The proposed renewal extends the immunocytochemical studies of gonadotropin storage in the rat anterior pituitary to focus on the cellular and subcellular events associated with joint storage of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the same cells or organelles. Joint storage of gonadotropins will be further tested by applying stains for alpha and beta chains to serial sections of pituitaries prepared after cryoultramicrotomy or rapid freezing combined with freeze drying. This will allow more precise localization of gonadotropin molecules and subunits unaltered by chemical fixatives or dehydrating agents. The techniques also promote the localization of molecules at sites of synthesis or packaging. Once optimum preparative conditions are established, they will be applied to a study of changing storage patterns throughout fetal development, the estrous cycle, and after castration. The techniques to be used include radioimmunoassay for gonadotropins and immunocytochemical stains for whole FSH and LH and their alapha, and beta chains. The stains will employ more precise identifying markers such as protein A labelled colloidal gold as well as the peroxidase anti peroxidase complex solutions employed in previous studies. Additional techniques include cryoultramicrotomy, combined freeze-fixation, freeze drying, and stereology. The use of serial thick and thin sections will allow us to identify sites of joint storageof gonadotropins while stereologic techniques will yield information about the volume density ofgonadotropin containing organelles and cells. The use of a goniometer will allow a three-dimensional analysis of storage patterns in organelles on stereo pairs of electronmicrographs. We anticipate that this immunocytochemical and morpometric approach will provide more basic information about mechanisms used by gonadotropic cells to process gonadotropin molecules and their subunits.
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