ESP13.2, a member of the beta-defensin family,is a macaque sperm surface-coating protein involved in the capacitation process

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the beta-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the beta-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.     

Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida

Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm-zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm-free media and incubated 1 hr before fixation. There were no significant diffences between groups in the percentage of live, acrosome-reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 ± 16 and 81 ± 17, respectively) compared to control and dbcAMP treatment (15 ± 4 and 28 ± 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome-reacted sperm on the zona (15.2 ± 2.1 and 9.0 ± 0.6) than control or caffeine treatment (3.0 ± 1.4 and 2.4 ± 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effects as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction. © 1994 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 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.     

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.     

Effects of caffeine and dbcAMP on zona pellucida penetration by epididymal spermatozoa of cynomolgus monkeys (Macaca fascicularis)

Spermatozoa mature during epididymal transit, acquiring the abilities to swim progressively, fertilize oocytes, and produce viable offspring. In this study, we investigate the capacity of spermatozoa retrieved from the midcorpus and distal cauda regions of the epididymis of the cynomolgus monkey to penetrate homologous zone pellucida. Successful in vitro fertilization by ejaculated macaque sperm is dependent upon the addition of caffeine and dbcAMP. Therefore, the effect of these cyclic nucleotide mediators was also examined in this study. Results of sperm motion analysis indicate no difference in baseline values (without stimulators) for any motion parameter. With the addition of caffeine and dbcAMP, curvilinear velocity significantly increased only for the distal cauda sperm (P = 0.05). Amplitude of the lateral head displacement was significantly increased for distal cauda sperm (P < 0.01); although elevated above baseline, the increase observed after activation by corpus sperm was significantly lower than that achieved by cauda sperm (P < 0.05). The addition of caffeine and dbcAMP was an absolute requirement for zona penetration by both midcorpus and distal cauda sperm. With activation, zona penetration was significantly decreased for corpus sperm compared to cauda sperm (P < 0.001). These results suggest that cynomolgus monkey sperm reaching the midcorpus region of the epididymis have not completed all of the maturational changes requisite for successful fertilization; this immaturity is evidenced by decreased sperm motion and by impedance at the level of zona penetration. © 1996 Wiley-Liss, Inc.     

Beta-defensin 126 on the surface of macaque sperm mediates attachment of sperm to oviductal epithelia

Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.     

Separate and combined effects of caffeine and dbcAMP on olive baboon (Papio anubis) sperm

Background Improvement of baboon sperm capacitation is necessary for achieving high in vitro fertilization (IVF) rates in baboons. In this study, we evaluated separate and combined effects of caffeine and dbcAMP on baboon sperm capacitation. Methods Sixteen male baboons (n = 16) were electroejaculated. Each sperm sample was divided into two aliquots: one for chemical activation and the other untreated control. Group 1: dbcAMP (n = 6); Group 2: caffeine (n = 6) and Group 3: combination of caffeine and dbcAMP (n = 4). In each aliquot, sperm motility after 30 minutes of incubation was evaluated as well as zona pellucida (ZP) binding ability after overnight incubation with 4–5 ZP from unfertilized human oocytes. Results Sperm motility and ZP binding ability in all chemically activated groups increased significantly as compared to their respective controls (P < 0.05). Conclusion Combined and separate effects of caffeine and dbcAMP increases baboon sperm motility and ZP binding ability and may improve baboon IVF.     

Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa

Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca fascicularis) spermatozoa; instead, both have an absolute requirement for exogenous stimulation with caffeine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting criteria for automated analysis of macaque sperm exhibiting hyperactivated motility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteristics were assessed by computer-assisted motion analysis. Tyrosine phosphorylation of sperm tail proteins was determined by immunocytochemistry with PY-20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) >/= 150 microm/sec; amplitude of lateral head displacement (ALH) >/= 8.0 microm, and linearity (LIN) 相似文献   

The Carbohydrate Structure of DEFB126, the Major Component of the Cynomolgus Macaque Sperm Plasma Membrane Glycocalyx

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as β-defensin DEFB126. DEFB126 is one of the five β-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine β-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34–36 kDa to approximately 38–40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify β-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.     

Receptor function of mouse sperm surface galactosyltransferase during fertilization

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

Physiological state of bull sperm affects fucose- and mannose-binding properties

In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean +/- SD:167 +/- 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by capacitating washed sperm in medium containing 10 microg/ml heparin (50 +/- 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 +/- 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

Status of the rat acrosome during sperm-zona pellucida interactions

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.     

Time and process of sperm penetration into cumulus-free mouse eggs fertilized in vitro

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus-free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.     

Induction of acrosome reactions by the human zona pellucida

We have used two approaches to test the ability of the human zona pellucida to induce acrosome reactions in human sperm. First, nonviable human oocytes were incubated for 1 min in a suspension of capacitated sperm (of which fewer than 5% were acrosome-reacted) to allow binding of about 200 sperm per oocyte. Some of the oocytes were fixed immediately, and the remainder were fixed after a further 1-h incubation without free-swimming sperm. As determined by light microscopy, sperm on the zona were only 3 +/- 2% (avg. +/- SD) acrosome-reacted at 1 min, and the incidence increased to 46 +/- 15% during the next hour. Electron microscopy confirmed that most sperm on the zona at 1 min were acrosome-intact. A few sperm were in an early stage of the acrosome reaction. Acrosome reactions occurring on the zona during the subsequent hour appeared to be morphologically normal. Second, treatment of sperm in suspension with acid-disaggregated zonae (2 to 4 zonae/microliter) increased the incidence of acrosome-reacted sperm from 3 +/- 1% to 24 +/- 4%. We conclude that the human zona pellucida, or material intimately associated with it, can induce acrosome reactions in human sperm.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha- lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta- N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.     

猕猴精子优选及体外获能的研究

选择活率高的精子并进行体外获能是开展猕猴体外受精研究的必要程序, 是研究猕猴受精生物学的重要手段。本实验采用上浮法和Percoll 梯度离心法对猕猴精液进行了优选, 并对处理后的精子形态正常率、精子活率、密度及受精率作了比较, 发现二者差异不显著; 用dbcAMP 和咖啡因使精子获能, 发现只有两种获能剂同时存在才能使猕猴精子获能并使卵母细胞受精。结论为: 上浮法和Percoll 法都是有效的精子优选法, 对受精率的影响差异不显著; dbcAMP 和咖啡因在猕猴精子体外获能时缺一不可。