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.     

Round-Headed Spermatozoa: A Model to study the Role of the Acrosome in early events of Gamete Interaction: Rundkopfspermatozoen: Ein Modell zum Studium der Rolle des Akrosoms

Gamete interactions in mouse involves at least two steps: the first is the interaction of a spermatozoa receptor located in the plasma membrane and ZP3, a zona pellucida (ZP) glycoprotein. ZP3 also can induce the acrosome reaction, making possible the second step: a closer interaction between ZP2 and an inner acrosomal membrane receptor. Our aim was to study gamete interaction in round-headed spermatozoa to determine at which functional level fertility is impaired. These spermatozoa are predominant in some infertile male and are characterized by the absence of acrosome; they also present an abnormal pattern of chromatin condensation. Human ZP and zona free hamster oocytes were used to study gamete interaction. No binding to ZP was observed either with light or electron microscopy. Our findings suggest that the presence of the acrosome could be necessary for the sorting and right organization of plasma membrane proteins. Round-headed spermatozoa could also present a general alteration of membrane protein synthesis. The lack of fusion with zona-free hamster oocytes may be explained by an altered reorganization of plasma membrane proteins in the post acrosomal region as a result of the absence of the acrosome reaction in round headed spermatozoa.     

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

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

Inhibition of sperm-zona pellucida binding by a 55 kDa pig sperm protein in vitro

This study was conducted to evaluate the role of a 55 kDa pig sperm protein on the oocytesperm binding process, and its location in situ. For this purpose, in vitro matured oocytes were incubated with isolated and purified protein, and incubated with capacitated spermatozoa. In addition, capacitated sperm were incubated with anti-55 kDa antiserum and later with mature oocytes. Immunolocalization assays were performed using non-capacitated, capacitated and acrosome reacted sperm, which were incubated independently with anti-55 kDa protein antibodies and analyzed under fluorescence light microscopy. The 55 kDa protein concentrations correlated negatively with the amounts of sperm bound to the zona pellucida (ZP); the presence of the anti-55 kDa protein totally inhibited this binding. The immunolocalization assays revealed that fluorescence was located preferentially at the apical edge of the head in capacitated sperm, but not in acrosome reacted sperm. It would appear that the 55 kDa protein binds specifically to the oocyte ZP, and that it may be responsible for primary gamete binding during 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.     

Acrosome-reacted human sperm in insemination medium do not bind to the zona pellucida of human oocytes

In the literature there is still confusion whether acrosome-reacted sperm in medium can initiate primary binding to human zona pellucida (ZP). The ability of acrosome-reacted sperm to bind to ZP in vitro can be deduced by measuring the acrosome reaction (AR) of ZP-bound sperm compared with sperm in medium after incubation under different conditions inhibiting the ZP-induced AR. Motile sperm from fertile men, normospermic men and infertile men diagnosed with disordered ZP-induced AR (DZPIAR) were selected by swim-up (2 x 10(6) in 1 mL medium) and incubated for 1-2 h with four oocytes from failed in vitro fertilization (IVF). The acrosome status of sperm was assessed using pisum sativum agglutinin labelled with fluorescein. The ZP-induced AR was inhibited in experiments using sperm from DZPIAR patients, hyper-osmotic medium (400 mOsm/kg) and medium containing soybean trypsin inhibitor (SBTI; 4 mg/mL). Pre-treatment with calcium ionophore was used to create a sperm population with elevated AR. In all experiments with factors inhibiting the ZP-induced AR, the AR was significantly lower for ZP-bound sperm compared with sperm in medium: DZPIAR patients 4% vs. 15%, hyper-osmotic medium 3% vs. 12%, SBTI 2% vs. 12% and SBTI 3% vs. 23% after treatment with calcium ionophore. In conclusion, acrosome-reacted sperm in vitro have significantly reduced, in fact probably zero ability to bind to the ZP.     

