Sperm binding activity of the zona pellucida of immature mouse oocytes

SummaryThe zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.

Download Full-text

Sperm-binding regions on bovine egg zona pellucida glycoprotein ZP4 studied in a solid supported form on plastic plate

PLoS ONE ◽

10.1371/journal.pone.0254234 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254234

Author(s):

Kamila Dilimulati ◽

Misaki Orita ◽

Ganbat Undram ◽

Naoto Yonezawa

Keyword(s):

Zona Pellucida ◽

Binding Sites ◽

Insect Cells ◽

Competitive Inhibition ◽

Expression System ◽

Binding Activity ◽

Mammalian Oocyte ◽

Sperm Binding ◽

Bovine Sperm ◽

Binding Mechanisms

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–oocyte interactions. The bovine ZP consists of the glycoproteins ZP2, ZP3, and ZP4. Sperm-binding mechanisms of the bovine ZP are not yet fully elucidated. In a previous report, we established the expression system of bovine ZP glycoproteins using Sf9 insect cells and found that the ZP3/ZP4 heterocomplex inhibits the binding of sperm to the ZP in a competitive inhibition assay, while ZP2, ZP3, ZP4, the ZP2/ZP3 complex, and the ZP2/ZP4 complex do not exhibit this activity. Here, we show that bovine sperm binds to plastic plates coated with ZP4 in the absence of ZP3. We made a series of ZP4 deletion mutants to study the sperm-binding sites. The N-terminal region, Lys-25 to Asp-136, and the middle region, Ser-290 to Lys-340, of ZP4 exhibit sperm-binding activity. These results suggest that among the three components of bovine ZP glycoproteins, ZP4 contains the major potential sperm-binding sites, and the formation of a multivalent complex is necessary for the sperm-binding activity of ZP4.

Download Full-text

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1431 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1431-1440 ◽

Cited By ~ 87

Author(s):

D J Miller ◽

X Gong ◽

G Decker ◽

B D Shur

Keyword(s):

Zona Pellucida ◽

Binding Sites ◽

Binding Activity ◽

Cortical Granule ◽

Cortical Granules ◽

Sperm Binding ◽

Competitive Inhibitors ◽

Glycosidase Inhibitor ◽

Egg Coat

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

Download Full-text

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽

10.1530/reprod/119.1.15 ◽

2000 ◽

pp. 15-23 ◽

Cited By ~ 15

Author(s):

K Jewgenow ◽

M Rohleder ◽

I Wegner

Keyword(s):

In Vitro Fertilization ◽

Zona Pellucida ◽

Antigenic Determinants ◽

Vitro Fertilization ◽

Contraceptive Vaccine ◽

Sperm Binding ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Download Full-text

Faculty Opinions recommendation of Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13396969.14765074 ◽

2011 ◽

Author(s):

Dolores Lamb ◽

Alexander W Pastuszak

Keyword(s):

Zona Pellucida ◽

Human Sperm ◽

Sialyl Lewis X ◽

Lewis X ◽

Sperm Binding ◽

Sialyl Lewis

Download Full-text

Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment

Zygote ◽

10.1017/s0967199400003646 ◽

1997 ◽

Vol 5 (3) ◽

pp. 213-217 ◽

Cited By ~ 3

Author(s):

J. Fulka ◽

N.L. First ◽

C. Lee ◽

J. Fulka ◽

R.M. Moor

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Germinal Vesicle ◽

The Other ◽

Immature Oocytes ◽

Mouse Oocytes ◽

Germinal Vesicles ◽

Immature Mouse ◽

Condensed Dna ◽

Metaphase Ii Oocytes

SummaryImmature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

Download Full-text