In vitro tests for essential sperm functions using the phyto-oestrogen genistein as a test substance

Sperm motility, binding of spermatozoa to the zona pellucida and induction of the acrosome reaction are prerequisites for successful oocyte fertilization. Examination of the physiological and nonphysiological effects of particular compounds on sperm functions requires high-quality in vitro test systems. In this short methodological overview, a reliable combined in vitro test system with bovine gametes is described. The purpose of the study was to evaluate whether aliquots of pooled post-thaw spermatozoa are suitable for examination of environmental substances that affect essential sperm functions. The combined test system includes a number of known methods for the assessment of sperm vitality and motion parameters, acrosomal status, inducibility of acrosome reaction and sperm zona pellucida binding. First observations indicate that genistein inhibits the induction of acrosomal exocytosis and binding of spermatozoa to the zona pellucida. Motility parameters and the viability of bovine spermatozoa were not affected by this substance. It is concluded that genistein, a phyto-oestrogen which is abundant in several plants, can be used as a test substance for the evaluation of effects upon essential bovine sperm functions in vitro.     

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.     

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P 相似文献   

Participation of superoxide anion in the capacitation of cryopreserved bovine sperm

Mammalian spermatozoa must undergo a preparation period known as capacitation to become capable of fertilizing oocytes. Controlled amounts of reactive oxygen species (ROS), such as superoxide anion (O2.-) and hydrogen peroxide (H2O2) have been shown essential for capacitation and acrosome reaction. The presence of an oxidase in the sperm plasma membrane has been suggested. The objective of the present study was to provide evidence for the production of O2.- by capacitating cryopreserved bovine spermatozoa. Percentages of capacitation and acrosome reaction were determined by the chlortetracycline assay. The effect of several nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors on capacitation was also studied. H2O2 production was determined by the fluorometric assay using the p-hydroxyphenylacetic acid-horseradish peroxidase system. Superoxide dismutase (SOD) activity was determined spectrophotometrically at 480 nm. Heparin-dependent capacitation was inhibited by all NADPH oxidase inhibitors tested (p < 0.05). Significant levels of H2O2 were produced during capacitation with heparin; such production was inhibited by diphenyleneiodonium, one of the NADPH oxidase inhibitors. The addition of catalase to the incubation medium failed to modify the capacitation rate; inhibition was only observed when SOD was present (p < 0.05). Endogenous SOD activity was diminished during heparin-dependent capacitation (p < 0.05). Similar levels of acrosome reaction induced by lysophosphatidylcholine were obtained in both heparin and O2.--dependent capacitation. Overall results suggest the participation of a sperm oxidase in bovine sperm capacitation. H2O2, generated by O2.- dismutation, failed to participate in capacitation, although this ROS may have been able to decrease endogenous SOD activity. Exogenous O2.- promotes physiological capacitation in cryopreserved bovine sperm, thus allowing the acquisition of fertilizing capacity.     

蛋白激酶C对精子活力和顶体反应的影响

蛋白激酶C(PKC)位于精子头部赤道段和尾部主段。外源性PKC激动剂可增强精子鞭毛的活力 ,而PKC抑制剂如癌基因抑活药 (staurosporine)则能抑制这种作用。精子中PKC的含量与精子活力呈显著正相关 (r =0 .9,P <0 .0 0 1)。精子与透明带 (ZP)结合刺激顶体反应的发生 ,导致顶体释放多种水解酶以及精子暴露出新的膜区域。ZP与精子质膜上的受体结合后 ,可以调节腺苷酸环化酶 (AC)的活性 ,使cAMP浓度升高并激活蛋白激酶A(PKA)。PKA能激活位于顶体外膜的电压依赖性钙通道 ,后者使顶体内部的Ca2 + 释放至胞质中 ,磷脂酶C(PLC)因Ca2 + 浓度升高而激活并水解磷脂酰肌醇二磷酸 ,其水解产物激活PKC ,后者使精子质膜上电压依赖性钙通道 (L型 )开放 ,胞质中第二次较大幅度Ca2 + 浓度的上升导致质膜溶解及顶体反应发生。推测PKC参与调节精子的活力和顶体反应     

