Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.     

Evaluation of the acrosome reaction in human spermatozoa: comparison of cytochemical and fluorescence techniques

The acrosome reaction, which is essential for fertilization, includes fusion and vesiculation of the plasma membrane with the outer acrosomal membrane of spermatozoa, thereby releasing the acrosomal content. Determination of the ability of spermatozoa to undergo the acrosome reaction has proved to be a useful parameter in evaluation of infertile patients. The objective of this study was to compare cytochemical techniques, such as double stain (Giemsa/trypan blue) and triple stain (Bismarck brown/rose bengal/trypan blue), with a fluorescence method using Pisum sativum agglutinin fluorescein conjugate and Hoechst dye N degrees 33258 (double fluorescence). Whereas the cytochemical methods are easy to perform in general laboratories, the fluorescence technique requires special and costly instrumentation. In semen obtained from fertile donors, spermatozoa were selected by the swim-up technique and the acrosome reaction was induced by incubation at low temperature. The percentages of vital and acrosome-reacted spermatozoa were determined after incubation at 4 degrees C and at room temperature. No statistically significant difference was found between double fluorescence (viability 86.3%, acrosome reaction 14.7%) and triple stain (viability 85.3%, acrosome reaction 17%) (P > 0.05). On the other hand, the double stain technique showed different values for viability (70.3%) and acrosome reaction (42.5%) (P < 0.05). In conclusion, triple stain yielded results similar to those obtained by the fluorescence technique in evaluating the acrosome reaction and can therefore easily be used in general or research laboratories.     

A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters

Acrosome reaction capacity was tested on semen samples from 53 fertile and 26 subfertile men. Preparations were divided into two aliquots after 3 or 24 hours of culture. One aliquot received 10 mumol/L calcium ionophore A23187 in dimethyl sulfoxide (DMSO) and the other received DMSO alone. Acrosome reactions were scored on ethanol-permeabilized smears using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum lectin. The following factors were analyzed: the spontaneous reaction rates (control); induced reaction rates (ionophore-challenged); and the difference between the two, being the proportion of spermatozoa in the population capable of reacting in response to calcium influx (acrosome reaction to ionophore challenge [ARIC]). While spontaneous reactions bore no relation to fertility, induced reactions and ARICs were significantly reduced or absent in subfertile men, indicating acrosomal dysfunction as a likely cause of fertilization failure. The test was shown to have a predictive value for fertility comparable to that of the hamster ovum sperm penetration assay and to be a simple and cost-effective addition to existing semenology.     

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.     

Differential activities of malate and isocitrate NAD(P)-dependent dehydrogenases are involved in the induction of capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm catabolic processes produce energy for capacitation and acrosome reaction induction required for oocyte fertilization. The aim was to determine metabolic enzymes' activities and their participation in the supply of energy and generation of the redox state to acquire fertilizing capacity. Capacitation was induced with heparin and quercetin, and the acrosome reaction with progesterone. Enzymatic activities were determined spectrophotometrically. The chlortetracycline and differential-interferential contrast microscopy/tryptan blue techniques were used to evaluate capacitation and acrosome reaction, acrosomal integrity and sperm viability respectively. A 2 : 1 and 3 : 1 ratio were obtained for isocitrate dehydrogenase (IDH)-NADP/NAD and malate dehydrogenase (MDH)-NADP/NAD activities respectively. MDH-NADP activity remained constant with different treatments, unlike MDH-NAD activity, which diminished with both capacitation inducers and in acrosome-reacted spermatozoa previously treated with heparin (P < 0.05). IDH-NADP decreased its activity 50% in spermatozoa capacitated with heparin and acrosome reacted with progesterone (P < 0.05). Capacitation and acrosome reaction processes induced with heparin and progesterone, respectively, involve a differential oxidative metabolism, with the participation of MDH-NAD(P) and IDH-NAD(P) enzymes, whose activities would be linked to the malate-aspartate, lactate-pyruvate and isocitrate cytosolic-mitochondrial shuttles. These enzymes play a major role in supplying reduction equivalents and/or energy required for capacitation and acrosome reaction in cryopreserved bovine spermatozoa.     

