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.     

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction

Summary. Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml⁻¹ of pentoxifylline at 37 °C, 5% CO₂ for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 × 10⁶ cells ml⁻¹. One hundred microlitres of each sperm suspension was then deposited under oil and 30–40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.     

Influence of progesterone and GABAA receptor on calcium mobilization during human sperm acrosome reaction.

At fertilization, mammalian sperm has to undergo morphological changes of acrosome, namely acrosome reaction (AR), during which a dramatic increase of cytosolic calcium in consequence of extracellular calcium influx induces acrosomal exocytosis. It has been reported that progesterone is capable of inducing mammalian sperm AR. Several authors insisted that the agent, which was so far understood to bind with intracellular receptor, might act as an agonist against cell surface r-aminobutyric acid with type A (GABAA) receptor. The mode of action is, however, still in controversy. To investigate whether progesterone-induced AR is mediated by GABAA receptor, the present study examined pharmacologically the actions of progesterone on the morphological changes of acrosome and calcium mobilization during human sperm AR. Progesterone (15 microM) stimulated AR and increased cytosolic calcium, and the AR rate was further promoted by the coexistence of GABA (15 microM). Then these phenomena were suppressed by an antagonist of GABAA receptor (bicuculline, 10 microM), a blocker of GABAA receptor-coupled chloride channel (picrotoxin, 200 microM) and an antagonist of receptor-operated calcium channel (Lantan, 250 microM), respectively. These results indicated that the complex work of GABAA receptor-chloride channel and receptor operated calcium channel might participate in progesterone-induced AR and the transient increase of cytosolic calcium.     

Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs

Human spermatozoa were exposed to a monoclonal antibody (C11H), which recognizes sperm acrosin. The antibody was presented to the sperm during capacitation and/or insemination, and its effect on penetration was tested using zona-free hamster eggs. An inhibitory effect on penetration was observed when the antibody was present during insemination but not when it was included only in the capacitation medium. As judged by immunofluorescence microscopy, most of the sperm bound to the egg surface were devoid of acrosomal staining. Some of the bound sperm were stained at their equatorial segments. Sperm that had penetrated the ooplasm did not exhibit immunofluorescence.     

IgA抗精子抗体对精子顶体反应的影响

目的 探讨精浆中IgA抗精子抗体对人精子顶体反应的影响。方法 利用免疫珠法（IBT）筛选出IgA抗精子抗体阳性精浆标本同正常人精子孵育，以孕酮诱发精子顶体反应；以特异性荧光标记物．络合异硫氰酸荧光素的花生凝集素（FITC-PNA）标记精子顶体，通过流式细胞仪检测精子顶体完整性。结果 与IgA抗精子抗体阳性精浆孵育的精子，其孕酮诱发的顶体反应发生率明显低于正常精浆及精子培养液组（P〈0．01），正常精浆组及精子培养液组间无显著性差异（P〉0．05）；IgA抗精子抗体阳性精浆组、正常精浆组、精子培养液组自发顶体反应的发生率无显著性差异（P〉0．05）。结论 免疫性不育患者精浆中的IgA抗精子抗体可以明显抑制孕酮诱发的顶体反应的发生，可能是导致不育的原因之一。     

Zona pellucida mediated acrosome reaction and sperm morphology

Summary. Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per μl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoo-spermic (normal sperm morphology 5–14%; n = 25) and severely teratozoospermic (normal sperm morphology <4%; n = 4) groups. The mean percent normal sperm were 15.8 ± 0.9, 10.4 ± 0.7 and 2.7 ± 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (± SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% ± 0.9 and 19.0% ± 0.9 (P = 0.001), compared to 36.8% ± 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.     

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.     

Controlling the swimming speed of human sperm by varying the incubation temperature and its effect on cervical mucus penetration

The objective of the present experiments was to study the effect of sperm velocity as a single variable on the ability of sperm to penetrate cervical mucus in a modified Kremer test. Sperm incubated at 13, 22 and 37 degrees C exhibited progressive velocities of 25 +/- 1.7, 40 +/- 2.1 and 56 +/- 2.1 microns sec-1 (mean +/- SEM, n = 6) respectively, but the percentage of progressively motile sperm, their lateral head displacement and the viscoelastic properties of cervical mucus remained comparatively unchanged over this temperature range. The number of sperm which penetrated the mucus and the percentage of successful collisions were correlated strongly with the average velocity of the sperm population (r = 0.82 and r = 0.72 respectively). It is concluded that sperm velocity has an important influence on the penetration of cervical mucus because it governs the frequency of collisions with the mucus interface and is determined by the thrust generated by the flagellum which also determines the ability of the sperm to traverse the mucus interface.     

