Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium oscillations.
(1/949)
Immature oocytes of many species are incompetent to undergo cortical granule (CG) exocytosis upon fertilization. In mouse eggs, CG exocytosis is dependent primarily on an inositol 1,4,5-trisphosphate (IP3)-mediated elevation of intracellular calcium ([Ca2+]i). While deficiencies upstream of [Ca2+]i release are known, this study examined whether downstream deficiencies also contribute to the incompetence of preovulatory mouse oocytes to release CGs. The experimental strategy was to bypass upstream deficiencies by inducing normal, fertilization-like [Ca2+]i oscillations in fully grown, germinal vesicle (GV) stage oocytes and determine if the extent of CG exocytosis was restored to levels observed in mature, metaphase II (MII)-stage eggs. Because IP3 does not stimulate a normal Ca2+ response in GV-stage oocytes, three alternate methods were used to induce oscillations: thimerosal treatment, electroporation, and sperm factor injection. Long-lasting oscillations from thimerosal treatment resulted in 64 and 10% mean CG release at the MII and GV stages, respectively (P < 0.001). Three electrical pulses induced mean [Ca2+]i elevations of approximately 730 and 650 nM in MII- and GV-stage oocytes, respectively, and 31% CG release in MII-stage eggs and 9% in GV-stage oocytes (P < 0.001). Sperm factor microinjection resulted in 86% CG release in MII-stage eggs, while similarly treated GV-stage oocytes exhibited < 1% CG release (P < 0.001). Taken together, these results demonstrate a deficiency downstream of [Ca2+]i release which is developmentally regulated in the 12 h prior to ovulation. (+info)
A sialoglycoprotein, gp20, of the human capacitated sperm surface is a homologue of the leukocyte CD52 antigen: analysis of the effect of anti-CD52 monoclonal antibody (CAMPATH-1) on capacitated spermatozoa.
(2/949)
In this study we performed N-terminal sequence analysis of gp20, a 20 kDa sialoglycoprotein on the human sperm surface previously identified by radiolabelling of the sialic acid residues of sperm surface. We found 100% identity with the N-terminus of CD52, an antigen expressed on almost all human leukocytes. We also show that, like CD52, gp20 behaves as a glycosylphosphatidylinositol (GPI)-anchored protein and that anti-gp20 antiserum reacts with an antigen on leukocytes of the same molecular weight as CD52. Using CAMPATH-1, the monoclonal antibody against CD52, in fluorescent staining of capacitated spermatozoa, Western blot analysis and the zona-free hamster egg penetration test, we found that the effect of this antibody was different from that of our anti-gp20. Western blot analysis revealed a well-defined 20 kDa band with anti-gp20, whereas a 14-20 kDa band was detected with CAMPATH-1. Anti-gp20 stained the equatorial region of the sperm head, whereas CAMPATH-1 stained the tail in immunofluorescence analysis of capacitated spermatozoa. A dose-dependent inhibitory effect was seen with CAMPATH-1, similar to that previously detected with anti-gp20, in a zona-free hamster egg penetration test. However, with CAMPATH-1 agglutination of motile spermatozoa was detected, and this was not present with anti-gp20. This suggests that the epitopes recognized by the two antibodies are different. (+info)
Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.
(3/949)
The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster (approximately 70 kDa, pI 5) and a lower molecular weight (approximately 35-45 kDa, pI 5.5) protein cluster were released from the oolemmal surface as a result of PI-PLC treatment. It is likely that these GPI-anchored egg surface proteins are required for sperm-egg binding and fusion. (+info)
Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides.
(4/949)
The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes. ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. Antisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or present in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrated that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies react with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These results show that antibodies against synthetic ZP2 peptides react with ZP2 protein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possible functional importance for sperm-oocyte interaction. (+info)
Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida.
(5/949)
The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]i increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+]i occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]i was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle approximately 20 s after the onset of the [Ca2+]i rise. High-resolution imaging revealed that the [Ca2+]i rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]i rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+]i rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR. (+info)
Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48-micron diode laser beam.
(6/949)
PURPOSE: Our purpose was to test whether zona pellucida (ZP) drilling using a 1.48-micron diode laser beam on bovine IVM/IVF/IVC blastocysts is effective for embryo hatching. METHODS: Blastocysts produced in vitro at day 7 after IVF were divided into control and laser-drilled groups, respectively. RESULTS: When the rates of in vitro development of bovine embryos were examined, the average cleavage rate (> or = two-cell) was 82.3% and the blastocyst rate at day 7 after IVF was 32.5%. Using these blastocysts, when the laser drilling effect was investigated at 48 hr after treatment, the total hatching rate in the laser-drilled group (98.0%) was significantly higher than that in the control group (60.0%) (P < 0.001). Especially, the hatched rate of the laser-drilled group (68.0%) was significantly enhanced compared with that of the control group (30.0%) (P < 0.001). CONCLUSIONS: These results demonstrated that laser ZP drilling on bovine IVM/IVF/IVC blastocysts can significantly increase the hatching rate. (+info)
A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida.
(7/949)
In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone. (+info)
Localization of neutral N-linked carbohydrate chains in pig zona pellucida glycoprotein ZPC.
(8/949)
Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reported that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N-glycosylation sites of ZPB. In this study, we focused on the localization of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the main neutral N-linked chains are not susceptible to the enzyme. N-glycanase digestion indicated that ZPC has three N-glycosylation sites. Three glycopeptides each containing one of the N-glycosylation sites were obtained by tryptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N-linked chains from each glycopeptide were characterized by two-dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB. (+info)