Evidence for the involvement of sperm angiotensin converting enzyme in fertilization

Recently it has been observed that ejaculated human sperm possess high angiotensin converting enzyme (ACE) activity and that this enzyme is released during the process of capacitation. This observation raises the possibility that ACE may be involved in the fertilization process. To verify this hypothesis, we tested the effects of a potent ACE inhibitor, Captopril, on acrosome reaction induced by capacitating medium (3.5% HSA-added BWW) and on ability of human capacitated spermatozoa to penetrate zona-free hamster oocytes. Addition of Captopril (100 nmol l-1) modified neither sperm motility nor viability at any time considered, but significantly reduced the acrosome reaction percentages of sperm incubated in capacitating medium. Furthermore, Captopril significantly reduced the percentage of penetrated oocytes. The mean penetration rates both in the absence and presence of Captopril were 65.5 +/- 4.9% and 26.9 +/- 2.3% (P less than 0.001) respectively. These findings provide evidence that sperm release of ACE during capacitation may have a physiological role in the regulation of the mechanisms that allow sperm acrosome reaction and thus fertilizability.     

Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine

Identification of norepinephrine (NE) within the microenvironment of the bovine oviduct suggests a potential role for catecholamines in the events surrounding fertilization. Previous studies have shown that the catecholamines capacitate and induce the acrosome reaction in spermatozoa from several species. The current project was undertaken to investigate the role of catecholamines in bovine sperm capacitation and the acrosome reaction. Freshly ejaculated bovine spermatozoa were incubated in NE (0-1000 ng/mL) and induced to acrosome-react with lysophosphatidylcholine (LPC). Additionally, spermatozoa capacitated with heparin were incubated with NE (0-1000 ng/mL) to assess its ability to induce the acrosome-reaction in capacitated spermatozoa. Concentrations of NE were chosen on the basis of physiological concentrations previously determined for bovine oviductal fluid. NE at concentrations of 10 and 20 ng/mL capacitated bovine spermatozoa after 2 hours of incubation. Additionally, spermatozoa incubated for 2 hours with heparin were induced to acrosome-react with 10 and 20 ng/mL NE. Interestingly, higher concentrations of NE inhibited both capacitation and the acrosome reaction. Incubating spermatozoa with dopamine or epinephrine did not result in capacitation or the acrosome reaction, suggesting that the action of NE was specific to that catecholamine. The ability of NE to capacitate or induce the acrosome reaction appears to be dependent on the presence of another membrane-destabilizing factor. Although adrenergic receptors have not been identified on spermatozoa from any species, the action of NE on spermatozoa may be a receptor-mediated event. This study suggests a possible function for oviductal catecholamines in sperm preparation prior to fertilization.     

Synchronous assay for human sperm capacitation and the acrosome reaction

A synchronous acrosome reaction system was established for human spermatozoa. Seminal plasma is removed from the spermatozoa by centrifugation and the washed spermatozoa are capacitated in a modified BWW medium (without exogenous substrates) containing 35 mg/ml human serum albumin for 3 h at 37 C. Subsequently, 10 microM ionophore A23187 (final concentration) is added, the mixture incubated for 15 min at 37 C and the percent acrosome reaction determined by a modified triple stain technique (trypan blue stain omitted). Since no significant decrease in sperm motility occurs during incubation or after ionophore treatment, a vital stain does not need to be employed, allowing the use of any acrosome detection technique. The average percentage of acrosome-rejected spermatozoa after ionophore treatment (40 +/- 10%) was about 2- to 3-fold higher than that seen without ionophore treatment. Ionophore treatment for 15 min failed to stimulate the acrosome reaction in spermatozoa incubated for less than 3 h. Additionally, the presence of substrates in the BWW medium, higher sperm numbers, increased ionophore concentrations or longer incubation periods did not enhance the induction of the acrosome reaction. Ionomycin, a more specific calcium ionophore than A23187, produced essentially the same results as A23187 but tended to decrease sperm motility. This synchronous acrosome reaction system for human spermatozoa is relatively simple and can be used to study the effect of modulators on capacitation and/or the acrosome reaction.     

