 1. Experiment 1: Effect of the staining method (On-smear and In-suspension),

different antifades and 24-h storage on the acrosomal staining patterns (Method ×

Antifade × Storage)

Spermatozoa that were washed and fixed with 3% PFA for 30 min were divided into 2

portions and stained by the On-smear method or the In-suspension method including

permeabilization with 1% Triton X-100 for 5 min. Spermatozoa were mounted using the three

different antifades, examined for the acrosomal integrity immediately (0 hr) and 24 hr later,

and the percentage of spermatozoa showing each pattern was determined.

2. 2. 6. 2. Experiment 2: Effect of different levels of Triton X-100 on the staining patterns

of acrosomes using the In-suspension method (Triton X-100)

Spermatozoa that were washed and fixed with 3% PFA for 30 min were stained using the

In-suspension method, including permeabilization with different concentrations of Triton X-

100 (0, 0.1, 0.5, 1, and 2%), in 2-ml vials for 5 min at RT. Stained spermatozoa were mixed

with ProLong® and examined for acrosomal integrity immediately, and the percentage of

spermatozoa showing each pattern was obtained.

2. 2. 6. 3. Experiment 3: Effect of extended times (30 min, 6, 24, and 48 h) for fixation

with 3 % PFA on the staining patterns of acrosomes by the In-suspension method

(Prolonged fixation)

Washed spermatozoa were fixed with 3% PFA in vials for 30 min, 6, 24, and 48 h, washed

in vials and then permeabilized in vials with 1% Triton X-100 for 5 min at RT. The

permeabilized spermatozoa were washed and stained with FITC-PNA for 30 min at RT in vials

then spermatozoa were washed and mixed with ProLong®. Then spermatozoa were

16

immediately examined for the acrosomal integrity, and the percentages of spermatozoa

showing each pattern was obtained.

2. 2. 6. 4. Experiment 4: Effect of shortened fixation times (10, 20, and 30 min) with

different concentrations of PFA on the staining patterns of acrosomes using the In-

suspension method (Shortened fixation)

Spermatozoa that were washed and fixed with 1, 2, and 3% PFA for 10, 20, and 30 min were

permeabilized with 1% Triton X-100 for 5 min at RT and were later stained using the In-

suspension method. Stained spermatozoa were mounted with ProLong® and were examined

for acrosomal integrity immediately (0 hr of storage), and the percentage of spermatozoa

showing each pattern was determined.

2. 2. 7. Statistical analyses

Results are presented as the mean ± SEM. The data obtained in Experiments 1 and 3 were

subjected to repeated measures using a two-way analysis of variance (ANOVA). When the

main effect was significant, the averages of one factor across the other were further compared

using Tukey’s multiple comparison test. When a significant interaction was found, comparison

was made among the different treatments by Bonferroni’s multiple comparison test. A one-

way ANOVA was used for Experiments 2 and 3, and when the results of the one-way ANOVA

were significant, Tukey’s multiple comparison test was performed to compare different

treatments. Differences with P < 0.05 were considered to be statistically significant. All

analyses were performed using a statistical software program (GraphPad Prism Version 6.0;

GraphPad Software, San Diego, CA, U. S. A.).

17

2. 3. Results

2. 3. 1. Experiment 1. – Method × Antifade × Storage

Because of complexity, the results of Experiment 1 were divided into subsets (Tables 2. 1–

2. 3) to analyze by 2-factor ANOVA. Therefore, certain parts of Tables 2. 1–2. 3 represent the

same values.

2. 3. 2. Effects of the staining condition and the antifade agent (Table 2. 1) – Method ×

Antifade

Two-factor ANOVA of the staining method × the antifade agent revealed that there was no

significant interaction between the two on any of the staining patterns, but the antifade agent

had a significant effect on the percent levels of Pattern III, and the staining condition impacted

the percent levels of Pattern V. The average percentage of intact acrosomes across the antifade

agents was higher for the In-suspension method (41.8 ± 0.5%) than for the On-smear method

(36.5 ± 0.6%), although the difference was not statistically significant (P = 0.33). Percent

Pattern III averaged across the SlowFade® condition was significantly lower than for both

DABCO and Prolong® (P < 0.05, respectively, Tukey’s test), and percent Pattern V averaged

across the antifade agents was significantly lower for the In-suspension method (10.8 ± 0.8%)

than for the On-smear method (13.1 ± 0.6%).

2. 3. 3. Effects of the antifade agent and sample storage for 24 hr using the On-smear

method (Table 2. 2) – Antifade × Storage (On-smear)

Two-factor ANOVA evaluating the antifade agent × storage for 24 hr with the On-smear

method revealed that there was a significant interaction between these 2 factors (P < 0.05) on

the percentage of intact acrosomes and on percent Pattern IV with a significant decrease in the

percentage of intact acrosomes with DABCO after storage for 24 hr (P < 0.05, Bonferroni’s

18

test). The percentage of intact acrosomes after storage for 24 hr was significantly lower for

DABCO than for SlowFade® and Prolong® (P < 0.05, Bonferroni’s test, respectively). Exactly

the same tendency was seen for percent Pattern IV, where a significant interaction was found

between the antifade agent and storage, with a significant increase being observed with

DABCO after storage (P < 0.05, Bonferroni’s test). Percent Pattern IV after storage for 24 hr

was significantly higher for DABCO than for SlowFade® and Prolong® (P < 0.05,

respectively; Bonferroni’s test).

2. 3. 4. Effects of the antifade agent and sample storage for 24 hr by the In-

suspension method (Table 2. 3) – Antifade × Storage (In-suspension)

A two-factor ANOVA evaluating the antifade agent × storage for 24 hr using the In-

suspension method revealed that there was no significant interaction and that there was a

significant main effect of the storage on the percentage of intact acrosomes and of the antifade

on percent Pattern IV. The percentage of intact acrosomes at 0 hr of storage (42.3 ± 0.6%)

averaged across the antifades significantly decreased after 24 hr (37.8 ± 1.6%) in the In-

suspension method, while the percent Pattern IV averaged across storage time was the highest

for Slowfade® (5.6 ± 1.8%), intermediate for DABCO (3.2 ± 1.1%) and the lowest for

Prolong® (2.4 ± 0.7%; P < 0.05, respectively, Tukey’s test).

Notably, when using Prolong®, there was a clearer background during the assessment of

the acrosome conditions (data not shown).

2. 3. 5. Experiment 2 (Fig. 2. 2) – Triton X-100

When fixed spermatozoa were stained with FITC-PNA without permeabilization (0% Triton

X-100 for 5 min), the percentage of intact acrosomes was virtually 0% and significantly lower

than for 0.1–2% Triton X-100. Percent Pattern III was significantly higher at 0.1–2% than at

19

0% (P < 0.05), and percent Pattern VII was significantly lower at 0.1–2% than at 0% (P < 0.05).

There was no significant difference in the percentage of intact acrosomes, percent Pattern III

and percent Pattern VII at 0.1–2% Triton X-100. Although not significant, the percentage of

intact acrosomes was the lowest (33.4 ± 5.5%) at 2% Triton X-100 and the highest (43.7 ±

6.5%) at 0.1% Triton X-100 (Fig. 2. 2).

2. 3. 6. Experiment 3 (Fig. 2. 3) – Prolonged fixation

When spermatozoa were fixed with 3% PFA for different times (30 min–48 hr) and

permeabilized with 1% Triton X-100 for 5 min, per cent intact acrosome was decreased with

time for fixation, and fixation for 24 and 48 hr resulted in a significant decrease in per cent

intact acrosome and a significant increase in per cent Pattern IV (P < 0.05; Fig. 2. 3).

2. 3. 7. Experiment 4 (Fig. 2. 4) – Shortened fixation

The two-way ANOVA evaluating PFA concentration (1, 2, and 3%) × time (10, 20, and 30

min) for fixation revealed that there was no significant interaction for any of the patterns but

that there was a significant main effect of the concentration of PFA on the percentage of intact

acrosomes and on percent Pattern V (P < 0.05). At 10 min of fixation, 3% PFA had a

significantly higher percentage of intact acrosomes and lower percent Pattern V than 1% PFA,

with 2% PFA being intermediate (P < 0.05). At 20 min of fixation, 1% PFA had a significantly

lower percentage of intact acrosomes and a significantly higher percent Pattern V than 2 and

3% of PFA (P < 0.05). At 30 min of fixation, 1% PFA had a higher percent Pattern V (P <

0.05) than the other concentrations, while the percentage of intact acrosomes showed no

significant differences among the three concentrations.

20

Actual percentages of intact acrosomes are shown in the Fig. 2. 3, although not statistically

significant the highest percentage of intact acrosomes (50.1%) was seen with 2% PFA for 30

min.

21

2. 4. Discussion

The present study evaluated the validity of staining frozen-thawed bull spermatozoa with

FITC-PNA in suspension or in a smeared state when the staining patterns of the acrosomes

were categorized according to Harayama et al. (2010), rather than by our previous method of

categorization (Almadaly et al., 2012). I simultaneously determined the possibility of staining

acrosomes under gentler conditions to minimize artifactual damage.

The results of Experiment 1 (Table 2. 1) showed that the average percentage of intact

acrosomes across the antifade agents was not significantly different between the In-suspension

method and the On-smear method but that % Pattern V was significantly lower with In-

suspension method than with On-smear method. These results suggest that if stained samples

are examined immediately without storage, as is usually performed, the In-suspension method

may stain spermatozoa under gentler conditions, indicating that this method may be better for

determining the acrosomal status of the spermatozoa. This finding was contradictory to the

previous findings that on-smear staining may be better than that in a suspension (Almadaly et

al., 2012). The reason for the discrepancy may be due to differences in the categorization

criteria; in the previous report, slightly damaged acrosomes were categorized into ‘E1’, which

was recognized as damaged, whereas in this study, slightly damaged acrosomes were

categorized into Pattern II, which is recognized as intact according to Harayama et al. (2010).

