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The sections in this article are: General Introduction Oogenesis and Spermatogenesis Oogenesis: Primordial Germ Cells to Eggs Spermatogenesis: Primordial Germ Cells to Spermatozoa Final Preparation of Gametes for Fertilization Eggs: “Meiotic Maturation” Regulatory Aspects of Oocyte Maturation Sperm: “Capacitation” Sperm Activity States Binding of Sperm to Eggs Structure of the Zona Pellucida Identification of a Mammalian Sperm Receptor Other Mammalian Sperm Receptors The Acrosome Reaction Anatomy of the Acrosome Stages of Exocytosis Functions of the Acrosome Reaction during Fertilization Site of the Acrosome Reaction Mechanisms of the Acrosome Reaction Initiators of Exocytosis Conclusions Signaling at the Sperm Plasma Membrane Mediators of Receptor‐Activated Second‐Messenger Production in Sperm Second‐Messenger Production Ca 2+ and Ca 2+ Channels Internal pH Downstream Effectors of Receptor Activation Gamete Fusion and Cortical Granule Exocytosis Cortical Granules Gamete Membrane Fusion and the Receptor Question From Oolemma to Cortical Granule—Signal Transduction Pathways
Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-huIgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.
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