γ-氨基丁酸诱发人精子顶体反应及其受精能力

本文的目的是为了探讨γ－氨基丁酸是否可激发人精子顶体反应,受主其可能的作用方式。实验采用金霉素荧光染色法,胞内游离Ｃ６２＋测定和精子穿透去透明带仓鼠卵试验,分别评价ＧＡＢＡ诱发人精子ＡＲ及其穿卵能力。结果表明;ＧＡＢＡ可诱发人获能精子发生ＡＲ,且随精子获得进程而显著增加,并存在明显的量效关系,该作用可被Ｐ４加强。     

Bicarbonate: carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reactions

The bicarbonate: CO2 (HCO3-:CO2) concentration dependencies of hamster sperm motility, spontaneous acrosome reactions, and zona penetration (used to assay the zona-induced acrosome reaction) were examined. A cross-over experimental design was used to segregate effects on early stages of capacitation, spanning the first 5 h of incubation, from those on acrosome reactions and zona penetration during the last 1 h. After 5 h, HCO3-:CO2 concentrations were increased, decreased, or kept the same for 1 h. Compared to no HCO3-:CO2, as little as 2.9 mM: 0.6% HCO3-:CO2 increased the sperm motility index (MI) by 2.7-3.6 times. When HCO3-:CO2 was continuously present, both progressive and hyperactivated motility were stimulated by HCO3-:CO2 in a dose-dependent manner by 3-4 h, well before completion of capacitation. Stimulation of acrosome reactions or zona penetration, by addition of HCO3-:CO2 to sperm for 1 h late in capacitation, depended mainly on levels of HCO3-:CO2 present earlier in capacitation. When 25 mM: 5% HCO3-:CO2 was added only at 5 h, responses were significantly lower than with sperm treated continuously with the same concentration of HCO3-:CO2, being 2.5 times lower for MI, 2 times lower for acrosome reactions, and 6.3 times lower for zona penetration. In contrast, decreasing HCO3-:CO2 to suboptimal levels after 5 h did not decrease any 6-h sperm responses significantly. The average maximal and one-half maximal preincubation HCO3- concentrations for all responses were 34.2 +/- 1.0 and 9.2 +/- 0.3 mM, respectively. Zona penetration and hyperactivation were highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation-related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5′-nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.     

Both taurine and albumin support mouse sperm motility and fertilizing ability in vitro but there is no obligatory requirement for taurine

When mouse spermatozoa were washed immediately upon release from the epididymis, preincubated for up to 120 min in PVA-containing, albumin-free medium and assessed for their ability to fertilize cumulus-intact eggs in vitro, they were poorly fertile in comparison with their unwashed counterparts in the same medium. Fertilizing ability could be significantly improved by introducing taurine or albumin or by washing a second time at the end of preincubation. The most effective treatment was provided by the continuous presence of low concentrations (0.05-0.1 mg/ml) of BSA, similar to the amount of albumin detected in the supernatants removed during washing. There was no evidence that acrosome loss was inhibited by washing; rather, it was enhanced by the removal of a surface component which inhibits the acrosome reaction. The presence of taurine did not further increase this response. Motility, reduced in washed suspensions, was improved by the presence of taurine or albumin and experimental results suggest that this was a major factor in the improvement of fertilizing ability after introduction of these compounds. Although taurine, hypotaurine and albumin were all found in the sperm washings and thus would be present in unwashed, fertile samples, low concentrations of albumin were able to maintain full fertilizing ability. Therefore, unlike hamster spermatozoa, mouse spermatozoa would not appear to have an obligatory requirement for a motility stimulating factor such as taurine.     

Stimulating effect of pyroglutamylglutamylprolineamide,a prostatic,TRH-related tripeptide,on mouse sperm capacitation and fertilizing ability in vitro

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin-releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome-intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome-intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide-treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley-Liss, Inc.     