Earlier studies suggested that when mouse spermatozoa were spotted onto a slide followed

by drying, fixation and staining with FITC-PNA, acrosomes were better stained compared to

those that were smeared and dried (Lybaert et al., 2009). Similarly, examination by differential

interference phase contrast microscopy of a fixed, wet-mounted sample of bull spermatozoa

revealed more major abnormalities than a dry-mounted sample (Freneau et al., 2010). When

frozen-thawed bull spermatozoa were smeared without fixation and later stained with Naphthol

Yellow S and Erythrosin B, the percentage of spermatozoa showing intact acrosomes were

22

slightly lower than when live, wet spermatozoa or spermatozoa fixed with glutaraldehyde were

examined under differential interference contrast microscopy, but this difference was not

statistically significant (Steinholt et al., 1991). Similar to these studies, the results of the present

study showed that staining spermatozoa in suspension was more suitable than on a smeared

preparation.

In addition, the observation that percent Pattern III (severely disordered acrosomes with

highly bright fluorescence) for SlowFade® was significantly lower than the other 2 agents at 0

hr of sample storage (Table 2. 1) suggested that if the stained samples are examined

immediately without storage, SlowFade® may be the best of the three antifade agents examined.

The increase in percent pattern IV after storage of stained samples either on the smear (Table

2. 2) or in sperm suspension (Table 2. 3) indicated that storage of the stained samples with an

antifade agent, regardless of the staining methods, may simply attenuate fluorescence without

any damage to sperm acrosomes, and thus storage of stained samples is not recommended.

A significant interaction between the antifade agent and the sample storage for 24 hr on the

percentage of intact acrosomes and the percent Pattern IV by the On-smear method (Table 2.

2) suggests that depending on the antifade used, storage of the stained sample on the slide with

an antifade may attenuate fluorescence more severely. A significantly lower percentage of

intact acrosomes and higher percent Pattern IV with DABCO than with the other 2 agents

(Table 2. 2) suggested that DABCO should be avoided if the stained sample were to be stored

on the slide with an antifade for up to 24 hr.

By contrast, the results of the In-suspension method (Table 2. 3) suggest that when

spermatozoa are stained by the In-suspension method, storage of the stained sample in

suspension with an antifade agent may not be recommended, as characterized by the decrease

in the percentage of intact acrosomes. Nevertheless, the lower percent Pattern IV with Prolong®

23

suggests that this antifade agent is the best to use if the stained sample is to be stored in

suspension with an antifade agent for up to 24 hr.

SlowFade® is essentially designed to keep a histological section or a cell preparation stained

with a fluorescent probe for immediate examination or for examination after storage for a

relatively short period of time (2–3 weeks, manufacturer’s instructions). It was shown that

SlowFade® has a quenching effect on the initial fluorescence but retards fading fluorescence

well (Longin et al., 1993). For longer storage of fluorescent samples, Prolong® is suitable to

keep fluorescent preparation for up to several months (manufacturer’s instructions), but this

antifade is essentially designed for sealing a histological section on the slide because this agent

solidifies after being applied to the preparation (manufacturer’s instructions). In agreement

with the manufacturer’s instructions, SlowFade® was the best when stained spermatozoa were

examined immediately after staining (Table 2. 1), while Prolong® was the best when the

samples stained by the In-suspension method were kept for 24 hr (Table 2. 3).

The results of Experiment 2 (Fig. 2. 2) indicate that at least 0.1% Triton X-100 may damage

spermatozoa to a some extent. A significantly higher percent Pattern VII (acrosomes with

almost no fluorescence) at 0% Triton X-100 compared to the other concentrations indicates

that without permeabilization, FITC-PNA does not enter the acrosome and cannot bind to the

molecules present inside but that it can enter and stain damaged acrosomes. This situation is

similar to the reports where un-permeabilized spermatozoa were stained with FITC-PNA and

damaged acrosomes were detected by the fluorescence-positive spermatozoa (Gürler et al.,

2015; Prathalingam et al., 2006). Conversely, the percentage of all patterns between 0.1 and

2% Triton X-100 suggests that the concentration of Triton X-100 may be suitable within that

range for spermatozoa permeabilization. Treatment with 1% Triton X-100 for 5 min has been

used to permeabilize frozen-thawed bull spermatozoa (Almadaly et al., 2012; Harayama et al.,

2010) and in human spermatozoa, treatment with 1% for 10 min has been successfully

24

employed (Mahony et al., 1993). The present study clearly shows that the concentration can be

reduced to 0.1% such that damage to bull spermatozoa can be minimized.

Because the effect of time was not significant in Experiment 3 (Fig. 2. 3), it was considered

that the effects of PFA concentration should be compared within the same time frames (10, 20,

or 30 min) for fixation. The results showed that at 10 min of fixation, 3% PFA was the best,

2% PFA was intermediate and 1% was worse than other treatments in terms of the percentage

of intact acrosomes and percent Pattern V (Fig. 2. 3), suggesting that fixation time can be

shortened to 10 min from 30 min if 3% PFA is used. At 20 min of fixation, 2% and 3% PFA

had similar percentages of intact acrosomes and percent Pattern IV, which were both

significantly different from 1%, suggesting that the fixation time can be shortened to 20 min if

2 or 3% PFA is used. The observation that at 30 min of fixation there was no significant

difference among 1, 2, and 3% PFA in the percentage of intact acrosomes with a significant

increase in percent Pattern V at 1% PFA suggests that if spermatozoa are fixed for 30 min, the

concentration of PFA to be used can be reduced to 2% from 3%. Taken together, options of the

improved fixation condition were 10 min with 3% PFA, 20 min with 2 or 3% PFA, and 30 min

with 2% PFA. Because the highest percentage of intact acrosomes was achieved by the

condition of 30 min with 2% PFA (Fig. 2. 3), this condition is proposed as an improved one.

From the results obtained, the following new protocol is proposed: spermatozoa are fixed

with 2% PFA for 30 min, permeabilized with 0.1% Triton for 5 min, stained with FITC-PNA,

mounted with an antifade agent SlowFade® and immediately examined according to Harayama

et al. (2010) categorization. In all steps, spermatozoa are handled in suspension. Because it has

been shown that the staining patterns by FITC-PNA of frozen-thawed bull spermatozoa used

in this study are related to bull fertility (Harayama et al., 2010), it is expected that the staining

protocol modified in this study will be a useful tool to examine bull spermatozoa routinely for

this purpose.

25

Table 2. 1. Effect of antifade reagents and staining method on the staining patterns of frozen-thawed Japanese Black bull sperm acrosomes stained with FITC-PNA at 0 hr of sample storage (n = 6).

Pattern Method % Patterns with an antifade reagent Mean ± SEM DABCO SlowFade® ProLong® I+II

(Intact acrosome)

On-smear 36.2 ± 5.8 37.7 ± 5.5 35.7 ± 4.1 36.5 ± 0.6 In-suspension 41.1 ± 7.3 41.4 ± 8.3 42.8 ± 7.9 41.8 ± 0.5 Mean ± SEM 38.7 ± 2.5 39.5 ± 1.9 39.2 ± 3.5

IIIA On-smear 40.3 ± 3.5 39.2 ± 4.5 42.6 ± 3.9 40.7 ± 1.0

In-suspension 37.8 ± 2.2 33.7 ± 3.0 38.0 ± 2.2 36.5 ± 1.4 Mean ± SEM 39.0 ± 1.2b 36.4 ± 2.7a 40.3 ± 2.3b

IV On-smear 0.6 ± 0.6 0.4 ± 0.3 0.2 ± 0.2 0.4 ± 0.1

In-suspension 2.1 ± 1.3 3.9 ± 2.5 1.7 ± 1.4 2.6 ± 0.7 Mean ± SEM 1.4 ± 0.8 2.1 ± 1.7 0.9 ± 0.8

VB On-smear 14.2 ± 5.0 13.2 ± 5.0 12.1 ± 4.5 13.1 ± 0.6

In-suspension 11.1 ± 5.4 12.1 ± 5.8 9.3 ± 4.7 10.8 ± 0.8* Mean ± SEM 12.6 ± 1.6 12.6 ± 0.6 10.7 ± 1.4

VI On-smear 8.8 ± 1.3 9.6 ± 2.0 9.4 ± 1.9 9.3 ± 0.3


VII On-smear 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0


A: A significant main effect of antifade (P < 0.05, Two-way ANOVA).

B: A significant main effect of method (P < 0.05, Two-way ANOVA).

No significant interaction was found in any of the patterns (P > 0.05, Two-way ANOVA).

a, b: Different superscripts denote a significant difference among the antifades (P < 0.05,

Tukey test).

*: Significantly different from smear (P < 0.05, Two-way ANOVA).

26

Table 2. 2. Effect of antifade reagents and sample storage time on the staining patterns of frozen-thawed Japanese Black bull sperm acrosomes stained with FITC-PNA by On-smear method (n = 6).

