Abstract
Vertebrate eggs have an extracellular matrix surrounding the egg, termed zona pellucida in mammals and vitelline envelope in amphibians that regulate the entry of sperm into the egg. Sperm-binding glycoprotein ZPC has been rapidly changing within mammalian species and has been thought to be involved in driving speciation events. Rapid amino acid changes at the sperm-binding sites within the protein can change ability of sperm to bind to and fertilize the egg. This can cause a prezygotic block to fertilization. We investigated the ZPC gene family in two closely related frog species Xenopus laevis and Xenopus bolrealis (diverged from each other about 30 million years ago) to examine how rapidly the ZPC protein is evolving compared to mammalian ZPC genes. Four and five unique cDNAs were found within ovary cDNA libraries from the frog species X.laevis and X.borealis respectively, indicating that this gene has undergone duplication event in both frog species. In the present study, we tested the hypothesis that X.laevis and X.borealis have the same complement of ZPC cDNAs (coding for sperm binding protein) expressed within their ovaries; and amino acid sites encoded in the ZPC cDNAs show evidence of having been subjected to positive Darwinian selection toward their 3’ ends. New hypothesis could then be formulated as to where possible sperm¬_binding sites are located given to the location of positively selected amino acid positions. The objectives of this study included 1) cloning unique ZPC genes within X.borealis and X.laevis cDNA libraries 2) determining the ZPC genes relationship ( orthologus versus paralogus) 3) performing phylogenetic evolutionary analyses to further understand the evolution rate of ZPC genes in two frog species, and 4) comparing the sites under positive selection in frog ZPC with the ones identified in mammalian ZPC genes. In order to analyze the Xenopus ZPC sequences, cDNAs from two were PCR amplified from ovary cDNA libraries, cloned, sequenced and assembled into partial length(middle section and 3’ ends) cDNAs. We then analyzed ZPCs genes to determine phylogenetic relationships by performing multiple alignments and generating two different tree topologies (Neigbor joining and Maximum likelihood parsimony trees). Finally the molecular evolution of the sites was statistically evaluated using PAML (Phylogenetic Analysis by Maximum Likelihood) software.