GONADOTROPIN RECEPTOR REGULATION IN VIVO AND IN VITRO

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During the previous grant period our laboratory identified several Transforming Growth Factor-Beta (TGF-beta) sub-types in the ovary and demonstrated significant interrelationships between TGF-B and Follicle- Stimulating Hormone (FSH) in regulating ovarian cellular function. We also detected growth inhibitory activity in the conditioned culture medium of porcine granulosa cells (GC) which was not TGF-beta and by molecular weight estimate does not represent any of the known growth factors or their binding proteins. Specifically, current results indicate the presence of a putative small-molecular-weight polypeptide inhibitory to 3H-thymidine uptake/cell proliferation and activity which induces apoptosis (programmed cell death) in these cells. Since our knowledge of the locally acting biochemical factors regulating the cessation of ovarian follicular development is virtually non-existent, the isolation and identification of new/novel factors which inhibit follicle cell growth and/or induce programmed cell death would be very significant research advance in this area. Accordingly, the specific aims of this proposal are to elucidate at the molecular level the characteristics of the causative effector(s): 1) to determine whether the same factor or different factors are responsible for the growth inhibitory and apoptotic activities; 2) to isolate the putative growth inhibitory polypeptide and clone the gene for it - a) isolate by preparative and analytical HPLC, b) determine amino acid sequence, c) generate antibodies, d) screen cDNA libraries with antibodies or labelled oligonucleotides, and e) determine nucleotide sequence; 3) to analyze genomic DNA and mRNA with respect to species and cell distribution - a) prepare labelled cDNA probes, b) Southern blot analysis of genomic DNA, c) Northern blot analysis of mRNA, including that from human ovarian cells, to determine transcript(s) size and patterns of cellular expression. The long term objective of these studies is to provide a basic understanding of ovarian cellular function which will eventually be applicable to the problem of fertility regulation.