Pattern Storage time % Patterns with an antifade reagent Mean ± SEM DABCO SlowFade® ProLong® I+IIA

(Intact acrosome)

0 hr 36.2 ± 5.8 37.7 ± 5.5 35.7 ± 4.1 36.5 ± 0.6 24 hr 24.0 ± 5.6*, a 33.5 ± 4.7b b5.4± 35.2 30.9 ± 3.5

Mean ± SEM 30.1 ± 6.1 35.6 ± 2.1 35.5 ± 0.3

III 0 hr 40.3 ± 3.5 39.2 ± 4.5 42.6 ± 3.9 40.7 ± 1.0

24 hr 42.7 ± 5.6 41.5 ± 4.5 43.4 ± 3.0 42.5 ± 0.6 Mean ± SEM 41.5 ± 1.2 40.3 ± 1.2 43.0 ± 0.4

IVB 0 hr 0.6 ± 0.6 0.4 ± 0.3 0.2 ± 0.2 0.4 ± 0.1

24 hr 10.0 ± 3.3*, a 3.2 ±1.5b 0.1 ± 0.1b 4.5 ± 2.9 Mean ± SEM 5.3±4.7 1.8 ± 1.3 0.2 ± 0.0

V 0 hr 14.2 ± 5.0 13.2 ± 5.0 12.1 ± 4.5 13.1 ± 0.6

24 hr 13.3 ± 5.2 13.0 ± 5.0 12.8 ± 5.2 13.0 ± 0.1 Mean ± SEM 13.7 ± 0.5 13.1 ± 0.1 12.4 ± 0.3

VI 0 hr 8.8 ± 1.3 9.6 ± 2.0 9.4 ± 1.9 9.3 ± 0.3

24 hr 10.0 ± 1.6 8.8 ± 2.2 8.5 ± 1.2 9.1 ± 0.5 Mean ± SEM 9.4 ± 0.6 9.2 ± 0.4 8.9 ± 0.4

VII 0 hr 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

24 hr 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Mean ± SEM 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

A,B: Significant interaction between the antifade and the sample storage (P < 0.05, Two-way ANOVA). *Significantly different from 0 hours (P < 0.05, Bonferroni’s test).

a, b: Different superscripts represents significant differences within the same staining pattern (P

< 0.05, Bonferroni’s test).

27

Table 2. 3. Effect of antifade reagents and sample storage time on the staining patterns of frozen-thawed Japanese Black bull sperm acrosomes stained with FITC-PNA by In-suspension method (n = 6).

Pattern Storage time % Patterns with an antifade reagent Mean ± SEM DABCO SlowFade® ProLong® I+IIA

(Intact acrosome)

0 hr 41.1 ± 7.3 41.4 ± 8.3 42.8 ± 7.9 42.3 ± 0.6 24 hr 37.6 ± 8.6 35.0 ± 7.9 40.7 ± 7.4 37.8 ± 1.6*

Mean ± SEM 39.4 ± 1.8 39.0 ± 3.9 41.7 ± 1.0

III 0 hr 37.8 ± 2.2 33.7 ± 3.0 38.0 ± 2.2 36.5 ± 1.4

24 hr 36.7 ± 5.1 37.6 ± 3.0 38.6 ± 2.9 37.6 ± 0.5 Mean ± SEM 37.3 ± 0.6 35.7 ± 1.9 38.3 ± 0.3

IVB 0 hr 2.1 ± 1.3 3.9 ± 2.5 1.7 ± 1.4 2.6 ± 0.7

24 hr 4.3 ± 2.3 7.4 ± 3.1 3.1 ± 1.7 4.9 ± 1.3 Mean ± SEM 3.2 ± 1.1ab 5.6 ± 1.8a 2.4 ± 0.7b

V 0 hr 11.1 ± 5.4 12.1 ± 5.8 9.3 ± 4.7 10.8 ± 0.8

24 hr 12.3 ± 5.7 11.3 ± 5.8 10.6 ± 5.9 11.4 ± 0.5 Mean ± SEM 11.7 ± 0.6 11.7 ± 0.4 9.9 ± 0.6

VI 0 hr 7.9 ± 1.7 9.0 ± 2.0 8.3 ± 1.9 8.4 ± 0.3

24 hr 9.2 ± 2.8 8.5 ± 1.1 6.9 ± 1.4 8.2 ± 0.7 Mean ± SEM 8.5 ± 0.6 8.8 ± 0.3 7.6±0.7

VII 0 hr 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

24 hr 0.0 ± 0.0 0.3 ± 0.3 0.2 ± 0.2 0.2 ± 0.1 Mean ± SEM 0.0 ± 0.0 0.1 ± 0.1 0.1 ± 0.1

A: A significant main effect of storage (P < 0.05, Two-way ANOVA).

B: A significant main effect of antifade (P < 0.05, Two-way ANOVA).

*: Significantly different from 0 hours (P < 0.05, Main effect of time by Two-way ANOVA).

a, b: Different superscripts differ significantly (P < 0.05, Tukey test).

No significant interaction was found in any of the patterns (P > 0.05, Two-way ANOVA).

28

I II III IV

V VI VII

Fig. 2. 1. Staining patterns of the acrosome with FITC-PNA.

Pattern I: normal acrosome, Pattern II: slightly disordered acrosome (Patterns I and II are

considered as intact), Pattern III: severely disordered acrosome with highly bright fluorescence,

Pattern IV: acrosome with less fluorescence in the whole part, Pattern V: severely deformed

acrosome with less fluorescence in the anterior region, Pattern VI: acrosome with fluorescence

only along its outline, and Pattern VII: acrosome with almost no fluorescence.

29

Fig. 2. 2. Effect of permeabilization using different concentrations of Triton X-100 for 5 min

on the staining patterns of frozen-thawed Japanese Black bull sperm acrosomes stained with

FITC-PNA by the In-suspension method. Spermatozoa were fixed prior to staining with 3%

PFA for 30 min (n=6).

a, b: Different superscripts denote a significant difference (P < 0.05, One-way ANOVA; P <

0.05, Tukey’s test).

30

Fig. 2. 3. Effect of different times for fixation with 3% PFA on the staining patterns of Japanese

Black Bull sperm acrosomes stained with FITC-PNA by the In-suspension method. Fixed

spermatozoa were permeablized with 1% Triton X-100 for 5 min.

a, b: Different superscripts denote a significant difference (One-way ANOVA, P < 0.05;

Tukey’s test, P < 0.05).

31

Fig. 2. 4. Effect of different times for fixation with different concentrations of PFA on the

staining patterns of frozen-thawed Japanese Black bull sperm acrosomes stained with FITC-

PNA by the In-suspension method. Fixed spermatozoa were permeablized prior to staining

with 1% Triton X-100 for 5 min (n=3).

No significant interaction was found in any of the patterns. A significant main effect of the

concentration of PFA was found for % intact acrosome (Patterns I + II) and % Pattern V (P <

0.05, One-way ANOVA).

Different superscripts denote a significant difference among different concentrations of PFA

within the same time frame of 10 min (a, b), 20 min (A, B), and 30 min (x, y) (P < 0.05, Tukey’s

test, respectively).

32

Chapter 3 Differential responsiveness of cryopreserved Japanese Black bull spermatozoa to calcium and calciumionophore A23187

and its relation to sperm traits and acrosomal integrity

33

3. 1. Introduction

AI is an essential technique to produce offspring or embryos for transfer in cattle industry.

Sires are used to prepare cryopreserved semen for AI, and for this purpose, conventional semen

analyses are routinely performed. However, recently some bulls have shown subfertility even

with outcomes from standard semen analyses (semen volume, sperm concentration, motility,

viability and normal morphology) being within normal range (Kishida et al., 2015; Murase et

al., 2001a, 2001b). Thus standard semen analyses sometime fails in detecting subfertile bulls,

and therefore a novel, accurate method is demanded for estimating bull fertility (Reviewed by

Harayama et al., 2017).

At fertilization, exocytosis of the acrosome, the so-called AR, releases enzymes that aid

spermatozoa to penetrate oocyte vestments, cumulus oophorus and the ZP, and is an essential

process for fertilization (Visconti et al., 1998; Stival et al., 2016). Spermatozoa must possess

intact acrosomes for successful fertilization at the time when they are deposited or inseminated

into the female reproductive tract before reaching the fertilization site and also must undergo

exocytosis at the right time and the right site. Impairment of this function may cause

fertilization failure potentially leading to infertility or subfertility, and thus assessment of

acrosomal integrity of spermatozoa in semen and the ability to undergo the AR are a powerful

tool to estimate bull subfertility/infertility.

The AR is induced by physiological stilumi such as progesterone and ZP (Shi and Roldan,

1995) but can also be induced in vitro by calcium (Ca2+) and calcium ionophore A23187. In

human spermatozoa, the AR induced by A23187 was reduced in subfertile men (Cummins et

al., 1991) and was associated with ZP-free hamster egg penetration (Francavilla et al., 1995)

and in-vitro fertilization rate (Tello-Mora et al., 2018; Xu et al., 2018). In domesticated animals,

it was shown that rate of increase in induced AR by A23187 (Whitfield and Parkinson, 1995)

or proportion of acrosome-reacted spermatozoa after incubation with A23187 for 60 min

34

(Januskauskas et al., 2000) was correlated with conception rates. These reports in domesticated

animals used only fixed time of stimulation to measure the ability of spermatozoa to undergo

the AR, and the measured ability was not examined for a possibility to detect subfertility or

infertility but was used to correlate conception rates basically of fertile bulls. Because there are

subfertile bulls that are not detected by standard semen analyses as mentioned above, new

method was required to detect those subfertile bulls. Although we have shown previously time-

course changes in the AR induced by A23187 in subfertile and fertile bulls, the information

was from only the limited number of bulls (2 subfertile and 2 fertile bulls) (Murase et al.,

2001b), and therefore, general picture of responsiveness to A23187 stimulation in more sperm

samples was required as basic knowledge to utilize AR responsiveness for detecting subfertile

bulls.

On the other hand, FITC-PNA, which specifically binds to sugar galactosyl β-1,3 N-

acetylgalactosamine in acrosomal membranes (Mortimer et al., 1987), has been used as a probe

to assess acrosomal integrity (Almadaly et al., 2012; Harayama et al., 2010; Kishida et al.,

2015; Lybaert et al., 2009; Muiño et al., 2007; Nagy et al., 2003; Silva and Gadella, 2006). In

particular, the report by Harayama et al. (2010) showed that categorization according to the

staining pattern with FITC-PNA was related to bull subfertility.

A recent study has suggested that the genes involved in the fertilizing ability including

sperm motility, the AR and sperm biology are related to sire conception rate in Jersey cattle

(Rezende et al., 2018). Thus it is important to evaluate acrosomal status and the ability to

undergo the AR in bull spermatozoa for assessing bull fertility. In bulls, in order to develop

new methods to detect bull subfertility, it would be of importance to reveal the relationship

between these new sperm functional tests of acrosomal integrity and the ability of spermatozoa

to undergo the AR and between the two new parameters and the conventional methods

including sperm motility, viability and morphology.

