Rabbit zona pellucida composition: A molecular, proteomic and phylogenetic approach

Abstract

The zona pellucida (ZP) participates in sperm–egg interactions during the first steps of 22 fertilization. Recent studies have shown that the ZP matrix of oocytes in several species is 23 composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, rather than the three 24 described in mouse, pig and cow. In this study, investigations were carried out to unveil a Q425 fourth glycoprotein in the rabbit (Oryctolagus cuniculus) ZP. Using total RNA isolated from 26 rabbit ovaries, the complementary deoxyribonucleic acid (cDNA) encoding rabbit ZP1 was 27 amplified by reverse transcribed polymerase chain reaction (RT-PCR). The ZP1 cDNA 28 contains an open reading frame of 1825 nucleotides encoding a polypeptide of 608 amino 29 acid residues. The deduced amino acid sequence of rabbit ZP1 showed high identity with 30 other species: 70% identity with human and horse ZP1, and 67% identity with mouse and rat 31 ZP1. At the proteomic level, peptides corresponding to the four proteins were detected by 32 mass spectrometry. In addition, a molecular phylogenetic analysis of ZP1 showed that 33 pseudogenization of this gene has occurred at least four times during the evolution of 34 mammals. The data presented in this manuscript provide evidence, for the first time, that 35 the rabbit ZP is composed of four glycoproteins.