The egg coat zona pellucida 3 glycoprotein - evolution of its putative sperm-binding region in Old World murine rodents (Rodentia: Muridae).

In eutherian mammals, before fertilisation can occur the spermatozoon has to bind to, and penetrate, the egg coat, the zona pellucida (ZP). In the laboratory mouse there is good evidence that the primary sperm-binding site is a protein region encoded by Exon 7 of the ZP3 gene and it has been proposed that binding is species specific and evolves by sexual selection. In the present study we investigate these hypotheses by comparing Exon 6 and 7 sequences of ZP3 in 28 species of murine rodents of eight different divisions from Asia, Africa and Australasia, in which a diverse array of sperm morphologies occurs. We found considerable nucleotide (and corresponding amino acid) sequence divergence in Exon 7, but not in Exon 6, across these species, with evidence for positive selection at five codon positions. This molecular divergence does not appear to be due to reinforcement to reduce hybridisation, nor does it correlate with divergence in sperm head morphology or tail length, thus it is unlikely to be driven by inter-male sperm competition. Other forms of post-copulatory sexual selection therefore appear to have resulted in the molecular divergence of this region of ZP3 in this highly speciose group of mammals.

Molecular evolution of the carboxy terminal region of the zona pellucida 3 glycoprotein in murine rodents.

In mammals, before fertilization can occur, sperm have to bind to, and penetrate, the zona pellucida (ZP). In the laboratory mouse, which has been used as a model system for fertilization studies, sperm-ZP binding has been found to be mediated by a region at the carboxy terminal, encoded by exon 7 of the Zp3 gene. This region shows considerable interspecific sequence diversity with some evidence of adaptive evolution in mammals, suggesting that it may contribute to species-specific sperm-ZP binding. However, in a previous study of sequence diversity of ZP3 of three species of Australian murine rodents, we found an identical protein sequence of the region encoded by exon 7. Here, we expand this earlier study to determine the sequence diversity of this region in 68 out of the 130 species of Australasian murine rodents. Maximum likelihood analyses, using representatives of both New Guinean and Australian taxa, provide evidence of positive selection at three codons adjacent to, or within, the putative combining-site for sperm of ZP3, but this was not evident when the analysis was restricted to the Australian taxa. The latter group showed low levels of both intra- and inter-generic sequence divergences in the region encoded by exon 7 of Zp3, with little evidence that this region contributes to species specificity of sperm-ZP binding. These findings suggest that the selective forces acting on the Zp3 exon 7 region during the evolution of the Australasian murine rodents have been variable, and that positive selection has only occurred in a few lineages.

cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae.

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.