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

Abstract

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.

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

“…Recently, we showed that bZP4 has a predominant binding activity for bovine sperm in a solid support assay [ 11 ]. The bZP4 polypeptide expressed in Sf9 cells included residues Lys-25 to Arg-464 and consisted of five regions: N-terminal ZP-N-like domain (Lys-25 to Pro-135, ZP-N1), trefoil domain (Asp-136 to Tyr-181), ZP-N domain (Gly-182 to Ala-289, ZP-N), hinge region (Ser-290 to Gln-311) and ZP-C domain (Pro-312 to Arg-464, ZP-C) ( Figure 1 A).…”

“…In pigs, ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex [ 10 ]. Recently, we showed that ZP4 binds to sperm in bovines [ 11 ]. The species difference in sperm-binding ZP glycoproteins suggests that sperm-ZP binding mechanisms are different amongst mammals.…”

“…The estimated protein molar ratio of ZP2/ZP3/ZP4 for native bovine ZP is 1:2:1 [ 18 ], whereas it is 1:6:6 for native porcine ZP [ 19 ], suggesting that the bovine and porcine ZP filamentous structures are different. Interestingly, ZP4 is responsible for ZP sperm-binding activity in bovines and pigs [ 10 , 11 ], suggesting that the sperm-ZP interacting mechanisms between these two species are conserved to some extent.…”

“…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 Lys-340, which consists of the flexible hinge region and the N-terminal part of the ZP-C domain (see Figure 1 A) [ 11 ]. The sperm-binding activity of bZP4(25–136) is a little higher than that of bZP4(290–340).…”

“…The sperm-binding activity of bZP4(25–136) is a little higher than that of bZP4(290–340). It was hypothesised that the bZP4(290–340) interaction with bZP3 enhances its sperm-binding activity [ 11 ]. Although these results document the dependency of sperm recognition on bZP4(25–136), it is still unknown which region of the bZP4(25–136) domain is involved in the sperm-binding activity.…”