Abstract
The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.
Highlights
The zona pellucida (ZP), a transparent coat surrounding mammalian oocytes, plays important roles in oogenesis, species-selective sperm recognition, blocking polyspermy and early embryonic development [1,2]
By developing a solid support assay, we recently demonstrated the sperm-binding activity of two regions of bZP4: the region extending from Lys-25 to Asp-136, which almost corresponds to the N-terminal ZP-N1 domain, and the one extending from Ser-290 to Lys340, which consists of the flexible hinge region and the N-terminal part of the ZP-C domain [11]
We showed that bZP4 has a predominant binding activity for bovine sperm in a solid support assay [11]
Summary
The zona pellucida (ZP), a transparent coat surrounding mammalian oocytes, plays important roles in oogenesis, species-selective sperm recognition, blocking polyspermy and early embryonic development [1,2]. In most species, capacitated sperm bearing an intact acrosome binds to the ZP (primary sperm-ZP binding). The sperm undergoes the acrosome reaction, which reinforces the binding to the ZP (secondary sperm-ZP binding). The acrosome-reacted sperm reaches the perivitelline space and binds to the oolemma.
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