Acrosome loss in human sperm incubated in vitro under capacitating conditions

Human sperm were incubated under capacitating conditions and, at selected points up to 24 h of incubation, motile cells were fixed and assessed with the electron microscope for the presence or absence of the acrosome. Two methods of sample preparation were compared. In the first, semen samples were washed, incubated and filtered through glass beads to select motile cells before fixation. In the second, motile sperm were allowed to swim up into medium; this produced greater than 95% motile cells which were then incubated and fixed as required. In both series of experiments a significant increase in acrosome loss with time was observed (P = 0.00013), although only 10.5% of cells had lost the acrosome after 24 h. It is concluded that overt acrosome loss occurs less frequently in human sperm than in those of commonly used laboratory animals.     

Acrosome reaction of spermatozoa with different morphology*

Summary. The acrosome reaction of human spermatozoa with different morphological disturbances was evaluated after application of the triple staining method. After triple staining, spermatozoa which display only a small or an undetectable acrosomal region in Papanicolaoustained semen smears, turn out to be unable to perform an acrosome reaction, whereas merely post-acrosomally hyperelongated spermatozoa show a normal ability for acrosome reaction.     

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.     

Acrosome reaction inhibitor released during in vitro sperm capacitation

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

硝普钢地冷冻复温人精子顶体反应的影响

本文目的是研究复温温度和硝普钠（SNP）对冷冻精子复温后成活率和顶体反应的影响和机制,结果表明：对冷冻精子37℃水浴复温效果优于41℃,高浓度SNP可减少精子成活率和顶体反应率,低浓度(0.2nmol/L)可促进精子成活率和顶体反应率,低浓度SNP可使Na^ -K^ -ATP酶活性增加,而对丙二醛（MDA）无影响。     

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.     

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

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

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.     

酸性磷酸酶检测评价人精子获能和顶体反应

目的 探讨酸性磷酸酶(ACP)检测评价人精子获能和顶体反应(AR)的可行性。方法 32名健康生育男性的精子悬液，于37℃下孵育lh获能，用孕酮在有无苯甲基磺酰氟(PMSF)下诱导AR15、30、45、60min，分别检测获能前后及AR各时间点的精子悬液上清中ACP活性，同时用CASA分析精子运动参数证实精子发生获能和AR。结果 精子获能前后的ACP活性无显著性差异；AR(有PMSF)各时间点的ACP活性显著高于获能前后及不加PMSF时的ACP活性；CASA分析证实，精子运动参数的改变与其发生获能和AR的运动特性一致。结论 ACP分析为一种简便易行、耗时少、能反映精子群体AR状态的较为客观的方法，但蛋白酶的潜在影响不容忽视。     

Effect of multiple centrifugations on the evaluation of the acrosome reaction in human spermatozoa

This study was undertaken to assess the effect of multiple centrifugations on the human acrosome reaction and in vitro fertilizing ability. Semen samples were obtained from sperm donors and from patients participating in our in vitro fertilization programme. Measured sperm samples were centrifuged twice (400xg for 5 min) before or after the induction of the acrosome reaction with calcium ionophore A23187 and before the insemination of oocytes. The samples obtained from the sperm donors were used to measure the acrosomal loss by means of indirect immunofluorescence (T6 monoclonal antibody) and the patient samples were used to assess fertilizing ability and cleavage. Samples centrifuged twice before the chemical induction of the acrosome reaction exhibited significantly elevated levels of acrosomal loss (mean = 46%) as compared to the controls (23%). However, the fertilization (P = 0.688) and cleavage (P = 0.187) rates were not significantly different between the controls and the centrifuged samples. The present study has shown that the centrifugation of motility enriched (swim-up) samples may also modify the acrosomal membrane complex, without compromising fertilizing potential.     

Comparative study of the kinetics of the acrosome reaction and survival of human spermatozoa in various media

The in-vitro kinetics of the acrosome reaction and the survival of human spermatozoa were studied under different capacitating conditions. Human preovulatory follicular fluid (FF), isotonic BWW (N-BWW) and hypertonic BWW (H-BWW) were tested. Motile sperm selected by migration in these media were examined after 1, 3, 5 and 22 h of incubation under 5% CO2. The kinetics of the reaction in the population of live, morphologically normal sperm was dependent on both the culture medium and time of incubation. In the first hour, the mean percentage of acrosome-reacted sperm in H-BWW and FF was significantly greater than in N-BWW. The proportion of reacted cells increased significantly after 3 h in N-BWW (P = 0.001), after 5 h in FF (P = 0.03) and after 22 h in H-BWW (P = 0.01). A significant decrease in sperm viability was registered at 3 and 22 h of incubation (P less than 0.002) in all media. These results demonstrate that both H-BWW and FF stimulate the acrosome reaction while survival is optimal in the latter.     

