The mammalian zona pellucida (ZP) is the unique extracellular glycoprotein matrix which is formed during the early stages of ovarian follicular development and plays a critical role in the fertilization and implantation processes. The P.I. of this proposal has made advances in analysis of ZP glycoproteins and has identified specific molecular probes encoding the mRNA encoding rabbit ZP proteins. These studies have shown that the 55Kd rabbit ZP protein produced by expression of the cDNA will elicit antibodies in cynomolgus monkeys and that these antibodies inhibit homologous sperm binding to monkey ZP. These studies have shown that this protein has sperm receptor properties in the rabbit and studies by other investigators have shown that the pig ZP sperm receptor is the homologue of this rabbit 55Kd ZP protein. Given these advances in the molecular analysis of ZP proteins it is proposed to: (1) Isolate the gene and a full length cDNA encoding the human homologue of the rabbit 55 kDa sperm binding protein. (2) Evaluate the functional roles for the human homologue of the rabbit 55Kd ZP sperm receptor by: (a) Expressing the full length cDNA in prokaryotic (non-glycosylated) and eukaryotic (glycosylated) protein expression systems; and (b) Test the expressed protein for: a) sperm receptor activity; and b) induction of the sperm acrosome reaction. (3) Carry out the structural and functional analysis of the gene encoding the human homologue of the rabbit 55Kd sperm receptor by determining the structure of the 5' and 3' regulatory flanking regions of the genomic sequence. (4) Use the human homologue genomic clone isolated in Specific Aim I to study the regulation of the transcription and expression of the human 55 kDa protein in vitro using the transgenic mouse system. In summary, these studies will allow us to evaluate the function of the human homologue and to study the regulation of transcription and expression of the gen encoding the message for this protein. They will also provide information on the expression of ZP proteins during ovarian development which will be important in designing a safe and effective contraceptive vaccine. Because there is no animal model for evaluating human sperm binding to the ZP the animal model generated in these studies may further provide a source of recombinant ZP protein as well as ZP for the clinical evaluate sperm in infertile patients.
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