Follicle-stimulating hormone (FSH) is a principal regulator of the development of ovarian follicles. With the increasing use of FSH preparations in the treatment of infertility and in assisted reproductive technologies as well as the increasing availability of species-specific FSH preparations, this gonadotropin has acquired vital importance in women's health. The overall objective of the proposed project is to understand the FSH receptor and its regulation. The specific goals of this project are to: 1) Determine the amino acid sequence of the porcine ovarian FSH receptor by completing the cloning, sequencing and expression of a cDNA for the porcine ovarian FSH receptor. The properties of the recombinant receptor will be compared with those of native receptor in porcine granulosa cells. 2) Generate polyclonal antibodies to synthetic peptides analogous to hydrophilic amino acid sequences of the putative extracellular domain deduced from the nucleotide sequence of the cDNA. Peptide-specific antibodies will be purified from the crude antisera by affinity chromatography. 3) Determine whether exposure of granulosa cells to FSH increases (up regulates) or decreases (down regulates) the number of FSH receptors using both anti-FSH receptor-specific antibodies and 125l-pFSH as receptor probes. Use of both ligand-binding assays and immunodetection techniques will enable us to distinguish between changes in binding activity and actual receptor content. Changes in immunoreactive receptor and in 125-l-pFSH-binding activity will be correlated with changes in receptor mRNA, assessed by hybridization analysis using the porcine FSH receptor cDNA. The effect of FSH on the rate of transcription will be determined by nuclear runoff transcription assay. Changes in the distribution of FSH receptor mRNA in the porcine ovary during the estrous cycle will be assessed by in situ hybridization. As part of the characterization of the peptides and antibodies, the abilities of each to inhibit binding of FSH, to activate or inhibit adenylyl cyclase, and to stimulate or inhibit progesterone production will be assessed. The results may identify possible hormone-binding sites on the receptor. Preliminary studies with an anti-rat FSH receptor peptide suggest that an antibody which binds to the porcine granulosa cell FSH receptor and stimulates progesterone production without inhibiting FSH binding has been generated. The proposed studies will advance the understanding of FSH regulation of granulosa cell function, thus, facilitating development of methods to increase fertility in women.
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