The mouse ovary serves as a paradigm for investigating the developmental biology of mammalian gonadogenesis, oogenesis and fertilization. Gonadogenesis: Mouse gestation occurs over 20 days. Germ cells, first detected in the developing embryo 7.5 days post coitus (dpc), migrate from the allantois to the genital ridge by 12.5 dpc. In XX females, the primitive gonad then differentiates into an ovary. Poly(A+) was purified from female and male genital ridges isolated 12-13 dpc. cDNA libraries have been made for each sex and subtractive cloning techniques are being used to identify female-specific gene products involved in early sex determination and gonadogenesis. Oogenesis: The expression of the zona pellucida, an ovary-specific extracellular matrix composed of three glycoproteins (ZP1, ZP2, ZP3), serves as a marker of oocyte growth and differentiation in the adult female. A 12bp DNA sequence 200bp upstream of the start of transcription of the Zp-2 and Zp-3 genes is necessary and sufficient for zona promoter activation. Using yeast genetics, efforts are underway to clone ZAP-1, the putative transcription factor that binds to this DNA element. These studies will provide molecular details of mechanisms involved in the coordinate, oocyte-specific expression of the zona genes. ZAP-1 will additionally provide a marker of early oocyte growth and differentiation. Fertilization: The three zona proteins are secreted and form an extracellular matrix that mediates the relatively species-specific events of fertilization. Using microinjection techniques to degrade endogenous zona transcripts and to introduce synthetic zona mRNAs, we are assessing protein-protein interactions in the assemblage of the zona matrix. To investigate the molecular biology of sperm-egg interactions mediated by the zona, we are using recombinant DNA techniques to express mouse and human zona proteins and to create mutant zona proteins, including mouse/human chimeric proteins. These will be tested for their ability to competitively inhibit fertilization, enabling establishment of structure-function correlations.