35

The present study aimed to reveal 1) the time-course changes in the AR triggered by Ca2+

and A23187 (Ca2+/A23187) in different bull sperm samples and 2) the relationship among the

conventional sperm traits (motility, viability, morphology, and the induced AR at a fixed time)

and the two new parameters of acrosomal integrity assessed by FITC-PNA staining and sperm

responsiveness to Ca2+/A23187 in order to obtain basic information for a development of more

accurate assessment methods to detect bull subfertility.

36


3. 2. 1. An animal use ethics statement

The experiments described here were approved by the Committee for Animal Research and

Welfare of Gifu University, number: 17125.

The use of cryopreserved semen was approved by the Animal Ethics Committee of the Gifu

Prefectural Livestock Research Institute, Japan.

3. 2. 2. Chemicals and reagents

All chemicals used in this study were purchased from Sigma Aldrich (Sigma-Aldrich,

Steinheim, Germany) and Wako Pure Chemicals Industries (Osaka, Japan) unless otherwise

stated.




semen of 21 ejaculates from 7 Japanese Black bulls of known fertility including one with low

sperm motility (Fert-A) and from 2 bulls of known subfertility (Sub-1 and Sub-2) were used in

this study. From the bulls used in this study, frozen semen from 3 bulls with 3 similar ages at

semen collection were selected and used to examine effect of age on sperm traits by an

experimental design of 3 bulls × 3 age groups (1–2 years, 2–6 years and 6–10 years).

For use, frozen straws were allowed to thaw in water at 39 °C for 1 min.

3. 2. 4. Media

The saline medium used for dilution and incubation of spermatozoa consisted of 142 mM

NaCl, 2.5 mM KOH, 10 mM glucose and 20 mM Hepes adjusted to pH 7.55 at 20 °C with

37

NaOH (Roldan et al., 1994; Roldan and Harrison, 1989). Saline medium containing 222 mM

sucrose in place of NaCl was used for washing spermatozoa (sucrose medium, Roldan et al.,

1994; Roldan and Harrison, 1989). Both media also contained 0.1 % (w/v) polyvinyl alcohol

(PVA; molecular weight 30,000–70,000) for staining with FITC-PNA.

Fixative used for staining with FITC-PNA was 12.5 % (w/v) PFA in 0.5 M Tris, adjusted to

pH 7.4 at 20 °C, aliquoted, and kept frozen at -30 °C until use.

FITC-PNA was dissolved in H2O. After adding 0.05 % sodium azide, the solution was

aliquoted, and the vials were wrapped with aluminum foils to protect them from light and stored

at -30 °C until use. For use, stock solution was diluted with PBS to 20 μg/ml according to

Almadaly et al. (2012).

3. 2. 5. Standard semen analysis of frozen-thawed spermatozoa

In frozen-thawed semen the percentage of total and progressive motility was determined

subjectively under a phase contrast microscope at 200 × magnification.

Viability was assessed by staining spermatozoa with propidium iodide according to

Harrison and Vickers (1990).

For morphology assessment, frozen-thawed semen was diluted with an equal volume of 0.16

M of NaCl, and spermatozoa were fixed with 1% glutaraldehyde by mixing the diluted semen

with an equal volume of 2% glutaraldehyde in 0.165 M sodium cacodylate buffer (pH 7.3 at

25 °C) at RT for a minimum of 30 min. Glutaraldehyde-fixed spermatozoa (2 μl) were applied

onto a glass slide and covered with a coverslip (18×18 mm) compressed gently to remove

excess fluid. A total of 200 spermatozoa were examined for sperm morphology under a phase

contrast microscope (BX41; Olympus, Tokyo, Japan) at 1000 × magnification, and percentage

of spermatozoa showing normal morphology (% normal morphology) was obtained.

38

3. 2. 6. Staining of acrosomes of spermatozoa in frozen-thawed semen with FITC-PNA

Frozen-thawed spermatozoa were resuspended in saline medium containing PVA by

centrifugation and later washed through 4 ml sucrose medium containing PVA. Washed

spermatozoa were stained as described by Harayama et al. (2010) with minor modifications.

Spermatozoa were fixed in vials with 3% PFA for 30 min. Fixed spermatozoa were

permeablized with 1% Triton X-100 for 5 min and then stained with 200 μl of FITC-PNA (20

μg/ml) at RT for 30 min. A portion was taken and mixed with an equal volume of an antifade

agent ProLong® Diamond (Thermo Fisher, West Sacramento, CA, U. S. A.). Stained

spermatozoa were examined under a phase contrast microscope at a magnification of 1000 ×

using fluorescence illumination (mirror unit U-MWB2: dichroic mirror DM500, excitation

filter, BP460–490 and emission filter BA520IF; Olympus, Tokyo, Japan). Two hundred

spermatozoa were examined on each slide, and the staining patterns of the acrosomes were

classified into seven categories as described in chapter 2. Percentage of spermatozoa showing

each pattern was obtained (% pattern I–VII), and the % of spermatozoa showing patterns I and

II were combined to give % intact acrosome and similarly the % of spermatozoa showing

patterns III to VII were combined to give % damaged acrosome.

3. 2. 7. Induction of the acrosome reaction with Ca2+ and Ca2+ ionophore A23187

Frozen-thawed spermatozoa were incubated for stimulation with calcium and calcium

ionophore A23187 as described previously (Almadaly et al., 2015; Murase et al., 2001b).

Briefly, frozen-thawed semen was centrifuged at 800 × g for 5 min, and the pelleted

spermatozoa were resuspended in saline medium containing PVA and PEG and overlaid onto

a sucrose medium containing PVA and PEG. The sample was centrifuged at 400 × g for 5 min

followed by 1000 × g for 10 min, and the supernatant was removed. The pelleted spermatozoa

in the sucrose medium were resuspended in the saline medium containing 3 mM of CaCl2 and

39

incubated in the presence or absence (DMSO vehicle control) of A23187 (1 μM) at 37 °C for

up to 60 min in air. Sperm concentration during incubation was adjusted to 2 × 107/ml. At

various intervals (0, 5, 10, 15, 30, and 60 min), subsamples were taken and fixed by mixing

with an equal volume of 2% glutaraldehyde in 0.165 M cacodylate buffer.

The fixed spermatozoa were smeared gently onto slides pre-coated with 1% poly-L-lysine

as an adhesive, allowed to air-dry, and then stained with naphthol yellow S and Erythrosin B

according to Bryan and Akruk, (1977) and Lenz et al., (1982). Briefly, slides were stained in

solution A of 0.1% naphthol yellow S (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) in

1% acetic acid for 30 min, blotted between layers of filter paper and rinsed in 1% aqueous

acetic acid for 10-15 seconds. Slides were then drained, stained in the equal mixture of solution

B (0.2% aqueous naphthol yellow S) and C (0.2% aqueous erythrosin B) for 18 min, and then

rinsed well in distilled water adjusted to pH 4.6-5.0 with acetic acid. Slides were blotted and

allowed to air-dry. Slides were rinsed with xylene and mounted with Entellan® new (Merck,

Darmstadt, Germany). Spermatozoa were then examined under a phase contrast microscope at

a magnification of ×1000, and acrosomes were categorized into 3 patterns; intact, intermediate,

and acrosome-reacted (Fig. 3. 1). Spermatozoa showing complete acrosome loss (Fig. 3. 1c)

were considered acrosome-reacted and percent of acrosome-reacted spermatozoa (% AR;

absolute values) was obtained. Then percent of AR was converted into the % relative AR with

the following formula:

(% AR at each time point of stimulation ˗ percentage at 0 min)/(100 % ˗ percentage at 0

min) × 100 (Murase et al., 2001b).

The % AR at 0 and 60 min of stimulation was used to examine tolerance of spermatozoa to

freezing-thawing followed by washing and resuspending while the % AR at 60 min of

stimulation was presented as a conventional parameter of induced AR. The % relative AR was

40

used to examine time-course changes in induction of AR in spermatozoa that have intact

acrosomes at the beginning of stimulation.

Time-dependent changes in the % relative AR were analyzed by Hillslope of Interpolation

(Asymmetric Sigmoidal, 5PL), and hillslope amount was obtained in each ejaculate. The

calculation was carried out by the software GraphPad Prism Version 6.0; GraphPad Software,

San Diego, CA, USA.


Correlation between % damaged acrosome with FITC-PNA and the conventional

parameters (standard semen analyses and % AR at 0 and 60 min) were analysed with Pearson

linear regression.

According to the hillslope amount of % relative AR, response curve of individual samples

was categorized into 3 groups (AR response group; see Results section), and comparison of all

data among the 3 AR response groups and among the 3 different bull age groups were carried

out by one-way ANOVA. When the results of the one-way ANOVA were significant, Tukey’s

multiple comparison test was performed to compare different treatments.

Differences with P < 0.05 were considered to be statistically significant.

All analyses were performed using a statistical software program (GraphPad Prism Version

6.0; GraphPad Software, San Diego, CA, USA).

41

3. 3. Results

3. 3. 1. Categorization of response to Ca2+/A23187

When hillslope amount (X) of the time-course changes in % relative AR was examined in

each semen sample, it was noticed that changes in the response of spermatozoa to Ca2+/A23187

could be categorized into the 3 groups of sigmoidal curve with X ≥ 0.6 (Group 1 in Fig. 3. 2a),

slow increase with 0.6 > X ≥ 0.1 (Group 2 in Fig. 3. 2b) and quick increase with X<0.1 (Group

3 in Fig. 3. 2c). The 3 different AR response groups were confirmed by the observation that

the averages of hillslope amount were significantly different among these groups (Fig. 3. 2d).