Evidence for the function of hyperactivated motility in sperm

After insemination, mammalian sperm undergo a striking change in flagellar beat pattern, termed hyperactivation. In low-viscosity culture medium, nonhyperactivated sperm flagella generate relatively symmetrical, low-amplitude waves, while hyperactivated sperm flagella generate an asymetrical beating pattern that results in nonprogressive movement. Since sperm encounter highly viscous and viscoelastic fluids in the female reproductive tract, the progress of hyperactivated sperm was compared with that of nonhyperactivated and transitional sperm in media of increasing viscosity. Hamster sperm obtained from the caudal epididymis were incubated in a medium that promotes capacitation. After 0, 3, and 4 h of incubation, the majority of the sperm exhibited, respectively, activated, transitional, and hyperactivated motility. At each of these time points, aliquots of sperm were removed from incubation and added to solutions of 0, 5%, 10%, 20%, and 30% Ficoll in medium. Samples containing mostly hyperactivated sperm (4 h) maintained higher swimming and flagellar velocities and were able to generate greater forces in response to increased viscous loading than activated sperm (0 h). Transitional sperm (3 h) showed an intermediate response. The paths of hyperactivated sperm through solutions of 20% and 30% Ficoll were considerably straighter than those made through medium alone. This is the first demonstration that hyperactivation can confer a mechanical advantage upon sperm in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix.     

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.     

Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction

Although the protein tyrosine kinase (PTK) inhibitor, genistein, has been widely used to investigate the possible involvement of PTK during reproductive functions, it is unknown whether it modulates sperm calcium channel activity. In the present study, we recorded T-type calcium currents (I(Ca,T)) in mouse spermatogenic cells using whole-cell patch clamp and found that extracellular application of genistein reversibly decreased I(Ca,T) in a concentration-dependent manner (IC(50) approximately 22.7 microM). To determine whether TK activity is required for I(Ca,T) inhibition, we found that peroxovanadate, a tyrosine phosphatase inhibitor, was ineffective in preventing the inhibitory effect of genistein. Furthermore, intracellular perfusion of the cells with ATP-gamma-S also did not alter the inhibitory effect of genistein. To further reveal the direct inhibitory mechanism of genistein on I(Ca,T), we applied into the bath lavendustin A, a PTK inhibitor structurally unrelated to genistein, and found that the current amplitude remained unchanged. Moreover, daidzein, an inactive structural analog of genistein, robustly inhibited the currents. The inhibitory effect of genistein on T-type calcium channels was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Genistein was observed to decrease sperm motility and to significantly inhibit sperm acrosome reaction (AR) evoked by zona pellucida. Using transfected HEK293 cells system, only Cav3.1 and Cav3.2, instead of Cav3.3, channels were inhibited by genistein. Since T-type calcium channels are the key components in the male reproduction, such as in AR and sperm motility, our data suggest that this PTK-independent inhibition of genistein on I(Ca,T) might be involved in its anti-reproductive effects.     

Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability

The ability of samples of semen from individual male fowl to form the products of lipid peroxidation during 5 h aerobic incubation at 40 degrees C varied between 0 and 8 nmol malonaldehyde/10(9) spermatozoa. Formation of higher concentrations of malonaldehyde was associated with a partial or complete loss of fertilizing ability whilst the fertilizing ability of samples producing low or negligible concentrations of malonaldehyde remained unimpaired. The semen of birds which showed a tendency to form high concentrations of malonaldehyde was not readily identifiable as abnormal by assessment of sperm motility, morphology or ATP content. Nor was the loss of fertilizing ability during aerobic incubation associated with an obvious change in these characteristics.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction

Gamete recognition in the mouse is mediated by galactosyltransferase (GalTase) on the sperm surface, which binds to its appropriate glycoside substrate in the egg zona pellucida (Lopez, L. C., E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, 1985, J. Cell Biol., 101:1501-1510). GalTase has been localized by indirect immunofluorescence to the dorsal surface of the anterior sperm head overlying the intact acrosome. Sperm binding to the zona pellucida triggers induction of the acrosome reaction, an exocytotic event that results in vesiculation and release of the outer acrosomal and overlying plasma membranes. Consequently, we examined the fate of sperm surface GalTase after the acrosome reaction. Contrary to our expectations, surface GalTase is not lost during the acrosome reaction despite the loss of its membrane domain. Rather, double-label indirect immunofluorescence assays show that GalTase is redistributed to the lateral surface of the sperm, coincident with the acrosome reaction. This apparent redistribution of GalTase was confirmed by direct enzymatic assays, which show that 90% of sperm GalTase activity is retained during the acrosome reaction. No GalTase activity is detectable on plasma membrane vesicles released during the acrosome reaction. In contrast, removal of plasma membranes by nitrogen cavitation releases GalTase activity from the sperm surface, showing that GalTase redistribution requires a physiological acrosome reaction. The selective redistribution of GalTase to a new membrane domain from one that is lost during the acrosome reaction suggests that GalTase is repositioned for some additional function after initial sperm-zona binding.     

Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse

Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.     

Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro

Significant differences in the frequency of spontaneous and induced acrosome reactions (identified by electron microscopy) were detected between 5 individual sperm samples. Maximal stimulation of the acrosome reaction was achieved with ionophore A23187, no differences being detected between the two preincubation times used (5 and 15 h). The cumulus oophorus and follicular fluid caused a similar increase in the proportion of acrosome-reacted spermatozoa after 5 h of preincubation. It is concluded that specific products of the cumulus and/or granulosa cells may contribute to the acrosome reaction-inducing activity of human follicular fluid.     

氧离子辐射对体外人精子自化学发光,运动性,顶体反应和存活率的影响

Effects of 16O+6 ion irradiation with different doses on human sperm spontaneous chemiluminescence (SCL), motility, acrosome reaction (AR) and viability were examined. Spermatozoa were irradiated with 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, or 64 Gy 16O+6 ion beam at the energy of 3.17 MeV/u. After irradiation, samples were analyzed by SCL measurement at 1, 2 and 3 h of incubation; motility was determined by the transmembrane migration method within 2 h of incubation; the percentage of AR and viability was evaluated by the triple-stain technique at 3.5 h of incubation. The results showed: sperm SCL was significantly increased with irradiation doses and the lowest effective dose was 0.5 Gy; compared with controls, the transmembrane migration ratio of spermatozoa progressively elevated with irradiation doses at 0.5, 1, and 2 Gy; the percentage of sperm AR markedly increased in 0.5-4 Gy irradiation and the optimal dose was 2 Gy, and then significant decreased with further increase of irradiation doses; the viability had no significant change within 0.25-8 Gy, but was progressively decreased at 16, 32 and 64 Gy. These data suggested that heavy ion at low doses increased motility and AR, whereas had deleterious effects at higher doses, which are associated with free radical reactions induced by heavy ion irradiation.     

Extracellular adenosine 5'-triphosphate alters motility and improves the fertilizing capability of mouse sperm

Extracellular adenosine 5'-triphosphate (ATPe) treatment of human sperm has been implicated in improving in vitro fertilization (IVF) results. We used the mouse model to investigate mechanisms of action of ATPe on sperm. ATPe treatment significantly enhanced IVF success as indicated by both rate of pronuclear formation and percentage cleavage to the 2-cell stage. However, ATPe did not increase the percentage of sperm undergoing spontaneous acrosomal exocytosis nor change the pattern of protein tyrosine phosphorylation normally observed in capacitated sperm. ATPe altered sperm motility parameters; in particular, both noncapacitated and capacitated sperm swam faster and straighter. The percentage of hyperactivated sperm did not increase in capacitated ATPe-treated sperm compared to control sperm. ATPe induced a rapid increase in the level of intracellular calcium that was inhibited by two distinct P2 purinergic receptor inhibitors, confirming that these receptors have an ionotropic role in sperm function. The observed motility changes likely explain, in part, the improved fertilizing capability when ATPe-treated sperm were used in IVF procedures and suggest a mechanism by which ATPe treatment may be beneficial for artificial reproductive techniques.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

Effect of secretory particles in bovine seminal vesicle secretion on sperm motility and acrosome reaction

Particles found in bovine seminal vesicle secretion were enriched by centrifugation. They varied in size and morphology and contained Mg2+,Ca2+-activated ATPase, aminopeptidase A, alanyl aminopeptidase, gamma-glutamyl transpeptidase and dipeptidyl peptidase IV activities. Hyperactivation of sperm motility and the acrosome reaction were induced by these particles in epididymal spermatozoa suspended in a modified Ringer medium. The hyperactivation, analysed with a microscopic slide test, started within minutes of exposure to membrane particles and continued for 3-4 h, during which time spermatozoa underwent the acrosome reaction. Acrosome staining, phase-contrast microscopy and transmission electron microscopy revealed that the acrosome reaction started within 60 min at 37 degrees C and affected up to 80% of spermatozoa in 4 h. These membrane particles differed from those reported previously in other species in enzyme composition, function and organ of origin.     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.