Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

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.

Zona pellucida family genes in Chinese pond turtle: identification, expression profiles, and role in the spermatozoa acrosome reaction

“…In contrast, chicken spermatozoa interact with ZP3 [53], as is also the case for the frog X. laevis , which may also involve another ZP protein, ZPD [54]. In humans, the AR also can be induced by purified ZP4 [55], and sperm–egg binding sites have been identified at the N terminus of the ZP4 protein in bovine [56, 57]. Researchers recently proposed that instead of a single protein, the supramolecular structure of ZP is responsible for sperm binding in mammals [58].…”

Glycosylation Research in Bovines-the Significance and Recent Updates

“…ZP in bovines contains three ZPG3 annotated bZP2, bZP3, and bZP4. In vitro results identified the N-terminal domain of bZP4 (ZP-N1) and the middle region of bZP4 as substantial for sperm binding but without evidence for N-glycosylation of ZP-N1 as necessary for this purpose (Dilimulati et al, 2022). Further research of the bZP4 middle region (including the hinge domain crucial for the bZP3-bZP4 complex formation, which is essential for sperm binding) showed species-specific sperm binding.…”

“…These findings significantly contribute to a better understanding of between-species differences in ZP functions. Identification of sperm-associated factors responsible for sperm-ZP interactions and validation via in vivo bovine fertilization research represents the primary research efforts to achieve reliability in bovine reproduction practice (Dilimulati et al, 2022).…”

N-Glycosylation Site in the Middle Region Is Involved in the Sperm-Binding Activity of Bovine Zona Pellucida Glycoproteins ZP3 and ZP4

“…The second region, extending from Ser-290 to Lys-340, comprises the flexible hinge region and the Nterminus of the ZP-C domain (Figure 1A,B) [3,18]. Bovine sperm binds to bZP4 (25-136) in a species-selective manner without requiring N-glycosylation [19]. Additionally, we identified three sites involved in sperm binding on bZP4 (25-136) using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins (Figure 1B) [19].…”