Stimulation of capacitation and the acrosome reaction in ejaculated buffalo (Bubalus bubalis) sperm and the effects of a sperm motility factor

The conditions for stimulation of in vitro capacitation and the acrosome reaction of ejaculated buffalo sperm has been determined. Washed ejaculated sperm were successfully capacitated in BWW medium supplemented with bovine serum albumin (BSA) and a sperm motility factor(s) (SMF(s) isolated from the adrenal glands of rats. The acrosome reaction was induced in capacitated sperm by introducing Ca++ ions (final concentration 5 mM) into the medium. Supplementation of BWW medium with SMF(s) in the presence of BSA significantly increased the percentage of sperm showing capacitation and the acrosome reaction. SMF(s) also significantly increased the percentage motility, the percentage forward motility and maintained a higher percentage of live sperm in BWW medium under the conditions used in this study. The significance of the present findings is discussed.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Immunolocalization of heat shock protein 70 in bovine spermatozoa

Heat shock protein 70 (HSP70) is part of a superfamily of molecular chaperones, which protect cells from chemical and heat shock. The objectives of this study were to determine the presence of HSP70 in bovine spermatozoa and its subcellular localization during different stages of spermatogenesis. Analysis of sperm proteins by Western blotting using a monoclonal antibody to the inducible form of HSP70 revealed a single immunoreactive band with an estimated molecular weight of 70 kDa in samples from 18 of 18 bulls. Using immunofluorescence microscopy and the same antibody, HSP70 was localized to the cytoplasm of prophase spermatocytes and elongating spermatids, to cytoplasmic droplets of caput epididymal spermatozoa, and to cytoplasmic droplets, acrosome, post-acrosomal region and middle piece of corpus and cauda epididymal spermatozoa. The pattern of distribution changed in freshly ejaculated spermatozoa as HSP70 was detected on the acrosome only. During capacitation and acrosome reaction, HSP70 was once again redistributed, and was localized to the equatorial segment, post-acrosomal region and middle piece. Thus, HSP70 is present in the spermatozoa of mature bulls and redistribution of the protein occurs during capacitation and the acrosome reaction.     

Acrosome reaction in dog sperm is induced by a membrane-localized progesterone receptor.

The aim of this study was to investigate whether the dog sperm acrosome reaction can be induced by progesterone and whether the action of progesterone is mediated by binding of progesterone to a receptor on the sperm plasma. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Ten mM progesterone increased the acrosome reaction in viable spermatozoa over time from 3 +/- 1% at 0 hours to 69 +/- 8% at 6 hours (six dogs). In freshly ejaculated sperm from six dogs, P-BSA-FITC staining was observed in 13 +/- 1% of the viable, acrosome-intact cells, as characterized by bright fluorescence over the entire apical region. The proportion of P-BSA-FITC-stained, viable, acrosome-intact cells increased to 84 +/- 11% following 7 hours incubation in a low-calcium medium. In contrast, the majority (72 +/- 3%) of fresh epididymal sperm already demonstrated bright P-BSA-FITC staining. Apparently, epididymal spermatozoa already possess the progesterone receptor. The receptor is masked at ejaculation and subsequently gradually exposed.     

Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa

Leptin is a polypeptide hormone with important roles in reproduction. It has been detected in human seminal plasma as well as on human ejaculated spermatozoa. This study aimed at studying the possible role of leptin in regulating human sperm functions. Immunofluorescent staining was used to study the expression of leptin and its receptor. The correlation between the concentration of leptin and soluble leptin receptor (ObRs) in seminal plasma as measured by enzyme-linked immunosorbant assay and sperm motility parameters measured by computer-assisted sperm analyais (CASA) was determined. The effects of recombinant leptin on human sperm motility, capacitation and acrosome reaction as measured by chlortetracycline staing were also studied. Leptin immunoreactivity was demonstrated at the equatorial and neck regions of human spermatozoa, whereas that of ObRs was shown up on the tail. After Percoll separation, spermatozoa with high density had more intense leptin immunoreactivity compared with those with low density. No significant correlation was found between seminal plasma concentration of leptin/ObRs and sperm motility parameters. After incubation with recombinant human leptin for either 3 h or overnight, there was no change in all the CASA motility parameters determined and percentages of capacitated and acrosome-reacted spermatozoa. We concluded that leptin does not have a significant effect on motility and capacitation/acrosome reaction in human ejaculated mature spermatozoa. Its role in male reproduction is yet to be determined.     

Epididymal protein CRISP1 plays different roles during the fertilization process

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.     

Effect of two complex platinum salts on human sperm motility and acrosome reaction

Hydrogen hexachloroplatinate, H2PtCl6, has been shown to induce the human sperm acrosome reaction in vitro. However, the molecular mechanism underlying this exocytic process has not been studied. Therefore, two structurally and chemically different platinum (Pt) compounds, the potent sensitizer sodium-hexachloro-platinate-(IV), Na2[PtCl6], and the nonimmunogenic tetraamineplatinum-(II)-chloride, [Pt(NH3)4]Cl2, were selected for the experiments. Their effects on human sperm function and second messenger pathways were investigated. Washed human spermatozoa were treated with different concentrations of both Pt salts (0.5-1000 microM) during or after capacitation for 3 h at 37 degrees C. In addition, spermatozoa were incubated with Pt salts in calcium-free medium or in the presence of the protein kinase A+C inhibitor H7. Sperm motility was evaluated by computer-assisted sperm analysis; acrosomal loss was detected by triple staining. Compared with the controls (6.6+/-2.4%), the percentages of living acrosome-reacted spermatozoa showed a significant dose-dependent increase (P<0.001) after 3 h of incubation with Na2[PtCl6] (7.9+/-4.2% for 0.5 microM 25.0+/-2.9% for 1 mM) and [Pt(NH3)4]Cl2 (7.9+/-3.9% to 21.0+/-5.8%). Sperm motility was markedly reduced in samples containing the highest concentrations of the Pt salts. The acrosome reaction was also significantly increased when spermatozoa had first been capacitated and then treated with both Pt salts. Calcium-free medium had no effect on the ability of both Pt salts to induce the acrosome reaction. However, incubation of Na2[PtCl6] in the presence of H7 tendentiously decreased the percentage of acrosome-reacted spermatozoa. In conclusion, complex Pt salts such as Na2[PtCl6] or [Pt(NH3)4]Cl2 influence human sperm functions by inducing the acrosome reaction during or after capacitation. This stimulatory effect is independent of calcium and seems to be dependent on protein kinase A or C.     

Induction of the acrosome reaction in bull spermatozoa with plasmin

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.     

Effect of spermine on sperm capacitation of guinea pig in vitro.

Spermine (Sp) 10(-5) mM had vigorous activity of guinea pig spermatozoa, while it completely abolished sperm forward motility (SFM) at a concentration of 10(-3) mM. There appeared to be a dose relationship to inhibition to motility. 2-Difluoromethylornithine 10 mM antagonized the Sp-induced inhibition of SFM after 3 h of incubation. Capacitation of a guinea pig sperm was inhibited by Sp in a concentration-dependent manner. The majority of acrosome-reacted sperm did not display hyperactivated motility. Precapacitated sperm were able to undergo the acrosome reaction (AR) in the presence of Sp. Moreover, Sp-mediated inhibition of capacitation was a reversible process. Once sperm capacitation was completed, Sp no longer inhibited AR. Before capacitation, the content of Sp in spermatozoa was 4.5 +/- 0.5 micrograms/5 x 10(7) cells, whereas in case of capacitated spermatozoa it was significantly decreased (2.1 +/- 0.4 micrograms/5 x 10(7) cells). The penetration of spermatozoa into the zona-free hamster eggs in the presence of Sp was markedly decreased, but it did not affect the fertilizability of ova as compared to the control. These results suggest that Sp may be an inhibitory agent of sperm capacitation in guinea pig in vitro, and it may also be involved in the modulation of capacitation.     

