This proposed research will continue the studies of subsets of pituitary gonadotropes that secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH). Gonadotropes are diverse when tests of their storage or secretory functions are made and the purpose of the ongoing research is to learn if such diversity can be correlated with a particular functional or maturational state during the estrous cycle. The experiments will be gin by separating small, medium and large gonadotropes with counterflow centrifugation in a new elutriation chamber. The cells will be taken from homogeneous groups of female rats at specific stages of the cycle. The first set of studies will determine if subsets of small gonadotropes release more gonadotropins prior to peak proestrous or estrous secretory activity than large gonadotropes. Secretion (LH or FSH) will be detected by radioimmunoassay or the reverse hemolytic plaque assay (RHPA). The results will be correlated with changes in storage patterns of LHbeta and FSHbeta antigens detected by immunoperoxidase or immunogold protocols, and LHbeta and FSHbeta mRNA detected by in situ hybridization and measured by Northern Blot assays. The second set of studies will determine if and when the small gonadotropes bind a biotinylated analog of gonadotropin releasing hormone (GnRH) and if estradiol enhances GnRH binding. If enhancement is evident, the data would indicate that some small gonadotropes may be precursors to the large gonadotropes. The third set of studies will compare the responses of small and large gonadotropes to activation of second messengers, to learn if small gonadotropes change their capacity to respond during the estrous cycle. Responses to be analyzed include LH and FSH secretion, LHbeta or FSHbeta subunit antigen storage, mRNA expression, and GnRH binding. GnRH receptors,beta subunit antigens or mRNAs will be detected by labeled avidin protocols. Changes in the amount of label will be quantified with image analysis systems that measure color or density. The studies will provide information about individual subsets of gonadotropes that will broaden our understanding of their responses to GnRH and steroids and better define the different cycles of activity expressed by the small and large gonadotropes..
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