The gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are heterodimeric pituitary glycoprotein hormones that directly regulate gametogenesis and gonadal steroid hormone biosynthesis in the testis and ovary. Transcription of the gonadotropin subunit genes, and biosynthesis, assembly, and secretion of the biologically active hormones are precisely controlled by the hypothalamic neuropeptide gonadotropin releasing hormone (GnRH), and gonadal steroid hormones and peptides. Altered regulation of gonadotropin production is a major cause of infertility. Conversely, the selective control of gonadotropin production has implications for both the treatment of infertility disorders and the development of effective novel contraceptives. The major goal of this project is to understand at the molecular level the DNA components and transcription factors that are responsible for the gonadotroph-specific expression and hormonal regulation of the FSHbeta subunit gene. DNA elements will be coarsely mapped initially by a deletional and mutational analysis of the human and mouse FSHbeta genes expressed in the pituitary glands of transgenic mice. Gonadotroph-specific expression will be confirmed by histological techniques and the hormonal regulation of the transgenes will be studied by analysis of FSHbeta mRNA levels with species' specific probes and by radioimmunoassay of the expressed FSH. Standard reproductive endocrinological experiments will be performed using transgenic mice and hpg hypogonadal mice genetically crossed with the hpg mice. A comparison of functionally important sequences conserved between human and mouse will direct future fine scale mapping of the core DNA regulatory elements. The mechanism of androgen inhibition of the human FSHbeta subunit gene at the pituitary level will be studied by a combination of in vivo nuclear run on studies and in vitro DNA-protein binding reactions using purified androgen receptor and competition experiments with known positively regulated androgen response elements. Inhibin and activin regulation of hFSHbeta will be studied using perfused columns of primary pituitary cell cultures derived from transgenic mice. Finally, we will attempt to derive a continuous, well differentiated gonadotroph cell line from pituitary tumors induced in transgenic mice with a temperature sensitive simian virus 40 large T antigen oncogene. Alternatively we will attempt to immortalize gonadotrophs using a powerful papilloma virus oncogene carried in a retroviral vector or by the expression of other oncogenes in the gonadotrophs of transgenic mice. A beta-subunit expressing gonadotroph cell line would be invaluable for further functional analysis of regulatory elements in the gonadotropin subunit genes, to characterize gonadotroph-specific transcription factors, and to explore the physiological mechanisms underlying the coordinated regulation of gonadotropin gene expression and the cell biology of gonadotropin biosynthesis and secretion.
