Human synthetic peptide vaccine for contraception targeting sperm

A vaccine targeting sperm is an interesting approach to contraception. The acceptability of a sperm antigen for contraceptive vaccine development is contingent upon its sperm-specificity, surface expression for antibody binding, and involvement in fertilization. Sperm-zona pellucida (ZP) binding is a pivotal step and constitutes an attractive site for immunointerception. Using phage display technology, we have identified a novel dodecamer peptide sequence, designated as YLP(12), that is present on sperm, and is involved in ZP binding and sperm capacitation/acrosome reaction in man/mouse. Extensive search in the human and mouse genome sequence databases did not indicate a complete identity with the YLP(12) nucleotide sequence. It is peptide mimetic with sperm receptor carbohydrate mimicking action that is involved in oocyte binding. The sperm-specific YLP(12) sequence is also involved in involuntary immunoinfertility in men, indicating its autoantigenicity, sperm-specificity, and involvement in fertility/infertility in humans. Vaccination of female animals with synthetic YLP(12) peptide causes a long-term reversible contraception by raising a sperm-specific immune response. The contraceptive effect could also be completely reversed voluntarily at any time by immunoneutralization of antibodies using intravaginal administration of the peptide. Thus, the novel synthetic YLP(12) peptide is an attractive candidate for contraceptive vaccine development, and diagnosis and treatment of immunoinfertility in humans.     

Monoclonal antibody against human sperm-specific YLP12 peptide sequence involved in oocyte binding

The monoclonalantibody (mAb) against YLP12 peptide was raised and its immunobiological properties were examined. In the Western blot procedure, the YLP12 mAb recognized a specific protein band of approximately 50 +/- 5 kD in human sperm extract and approximately 72 +/- 5 kD in human testis extract. The myeloma Ig control did not recognize these specific protein bands. In the immunofluorescence studies, the YLP12 mAb, and not the myeloma Ig, predominantly reacted with the acrosome regions of methanol-fixed human sperm. In the acrosome reaction assay, the YLP12 mAb showed a significant (p < .001) and a concentration-dependent inhibition of acrosome reaction. The myeloma Ig did not affect the acrosome reaction. There was no apparent effect of antibodies on sperm motility. Thus, the monoclonal antibody, if humanized by genetic engineering technology, may provide a useful immunocontraceptive agent.     

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.     

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.     

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.     

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.     

NYD-SP27, a novel intrinsic decapacitation factor in sperm

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO₃⁻, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca²⁺ mobilization in sperm, which can be mimicked by the PLC inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.     

Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction.

The acrosome reaction of spermatozoa may be analogous to various somatic cell exocytotic events that incorporate cascade reactions. One such cascade system involves the hydrolysis of a membrane-bound phospholipid; generation of the intracellular second messenger, diacylglycerol; and activation of protein kinase C, followed by the phosphorylation of a number of intracellular proteins. Stimulators of protein kinase C, phorbol diesters and synthetic diacylglycerols, were evaluated to determine if this system functions in the human sperm acrosome reaction. Phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-didecanoate caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa. Conversely, an inactive phorbol diester had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. The synthetic diacylglycerols, 1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-dioleoyl-sn-glycerol caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa, and to a similar extent as the phorbol diesters. A nonactivating isomer of 1,2-dioleoyl-sn-glycerol, 1,3-diolein, had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. Protein kinase C activation is a diacylglycerol-dependent and Ca2(+)-dependent process, and stimulation of the acrosome reaction by 1,2-dioctanoyl-sn-glycerol required the presence of calcium ions in the capacitation medium. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), prevented the diacylglycerol-induced acrosome reaction (P less than 0.01). These results support the hypothesis that protein kinase C, via activation by the intracellular second messenger diacylglycerol, has a role in the human sperm acrosome reaction.     

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.     