Role of ions and ion channels in capacitation and acrosome reaction of spermatozoa

Capacitation and acrosome reaction are important prerequisites of the fertilization process. Capacitation is a highlycomplex phenomenon occurring in the female genital tract, rendering the spermatozoa capable of binding and fusionwith the oocyte. During capacitation various biochemical and biophysical changes occur in the spermatozoa and thespermatozoal membranes. Ions and ion channels also play important roles in governing the process of capacitation bychanging the fluxes of different ions which in turn controls various characteristics of capacitated spermatozoa. Alongwith the mobilization of ions the generation of free radicals and efflux of cholesterol also plays an impo~.nt role in thecapacitation state of the spermatozoa. The generation of free radical and efflux of cholesterol change the mechano-dynamic properties of the membrane by oxidation of the polyunsaturated lipids and by generating the cholesterol freepatches. The process of capacitation renders the spermatozoa responsive to the inducers of the acrosome reaction. Theglycoprotein zona pellucida 3 (ZP3) of the egg coat zona pellucida is the potent physiological stimulator of the acro-some reaction; progesterone, a major component of the follicular fluid, is also an inducer of the acrosome reaction.The inducers of the acrosome reaction cause the activation of the various ion-channels leading to high influxes of calci-um, sodium and bicarbonate. The efflux of cholesterol during the process of capacitation alters the permeability of themembrane to the ions and generate areas which are prone to fusion and ve.siculation process during the acrosome reactioa. this review focuses mainly on effects of the ion and ion-channels, free radicals, and membrane fluidity changesduring the process of capacitation and acrosome reaction.     

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.     

Modulation of human sperm function by follicular fluid

Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.     

Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Platelet-activating factor induces acrosome reaction via the activation of extracellular signal-regulated kinase in human spermatozoa

Platelet-activating factor (PAF) affects capacitation, acrosome reaction and fertilisation potential of spermatozoa. This study investigated the underlying mechanism(s) through which PAF regulated sperm function. Our data demonstrated that PAF dose-dependently induced, whilst lyso-PAF (PAF precursor) showed no effect on acrosome reaction of capacitated human spermatozoa. Treatment with PAF for 90 min enhanced tyrosine phosphorylation and expression of extracellular signal-regulated protein kinases (ERK) 1 and 2 in human spermatozoa. Moreover, pre-treatment with the ERK inhibitor U0126 significantly and dose-dependently suppressed PAF-induced acrosome reaction. Therefore, PAF may be actively involved in the modulation of sperm acrosome reaction by interacting with ERK. The role of PAF in fertilisation warrants further investigation.     

冻存后大鼠精子活力变化与其结构的关系

目的　观察冷冻对大鼠精子活动能力的影响 ,并探讨其活动能力的变化与细胞膜完整性、DNA结构、线粒体鞘和顶体的关系。　方法　使用计算机辅助精子活动分析仪 (CASA)检测冻存前后大鼠精子活动力的变化 ;荧光素乙酰乙酸盐 (FDA)染色法检测大鼠精子细胞膜的完整性 ;双氢鲁丹明荧光反应观察精子线粒体鞘的变化 ;金霉素 (CTC)荧光检测法观察冻存对大鼠精子顶体的影响。　结果　复苏后精子的活动力下降 ,为冻存前的 39.7% ;FDA检测显示冻存后的大鼠精子细胞膜完整 ,未受到破坏 ;冻存后的大鼠精子线粒体鞘荧光强度下降 ,且荧光不连续、甚至消失 ;CTC荧光反应显示冻存后精子顶体反应 (AR)的类型发生改变 ,AR型精子比例明显下降 ,由冻存前的 6 8.6 %降至冻存后的 13.4 %。　结论　冻存后大鼠精子细胞膜的完整性未受到破坏 ;精子的线粒体鞘和顶体受到较明显的破坏。大鼠精子冻存复苏后活力下降与线粒体鞘和AR存在相关性。     