Mobility shift assay of calcium-binding proteins of mouse epididymal spermatozoa

The calcium-binding proteins (CBPs) of mouse epididymal spermatozoa were analysed by mobility changes in the presence of added Ca2+ in two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The expression patterns of relatively high molecular weight CBPs (Mr > 20 kDa) were different between caput and cauda epididymal spermatozoa. There was a constitutive expression of low molecular weight CBPs (Mr < 20 kDa) regardless of the epididymal region. Most of the CBPs disappeared after the acrosome reaction (AR) induced by Ca2+ ionophore A23187, suggesting that they originated from the acrosome and/or the plasma membrane overlaying the acrosome. Taken together, it can be suggested that changes in CBPs of spermatozoa are important features of sperm maturation during epididymal transit, and that they may be related to the fertilizing ability of mouse epididymal spermatozoa.     

No change in acrosome reaction of human spermatozoa during storage in cervical mucus*

Summary. Incubation of human spermatozoa in capacitation media for 24 h results in a significant increase in acrosome reacted spermatozoa as compared to preincubation (baseline) levels. By contrast, sperm samples recovered from the cervix for as long as 72 h after coitus only showed baseline percentage of acrosome reacted spermatozoa. These results indicate that spermatozoa do not undergo the acrosome reaction during cervical storage and suggest that cervical mucus suppresses the spontaneous acrosome reaction of spermatozoa that become capacitated in the cervix.     

The meaning of sperm capacitation. A historical perspective

It should be recalled that sperm capacitation was originally defined in 1952 as some physiological changes of the spermatozoa in the female genital tract before they are capable of penetrating and fertilizing the eggs. It was found further that capacitation can be achieved outside the female tract, first in the presence of biological fluids, and then in the absence of biological fluids. Later on it was found that capacitated rabbit uterine spermatozoa still have acrosome and that the acrosome reaction of rabbit spermatozoa occurred in contact with eggs in the oviduct. Thus, several authors separated acrosome reaction from capacitation and considered capacitation as a preparation for the acrosome reaction, even though the titles of their articles still implied that capacitation included acrosome reaction. During the past 30 years we have found many membrane changes on the molecular and immunological level in spermatozoa that prepare them for physiological changes such as "hyperactivation," and morphological changes such as "the acrosome reaction." These events lead to more vigorous motility and to the release of various enzymes for the penetration of the egg. Undoubtedly, further study will reveal more molecular, physiological, and morphological changes in the mammalian spermatozoa before they are capable of fertilization. There are definite changes before hyperactivation and acrosome reaction, but these changes are parts of capacitation, if we prefer to keep its original meaning. It is proposed here that in order to save further confusion, capacitation of spermatozoa should be defined as originally proposed, that is, to include all the events that lead to the development of the capacity of mammalian spermatozoa to penetrate eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acrosome reaction inhibitor released during in vitro sperm capacitation

Mammalian spermatozoa fertilize only after capacitation. The removal of decapacitation factors that inhibit the acrosome reaction (AR) is one of the events taking place during capacitation. In this report, human sperm were capacitated by 18-h incubation in Biggers, Whitten & Whittingham medium (BWW) medium and the proteins, on release, were analysed. After gel filtration by high-performance liquid chromatography a main peak with an approximate native molecular weight of 130 kDa was recognized by an antinormal seminal plasma antibody. This fraction was able to inhibit the follicular fluid as well as the progesterone-induced AR, when added to capacitated spermatozoa. Additionally, it reacted with an antibody directed against seminal plasma from vasectomized donors but not with an antibody against epididymal proteins. The AR inhibitory activity was heat-denatured, could be partially destroyed when treated with proteases, and bound to Concanavalin-A and wheat germ lectins. These results suggest that during in vitro capacitation, human spermatozoa release a glycoproteic decapacitation factor produced by accessory sex glands.     

