EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA*

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5–6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity

Hamster epididymal spermatozoa were washed and preincubated at extremely low sperm concentrations (100/ml or less) in a culture medium containing naturally-occurring sperm motility-stimulating substances. These substances were partially purified "sperm motility factor" (SMF) derived from hamster adrenal glands and catecholamines (epinephrine or isoproterenol). After preincubation for three hours, a small number (5 or less) of washed, cumulus-free hamster eggs was added to each sperm suspension. Many of these eggs were undergoing fertilization when examined two to three hours later. Fertilization was accomplished in vitro at sperm:egg ratios approaching 1:1, a situation comparable to that believed to exist in vivo. It appears that this demonstration will considerably enhance the potential of in vitro fertilization studies for providing useful information on mammalian gamete interactions.     

ELECTRON MICROSCOPIC OBSERVATIONS OF GUINEA PIG SPERMATOZOA PENETRATING EGGS IN VITRO*

Guinea pig ovarian oocytes matured in vitro were inseminated in vitro with capacitated, acrosome-reacted spermatozoa and sperm penetration through the zona pellucida and into the egg cytoplasm were examined. Sperm heads passing through the zona pellucida had already lost all their acrosomal elements except for the inner acrosomal membrane and the equatorial segment. It was often observed that the texture of the zona material around the sperm head was distorted, giving the impression that the zona pellucida was parted, at least partially, by a shearing force produced by the sperm head advancing through the zona. When eggs were freed from their zonae pellucidae and inseminated, the acrosome-reacted spermatozoa immediately bound to the egg surfaces and began to fuse with the eggs; whereas the spermatozoa with intact acrosomes failed to do so. Fusion began between the egg plasma membrane and the sperm plasma membrane at the central region of the sperm head. The anterior half of the sperm head was engulfed by the egg in a phagocytic fashion, while its posterior half was incorporated into the egg by a fussion between egg and sperm plasma membranes. Incorporation of the sperm tail into the egg was achieved by fusion between the sperm and egg plasma membranes.     

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm–zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1^−/− animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1^−/− mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1^−/− sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1^+/+ and Crisp1^−/− sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1^−/− sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.     

SPECIES-SPECIFIC ADHESION OF SPERMATOZOA TO THE SURFACE OF FIXED EGGS IN SEA URCHINS

When the spermatozoa of sea urchins are added to eggs which have been fixed with glutaraldehyde and washed thoroughly, the spermatozoa swarm around the eggs and adhere to the egg surface. The mode of sperm adhesion to the fixed egg is assumed, on the evidence of electron-microscopical studies, to be the same as that of adhesion to the intact egg at the initial stage of normal fertilization. The spermatozoa and fixed eggs of five species of sea urchins were combined and heterologous crosses were studied. Species-specific adhesion of sperm to fixed eggs was clearly demonstrated. There is a direct relationship between the cross-fertilization of living gametes and the binding capacity of spermatozoa and fixed eggs in so far as the employed five species are concerned.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Reproductive Effects of Two Neonicotinoid Insecticides on Mouse Sperm Function and Early Embryonic Development In Vitro

Acetamiprid (ACE) and imidacloprid (IMI) are two major members in the family of neonicotinoid pesticides, which are synthesized with a higher selectivity to insects. The present study determined and compared in vitro effects of ACE, IMI and nicotine on mammalian reproduction by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 µM or 5 mM) on spermatozoa during capacitation was performed, and in vitro fertilization (IVF) process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 µM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF) process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (P<0.05), in an order of nicotine>IMI>ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

DOES BATEMAN'S PRINCIPLE APPLY TO BROADCAST-SPAWNING ORGANISMS? EGG TRAITS INFLUENCE IN SITU FERTILIZATION RATES AMONG CONGENERIC SEA URCHINS

Evolutionary biologists generally invoke male competition and female choice as mechanisms driving sexual selection. However, in broadcast-spawning organisms sperm may be limiting and females may compete, in the Darwinian sense, for increased mating success. In this study, I investigate how species differences in egg and sperm traits result in different patterns of fertilization among three closely related sea urchins (Strongylocentrotus purpuratus, S. franciscanus, and S. droebachiensis). Field studies demonstrate that all three species achieve similar percentages of eggs fertilized when eggs and sperm are released simultaneously. However, when sperm must disperse before encountering eggs, differences arise among species such that those with the smaller eggs and faster but shorter-lived sperm achieve relatively fewer fertilizations than do species with larger eggs and slower but longer-lived sperm. A field hybridization experiment, field estimates of sperm dispersal, correlations of egg size to field rates of fertilization, laboratory studies of fertilization kinetics, and a simulation model all suggest that it is attributes of the egg (probably egg size) that are responsible for the differences. These patterns of fertilization match the species' patterns of dispersion; species that do well only when sperm and eggs are released in close proximity are more aggregated, species that do relatively well when sperm and eggs are released farther apart are more dispersed. These results are consistent with the notion that eggs of different species are adapted to maximize reproductive success under different degrees of sperm limitation and suggest that male competition and female choice may not be an appropriate dichotomy in broadcast-spawning organisms.     

Sperm Penetration and in vitro Fertilization of the Egg of the House Cricket Acheta domesticus

Summary

A study of sperm penetration of the egg of the house cricket, Acheta domesticus, using a fluorescent microscope technique, showed that sperm penetration of the chorion, prior to fertilization, is not restricted to a specialised area of the egg surface, such as the micropyle. An acrosomal filament is seen on penetrating sperm. Polyspermy (multiple sperm attachment) is seen under normal conditions.

Eggs fertilized in vitro developed to the 4 day (pre-katatrepsis) stage, but did not undergo katatrepsis. Development was confirmed by cytogenetic studies. The percentage of eggs showing cleavage nuclei (i.e. initial development) was 59% after in vitro fertilization and 5% in ovarian eggs incubated in a hypotonic medium.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

Evidence for nonmuscle myosin in bovine ejaculated spermatozoa

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin-myosin function. An actin-stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized-actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K⁺-EDTA ATPase activity. The sperm actin-myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.     

Fertilization and Subsequent Development of Cattle Oocytes after Reduced Incubation with Spermatozoa

We studied the influence of the duration of the joint incubation of cattle oocytes and spermatozoa (18 versus 1 h) on fertilization, cleavage, and embryonic development in vitrountil the blastocyst stage. Spermatozoa of a bull of the Britanofrizskaya breed were used in the experiments. It was shown in the first experimental series that after a long-term incubation with the spermatozoa, the percentage of penetrated eggs increased 71.7 and 56.0% (p< 0.05) after 18-hour and 1-hour incubation, respectively. However, no differences were found in the number of normally fertilized eggs: 46.5 and 39.0%, respectively. In the second experimental series, no significant differences were found in either the number of cleaving embryos (41.2 and 32.2%, respectively) or the capacity of cleaving embryos to develop in vitrountil the blastocyst stage (21.7 and 15.8%, respectively). Thus, reduction in the time of the joint incubation of cattle gametes upon in vitrofertilization to 1 h did not reduce the number of normally fertilized eggs and did not affect their capacity for subsequent in vitrodevelopment.     

Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis

Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

A profile of fertilization in mammals

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.     

Vasopressin Effectively Suppresses Male Fertility

Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10⁻¹¹–10⁻⁵ M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10⁻⁸ and 10⁻⁵ M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca²⁺ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10⁻¹¹ M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research findings will enable further study to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.