PITUITARY CELL CULTURE: ESTROGEN CONTROL OF FSH

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Follicle stimulating hormone (FSH), the primary trophic hormone for egg and sperm development in mammals, is composed of two protein subunits, alpha and beta. The hormone-specific beta subunit is the limiting subunit in pituitary gonadotrophs and its regulation appears paramount in controlling FSH production. 17 beta-Estradiol (E) and progesterone (P) rapidly decrease secretion of FSH from pituitary cultures of sheep, pigs, and humans; data from sheep indicate that E and P decrease transcription of both subunit mRNAs by > 85% within 2 hr (beta) and 12 hr (alpha). We propose to define those DNA sequences on the FSH beta subunit gene that mediate E- and P-induced inhibition. These sequences may be steroid receptor binding sites which cause negative regulation of transcription. Newly synthesized proteins do not seem to mediate FSH inhibition because inhibition is not altered by cycloheximide. Biologically functional sequences responsible for negative regulation will be located by their abilities to confer E- and P- dependent regulation upon reporter genes during their transient expression in a steroid-responsive human choriocarcinoma cell line (JAR cells). These cells naturally express two gonadotrophin genes related to FSH beta (alpha and hCG beta) and they can be made E- responsive by transfection with plasmids that express minigenes coding for the E receptor; similar plasmids for expressing P receptors will be available soon. Enriched primary ovine gonadotrophs will also be developed as a possible alternative to, or companion with, JAR cells for studying negative steroid regulation. The involvement of proteins, other than E or P receptors, in negative steroid action will be studied by examining binding of cellular proteins to E and P silencer DNA. Our studies are designed to gain a better understanding of 1) negative gene regulation in mammals (an area which is poorly understood, partly due to a lack of model systems to study) and 2) the actions of E and P which are important because of their essential roles in reproduction, their apparent involvement as transcriptional regulators of certain tumors, and their use as a model for steroid hormone action. Regulatory studies on the FSH beta subunit are also important because they define critical factors in controlling FSH synthesis/secretion and, therefore, gonadal function and fertility.