Usefulness of sperm penetration assay in fertility predictions

The competence of the sperm penetration assay (SPA) to predict male fertility, as determined by normal sperm morphology and the fertilizing potential, as shown by human in vitro fertilization (IVF), was investigated. A significant correlation was obtained between normal sperm morphology and the SPA (phi = 0.623). A weaker correlation was however obtained with human IVF (phi = 0.397). Notwithstanding this weak association, a positive SPA (greater than 10%) was highly predictive (95%) of human IVF success. In contrast, a negative SPA (less than or equal to 10%) was associated with a high rate of false-negatives (65%). The SPA does however warn that a male factor may be present, as the mean fertilization rate of this group of patients was markedly reduced. The preincubation period for the spermatozoa did not play a major role in the predictive ability of a SPA outcome.     

Influence of extended incubation time on Human sperm chromatin condensation,sperm DNA strand breaks and their effect on fertilisation rate

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double‐strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double‐strand breaks (r = ?0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.     

NO通过人精子顶体酶对顶体反应的影响

目的探讨NO通过精子顶体酶(acrosin)对项体反应(acrosome reaction,AR)的影响。方法采用BAEE/ADH法测定精子顶体酶的活性,以及通过FITC-PSA法检测精子顶体反应。结果NO供体SNP可诱导人精子表达顶体酶活性,同时促进入精子顶体反应；顶体酶抑制剂TLCK能抑制顶体酶活性,并可抑制SNP诱导的人精子顶体反应。结论NO可能通过调节人精子顶体酶的活性而诱导精子的顶体反应。     

Relationship between the sperm penetration assay and other tests of sperm function

Semen from 88 men of infertile couples and 33 fertile donors differed in seminal fluid analysis (sperm density and motility) (SFA) as well as in the penetration of hamster ova (SPA) and bovine cervical mucus (MPT). In the fertile group, significantly more subjects had adequate SFA, SPA, or MPT results than in the infertile group. When the two groups were subdivided into those with normal or those with abnormal SFA, no differences were noted in SPA, MPT, or postcoital test (PCT) scores. The SFA parameter most consistently reflected in the results of the SPA, MPT, and PCT was sperm density. This was most evident when the SFA was poor. The worst prognosticator of fertility was the SFA, with 30% of the fertile donors having an abnormal SFA. The worst prognosticator of infertility was the MPT, with 79% of the patients penetrating in the fertile range. The SPA was a significantly better predictor than either the SFA or MPT. SPA and MPT results were positively correlated only in the overall infertile group. The SPA, MPT, and PCT measure sperm qualities distinct from those revealed by the SFA, and from each other, and in combination provide the best assessment of fertility.     

体外诱导人精子顶体反应方法的比较

目的 比较人透明带糖蛋白、孕酮和钙离子载体A23187在体外诱导人精子顶体反应的效率和可行性.方法 通过精子上游法制备正常男性精子悬液,分别以人透明带糖蛋白、孕酮和钙离子载体A23187诱导精子发生顶体反应.随后,运用考马斯亮蓝染色判断精子顶体的状态,并统计精子的顶体反应率.结果 人透明带糖蛋白、孕酮和钙离子载体A23187三法诱导的人精子顶体反应率分别为:(32.45±6.92)%(n=11)、(42.41±19.19)%(n=22)和(68.24±8.85)%(n=43),经统计学分析各组间差异均有统计学意义(P<0.05).结论 三种方法 中,钙离子载体A23187诱导效果最好,且不同个体间差异较小;而孕酮诱导的精子顶体反应率在不同个体间差异较大.     

Relationship between the human sperm hypo-osmotic swelling test and sperm penetration assay

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay in the diagnosis of the infertile male. A good correlation between the HOS test and the sperm penetration assay (SPA) in fertile and normal semen samples was initially found, but subsequently, no significant correlation was demonstrated with fertile and infertile patients. To validate the potential clinical usefulness of the HOS test, we evaluated 92 ejaculates using the HOS test, SPA, and traditional semen parameters. The methodology originally described by Jeyendran et al (1984) was used for the HOS test. The SPA was performed by the original procedure using an 18-hour preincubation period, and for 28 ejaculates, a modified procedure using TEST-yolk buffer was performed. Values of 60% or more for the HOS and 1% or more for the SPA were considered positive, and less than 60% for HOS and 0% for SPA were considered negative when the standard SPA was performed. For the TEST-yolk buffered SPA, values of 20% or more were considered positive. The sensitivity of the HOS test was 87%, but the specificity was 36%. The association of the two tests over and above that expected by chance (Kappa) was only 0.23. Using logistic regression, both sperm count (P less than 0.001) and morphology (P less than 0.025) were significant predictors of the SPA classification, but the HOS test did not improve the predictive results (P greater than 0.50).(ABSTRACT TRUNCATED AT 250 WORDS)     