3. 3. 2. AR response group and conventional parameters

Next a relationship between the AR responsiveness (3 different AR response groups) and

the other parameters was examined. Comparison of conventional parameters including the

standard parameters and % AR at 0 and 60 min among the 3 groups did not reveal any

significant differences (P > 0.05; Fig. 3. 3).

Notably, two subfertile bulls (Sub-1 and Sub-2) showing normal results of standard

parameters and one fertile bull showing poor standard parameters of low % Total motility, %

+++ motility and viability (Fert-A) were all categorized into Group 3 (quick responder) of the

AR.

3. 3. 3. Acrosomal integrity and conventional parameters

There were significant correlations of % damaged acrosome by FITC-PNA with

conventional parameters including the standard semen parameters of %Total motility, +++

motility, viability, normal morphology and %AR at 0 and 60 min (Fig. 3. 4 a-d).

42

3. 3. 4. Acrosomal integrity and AR response group

Acrosomal integrity was then compared among the 3 AR response groups. There were no

significant differences in any of the % patterns I–VII and % intact acrosome (Patterns I and II)

among the 3 groups (P > 0.05; Fig. 3. 5).

Bull Fert-A, categorized into Group 3 of AR response group, was located at very low %

intact acrosome (Fig. 3. 5a) and % pattern I (Fig. 3. 5b) and at high % pattern V (Fig. 3. 5f) as

compared to other bulls. Bulls Sub-1 and Sub-2 behaved similarly to other fertile bulls except

Bull Fert-1.

3. 3. 5. Age

The conventional parameters including standard semen analyses and % AR at 0 and 60 min

and % damaged acrosome by FITC-PNA did not show any significant differences among the

3 different age groups (1–2, 2–6, and 6–10 years) (P > 0.05; Figs. 3. 6a–f).

On the other hand, there was no significant correlation between ages and % damaged

acrosome by FITC-PNA (Fig. 3. 7a). Alternatively, there was no significant difference in age

among the 3 AR response groups (P > 0.05; Fig. 3. 7b).

43

3. 4. Discussion

3. 4. 1. Responsiveness of spermatozoa to Ca2+ and A23187

When time-dependent changes in % relative AR was compared among the individual semen

samples, it was noticed that there were at least 2 different patterns of changes; sigmoidal with

slow start of increase and quick increase. Then hillslope of the changes in % relative AR was

applied in an attempt to categorized the sperm response objectively. The results clearly showed

that there were 3 different patterns of time-dependent changes in % relative AR, which were

sigmoidal curve, slow increase and quick increase. To the best of our knowledge, this is the

first study that has categorized the time-course changes in AR into 3 groups in frozen-thawed

bull spermatozoa.

In our previous study, addition of desalted and lyophilized seminal plasma (SP) brought the

quick increase in the % relative AR in bull spermatozoa cryopreserved without SP and

stimulated with Ca2+ and A23187 to a slow increase as a sigmoidal curve, suggesting that

addition of SP prevented cryocapacitation (Almadaly et al., 2015).

Earlier studies showed the AR triggered by Ca2+ and A23187 was rapidly evoked at 5 min

of stimulation in frozen-thawed Japanese Black bull spermatozoa, where response curve looked

oval (Murase et al., 2001b). Whereas, fresh ram or boar spermatozoa do not initiate AR until

diacylglycerol is generated, in which case response curve is sigmoidal (Roldan and Murase,

1994; Vazquez and Roldan, 1997).

Taking these into consideration, Group 1 spermatozoa displaying a sigmoidal curve were

similar to the spermatozoa cryopreserved with SP or fresh spermatozoa, and this suggests that

spermatozoa in this Group may be less cryocapacitated and rather near to the uncapacitated

state. Group 3 spermatozoa showed quickest increase and Group 2 spermatozoa were at an

intermediate stage, suggesting that capacitation process may be advanced in these groups.

Therefore, it is considered that the order of advance in cryocapacitation status of the frozen

44

semen may be Group 3<Group 2<Group 1, and this categorization may be an indicator of

capacitation status of cryopreserved bull spermatozoa. Because premature capacitation may be

one of the causes for subfertility in bulls (Kuroda et al., 2007), this finding may serve as a basic

knowledge leading to detection of bull subfertility.

The previous reports in human (Cummins et al., 1991; Francavilla et al., 1995), dogs (Szász

et al., 2000) and bulls (Beorlegui et al., 1997; Januskauskas et al., 2000; Whitfield and

Parkinson, 1995) have used the test with Ca2+/ A23187 to investigate mammalian sperm

function. However, time-dependent changes in induced AR have not been shown but response

of AR at a fixed time point was used. The observed 3 patterns of AR implied that it is better to

examine time-dependent changes by measuring % AR at multiple time points of stimulation in

order to characterize sperm functional status precisely.

There were 2 subfertile bulls (Sub-1 and Sub-2) showing normal standard semen analyses

results and 1 fertile bull showing motility lower than normal range (> +++ 30%, > total 60%)

included in the semen samples examined, and they were all categorized into Group 3 of AR

(Fig. 2 c). This implies a possibility to detect a subfertile bull with normal results of standard

analyses by the response of Group 3 i.e. quick responder. By contrast, when induced AR at a

fixed time was studied in fresh human spermatozoa (Tello-Mora et al., 2018) and in frozen-

thawed bull spermatozoa (Lessard et al., 2011), the reduced response was related to subfertility,

suggesting dysfunction of undergoing the AR as a possible causal factor for

subfertility/infertility. Taken together, it seems that there are two different likely causes for

subfertility/infertility, which are premature capacitation (cryocapacitation) detected as quick

response to Ca2+/A23187 and dysfunction of the AR detected as reduced response to

Ca2+/A23187. The more cryocapacitated spermatozoa are, the less time would be needed to

complete capacitation. Therefore, AI with frozen-thawed spermatozoa with advanced

cryocapacitation may need to be inseminated at a later stage of estrus while less cryocapacitated

45

spermatozoa may need to be inseminated at earlier stage. In boar spermatozoa, hypersensivity

to Ca2+ and A23187 has been suggested to be related to summer infertility (Murase et al., 2007).

Thus quick response to Ca2+ and A23187 may be one of the indicators of subfertility.

Particularly in bulls, semen for AI is cryopreserved and thus proportion of acrosome-damaged

spermatozoa in frozen-thawed semen may be higher (see Fig. 3. 4) than fresh spermatozoa, for

instance, human spermatozoa. In this regard, test to measured % AR induced at a fixed time is

useful in fresh spermatozoa, but for frozen-thawed spermatozoa, AR at a fixed time may not

necessarily be an ideal parameter because background AR is high and % AR after a fixed time

of stimulation is high in parallel, which was confirmed by the observation that % damaged

acrosomes by FITC-PNA was significantly correlated with % (absolute) AR at 0 and 60 min

of stimulation (Fig. 3. 6e and f). In this regard, % relative AR may be a better parameter because

this measures the ability of acrosome-intact spermatozoa to undergo AR in response to

Ca2+/A23187.

3. 4. 2. Comparison of responsiveness to Ca2+ and A23187 with conventional parameters

The % AR at 0 and 60 min were used as a parameter for tolerance to the changes in the

extracellular environment (i.e. removal of seminal plasma and extender and resuspension in a

medium containing Ca2+ by washing) and for the maximal ability of AR as a conventional

parameter, respectively. Failure in finding difference in standard parameters, % AR at 0 and

60 min among AR response Groups 1–3 of % relative AR suggests that responsiveness of

acrosome-intact spermatozoa to Ca2+/A23187 may probably reveal a different aspect of

function in independent from standard parameters, tolerance to environmental changes and

maximum ability to undergo AR (Fig. 3. 3). Evidence that motility was not correlated with %

acrosome-reacted spermatozoa after stimulation with A23187 in bulls (Beorlegui et al., 1997)

supports this idea.

46

3. 4. 3. Comparison of acrosomal integrity (FITC-PNA staining) with the conventional

parameters

It was considered that membrane was fragile in this particular sperm sample with low

motility and resulting in AR quick response.

The proportion of spermatozoa with damaged acrosome (unstimulated) reflected all of

standard parameters including sperm motility, viability, normal morphology, % AR at 0 and

60 min in this study. Work by others have shown that acrosomal integrity assessed by FITC-

PNA staining was correlated with motility (Panmei et al., 2015; Shivahre et al., 2015) and

viability (Shivahre et al., 2015). The relation of damaged acrosome percentage to % AR at 0

min suggests that fragile sperm sample charactrized by high % damaged acrosome may also

be vulnerable to their environmental changes while the relation to % AR at 60 min may indicate

that fragile sperm sample have higher responsiveness to Ca2+/A23187, suggesting unstable

membrane of spermatozoa displaying high proportion of damaged acrosome. Therefore, it

seems that spermatozoa characterized by high proportion of damage acrosome may accompany

damage of other functions.

3. 4. 4. Comparison of AR responsiveness with acrosomal integrity by FITC-PNA

Because time-dependent changes in % relative AR and acrosomal integrity assessment are

recognized as novel test of sperm quality, association between these two parameters were

examined in details to obtain basic information for development of new tests. Damaged

acrosome is also indicative of success/unsuccess of freezing/thawing (Silva & Gadella, 2006),

and in this regard, acrosomal integrity is an important parameter of sperm quality. Kishida et

al. (2015) described that a subfertile bull showed a lower % of intact acrosome and high

percentage of pattern III of FITC-PNA staining. Therefore, this study attempted to seek a

possible relationship between each staining pattern by FITC-PNA and AR response groups.

47

The results showed that there were no clear relationship between the AR responsiveness and

acrosomal integrity, suggesting that these 2 sperm traits are independent from each other and

both may be necessary to be carried out.