考马斯亮蓝染色法检测人精子形态和顶体反应

目的 :探讨考马斯亮蓝G2 5 0染色法在人精子畸形率、顶体完整率和顶体反应检测中的应用。　方法 :用瑞 吉染色法和考马斯亮蓝G2 5 0染色法分别对获能前后和孕酮诱导顶体反应后的人精子进行涂片染色 ,观察其形态 ,并计算精子畸形率、顶体完整率及顶体反应率。　结果 :瑞 吉染色法和考马斯亮蓝G2 5 0染色法在检测人精子畸形率和顶体完整率上差异无显著性 (P >0 .0 5 )。孕酮诱导顶体反应后的精子用考马斯亮蓝G2 5 0染色后呈现两种类型 :顶体区染成紫蓝色和不着色或染成淡紫色 ,后者随诱导时间延长而增加 ,可能代表了已发生顶体反应的精子。诱导 1h的顶体反应率为 (75 .1± 3.8) %。　结论 :考马斯亮蓝G2 5 0染色法可作为检测人精子形态和顶体反应的可靠而实用的方法     

Progesterone induces capacitation in human spermatozoa

Mammalian sperm acrosome reaction is a prerequisite for oocyte fertilization, and to date the mechanisms regulating this event are still unclear. Recent studies have demonstrated that human follicular fluid is able to induce an influx of extracellular calcium and acrosome reaction in capacitated spermatozoa. More recently these effects have been attributed to progesterone. The results of our study demonstrated that progesterone is able to trigger the capacitation, acrosome reaction, and fertilizability in human spermatozoa through a calcium mediated mechanism, suggesting a clinical use of this steroid in the treatment of spermatozoa for the different techniques of assisted fertilization.     

Measurement of induced acrosome reactions in human sperm using physiologic stimuli--relevance for the prediction of fertilization outcome

Fertilization failure following standard in vitro fertilization in couples with normozoospermic men is an as yet unexplained phenomenon. A wide range of gametic disorders as well as environmental factors might contribute to this pathologic condition. One crucial condition appears to be the inability of the spermatozoa to undergo the acrosome reaction (AR). A discriminative test to distinguish fertile from non-fertile spermatozoa would be of utmost interest. In a prospective study, semen samples from men with normal semen parameters and fertilization failure were compared with semen samples from men with normal semen parameters and normal fertilization as to their capacity to undergo the AR. AR was induced using calcium ionophore as well as the physiologic stimuli progesterone and prostaglandin E(1). Discriminance analyses were undertaken to help identify patients with probable fertilization failure. Our data show that in patients with fertilization failure, the capacity of spermatozoa to undergo induced AR is greatly reduced using both unphysiologic and physiologic stimuli. However, physiologic stimuli are more suitable to identify patients with fertilization failure. Using physiologic stimuli, a formula was established to identify patients likely to fail at fertilization.     

Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187

In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophore-treated group, the proportion of changes in live acrosome-intact and live acrosome-reacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.     

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。     

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.     

人精子顶体反应3种检测方法的比较与评价

目的:寻找简便和准确的精子顶体反应检测方法。方法:正常人精液标本液化后混合,分成6组,分别用金霉素染色法、考马斯亮蓝染色法和酸性磷酸酶检测法对孕酮诱导顶体反应前后的人精子进行形态学观察并且对顶体反应率进行数据统计。结果:经考马斯亮蓝和金霉素染色后,发生顶体反应的精子和没有发生顶体反应的精子均有明显的形态差异;经考马斯亮蓝染色、金霉素荧光染色和酸性磷酸酶测试法检验,孕酮诱导组与阴性对照组的精子顶体反应率均有明显差异(P<0.05)。结论:3种方法均可作为顶体反应的检测手段,但考马斯亮蓝染色更为简便和稳定。     

A new technique to evaluate the ability of cryoprotectors to prevent premature acrosome reaction in human spermatozoa