Early penetration of human sperm through the vestments of human eggs in vitro

Penetration of human sperm through the vestments of human oocytes during the first 3 h after insemination was investigated to determine the time taken for sperm capacitation, which precedes the acrosome reaction and fertilization. Twelve oocytes from women who became pregnant by IVF, recovered at laparoscopy after appropriate stimulation, were examined by electron microscopy. Follicular development was controlled by administration of clomiphene citrate, hMG, and hCG. Oocytes were cultured in Whittingham's T6 medium for 7-14 h before insemination and were fixed 1-3 h after insemination with preincubated sperm obtained from fertile men. All oocytes had matured and eight were normally fertilized 2-3 h after insemination. The acrosome reaction had already begun 1 h after insemination when 10-30% of sperm had reacted. Sperm-oocyte membrane fusion occurred 2 h after insemination, and sperm decondensation and pronuclear formation were in progress 3 h after insemination. Variable numbers of sperm (30-60%) had reacted acrosomes after 2-3 h of insemination. Many sperm penetrating the cumulus were intact or had partially reacted acrosomes. Intact, partially and fully reacted sperm were found at the surface of the zona. Intact sperm were bound to the zona by their plasma membranes. Sperm penetrating the zona had reacting or reacted acrosomes exposing their inner acrosome membranes. Those approaching the perivitelline space had a persistent equatorial vestige of the acrosome with intact plasma membrane. The acrosome reaction involved multiple fusions of the sperm plasma and outer acrosome membranes, resulting in vesiculation. This study shows that human sperm could complete capacitation and initiate the acrosome reaction within 1 h of insemination in vitro.     

Effect of zinc on human sperm motility and the acrosome reaction

This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 microM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 microM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was greater than or equal to 100 microM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed.     

Changes in sperm phosphotyrosine proteins by human follicular fluid in mice

The effects of follicular fluid on the acrosome reaction (AR) and phosphorylation of tyrosine residues of the sperm proteins were examined in mouse epididymal sperm. Human follicular fluid (hFF) increased AR in the capacitated sperm. Genistein, a receptor tyrosine kinase (RTK) inhibitor, inhibited spontaneous AR. When the genistein was primed, hFF-induced AR was attenuated but the A23187-induced AR was not, suggesting that potentiation of AR by hFF attributed to the activation of RTK upstream the mobilization of Ca2+. Phosphotyrosine proteins of Mr 27 to 116 kDa were markedly increased in capacitated sperm but this increase was abrogated by genistein. hFF increased tyrosine phosphorylation of Mr 56 kDa protein with genistein sensitive manner, suggesting that 56 kDa phosphotyrosine protein might be involved in capacitation and AR by follicular fluid.     

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.     

Potassium increases intracellular calcium simulating progesterone action in human sperm

Progesterone (P) and zona pellucida are known to induce acrosome reaction in human sperm by increasing cytosolic calcium. High concentrations of potassium ions (K+) improve the rate of acrosome reaction in human sperm in vitro. This article determined whether the effect of K+ on the acrosome in human sperm is mediated by increasing intracellular calcium ([Ca2+]i). The effect of K+ on [Ca2+]i was examined by using Fura 2 as the fluorescent indicator. The effect of K+ and P on [Ca2+]i in sperm and the involvement of ion channels was compared. Motile sperm were collected by the swim-up method from semen of healthy volunteers and capacitated overnight in BWW containing 0.5% BSA. Incubation of capacitated sperm with different concentrations of potassium chloride (1.25-20 mM) resulted in dose-dependent increase in [Ca2+]i similar to that observed with P. The increase in [Ca2+]i by K+ and P was blocked by the addition of EGTA, a Ca2+ chelator. K+-induced change in [Ca2+] was not altered by the addition of dihydropyridine derivatives. The combined treatment of K+ (20 mM) and P (0.75 microg/mL) caused an additive effect on the increase in [Ca2+]i. It would appear that human sperm plasma membrane possess different Ca2+ channels responsive to P and K+.     

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.     

Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions

Summary.  The presence of components of the renin angiotensin system (RAS) and specific receptors of angiotensin II in the female and male reproductive tract supports the hypothesis that reproductive functions may be controlled by RAS. Therefore, the present study investigated the influence of ACE and angiotensins on sperm functions and the sperm–egg interaction.
The experiments did not indicate direct effects of ACE on the capacitation process or acrosome reaction. Release of ACE from human spermatozoa during capacitation was not related to their ability to undergo acrosome reaction after stimulation with ionophore. Therefore, ACE release does not seem to be a useful clinical marker for human sperm capacitation. However, decreased binding of human spermatozoa to the oolemma of zona-free hamster oocytes after inhibition of ACE by captopril indicates that kininase II is involved in sperm–egg interactions. In contrast to other studies, incubation with captopril had no influence on sperm binding to the zona pellucida. Because effects of ACE on sperm–egg interactions but not on capacitation or acrosome reaction were observed, several experiments were performed to study the influence of substrates and products on the acrosome reaction. Angiotensin II induced the acrosome reaction dose-dependently, whereas angiotensin I had no effect on the acrosome reaction. The effect of angiotensin II on acrosome reaction seems to be calcium-dependent and mediated by protein kinases. Since a specific type 2 angiotensin II receptor inhibits the acrosome reaction induced by angiotensin II, this subtype of receptors may be present at the surface of sperm heads. Another clue for the presence of type 2 receptors on human spermatozoa is the finding that pertussis toxin did not inhibit the angiotensin II induced acrosome reaction. In contrast to type 1 angiotensin II receptors, type 2 receptors are known to be G-protein independent.     

Lipid transfer activity in human follicular fluid: relation to human sperm capacitation

A potentially important event during sperm capacitation is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and high density lipoprotein have been proposed as lipid acceptors. The authors propose that lipid transfer may be involved in capacitation as a specific mediator in the sequence of events leading to sperm membrane cholesterol loss. We present the first direct evidence of lipid transfer activity (LTA) in human follicular fluid (HFF). The redistribution of 14C-cholesteryl ester among human plasma lipoproteins was used as a measure of LTA (% Transfer [%T]). The HFF was fractionated by S-300 gel filtration chromatography and assayed for LTA. Three peaks of activity were consistently eluted from the column. Each peak of LTA also stimulated human sperm to penetrate zona-free hamster oocytes after short capacitating incubations. The peak with highest LTA (12.75 +/- 1.11%T) with an Mr approximately 68,000, gave the greatest stimulation (penetration index, PI: 3.34 +/- 0.96 fold increase above control, n = 4). The HFF also showed a significant dose response for both LTA and PI, whereas bovine serum albumin did not. These results demonstrate the existence of LTA in HFF and suggest that a specific lipid transfer protein may have a role in human sperm capacitation or acrosome reaction.     

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.     

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.     

RhoGDIα在人睾丸、精子中表达及其与体外受精成功率相关性的初步分析

目的:观察Rho特异性的GDP解离抑制因子α(RhoGDIα)在人睾丸、精子中的表达及定位并比较RhoGDIα在正常生育男性和体外受精(IVF)不育患者精子中的表达差异。方法:通过免疫组化方法观察RhoGDIα在人睾丸中的定位;通过免疫荧光方法观察RhoGDIα在人精子获能前、获能后、顶体反应后的定位;收集正常男性精液标本(10例),高受精率(≥60%,12例)和低受精率(<60%,13例)的IVF不育患者的精液标本,Percoll细胞分离液分离精液标本,排除生精细胞和白细胞,分别通过免疫荧光和Western印迹方法,检测RhoGDIα的表达。结果:免疫组化结果显示RhoGDIα存在于人睾丸各级生精细胞中,并在长形精子细胞高表达。免疫荧光结果显示RhoGDIα在人精子的顶体和尾部有较强表达,并且随着获能的发生,在顶体上的表达减弱,当顶体反应发生后,顶体上的表达完全消失。Western印迹结果显示不易受精的IVF患者精子中RhoGDIα的表达(0.66±0.18)显著低于正常组(1.13±0.21)和易受精的IVF患者组(0.97±0.17)。结论:RhoGDIα定位于人精子的顶体和尾部,可能参与了精子运动,获能及顶体反应过程。RhoGDIα在受精率低下患者精子中表达显著降低,提示RhoGDIα可能成为一个新的男性不育的诊断指标,并且可能成为IVF供精选择的一个指标。