Effect of gossypol acetate on guinea pig epididymal spermatozoa in vivo and their susceptibility to capacitation in vitro

To determine the effects of gossypol acetate on guinea pig epididymal and vas deferens sperm maturity and in vivo susceptibility to in vitro capacitation and the acrosome reaction, we examined spermatozoa removed from 37 animals fed gossypol acetate (10-15 mg/kg/day) for 5 to 9 weeks, and 15 vegetable oil-fed, age-paired control animals. In gossypol-treated, reproductively immature guinea pigs, the number of spermatozoa in the epididymis was markedly reduced (P less than 0.01) compared to controls, whereas the presence of spermatids and spermatocytes increased in the epididymis with the duration of gossypol administration. In sexually mature guinea pigs (given 15 mg/kg/day for 5 weeks), the epididymal sperm survival and forward motility were decreased significantly (P less than 0.025 and P less than 0.01, respectively), although the density of mature spermatozoa was the same as in control animals. The percentage of induced acrosome reactions (26.4 +/- 12%) was almost three-fold lower than that of control animals (72.8 +/- 4.6%). Also, in 31.5 +/- 3.8% of spermatozoa from gossypol-treated animals, as compared to only 2.4 +/- 0.7% of controls, the cytoplasmic droplet failed to migrate to its proper position in the midpiece and was retained in the neck region. With a few exceptions, spermatozoa from both experimental and control groups had comparable patterns of freeze-fractured membrane differentiations. Susceptibility to the induced acrosome reactions and the position of the retained cytoplasmic droplet reversed within 3 weeks after the end of gossypol feeding. This study helps establish the suitability of the guinea pig for studies on gossypol-induced infertility.     

Activation of protein kinase A during human sperm capacitation and acrosome reaction

Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during sperm capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for capacitation and PKA activation and/or that the cAMP concentration measured in whole spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.     

Capacitation and the acrosome reaction in sperm from men with various semen profiles montored by a chlortetracycline flrorescence assay

Sperm obtained from groups of men with various semen profiles were incubated for 8 h in BWW medium containing human serum albumin to promote capacitation. Capacitation and the acrosome reaction were monitored by a chlortetracycline (CTC) fluorescence assay. Four distinct CTC patterns were observed on the sperm head. No significant difference was observed in the time-course curve of these CTC patterns in sperm obtained from normozoospermic, asthenozoospermic and oligozoospermic men. Spontaneous and A23187-induced acrosome reactions were also comparable in these groups. However, in sperm obtained from teratozoospermic and polyzoospermic men, the increase in CTC pattern associated with capacitation appeared slower and sluggish. In these two groups, the induced acrosome reaction was also significantly lower when compared to that in the other three groups of men. In polyzoospermia, the spontaneous acrosome reaction was significantly lower when compared to all the other groups. Fresh sperm would not undergo the acrosome reaction following A23187 treatment. The results of this study indicate sluggish (defective) capacitation and inability of capacitated sperm to undergo induced acrosome reaction in teratozoospermic and polyzoospermic men as evaluated by the CTC method.     

The Kinetic of Acrosome Reaction – an Additional Sperm Parameter?

Spermatozoa of six men with normozoospermia, oligozoospermia and teratozoospermia were capacitated in vitro in modified Tyrode's medium und evaluated for acrosome reaction using the recently described triple-stain technique. The resulting kinetics illustrate the different capacitation behaviour and ability of spermatozoa to undergo acrosome reaction. The data were completed by the movement characteristics of spermatozoa during the incubation period. The kinetic of acrosome reaction seems to be an important functional sperm parameter that may be useful to discriminate between fertile and infertile spermatozoa.