3. 4. 5. Bull age and sperm traits

Relationship between age and sperm traits have been implicated (Al-Qarawi, 2005; Foote,

1978) but this study did not show any age effect on sperm traits, possibly due to the limited

variation of age of bulls. Within the age examined in the present study (1-10 years) none of the

parameters including standard semen parameters and % AR at 0 and 60 min was associated

with age. The 2 new methods (PNA staining and time-course of AR) were not associated with

age (1-14 <) either. These results suggest that the differences in the parameters observed were

not dependent upon age at which semen was collected within the bulls used. Therefore, at least

age could be ruled out as a factor responsible for the observed relationship between the %

damaged acrosomes and for the conventional parameters and the difference in the

responsiveness to Ca2+/A23187.

In conclusion, to my best knowledge this study is the first to show different patterns of time-

dependent changes in the AR triggered by Ca2+ and A23187 in frozen-thawed bull spermatozoa.

The results suggest that responsiveness of AR may be independent from motility, viability,

normal morphology, or acrosomal integrity of frozen-thawed bull spermatozoa. Because

acrosome-damaged spermatozoa cannot participate in fertilization, the relative AR used in this

study that reflects the ability of acrosome-intact spermatozoa to undergo the AR may be a

practical parameter for sperm function particularly in bulls where cryopreserved spermatozoa

are used for AI. A combined usage of acrosomal integrity assessed by FITC-PNA staining and

AR responsiveness may provide a poweful information on bull sperm function. These

parameters will become a useful tool to assess the semen quality to detect subfertile bulls.

48

Author would like to expect that the information provided in the present study may contribute

to development of diagnosis of human male subfertility and infertility.

49

(a) (b) (c)

Fig. 3. 1. Photographs of frozen-thawed bull spermatozoa stained by the double staining

method with Naphthol Yellow S and Erythrosin B.

Spermatozoa showing deep (purple) colour on the acrosomal region of the head with (a) or

without (b) dense apical ridge were considered as acrosome-intact. Spermatozoa showing

evenly faint (pink) colour on the whole head (c) were considered as acrosome-reacted.

50

Fig. 3. 2. Categorization of time-dependent changes in % relative AR into 3 patterns on the

basis of hill slope amount (d); sigmoidal (a), slow increase (b) and quick increase (c).

Lines in scatter graph (d) show median, maximum and minimum amount.

a, b and c: Different superscripts denote a significant difference (P < 0.05, One-way ANOVA;

P < 0.05, Tukey’s test).

51

Fig. 3. 3. Comparison of the conventional parameters of standard semen analyses including %

Total motility (a), % +++ Motility (b), % Viability (c), % Normal morphology (d), % AR at 0

min (e) and 60 min (f) of stimulation with Ca2+/A23187 among the 3 different AR response

groups. , Fert-1; , , Sub-1; , Sub-2.

Lines in scatter graphs show median, maximum and minimum amount.

52

Fig. 3. 4. Correlation between % damaged acrosome by FITC-PNA and the conventional

parameters of standard semen analyses including % Total motility (a), % +++ Motility (b),

% Viability (c), % Normal morphology (d) and % AR at 0 min (e) and 60 min (f) of stimulation

with Ca2+/A23187.

Dotted lines show the lower and upper confidence limit.

Y = -0.1608*X + 70.95r2 = 0.2090 P = 0.0372

Y = -0.2441*X + 48.24 r2 = 0.3962 P = 0.0022

Y = -0.3791*X + 106.5 r2 = 0.5764 P < 0.0001

Y = -0.3974*X + 67.79 r2 = 0.3925 P = 0.0024

Y = 0.7270*X + 2.091 r2 = 0.7314 P < 0.0001

Y = 0.2811*X + 69.16 r2 = 0.4453 P < 0.001

53

Fig. 3. 5. Comparison of %intact acrosome (I+II (a)) and %patterns I-VII (b-h) of acrosomal

integrity by FITC-PNA among the 3 different AR response groups.

, Fert-1; , , Sub-1; , Sub-2.

Lines in scatter graphs show median, maximum and minimum amount.

54

55

Fig. 3. 6. Comparison of 3 bulls in 3 different ages (1-2, 2-6, and 6-10 years) with the

conventional parameters of standard semen analyses including %Total motility (a), %+++

Motility (b), %Viability (c), %Normal morphology (d) and % AR at 0 min (e) and 60 min (f)

of stimulation with Ca2+/A23187, and %intact acrosome by FITC-PNA staining among 3

different AR response groups in 3 bulls × 3 age groups (1-2, 2-6, and 6-10 years).

56

Fig. 3. 7. Age (0-14 years) at semen collection and new parameters of %intact acrosome and

AR response group in 21 ejaculates from 9 bulls.

, Fert-1; , , Sub-1; , Sub-2.

Dotted lines show the lower and upper confidence limit.

Lines in scatter graph (b) show median, maximum and minimum amount.

Y = -0.3177*X + 85.50 r2 = 0.02470 P = 0.4963

Age

(Mon

th)

57

Chapter 4 Relationship of standard analyses with hyperactivation, AR and acrosome condition with FITC-PNA in frozen-

thawed bull spermatozoa

58

4. 1. Introduction

Immediately after ejaculation, mammalian spermatozoa initiate flagellar beating, and move

rapidly in a forward direction. These motile gametes are not, however, able to fertilize oocytes

until they complete various capacitation-associated changes that are normally initiated within

the environment specific to the female reproductive tract (Yanagimachi, 1994). Capacitation is

achieved most effectively when the spermatozoa continuously transit through the uterus and

oviduct (Hunter and Rodriguez-Martinez, 2004).

The physiological changes that confer on sperm the ability to fertilize are collectively called

‘capacitation’ that mammalian spermatozoa need to undergo for successful fertilization

(Yanagimachi, 1994). This change includes the AR and hyperactivation. The AR takes place

following capacitation of sperm head, and hyperactivation is related to dramatic changes in

motility of the flagellum. Capacitation, hyperactivation, and the AR are essential processes for

mammalian spermatozoa to fertilize the oocyte. Decrease in any of these functions might cause

failure in fertilization leading to possible infertility or subfertility. Similar cases have also been

reported in humans (Luconi et al., 2006; Munire et al., 2004).

Once capacitation progresses beyond a critical point, dramatic reactivation of flagellar

movement occurs that frees spermatozoa from the epithelial cells, allowing the spermatozoon

to move up through the mucus in the lumen to the ampulla of the oviduct. Such

‘‘hyperactivation’’ of sperm motility is characterized by highly asymmetric bending patterns

that often make the sperm travel along a circular trajectory (a ‘‘partial-type’’ of

hyperactivation). Extremely asymmetrical bends of the middle piece in fully hyperactivated

spermatozoa produce intense twisting or figure-eight movements (‘‘full-type’’

hyperactivation) (Marquez and Suarez, 2004; Suarez, 2008).

Thus, hyperactivation and the AR are possible targets to assess as fertilizing functions

of spermatozoa. Japanese Black bulls of known subfertility with normal results of standard

59

semen analyses showed slower and lower response to calcium and the calcium ionophore

A23187 for the AR (Murase et al., 2001b). On the other hand, previous studies showed that

hyperactivated motility of frozen-thawed bull spermatozoa can be induced by a cell-permeable

cAMP analog, sp-5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3',5'-

monophosphorothioate (cBiMPS) (Murase et al., 2010; Harayama et al., 2010), and inducibility

of hyperactivated motility by cBiMPS was shown to be related to bull subfertility (Murase et

al., 2010). These suggest the importance of assessing the fertilizing abilities of spermatozoa in

adjacent to standard semen analyses including motility and progressive motility. It is expected

that decrease in any one of these 2 functions might cause fertilization failure but it was unclear

whether these functions are interrelated together.

The present chapter aimed to reveal the relation of standard semen analyses with

hyperactivation, AR and acrosome condition with FITC-PNA in different bulls in order to gain

fundamental knowledge to develop new functional tests of frozen-thawed bull spermatozoa.

60


4. 2. 1. Chemicals and reagents

All chemicals used in this study were purchased from Sigma Aldrich (Sigma-Aldrich,

Steinheim, Germany), and Wako Pure Chemicals Industries (Osaka, Japan) unless otherwise

stated.

4. 2. 2. Media

All media for standard semen analyses, FITC-PNA staining, AR and double staining was

prepared same as in Part 3.2.2. Brackett and Oliphant (BO)-Hepes medium, BO medium

modified by adding Hepes instead of sodium HCO3 , was used for incubation of spermatozoa

in the experiments of hyperactivation. It consists of 112 mM NaCl, 4.02 mM KCl, 2.25 mM

CaCl2, 0.52 mM MgCl2 , 0.83 mM NaH2PO4 , 37 mM Hepes, 13.9 mM glucose, 1.25 mM

sodium pyruvate, 100 IU/ml potassium penicillin G, and 0.1% (w/v) PVA, pH 7.55, at 20°C.

For washing spermatozoa, BO-Hepes medium that did not contain CaCl2 was used. BO-Hepes

medium that contains 10% sucrose medium (BO-Hepes/sucrose) was used for adjustment of

sperm concentration before incubation with cBiMPS. A stock solution of 10 mM cBiMPS

(Biomol International, L.P., Plymouth Meeting, PA, USA) in dimethyl sulfoxide (DMSO) was

prepared, and then added to the incubation medium to give a final concentration of 100 μM.




spermatozoa of 5 Japanese Black bulls were used in this study. This study was approved by the

Committee for Animal Research and Welfare of Gifu University, number: 17125. The use of

cryopreservation semen was approved by the Animal Ethics Committee of the institute.

61

4. 2. 4. Standard semen analysis, acrosomal integrity condition with FITC-PNA,

induction and assessment of AR with Ca2+ and Ca2+ ionophore A23187 and acrosome

double staining procedure

Standard semen analysis, acrosomal integrity condition with FITC-PNA, induction and

assessment of AR with Ca2+ and Ca2+ ionophore A23187 and acrosome double staining

procedure were explained in parts 3. 2. 4 (Media) – 3. 2. 7 (Induction of the AR with Ca2+ and

Ca2+ ionophore A23187).