Acrosome reaction (AR) induced by low temperature has been used to evaluate sperm function; it correlates adequately with the fertilization percentages in vitro. In this study, the technique of AR induction by low temperature was used to evaluate the effect in the protection of the acrosome by cryopreservatives normally used in human semen cryopreservation. Donor sperm selected by use of the migration sedimentation technique was incubated in human tubal fluid medium, added to dimethyl sulphoxide 1 m, ethylene glycol 0.75 m, glycerol 1 m, incubated at 4 degrees C and 20 degrees C (as a control) for 18 h, and then for 3 h at 37 degrees C in a cell incubator. The AR was evaluated by triple stain in 100 viable spermatozoa. The effect of cryopreservatives on acrosome preservation in samples incubated for 18 h at 4 degrees C was as follows: 78% intact acrosome for glycerol, 77.8% intact acrosome for dimethyl sulphoxide and 96.2% intact acrosome for ethylene glycol (P < 0.0025 compared with glycerol and dimethyl-sulphoxide). The sperm samples incubated with cryopreservatives for 18 h at 20 degrees C did not show an increase in the percentage of AR in samples incubated with glycerol and ethylene glycol, while a significant variation was observed in the sample incubated with dimethyl sulphoxide (P < 0.001). Additional incubation for 3 h at 37 degrees C significantly increased the AR only in the sample incubated with glycerol (P < 0.001). Acrosome preservation is essential in the fertilization process and the evaluation of acrosome reaction induction by low temperature test was satisfactory. This test proves that ethylene glycol presents a greater protective effect on the acrosome preservation of human spermatozoa.     

Acrosomal status of human spermatozoa after follicular fluid or calcium ionophore challenge in relation to semen parameters and fertilizing capacity in vitro

Summary.  The proportion of spermatozoa that undergo spontaneous acrosome reaction in vitro is relatively low. The proportion can be enhanced by incubation with either biological inducers such as follicular fluid or chemicals like calcium ionophore. It has been suggested that improper acro-somal reaction may be a cause of fertilization failure in vitro. The objectives of the present study were to assess the acrosomal status of human sperm following follicular fluid or calcium ionophore treatment and to analyse the relationship between spontaneous and induced acrosome reaction and fertilization rates in vitro by standard in vitro fertilization (IVF) technology. In all, 53 semen samples (22 normal and 31 subnormal) were studied. The effect of calcium ionophore A 23187 and follicular fluid was assessed using the fluorescence activated cell sorter. IVF results were evaluated in relation to the acrosome status of the sperm samples. Our results demonstrate that the effect of follicular fluid on the acrosomal status correlated positively with the effect obtained by the calcium ionophore (Pearson's correlation r = 0.45). A significantly higher percentage of maximal acrosome change ( P <0.02) was found in cases where fertilization occurred (19/27), than in sperm samples that did not achieve fertilization in vitro (8/27). The present finding that follicular fluid induced acrosome reaction can serve as a predictive tool which is as good as the ionophore treatment for assessing IVF outcome, supports the use of this method for clinical purposes.     

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of ⁴⁵Ca²⁺ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of ⁴⁵Ca²⁺ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll® inhibited, influx of ⁴⁵Ca²⁺ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca²⁺ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.     

人精子顶体反应的检测及其与穿卵试验的相关性

选择正常生育男子（生育组）、输精管结扎再吻合（吻合组）、不育症患者（不育组）各２５例的精液进行人精子顶体蛋白酶及酸性磷酸酶测定,顶体组织化学三色染色和人精子穿透去透明带金黄仓鼠卵试验,比较不同生育条件下精子功能和生育能力的相关性。结果显示,精子获能后顶体反应率明显高于获能前（Ｐ＜０．０１）。生育组顶体反应率和穿透试验高于吻合组和不育组（Ｐ＜０．０１）。结果表明,精子穿卵试验结合精子顶体反应测定可作为客观评价人类精子受精能力的依据     

