The development and coordinated function of the reproductive tract are dependent on the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). These two glycoprotein hormones are composed of a common alpha subunit and differing beta subunits that confer receptor, and therefore biological specificity. Transcriptional regulation of the individual subunit genes in response to a myriad of hypothalamic and gonadal factors plays a central role in the certain forms of human infertility, the design of new contraceptive methods, and the production of gonadotropins from the relatively common """"""""null cell"""""""" pituitary adenomas. A detailed analysis of the cis-acting regulatory elements of the gonadotropin beta subunit genes and identification of their cognate nuclear binding proteins has been hampered by the unavailability of appropriate cell lines, however. The overall goal of this proposal is to define the mechanism of cell-specific and hormonally regulated expression of the gonadotropin genes. We will characterize initially the basal and hormonal regulation of human FSH beta gene expression in transgenic mice to test whether the human beta gene is expressed at levels comparable to the endogenous mouse gene and if it is regulated directly by gonadotropin releasing hormone and gonadal steroid hormones. A series of deletional mutants and FSH-reporter fusion genes will be introduced into transgenic mice to identify within 300 bp the FSH beta gene regulatory elements that are required for gonadotrope- specific expression and to determine their overlap with hormonal regulatory elements. We will establish lines of transgenic mice that develop well- differentiated gonadotropin-producing tumors to be used directly as a source of nuclear protein extracts and for the development of an immortalized glycoprotein beta subunit expressing cell line. Conditionally immortalized gonadotropes will be produced using either a temperature- sensitive SV40 T antigen or wild-type T antigen whose expression can be regulated in vitro and by the lac operator/repressor system. A cell line will be used for a more detailed analysis of the cis-acting regulatory elements of the hFSH beta gene and would provide a valuable resource for studying the cell biology of gonadotropin biosynthesis, assembly, and secretion. Finally, a multipronged approach of in vitro and in ovo transcription assays, and expression cloning techniques will be employed to characterize the gonadotrope-specific transactivator of the hFSH beta subunit gene and clone a cDNA encoding the protein. The new reagents resulting from this work will be useful for understanding the coordinated regulation of all the gonadotropin subunit genes in addition to FSH beta.