4. 2. 5. Incubation of spermatozoa with cBiMPS

The washed spermatozoa were pre-warmed for exactly 5 min at 38.5 °C before resuspended

in the incubation medium (BO-Hepes) to adjust a final sperm concentration of 1 × 108 /ml.

Washed spermatozoa were incubated in a water bath (38.5 °C) in air for 180 min. The

incubation medium contained 2.25 mM CaCl2 and 0.1 mM cBiMPS during incubation. To the

control samples without cBiMPS, the same volume of DMSO was added to equalize the

concentration of solvent. During incubation sperm suspensions were gently mixed well and

then 10 μl of them were recovered for the use in assessment of sperm motility. Sperm motility

was subjectively assessed at specific intervals of 0, 30, 60, 120, and 180 min of incubation.

Briefly, 10 μl of sperm suspension were taken and applied onto a glass slide prewarmed at

38.5°C and covered with coverslip (18 mm × 18 mm), then sperm motility was examined at

200x magnification with a phase contrast microscope (Olympus BX41, Tokyo, Japan),

equipped with an automatic warming plate for total and progressive motility, circular and

whiplash movement. % total motility includes all motile spermatozoa irrespective of their

progressive motility. The swimming patterns of the motile spermatozoa were classified into 4

categories as following: straight forward swimming (% progressive motility), non-linear

swimming (% non-linear motility), circular swimming with tails beating asymmetrically (%

62

circles), and spermatozoa staying in a local area with vigorous whiplash flagellar beating (%

whiplash). The latter 2 patterns were considered to be hyperactivated motility and the

percentage of each pattern to the total motile spermatozoa was calculated and expressed as %

C and % W, respectively. Swimming spermatozoa with symmetrical flagellar beating were

considered to have activated motility and were not considered to be hyperactivated.


According to the hillslope amount of % relative AR, response curve of individual samples

was categorized into 3 groups.

All analyses were performed with using a statistical software program (GraphPad Prism

Version 6.0; GraphPad Software, San Diego, CA, USA).

63

4. 3. Results

Fig. 4. 1 shows the response of spermatozoa to calcium and calcium ionophore in different

bulls. Bull A and C responded very quickly and were categorized into Group 3 of AR response

group based on hillslope of the response curve (quick responder, Chapter 3). Bulls D and E

responded slowly and were categorized into Group 2 (slow responder, Chapter 3). However

Bull B was categorized into Group 2 with its maximum response being vey low (19.6 % after

60 min).

Fig. 4. 2. shows % Non-linear, progressive motility, circullar and whiplash movement of

spermatozoa in response to cBiMPS (Fig. 4. 2. a, c, e, g, and i), and without cBiMPS (Fig. 4.

2. b, d, f, h, and j). In bulls D and E higher % C and % W were observed but in bull B circular

and whiplash movement were not observed. For bulls A and C, % C and % W were low. In

bulls C, D, and E without adding cBiMPS after 180 min, stable % total and progressive motility

were observed. In bulls D and E with adding cBiMPS high % C and % W could be observed

after 180 min but in bull C it was not as high as bull D and E. On the other hand, bulls A and

B without adding cBiMPS after 180 min, showed % progressive motility decreased

dramatically and it was not observed high % C and W after 180 min.

Table 4. 1. shows the results of standard semen analyses (% total motility, progressive

motility, viability, and normal morphology), % circular movement at 60 min and AR group on

the basis of hillslope by interpolation asymetrical sigmoidal analyses (See 3.3). Percentages of

total motility and +++ motility were below normal range (60 % of total motility and 30 % of

+++ motility) in bull A and B, and % viability was below 50% in bull A, B and C. Normal

morphology were nearly the same in all the bulls. Percentage of motility, +++ motility and

viability were high in bull D and E. As to hyperactivated motility, bull A, B and C showed very

low % C with no circular movement being observed in bull B and C after 60-min incubation

with cBiMPS, whereas in bull D and E % C was higher than others.

64

Table 4. 2. shows the different patterns of acrosome condition with FITC-PNA (Table 4. 2).

Bulls D and E had higher % intact acrosome in comparison with other bulls.

65

4. 4. Discussion

In this study, standard semen analyses, acrosome condition with FITC-PNA, AR and

hyperactivation were assessed for 5 different bulls (A- E; Table 4. 1).

Results showed similar % total motility and normal morphology in different bulls, while for

bulls A and B, % progressive motility and viability was lower than others. These results showed

that for assessment of Japanese black bulls all standard semen analyses are necessary for

detecting subfertility.

Previous results demonstrated that the fertile bulls clearly show that the cAMP analogue

cBiMPS can induce hyperactivation as indicated by a circular swimming pattern and a

subsequent whiplash motion of the flagellum in frozen-thawed Japanese Black bull

spermatozoa and strongly suggest that bull sperm hyperactivation may mediate a cAMP/PKA

pathway (Murase et al., 2010). In the current study, different response to cBiMPS in different

bulls were observed. After 60 min of incubation with cBiMPS circular movement could not be

observed in bulls B and C. The % C for Bull A was relatively low (16.7%) in comparison to

bulls D and E with % C being higher than other bulls (41.7 and 33.3%, respectively; Table 4.

1).

Acrosome condition also was also not similar in different bulls. In bulls A and B intact

acrosome were low (20.5 % and 24.5 %; respectively), and in bulls D and E were high (55.5 %

and 75.2).

In response to calcium and A23187 different bulls showed different response. Bull A and C

categorized in Group 3 and bulls B, D, and E categorized in Group 2. However, bull B was

categorized in Group 2 but after 60 min the % AR was very low in comparison with other bulls

(Fig. 4. 1b).

Hyperactivated motility plays a role in releasing sperm from the epithelium of the oviduct

reservoir and helps the sperm to penetrate the ZP; thus, it has an important role in the

66

fertilization process (Suarez, 1996). Inhibition of hyperactivation prevents fertilization (Stauss

et al., 1995).

The in vivo sperm capacitation occurs during migration in the reproductive tract of the

female, whereas in vitro capacitation requires the exposition of fresh or freeze semen to specific

capacitating agents (Liu et al., 2012). Capacitation process is a prerequisite step for sperm to

bind to the ZP, it tested acrosomic reaction in response to natural agonistics and express hyper-

motility, a special movement that allows spermatozoa to move in the viscose fluid of the

oviduct and get into the ZP (Hiromi et al., 2000).

Results of standard semen analyses, acrosome condition with FITC-PNA, % AR and

hyperactivation showed differences in bulls. These parameters are in order to detect abnormal

traits when motility is normal and independent form each other, it seems all of them are

neccessary for precise assessment of sperm functional status.

Bull A, B, and C were thus thought to be abnormal in particular traits. Two of these bulls,

A and C, were categorized into Group 3 of AR response group, while bull B was into Group 2.

This shows the 2/3 of bulls showing abnormal hyperactivation are classified to quick responder

of AR, implying hyperactivation and AR connected features. However, bull B was exceptional

because hyperactivation was low but AR was similar to normal bulls (D and E). Thus it seems

that hyperactivation and AR tests may measure different aspects of function in spermatozoa.

Bull A and B showed both abnormal results of conventional tests (motility and +++ motility)

and of hyperactivation test, while bulls D and E showed normal results of both conventional

tests and hyperactivation. In these bulls, % damaged acrosomes by FITC-PNA seemed to

correlated with conventional test results, as seen in Chapter 3. Thus, conventional tests,

acrosomal integrity by FITC-PNA and hyperactivation appeared to inter related to each other.

However, in bull C, conventional test results (motility, +++ motility, and morphology) were

normal but hyperactivation and % intact acrosomes were decreased. This case implies that

67

hyperactivation test and FITC-PNA test can detect abnormal traits in spermatozoa showing

normal motility.

In conclusion, it seemed that hyperactivation and AR are independent from each other but

that somehow hyperactivation was relatively in parallel to acrosomal integrity. The information

provided in this chapter may be useful to develop further tests to assess bull sperm function to

detect subfertile bulls.

69

Tab

le 4

. 1. %

Tot

al m

otili

ty, P

rogr

essi

ve m

otili

ty, V

iabi

lity,

Nor

mal

mor

phol

ogy,

Circ

ular

mov

emen

t (%

C) a

n A

R

grou

p in

the

5 bu

lls.

Bul

l %

Tot

al

mot

ility

%

Pro

gres

sive

m

otili

ty

% V

iabi

lity

% N

orm

al

mor

phol

ogy

% C

at 6

0 m

in

Hill

slop

e A

R g

roup

A

50

15

29.5

90

16

.7

0.06

3

B

50

20

19.0

87

0.

0 0.

11

2 C

60

30

35

.0

89

0.0

0.04

3

D

70

40

53.5

88

41

.7

0.13

2

E 60

40

59

.0

93

33.3

0.

19

2

Tab

le 4

. 2. %

Spe

rmat

ozoa

show

ing

Patte

rn I-

VII

by

FITC

-PN

A st

aini

ng in

the

5 bu

lls.

Bul

l I

II

I+II

II

I IV

V

V

I V

II

% d

amag

ed a

cros

ome

(III

~VII

) A

15

.0

5.5

20.5

67

.3

0 6.

4 5.

9 0

79.6

B

22

.5

2.0

24.5

69

.0

0 2.

5 4.

0 0

75.5

C

34

.6

3.7

38.3

56

.5

0 2.

3 2.

8 0

61.6

D

52

.5

3.0

55.5

40

.1

0 1.

9 2.

5 0

44.5

E

68.8

6.

4 75

.2

23.8

0

1.0

0.0

0 24

.8

68

69

Fig. 4. 1. Time-dependent changes in % relative AR of frozen-thawed Japanese Black bull

spermatozoa stimulated with Ca2+/A23187 (DMSO for control to A23187) in the 5 bulls

used.

W: with ionophore A23187; W/O: without ionophore A23187.

