PARENT SESSION

(W315) CHARACTERIZATION OF THE ZONA PELLUCIDA BINDING PROTEIN (ZPBP) GENE FAMILY.

Lin, Yi-Nan¹, Yan, Wei², Matzuk, Martin¹, ¹ Baylor College of Medicine, Houston, TX² University of Nevada, School of Medicine, Reno, NV

ABSTRACT- To study the etiology of infertility and to identify potential contraceptive targets, we combined in silico approaches and traditional expression analyses to identify conserved genes with germ cell-specific expression. One of the identified genes is the paralog of zona pellucida binding protein (ZPBP1 or sp38), and we named it as Zpbp2. Zpbp2 was shown by Northern blot and RT-PCR analysis to be testis-specific. In situ hybridization demonstrated that Zpbp2 is expressed from the late pachytene spermatocyte stage to the round spermatid stage. The mouse Zpbp2 gene contains eight exons, and three alternative transcription products were detected by RT-PCR. The full-length mouse Zpbp2 sequence encodes a 326-amino acid ORF with N-terminal signal peptide. Mouse ZPBP2 is 73% identical to the orthologous human ZPBP2. Since 15 conserved cysteines are shared between ZPBP1 and ZPBP2, it is likely that these paralogous proteins share similar protein conformations and protein-protein interactions. Besides various model mammals, both ZPBP1 and ZPBP2 were identified in chicken testis cDNA library, suggesting their conserved roles in reproduction. Partial alignment of ZPBP with Xenopus draft genomic sequences further suggested its existence in amphibian lineage. The roles of ZPBP1 in sperm-egg interaction were first suggested from its in vitro binding activity to ZP2 and its subcellular localization in acrosome. Knockout studies have previously clarified the roles of several genes, such as proacrosin and beta-1,4-galactosyltransferase, which were originally thought to be important in sperm-zona interaction. Thus we are using knockout mice as loss-of-function models to elucidate if both ZPBP genes are involved in sperm-egg interaction, and ZPBP double knockout mice will likely provide molecular insight into this interaction. Functional analysis is also being performed to dissect the roles of ZPBP proteins in sperm-egg interaction. This study is supported in part by the National Institutes of Health Specialized Cooperative Centers Program in Reproduction Research (HD07495).

KEY WORDS: sperm, acrosome, knockout, sp38