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 egg yolk medium on the acrosome reaction of human spermatozoa

Preincubation of human spermatozoa in an egg yolk medium (TESTY) at 5 C, followed by washing at 37 C by centrifugation and resuspension in a standard medium (BWW), enhanced the percentage of spermatozoa that underwent the acrosome reaction and increased sperm penetration into zona-free hamster oocytes, as compared with BWW treatment only. The difference in the occurrence of the acrosome reaction between the two treatment protocols was present whether the spermatozoa were incubated for 3 or for 18 h. The increase in acrosome reaction occurred only when spermatozoa were washed after TESTY treatment. Washing at 5 C was not as effective as washing at 37 C. No increased loss of acrosomes was observed when BWW-treated spermatozoa were subjected to the washing procedure. Ionophore A23187 stimulated the acrosome reaction of BWW-treated but not of TESTY-treated spermatozoa, whether or not they were washed before ionophore treatment. In the absence of egg yolk, the medium (TEST) caused only a small enhancement in the acrosome reaction as compared with BWW, but an increase occurred upon addition of ionophore A23187. We conclude that treatment with TESTY enhances the capacitation/acrosome reaction of human spermatozoa and that the removal of egg yolk after incubation, as well as the temperature shock, contribute to this effect.     

Evaluation and assessment of semen for IVF/ICSI

Evaluation and assessment of semen is very important for both diagnosis of male infertility and selection of patients for treatment with IVF or ICSI. In standard IVF, sperm function is essential for normal fertilization: sperm must be able to bind to zona pellucida (ZP), undergo the acrosome reaction and penetrate the ZP and fuse with the oolemma before fertilization takes place. In contrast, most sperm functions are not required for fertilization in ICSI since sperm bypass the ZP and oolemma by injection of a single sperm directly into cytoplasm of oocyte. Therefore, the clinical decision on treatment of patients with either IVF or ICSI is mostly dependent on results of sperm tests. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability since many patients with subtle sperm defects can not be detected. More advanced sperm function tests are required to detect sperm defects that may lead to failure of fertilization in standard IVF. In the last 15 years w     

Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility

A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia.     

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

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

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。     

碳酸氢盐在小鼠精子体外获能和顶体反应中的作用

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

Remodeling of the plasma membrane in preparation for sperm–egg recognition: roles of acrosomal proteins

The interaction of sperm with the egg''s extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step.     

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution.

The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.     

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

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

Oocyte Penetration and Acrosome Reactions of Human Spermatozoa I: Influence of Incubation Time and Medium Composition

Washed sperm suspensions were evaluated for their ability to penetrate zona-free hamster oocytes and to exhibit an acrosome reaction in vitro. The percentage of acrosome reactions (AR%) was found to increase with incubation time and increased bovine serum albumin (BSA) concentration. We also found, however, that a remarkably high number of live, unreacted spermatozoa persisted during prolonged incubation in sperm samples obtained from some fertile men. After 22 hrs incubation, the motility of the spermatozoa was higher when the medium was supplemented with 10% human cord serum instead of 3.0% BSA. Penetration rates in hamster oocytes were not significantly different with 10% serum or 3.0% BSA as medium-supplementation. The addition of human instead of bovine serum albumin did not apparently affect the oocyte penetration rate of the spermatozoa.     

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.     

Effect of divalent ions in acrosome reaction induced by glycosamineglycans in porcine spermatozoa

Magnesium, calcium, and zinc at the concentration of 10 microM are capable of inducing a "true" acrosome reaction in the pig spermatozoa judged by the criteria of the fusion of the acrosome and the plasmatic membrane at the anterior region or the sperm nucleus. The optimal percent of acrosome reaction reached by any of the ions tested as a whole was 50%. When glycosamineglycan sulfate (GAGs) plus 10 microM of Mg++, Ca++, or Zn++ was added, they reach to 70-80% of acrosome reaction. At the electrom microscope, thin sections taken from pig spermatozoa treated with ions, GAGs, or ion + GAGs under optimal experimental conditions revealed the same pattern of acrosomal reaction. Results suggest the important role that divalent cations play in general in the induction of the acrosome reaction and question the so-called essential role of calcium ions.