%A

R

70

%++

+ M

otili

ty, N

on-li

near

,%

C a

nd %

W

%++

+ M

otili

ty, N

on-li

near

,%

C a

nd %

W

71

Fig. 4. 2. Time-course changes in the percentages (%) of Circullar movement (% C), Whiplash

movement (% W), +++ Motility and Non-linear motility (% Non-linear) of frozen-thawed

spermatozoa stimulated with 0.1 mM cBiMPS (a, b, c, d, and e), and without cBiMPS (f, g, h,

i, and j) in the 5 Japanese Black bulls (Bull A-E) used.

72

Chapter 5

General conclusion

73

In this thesis the methodology for assessment of sperm acrosome with FITC-PNA were

improved. The best concentration and time of fixation with PFA, the best concentration of

Triton X-100 for permeabilization and the best antifade agent to prevent fading FITC-PNA

were found. Also it was found that In-vial staining is better than On-smear method for acrosome

staining with FITC-PNA and stained spermatozoa could be stored till 24 hr for assessment.

It was found that acrosome conditions may be one of the best parameters to predict fertility.

Acrosome exocytosis and hyperactivation may reflect premature capacitation status of frozen

thawed spermatozoa, which would cause higher response to the agonists (cBiMPS and

A23187) and because higher incidence of acrosome damage may be a good indicator of

tolerance of spermatozoa against freezing-thawing, which is considered to decrease both

acrosome integrity and the fertilizing functions. Spermatozoa were categorized according to

their time course changes in response to A23187 into 3 groups (slow, intermediate, and quick

response).

The impact of the results will be the establishment of a highly effective diagnostic parameter

of frozen-thawed bull semen for the as yet unidentified cases where standard semen parameters

are within normal range but fertility is lowered. The method will definitely contribute to

improve bovine reproduction.

For finding AI subfertile bulls, it is necessary that the standard semen analyses (Total

motility, +++Motility, Viability, and Normal morphology) were evaluated but it is not enough

for detecting subfertile bulls. In this thesis, new parameters were assessed (Acrosome condition,

Time-course changes of AR, and Hyperactivation) for detecting the sperm characterestics.

Further studies will be required to establish methodology required for detecting subfertile bulls

when standard parameter such as motility is normal.

74

Chapter 6

Summary

75

Artificial insemination (AI) using frozen-thawed semen is one of the most important

technique in cattle breeding programs. Bull semen for AI use is conventionally assessed by

routine standard semen analyses such as total motility, progressive motility, viability and

normal morphology. However some of the bulls of subfertility after AI show the results of

standard semen analyses to be normal. Thus, this study aimed to gain basic information in an

attempt to establish novel assessment methods of bull spermatozoa to detect subfertile bulls

accurately, and analyzed the interrelationship among the conventional sperm parameters and

the three novel analyzing methods including acrosomal integrity assessed by a fluorescent

probe, fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) and the acrosome

reaction (AR) in the sperm head induced by the calcium ionophore A23187 and hyperactivation

in the flagellum induced by a cell-permeable cAMP analog, sp-5,6-dichloro-1-β-D-

ribofuranosylbenzimidazole-3',5'-monophosphorothioate (cBiMPS) in frozen-thawed

Japanese Black bull spermatozoa.

The Chapter 2 aimed to improve a protocol to stain sperm acrosomes with FITC-PNA for

gentler staining conditions that have low artifact damage to spermatozoa to be stained.

Spermatozoa washed and fixed with paraformaldehyde (PFA) in suspension were

permeabilized with Triton X-100, stained with FITC-PNA, mounted with different antifade

agents (DABCO, SlowFade® and ProLong®) in suspension (In-suspension) or on smear (On-

smear) and examined for the staining patterns under a fluorescence microscope immediately or

after storage for 24 h. The results of Experiment 1 examining effect of method × antifade ×

storage indicate 1) that In-suspension method is better than On-smear method, 2) that if

spermatozoa are stained by In-suspension method and examined immediately, the best antifade

agent may be SlowFade®, 3) that if samples were to be stored after staining by On-smear

method, DABCO should be avoided, 4) that if spermatozoa are stained by In-suspension

method, storage of stained sample may not be recommended as shown by the decrease in per

76

cent intact acrosome, 5) that if samples were to be stored after staining by In-suspension

method, ProLong® may be the best antifade agent. Results of Experiment 2 examining effect

of Triton X-100 showed that the concentration of Triton X-100 can be reduced to 0.1% from

usual 1%. The results of Experiments 3 and 4 examining effect of prolonged and shortened

fixation showed that the best fixation condition was with 2% PFA for 30 min. It is expected

that the staining protocol modified in this Chapter would be a useful tool to examine bull sperm

acrosomal integrity.

The Chapter 3 investigated the time-dependent changes in AR of different sperm samples

and the relationship among the standard semen analyses, acrosomal integrity by FITC-PNA

and the time-dependent changes in AR in order to gain basic information to establish new tests

to detect subfertile bulls. Motility, viability, normal morphology and percentage of AR (% AR)

at 0 and 60 min of stimulation with A23187 were examined as conventional parameters, and

acrosomal integrity by FITC-PNA and the time-dependent changes up to 60 min in the

percentage of acrosome-reacted spermatozoa to acrosome-intact spermatozoa at the beginning

of stimulation (% relative AR) were analysed. Changes in % relative AR were categorized into

3 distinct patterns (AR response groups) by hillslope analyses of the response curve; sigmoidal

curve, slow increase and quick increase. Neither the conventional parameter nor acrosomal

integrity was significantly different among the 3 AR response groups. Acrosomal integrity was

significantly correlated with all of the conventional parameters. Within the used samples, age

of the bull was not related to conventional parameters, acrosomal integrity nor the AR response

group. In addition, two bulls of known subfertilitywith normal standard parameters and one

fertile bull with low motility were all classified into Group 3. These results suggest that the

responsiveness of AR can distinguish functional status of frozen-thawed bull spermatozoa and

is independent from motility, viability, normal morphology or acrosomal integrity. A combined

77

usage of acrosomal integrity assessed by FITC-PNA staining and AR responsiveness is

expected to provide a powerful information on the semen quality to detect subfertile bulls.

The Chapter 4 aimed to examine the inter-relationship among standard semen analyses,

hyperactivation induced by cBiMPS, AR induced by A23187 and acrosomal inegrity assessed

by FITC-PNA in 5 different bulls (Bull A, B, C, D and E). Hyperactivated motility was

expressed as % spermatozoa showing circular movement (% C) and % spermatozoa showing

whiplash movement, and % C at 60 min of stimulation was used as a parameter for

hyperactivation ability. The AR was induced, and the bulls were categorized into the 3 AR

response group according to the criteria developed in the previous Chapter. Bull A and B

showing low sperm motility and Bull C of known subfertility with normal sperm motility had

low % intact acrosome and very low % C. On the other hand, Bull D and E with normal, high

motility had high % intact acrosome and showed high % C, implicating that somehow

hyperactivation was relatively in parallel to acrosomal integrity. Bull A with low motility and

Bull C of known subfertility were classified into Group 3 of the AR response group, and Bull

B with low motility into Group 2, and Bull D and E with high motility into Group 2. It appeared

from these results that motility might be related to hyperactivation relatively well, but as seen

in Bull C, there is an exception with normal motility but low hyperactivation. The results

seemed in favor for the idea that Group 2 may include normal bulls because 1) Bull D and E

were classified into Group 2 and 2) in the previous Chapter, bulls of known subfertility were

classified into Group 3. However, because Bull B showed low hyperactivation but was

classified into Group 2, it was considered that the ability to undergo hyperactivation might be

independent from AR responsiveness.

In conclusion, acrosomal integrity by FITC-PNA staining and the ability to undergo

hyperactivation and the AR responsiveness may probably reflect different aspects of sperm

functions. It is expected that the information provided may serve as basic knowledge to

78

establish novel assessment methods to detect subfertile bulls with normal standard semen

analysis results.

79

Acknowledgements

First and foremost, I would like to thank my supervisor, Prof. Dr. Tetsuma Murase, D.V.M.,

Ph.D. from Laboratory of Veterinary Theriogenology, Gifu University, for the patient

guidance, encouragement and advice he has provided throughout my time as his student. His

challenges brought this work towards a completion. It is with his supervision that this work

came into existence. I would like to thank him for encouraging my research and for allowing

me to grow as a research scientist. His advice on both research as well as on my career have

been priceless.

I would like to express my appreciation to Associate Prof. Dr. Masaki Takasu for his

encouragement during my doctoral study.

I must express my gratitude to my primary assistant advisor Prof. Dr. Yasuo Inoshima,

D.V.M., Ph.D. Gifu University and my secondary assistant advisor Tomomi Tanaka, D.V.M.,

Ph.D. Tokyo University of Agriculture and Technology for their constant support, guidance

and motivation. I benefited greatly from many fruitful discussions with them.

This work would not have been possible without the financial support of Japan Ministry of

Education, Culture, Sports, Science and Technology (Monbukagakusho scholarship). I am

thankful to them for supporting me to pursue my studies here in Japan. I would like to express

my sincere gratitude to Hida Beef Cattle Research Department, Gifu Prefectural Livestock

Research Institute, Japan, for donating of frozen straws of Japanese Black bull semen.

My appreciation and gratefulness to the friendly staff of the United Graduate School of

Veterinary Science, for their kind support. To my friends of the Theriogenology laboratory, I

thank them for their companionship and for providing a so pleasurable and friendly working

atmosphere. The moments of leisure shared together helped to overcome some more difficult

moments. I am also truly thankful for their always prompt help, whenever I needed it.

80

A special warm thank to Prof. Dr. Hiroshi Harayama from the University of Kobe. His

significant scientific comments and suggestions enhance this work.

Nobody has been more important to me in the pursuit of this project than the members of

my family. I would like to thank my parents, whose love and guidance are with me in whatever

I pursue. Most importantly, I wish to thank my loving and supportive wife, Fereshteh, who

provided unending inspiration. This thesis is heartily dedicated to her.

81

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