γ-氨基丁酸对抗精子抗体阳性患者精子顶体反应的影响及机制探讨

目的 研究γ-氨基丁酸(gamma-aminobutyric acid,GABA)对正常人类精子及抗精子抗体(AsAb)阳性精子顶体反应的影响及其机制。方法 两组各18例采用三色染色法检测精子顶体反应率。结果 GABA增加正常及AsAb阳性患者精子顶体反应率,与对照组比较,差异显著(P<0.01);GABA可使精子的Na~ —K~ —ATPase,Ca~(2 )—ATPase的活性增加(P<0.01;P<0.05);并且显著减少精子中的MDA含量(P<0.01),抑制氧自由基的产生。结论 GABA可明显提高正常人及AsAb阳性患者精子顶体反应。     

Induction of the acrosome reaction and zona-free hamster oocyte penetration by a bull with complete teratospermia versus a half brother with normal sperm.

A fertile bull producing normal sperm and a sterile half brother exhibiting 100% teratospermia were available to study an induced sperm acrosome reaction and oocyte penetration. Pedigree analysis indicated that this condition was inherited. Experiments were undertaken to study the induction of the acrosome reaction using dilaurylphosphatidylcholine (PC12) liposomes, because this procedure was previously established to be highly correlated with bull fertility. The sperm from each bull were incubated with several PC12 concentrations for varying time periods. The initial percentages of sperm from the sterile bull with intact, partially intact, and lost acrosomes were 67%, 18%, and 14%, respectively, vs 82%, 13%, and 5% for the fertile bull (P < .05). After incubation for 15 minutes with 50 microM PC12 liposomes the corresponding values were, respectively, 51%, 26%, and 19%; and 60%, 28%, and 12%. Thus, the differences after induction of the acrosome reaction, although significant (P < .05), were small. The number of sperm adhered to each oocyte averaged 22 and 10, respectively, for the fertile and sterile bulls, whereas 74% of the fertile bull sperm and only 11% of the sterile bull sperm penetrated oocytes. Mixing the sperm-oocyte complex during incubation and increasing the sperm concentration during incubation to compensate for differences in sperm motility did not markedly affect oocyte penetration by teratogenic sperm, which is consistent with this bull being sterile. In other studies, microinjection of this type of sperm was demonstrated to induce fertilization, so the consequences of using sperm with hereditary defects in assisted reproductive programs to overcome human male sterility may be a concern.     

Roles of tumor necrosis factor alpha on sperm acrosin activity and acrosome reaction

Aim: To study the roles of tumor necrosis factor alpha (TNF-a)on the sperm acrosin activity and acrosome reaction. Methods:The sperm acrosin activity was tested by the method of BAEE/ADH Unity and the acrosome reaction by the Triple-stain technique. Results: TNF-a decreased the sperm acrosin activityand acrosome reaction (P＜0.01, P＜0.01, respectively);     

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.     

壬基酚和镉离子在体外对小鼠精子顶体反应的影响

目的:研究壬基酚和镉离子在体外对小鼠精子顶体反应(AR)的影响。方法:从小鼠的输精管获得精子,体外培养使精子获能,加30μmol/L的A23187诱导精子顶体反应,然后使用不同浓度的壬基酚(10、20、30、60、100μmol/L),或者镉离子(500、2500、5000μmol/L)处理,对照组使用相应的载体溶剂处理。用FITC-PSA荧光染色法分析精子顶体反应。结果:当壬基酚浓度<30μmol/L时,小鼠精子顶体反应率与对照组比较没有显著差异(P>0.05),而当壬基酚浓度>60μmol/L时能够显著地抑制小鼠精子顶体反应发生率(P<0.01),并且观察到精子存活率随着壬基酚浓度增加而降低。与壬基酚作用不同,用镉离子对小鼠精子进行处理,在所选浓度内(500~5 000μmol/L)均对精子顶体反应无显著影响(P>0.05),且精子存活率与镉离子浓度变化无关。结论:壬基酚与镉离子对小鼠精子发生的作用是通过不同的途径来实现的,前者可以直接抑制顶体反应,而后者则与精子顶体反应无关。     

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.