The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex

Abstract

The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.

Cryo‐EM structure of native human uromodulin, a zona pellucida module polymer

“…At the same time, the fact that ZP3 precursors can already contain cross‐subunit αEFβ/βF′ interactions (Han et al , 2010) may facilitate their incorporation as dimers in the egg coat of species such as chicken (Han et al , 2010), humans (Zhao et al , 2004), and fish (Fig 5). In these and other heteropolymeric systems, the variable sequences of the interdomain linkers of different components are also expected to play a major role in determining how subunits incorporate into filaments (Suzuki et al , 2015).…”

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

“…At the same time, the fact that ZP3 precursors can already contain cross-subunit αEFβ/βF' interactions (Han et al, 2010) may facilitate their incorporation as dimers in the egg coat of species such as chicken (Han et al, 2010) , human ) and fish (Figure 5). In these and other heteropolymeric systems, the variable sequences of the interdomain linkers of different components are also expected to play a major role in determining how subunits incorporate into filaments (Suzuki et al, 2015) .…”

Comparative mRNA and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) provides novel insights into the genetic mechanisms for female reproductive success

“…Fertilization refers to the fusion of gametes that initiates the development of a new individual organism (Merriam-Webster 2017). During the process, the ZP proteins ( ZP1 to ZP4 ) in the zona pellucida function to surround the egg, bind the sperm and mediate species-selective sperm–oocyte interactions (Suzuki et al 2015; Avella et al 2016). ZP2 peptide beads have been shown to markedly inhibit the fertilization of ovulated eggs, prevent sperm binding in mice and humans (Gupta et al 2012; Avella et al 2016) and induce infertility in female mice (Avella et al 2016).…”