碳酸氢盐在小鼠精子体外获能和顶体反应中的作用

检验了精子获能和透明带（ＺＰ）及孕酮激发顶体反应是否需要ＨＣＯ３／ＣＯ２。小鼠精子分别在改良的Ｔｙｒｏｄｅ（ｍＴ－Ｂ２５，含２５ｍｍｏｌ／ＬＨＣＯ３／ＣＯ２）或ｍＴ－Ｈ（无ＨＣＯ３Ｈ／ＣＯ２，含２０ｍｍｏｌ／ＬＨｅｐｅｓ）中预培养９０ｍｉｎ后，以２步７０％和３５％ｐｅｒｃｏｌｌ／ｍＴ－Ｈ洗涤精子，并将精子重新悬浮于ｍＴ－Ｂ２５，ｍＴ－Ｂ１５ｍＴ－ＢＨ和ｍＴ－Ｈ中，用ＩＺＰ／μｌ或１５μｍｏｌ／Ｌ孕酮激发精子顶体反应。在上述ｍＴ诸培养基中，“Ｂ”精子（以ＣＴＣ荧光染色法确定）发生率为６１％～６７％；顶体反应均可达到４１．０士１．４％～４８．０±１．４％。表示精子一旦在ＨＣＯ３／Ｃ０２环境中获能，顶体反应就可在无ＨＣＯ３／ＣＯ２环境中发生。然而，精子预先在ｍＴ－Ｈ中培养，与上述相同方法处理精子，“Ｂ”型精子（２２％～３３％）明显低于前者，对ＺＰ或孕酮的刺激不起反应（１４．Ｏ士３．６～２４．７士０．６％），甚至将精子重新悬浮于ｍＴ－Ｂ２５中也不发生反应（２２．０±９．５％），表示这些精子并未获能。上述结果表明小鼠精子获能依赖于ＨＣＯ３／ＣＯ２，但顶体反应则否。     

Participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml^?1) or forskolin (1–75 μm ), a well‐known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2′,5′‐dideoxyadenosine (6–25 μm ). Spermatozoa capacitated with forskolin (25 μm ) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25‐μm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2′,5′‐dideoxyadenosine prevented forskolin‐induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25‐μm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.     

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.     

Antibodies to human sperm YLP12 peptide that is involved in egg binding inhibit human sperm capacitation/acrosome reaction

Recently, the authors reported a novel dodecamer peptide sequence, designated as YLP12 on human sperm, that is involved in binding to zona pellucida (ZP) of human oocyte [10]. This unique sequence is present on the acrosomal region of the human sperm cell and is expressed only in human testis/ sperm. The aim of the present study was to examine whether YLP12 sequence is involved in capacitation/acrosome reaction. Swim-up sperm were capacitated with anti-YLP12 Fab' antibodies or control Fab's (40 and 85 microg/mL) and then the acrosome reaction was induced with calcium ionophore. An average of 64-73% sperm underwent acrosome reaction when they were capacitated in the presence of 40-85 microg/mL of bovine serum albumin or control Fab's. A significant (p < .01 to < .001) reduction (58-75%) in the percentage of acrosome-reacted sperm was observed when the sperm were capacitated in the presence of YLP12 Fab's. These data indicate that the YLP12 peptide sequence is involved in sperm capacitation / acrosome reaction, and may find clinical applications in the diagnosis and treatment of male infertility and immunocontraception.     

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding

Various signalling pathways are involved in the regulation of sperm motility, capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility. However, no study as of yet has been carried out to determine the effect of sperm treatment with the PI3K inhibitor LY294002 on other sperm parameters. In the present study, we investigated the role of PI3K on human sperm motility, acrosome reaction and sperm-oocyte binding by using this inhibitor. We demonstrate that in vitro incubation of washed unselected spermatozoa with LY294002 increased the percentage motility and progressive motility in asthenozoospermia patients as evaluated by computer-aided sperm analysis. The compound furthermore did not influence the acrosome reaction, whilst it (further) slightly enhanced sperm-oocyte binding. Our results therefore imply that PI3K negatively affects sperm motility and oocyte binding and might suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for asthenozoospermia.     

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell‐to‐cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl‐ and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA‐Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA‐Gal) binding activity was similar to that of BSA‐Fuc, but was weaker. In acrosome‐reacted spermatozoa treated with the Ca²⁺ ionophore A23187, BSA‐zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA‐Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml^?1 BSA‐Fuc, in vitro sperm–ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm–egg ZP interactions. At the same concentration, BSA‐Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm–ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.     

Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination

The objective of the present study was to confirm the presence of prematurely capacitated spermatozoa in frozen-thawed bull semen and to investigate the relationship of premature capacitation to the fertility of the respective semen. Twenty batches of frozen semen from young AI bulls of the Swedish Red and White breed with known fertility (expressed as 56-day non-return rates; 56 d-NRR) were tested using a Chlortetracycline (CTC) assay to assess capacitation status in frozen-thawed spermatozoa. The status of capacitation, as evidenced in this experiment, was further tested based on the hypothesis that capacitated spermatozoa present in frozen-thawed semen should undergo the acrosome reaction (AR) on co-incubation with homologous zona pellucida (ZP) glycoproteins. The percentage (mean +/- SEM) of uncapacitated, capacitated and acrosome-reacted spermatozoa in the frozen-thawed semen (n = 20) were 49.3 +/- 11.9, 36.3 +/- 8.3 and 14.2 +/- 11.9, respectively. On co-incubation with ZP, there was a significant increase (p = 0.001) in the proportion of spermatozoa undergoing the AR compared to the control with a concurrent decrease in the proportion of capacitated spermatozoa, suggesting that a proportion of capacitated spermatozoa were undergoing the AR. The proportion of viable, uncapacitated spermatozoa present in the frozen-thawed semen was correlated to the 56 d-NRR (n = 20, r = 0.5, p = 0.03). In conclusion, a proportion of spermatozoa in frozen-thawed semen was capacitated and the proportion of viable, uncapacitated spermatozoa present in semen was positively correlated to fertility.     

Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa

The aim of this study was to evaluate the effects of Ca2+, BSA, NaHCO3 and PVA on the capacitation-associated time-dependent increase in protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in hamster spermatozoa. Hamster spermatozoa when incubated in TALP, a medium that assists capacitation, showed a time-dependent increase in protein tyrosine phosphorylation that correlated with the capacitated state of the spermatozoa. An absence of Ca2+ or NaHCO3 in the capacitation medium delayed the phosphorylation of the proteins, but without both there was a significant decrease in the phosphorylation of the proteins throughout the period of capacitation. An absence of bovine serum albumin also caused a decrease in the phosphorylation of the proteins but this did not occur if polyvinyl alcohol was substituted for it in the medium. The percentage hyperactivation was not affected in the absence of bovine serum albumin if the medium contained polyvinyl alcohol. However, it was delayed in the absence of NaHCO3 and inhibited in the absence of Ca2+. The absence of NaHCO3 or bovine serum albumin had no effect on the acrosome reaction. These results show that hamster spermatozoa undergo capacitation-associated protein tyrosine phosphorylation similar to that of the spermatozoa of other mammals. However, hamster spermatozoa are unique in that the capacitation-associated protein tyrosine phosphorylation is not absolutely dependent on the presence of Ca2+ and NaHCO3. As far as we know, this study is the first to provide evidence that capacitation-associated protein tyrosine phosphorylation is linked to hyperactivation in hamster spermatozoa.     

Extracellular signal-regulated kinase activation involved in human sperm-zona pellucida binding

In a previous study involving the inhibition the mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), we found that the very specific MAPK kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced acrosome reaction. As an intact acrosome on the spermatozoa is a prerequisite in ensuring tight binding to the ZP, we investigated the zona binding potential of spermatozoa after PD098059 treatment of sperm, followed by exposure to solubilised human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa, exposed to solubilised ZP, bound significantly more to the ZP, as compared to control spermatozoa also exposed to solubilised ZP (26.5 +/- 3.7 vs. 13.8 +/- 2.8, P < 0.05). No significant differences in binding to the ZP were observed between PD098059 treated and untreated sperm populations after A23187 exposure. These results can be interpreted to support the idea that the ZP-induced AR is the physiologically relevant exocytotic event, as it is the ZP-induced AR, and not the spontaneous (culture medium) or A23187 induced AR, that appears to be mediated through an ERK-mediated signal transduction process.