The meaning of sperm capacitation. A historical perspective

It should be recalled that sperm capacitation was originally defined in 1952 as some physiological changes of the spermatozoa in the female genital tract before they are capable of penetrating and fertilizing the eggs. It was found further that capacitation can be achieved outside the female tract, first in the presence of biological fluids, and then in the absence of biological fluids. Later on it was found that capacitated rabbit uterine spermatozoa still have acrosome and that the acrosome reaction of rabbit spermatozoa occurred in contact with eggs in the oviduct. Thus, several authors separated acrosome reaction from capacitation and considered capacitation as a preparation for the acrosome reaction, even though the titles of their articles still implied that capacitation included acrosome reaction. During the past 30 years we have found many membrane changes on the molecular and immunological level in spermatozoa that prepare them for physiological changes such as "hyperactivation," and morphological changes such as "the acrosome reaction." These events lead to more vigorous motility and to the release of various enzymes for the penetration of the egg. Undoubtedly, further study will reveal more molecular, physiological, and morphological changes in the mammalian spermatozoa before they are capable of fertilization. There are definite changes before hyperactivation and acrosome reaction, but these changes are parts of capacitation, if we prefer to keep its original meaning. It is proposed here that in order to save further confusion, capacitation of spermatozoa should be defined as originally proposed, that is, to include all the events that lead to the development of the capacity of mammalian spermatozoa to penetrate eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of the male factor to unexplained infertility: a review

The more exhaustive the evaluation of couples with unexplained infertility, the more likely is the opportunity for detecting the aetiological factor responsible for infertility. Transport of spermatozoa through the upper genital tract and their ability to fertilize the oocyte are two obscure areas for the conventional evaluation of infertility. Although research in the former area is limited, there is indirect evidence that impaired sperm transport could be one of the causes of infertility in some couples with otherwise unexplained infertility. On the other hand, the availability of sperm function tests and the correlation of their results with in-vitro fertilization rates have allowed the detection of a previously hidden male factor in couples with unexplained infertility. It has been demonstrated that couples suffering unexplained infertility have significantly lower in-vitro fertilization rates in comparison with patients with tubal problems. These results can be explained because several case control studies in patients with unexplained infertility have reported defects in capacitation and sperm motion characteristics, binding of the spermatozoa to the zona pellucida, acrosome reaction, acrosin activity of the spermatozoa, and the ability of the spermatozoa to penetrate zona-free hamster cocytes. These observations suggest that methods for assessing the fertilizing capacity of the spermatozoa have to be incorporated in the evaluation of couples with unexplained infertility in order to amplify the scope of the workup and to better decide the appropiate treatment for these couples.     

Proteasomes in human spermatozoa

In the present study we describe the localization of proteasomes in human spermatozoa by means of immunolabelling with different monoclonal and polyclonal antibodies detected by confocal microscopy. Western blotting confirmed the specificity of the antibodies and has shown that proteasomes are present in spermatozoa and in seminal fluid. In spermatozoa proteasomes are concentrated in the neck region where the centrioles are located. Some labelling was also detected at the periphery of the head, but no proteasomal antigens were detected in either the nucleus or associated with the flagellum. Proteasome inhibitors did not affect the motility of the spermatozoa, acrosome reaction nor zona binding. It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.     

Oocyte Penetration and Acrosome Reactions of Human Sperms II: Correlation with other Seminal Parameters

Washed sperm suspensions of fertile men and men consulting for infertility were evaluated for their ability to penetrate zona-free hamster eggs and for their ability to exhibit an acrosome reaction in vitro. Furthermore, the swelling of the spermatozoa under hypoosmotic conditions as indication of their membrane integrity was determined. In the group of fertile men and in the group of patients with normal spermiogram, significantly more acrosome reactions were observed than in the group of infertile men with abnormal spermiogram parameters (p less than 0.05). This difference was still more significant when men with a positive hamster penetration test (H.O.P. test) and men with a negative H.O.P. test were compared (p less than 0.005). However, within the groups the level of acrosome reactions after incubation appeared to be highly variable. In a second series of experiments, working with semen obtained during our in vitro fertilization program, we found that the fertilization of human ova does not seem to be dependent on a strong progress of the acrosome reaction. Finally, swelling of the spermatozoa in a hypoosmotic medium was weakly correlated (r = 0.70, p less than 0.01; n = 73) with trypan blue exclusion. No significant correlations with other semen parameters, hamster ova test included, were found.