Zona-induced Acrosome Reaction Research Articles

How accurate is sperm morphology as an indicator of sperm function?

Sperm morphology has been consistently correlated with fertilisation success or failure. The clinical relevance of the percentage normal spermatozoa has been a widely discussed topic amongst infertility specialists and scientists. This study aimed to evaluate the role of sperm morphology as an indicator of additional sperm functions among 114 andrology referrals. The sperm functions that were investigated included chromatin packaging quality (CMA3 test (n = 109), zona-induced acrosome reaction (ZIAR test; n = 36), hemizona assay (HZI; n = 36) and progressive motility (n = 47). Chromatin packaging quality had a negative and significant (P = 0.0001, r = -0.74) correlation with the percentage normal spermatozoa, while progressive motility had a significant and positive correlation (P = 0.0001, 0.59). Accurate sperm morphology scoring as described by the WHO 2010 manual can therefore be used as an indicator of specific sperm functions.

Sperm GIRK2-Containing K+ Inward Rectifying Channels Participate in Sperm Capacitation and Fertilization

The GIRK2-containing inward-rectifying K+ ion channels have been implicated in mammalian spermatogenesis. While the Girk2 null mice are fertile, the male weaver transgenic mice carrying a gain-of-function mutation in the Girk2 gene are infertile. To establish the exact period of spermatogenesis affected by this mutation, we performed StaPut isolation and morphological characterization of the germ cells present in the weaver testis. Germ cells representing all periods of spermatogenesis were identified. However, no spermatozoa were present, suggesting that this mutation only affected the haploid phase of spermatogenesis. Real-time PCR studies performed on StaPut purified germ cells from wild-type mice indicated that the Girk2 transcripts were exclusively expressed in spermatids. Immunofluorescence studies of mouse and boar spermatids/spermatozoa localized the GIRK2 K+ containing channels to the acrosomal region of the sperm plasma membrane. During porcine in vitro fertilization (IVF), GIRK2-containing channels remained associated with the acrosomal shroud following zona-induced acrosome reaction. Fertilization was blocked by tertiapin-Q (TQ), a specific inhibitor of GIRK channels, and by anti-GIRK2 antibodies. Altogether, studies in two different mammalian species point to a conserved mechanism by which the GIRK2 inward-rectifying K+ ion channels support sperm function during fertilization.

Accurate sperm morphology assessment predicts sperm function

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

Sequential analysis of sperm functional aspects involved in fertilisation: a pilot study

The development of diagnostic techniques in andrology as a second level of approach to the diagnosis of male factor infertility has enthused the focus of researchers on the development of a sequential diagnostic programme for these men. Semen samples of 78 men form couples undergoing in vitro fertilisation therapy were used in the study. The semen samples were used to test sperm functional aspects known to interfere with fertilisation. These tests included semen profile, DNA integrity, apoptosis, chromatin packaging, acridin orange staining, zona binding capacity, zona-induced acrosome reaction (AR). Results were correlated with fertilisation outcome. Statistical analyses of the recorded data were carried out using a logistic regression analysis model on all sperm functional tests. A negative and significant association with the fertilisation rates was recorded for DNA damage (r = -0.56; P ≤ 0.0005). A positive significant correlation was recorded between fertilisation rates and sperm with normal DNA (r = -0.57, P ≤ 0.0004), and zona-induced AR (r = 0.33, P ≤ 0.002). Diagnostic andrology can be regarded as a mandatory part of the male factor patient's work-up schedule to assist clinicians with the most suitable therapeutic modality to follow.

PACAP-mediated sperm–cumulus cell interaction promotes fertilization

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus-oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus-oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: Functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

Sperm head membrane reorganisation during capacitation

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.

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 <or= 0.05). The zona-induced acrosome reaction for the three groups were 25.0 +/- 2% (normal group), 18.0 +/- 2% (G-patterns) and 14.1 +/- 0.6% (P-pattern) (P <or= 0.05). The number of hyperactivated sperm showed an increase in the ejaculates (1%), after capacitation (2%) and exposure to zona pellucida (5%) (P <or= 0.05). When divided into the three morphology groups, the number of spermatozoa with hyperactivated motility was more profound in men with normal morphology compared with G- and P-pattern men. Human zona pellucida is a strong inducer of hyperactivated motility among spermatozoa and the effect is more profound among sperm populations with normal morphology.

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.

Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat.

During mammalian fertilization, sperm adhere to the extracellular coat of the egg, or zona pellucida, in a species-specific manner. In mouse, evidence suggests that sperm recognize and bind to specific oligosaccharide ligands within the zona pellucida glycoprotein, ZP3, via beta1,4-galactosyltransferase I (GalT I), a lectin-like receptor on the sperm surface. Although in vitro experiments using isolated gametes lend support to this model, recent in vivo studies of genetically altered mice question whether ZP3 and/or GalT I are solely responsible for sperm-egg binding. In this regard, sperm from GalT I-null mice bind poorly to ZP3 and fail to undergo a zona-induced acrosome reaction; however, they still bind to the ovulated egg coat in vitro. In this report, we characterize a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat. Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and facilitates gamete adhesion; and a ZP3-dependent ligand that is present in the insoluble zona matrix and is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of oviduct-specific glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm, whereas the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally associated, sperm-binding ligand on the ovulated zona pellucida.

Relationship between zona pellucida–induced acrosome reaction, sperm morphology, sperm–zona pellucida binding, and in vitro fertilization

Objective To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm–zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. Design Prospective analytical study. Setting Academic training hospital. Patient(s) Randomly selected couples consulting for infertility. Intervention(s) Acrosome reaction response to solubilized human zona pellucida was recorded. Main outcome measure(s) We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). Result(s) Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm–zona binding (r = 0.57). Conclusion(s) ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.

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.

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: Fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca2+ changes monitored by Fluo-3

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, DapoxylTM (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 ± 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca2+ concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 ± 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca2+ transient as the precursor reaction to onset of the zona-induced acrosome reaction. Mol. Reprod. Dev. 55:335–349, 2000. © 2000 Wiley-Liss, Inc.

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3.

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 μg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 Wiley-Liss, Inc.

PH-20 but not acrosin is involved in sperm penetration of the macaque zona pellucida.

In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 microg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 microg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida.

Rearrangement of the PH‐20 protein on the surface of macaque spermatozoa following exposure to anti‐PH‐20 antibodies or binding to zona pellucida

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20 . The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction. Mol. Reprod. Dev. 50:207–220, 1998. © 1998 Wiley-Liss, Inc.

Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida.

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20. The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction.

Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida.

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.