Absence of Acrosome Reaction in Polyzoospermia*

Summary: Using the recently described triple-stain technique the time course of the acrosome reaction in a group of patients with polyzoospermia was evaluated and was compared to that of a group of normozoospermic men and a group of men with proven fertility. Polyzoospermia (> 250 Mill, spermatozoa/ml) has been repeatedly associated with subfertility. This investigation shows that most of the spermatozoa of polyzoospermic men do not undergo the acrosome reaction in vitro. This renders the spermatozoa incapable to penetrate the outer investments of the oocyte which might be one of the reasons for the reduced fertility observed in this patient group. Zusammenfassung: Fehlen einer Akrosomreaktion in vitro bei Männern mit Polyzoospermie Der Verlauf der Akrosomreaktion in vitro bei einer Gruppe von Pa-tienten mit Polyzoospermie wurde mit Hilfe der kürzlich beschriebenen Triple-Stain-Technik lichtmikroskopisch untersucht und mit einer Gruppe von Männern mit Normo-zoospermie und einer Kontrollgruppe von Männern mit erwiesener Fertilität verglichen. Polyzoospermie (> 250 Mill. Spermatozoen/ml) geht gehäuft mit reduzierter Fertilität einher. Die folgenden Untersuchungen zeigen, daß bei dem überwiegenden Anteil der Spermatozoen bei Polyzoospermie in vitro keine Akrosomreaktion abläuft. Die damit ver-bundene eingeschränkte Fähigkeit der Spermatozoen, die äußeren Eihüllen zu penetrieren, wird als einer der Gründe für die reduzierte Fertilität bei dieser Patientengruppe diskutiert.     

ATP-Content and Kinetics of Acrosome Reaction in Human Spermatozoa: Influence of Various Culture Media and Incubation Time

The ATP content and the kinetics of acrosome reaction of isolated human spermatozoa were investigated during an in vitro culture period of 3 hours in 3 serum-enriched media commonly adopted in IVF and GIFT procedures (Earle solution, Ham F10 and Menezo B2). The ATP concentration in spermatozoa did not change between 1 and 3 hr of incubation and no differences were seen using the three media under investigation. The percentage of acrosome reactions significantly increased during culture, to a similar extent in the three media. The results suggest that each of the three media is equally well suitable for human sperm culture in preparation of IVF and GIFT procedures.     

A new method for evaluation of the acrosome reaction in viable human spermatozoa

The acrosome reaction of human spermatozoa was induced by changes of temperature. Spermatozoa were collected from fertile donors and a patient group, and selected by the "swim-up" method. The spermatozoa were treated in two different ways: Protocol I: 24 hours at room temperature followed by additional incubation at 37 degrees C for 3 hours (control), and protocol II: 24 hours at 4 degrees C followed by additional incubation at 37 degrees C for 3 hours. The acrosome reaction of the viable spermatozoa was evaluated by a new method utilizing indirect immunofluorescence with anti-outer acrosomal membrane antibodies and exposure to a hypo-osmotic medium. In fertile donors as well as in the patient group, significant induction of the acrosome reaction (20%) was evident after exposure to low temperature (4 degrees C). The spontaneous rate of acrosome reaction in the control group was below 7%.     

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.     

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。     

Paramagnetic beads coated with Pisum sativum agglutinin bind to human spermatozoa undergoing the acrosome reaction

Summary. For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction.
Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 μM A23187 (20.3±6.7% [mean±SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1±8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1±12.5%, n = 10). Bead binding was significantly reduced by preincubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with α-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.     

Relation of human sperm acrosin activity and fertilization in vitro

The aim of the study described here was to determine the possible contribution of the acrosin activity test to routine semen analysis in enhancing the precision of the prognosis of IVF success in a group of patients in which the contribution of the egg factor to infertility was ruled out (20 cases) compared to a control IVF group (39 cases). Semen analysis, acrosin activity and acrosome ultrastructure were determined for all semen samples. The group with high fertilization rates was comprised of normozoospermic patients while the group with low fertilization rates was comprised of astheno-teratozoospermic patients. The mean acrosin level of the positive IVF group was significantly higher than that of the negative group (51.7 ± 33.2 and 28.6 ±13.7, respectively). Two parameters: per cent motile spermatozoa and acrosin level, were found to have a significant positive correlation with subsequent successful IVF ( r = 0.36, P < 0.006; r = 0.37, P < 0.004, respectively); and agenesis of the acrosome was found to have a significant negative correlation ( r = -0.33, P < 0.01). The ability of these parameters to correctly predict fertilization success was 59%, with 5% false positive, among which 15.4% was predicted solely by the acrosin level (above 54 μIU 10⁶ cells⁻¹) and 23% solely by per cent motile spermatozoa (above 50%). Abnormalities of the acrosome ultrastructure did not contribute further to the correct classification. The apparent clinical benefit of the acrosin level test is discussed.     

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.     

Zona pellucida as physiological trigger for the induction of acrosome reaction

Summary.  To evaluate the kinetics of acrosome reaction, sperm samples from four fertile donors were prepared by swim-up and incubated with solutions of human zonae containing 0.1, 0.15, 0.3, 0.5 and 1.0 zonae μl^-1. After 20, 40 and 60 min of incubation at 37 °C, aliquots were taken for evaluation of the acrosomal status. The results showed a distinct time- and dose dependence of the acrosome reaction induced by solubilized zona proteins. After 60 min of incubation in 1.0 zonae μl^-1, about 80% of the spermatozoa showed signs of acrosomal loss; about 40% were completely acrosome-reacted. In addition, zona-bound sperm showed the same ratios of acrosome-reacted spermatozoa in control experiments. The velocity of acrosome reaction was calculated by means of a double-reciprocal plot being 2.0–2.5% min^-1 for completely reacted spermatozoa and those showing signs of acrosome reaction. However, both subgroups differed considerably in their constants of equilibrium (K = 2.0 ZP μl^-1 and K = 0.2 ZP μl^-1, respectively). In nonreacted and partly reacted spermatozoa results might indicate a disturbed course of acrosome reaction or possibly the existence of different subpopulations in respect of sperm competition.