Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and Sacl restricted fragment was cloned in frama downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β—a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23–45 aa residues) and C-(300–322 and 324–347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3β and bonnet r-ZP3 but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates. Mol. Reprod. Dev. 47:140–147, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA λgt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115–1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429–444; K₄₃₄ replaced by R and I₄₃₆ by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2. Mol. Reprod. Dev. 50:229–239, 1998. © 1998 Wiley-Liss, Inc.     

Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey.

To investigate the role of polypeptide backbone vis-à-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system. The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione. SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E. coli and baculovirus expressed r-bmZP3, respectively. The r-bmZP3 purified from both E. coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay. In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E. coli expressed r-bmZP3 and vice-versa. Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues. Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E. coli expressed r-bmZP3 failed to have a significant effect. These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis.     

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.     

In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.     

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.     

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial,the brushtail possum,Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N-linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998. © 1998 Wiley-Liss, Inc.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa

Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N:-terminal signal sequence and the C:-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both alpha helical and beta sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization.     

哺乳动物精子中的ZP3结合蛋白研究进展

哺乳动物卵透明带糖蛋白ZP3(zona pellucida3)是介导精卵初级结合、诱发精子发生顶体反应的关键分子。目前已在精子中发现多种ZP3结合蛋白。95kD酪氨酸激酶受体可能通过其酪氨酸激酶活性介导ZP3诱发的顶体反应。β—1,4—半乳糖基转移酶与ZP3的糖基结合后,通过激活下游信号分子诱发顶体反应。精子蛋白sp56可能介导了顶体反应期间顶体基质与ZP之间的相互作用。透明带粘附素(zonadhesin)也是在顶体反应发生之后才与ZP发生相互作用。这些精子蛋白介导的下游信号事件将是下一步研究的热点。     

Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli.

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.     

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.     

Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.     

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

Distribution of α-D-mannose residues on zona pellucida and their role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cumulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost completely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4,spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Determining acrosomal status of the cynomolgus monkey (Macaca fascicularis) sperm by fluorescence microscopy

The acrosome of Macaca fascicularis sperm cannot be distinguished by conventional light microscopy, so determining whether sperm are acrosome-intact or-reacted is difficult. We describe methods for labeling the acrosomal region of sperm with two different probes: fluoresceinated Pisum sativum agglutinin and anti-sperm antiserum. Acrosome-intact sperm are much more heavily labeled in the acrosomal region than are acrosome-reacted sperm, providing a simple means of differentiating the two types of sperm. The two probes detect similar numbers of acrosome-reacted sperm following treatment with the divalent cation ionophore, A23187.