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.     

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.     

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.     

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.     

First steps in the development of a functional assay for human sperm using pig oocytes

The use of mammalian oocytes to assess human sperm functionality could be a helpful tool with potential applications in clinical and research programs. In an attempt to develop the pig model, the aim of the present work was to study the interaction between human spermatozoa and pig oocytes at the zona pellucida (ZP), the oolemma, and the ooplasm levels. In vitro matured pig oocytes and human spermatozoa from fertile and low-fertility donors were employed. The induction of the acrosome reaction by the ZP, the ability of the sperm to penetrate the oocyte after coincubation, and the male pronuclear formation after ICSI were evaluated. Human spermatozoa can bind to pig ZP and undergo the acrosome reaction (15% to 58%, depending on the individual); they are not able to fuse with the oolemma but they can decondense and form a male pronucleus (40%-100%) when injected into pig oocytes. In conclusion, this study shows that pig oocytes can be a useful model to assess human sperm functionality.     

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.     

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.     

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.     

The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.     

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P 相似文献   

蛋白激酶C对精子活力和顶体反应的影响

蛋白激酶C(PKC)位于精子头部赤道段和尾部主段。外源性PKC激动剂可增强精子鞭毛的活力 ,而PKC抑制剂如癌基因抑活药 (staurosporine)则能抑制这种作用。精子中PKC的含量与精子活力呈显著正相关 (r =0 .9,P <0 .0 0 1)。精子与透明带 (ZP)结合刺激顶体反应的发生 ,导致顶体释放多种水解酶以及精子暴露出新的膜区域。ZP与精子质膜上的受体结合后 ,可以调节腺苷酸环化酶 (AC)的活性 ,使cAMP浓度升高并激活蛋白激酶A(PKA)。PKA能激活位于顶体外膜的电压依赖性钙通道 ,后者使顶体内部的Ca2 + 释放至胞质中 ,磷脂酶C(PLC)因Ca2 + 浓度升高而激活并水解磷脂酰肌醇二磷酸 ,其水解产物激活PKC ,后者使精子质膜上电压依赖性钙通道 (L型 )开放 ,胞质中第二次较大幅度Ca2 + 浓度的上升导致质膜溶解及顶体反应发生。推测PKC参与调节精子的活力和顶体反应     

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.     

The zona pellucida-induced acrosome reaction of human spermatozoa involves extracellular signal-regulated kinase activation

Extracellular signal-regulated kinases (ERKs), belonging to the family of mitogen-activated protein kinases (MAPKs), are cytoplasmic and nuclear serine/threonine kinases involved in the signal transduction of several extracellular effectors. Recent evidence indicates the presence of p21 Ras and the phosphorylation of ERK1 and ERK2, suggesting the occurrence of the Ras/ERK cascade in mammalian spermatozoa. The present article describes the biological role of ERK during the acrosome reaction of human spermatozoa on stimulation with zona pellucida (ZP). The mitogen-activated protein-kinase inhibitor PD098059 was used as a pharmacological tool to study the involvement of extracellular signal-regulated kinases in the induction of the acrosome reaction in human spermatozoa. This compound significantly inhibited the acrosome reaction induced by both ZP and the calcium ionophore A23187. These results suggest that ERKs are involved in the signal transduction pathway through which ZP stimulation works during the process of fertilization.     

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.     

透明质酸受体精子的质量特性研究

目的:分析具有透明质酸受体精子的质量特征,寻找精子质量评估新指标。方法:用透明质酸包被载玻片检测活动精子-透明质酸结合率,分析其与精液常规、精子膜功能、精子受精功能和精子成熟障碍指标的关系。结果:头部具有透明质酸结合位点的精子,顶体完整性[(95.4±3.9)%]、线粒体膜高电位率[(97.8±2.1)%]明显高于原始活动精子[(68.8±6.2)%和(72.8±7.4)%,P均0.01];精子-透明质酸结合率与多项精液常规参数呈弱相关性(r=0.195~0.268,P均0.05),与诱发顶体反应率和精子正常形态率呈正相关(r=0.666、0.417,P均0.01),与精子核蛋白不成熟度、DNA碎片率和过量残留胞质率呈负相关(r=-0.266、-0.308、-0.218,P0.01、0.01、0.05)。结论:头部存在透明质酸受体的精子,其质膜结构、受精潜能、成熟度俱佳;用于检测精子透明质酸受体的精子-透明质酸结合试验,是独立的精子多重质量评价新指标。     

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。     

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.     

Modulation of human sperm function by follicular fluid

Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.     

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.