Gonadotropin releasing hormone (GnRH) plays an essential role in the regulation of reproductive physiology by the hypothalamic-pituitary axis. This project will examine mechanisms controlling GnRH gene expression and will provide insight into basic reproductive physiology as well as into heritable defects and other diseases affecting reproduction. Objectives of this project include elucidation of developmental controls determining male and/or female hypothalamic responses to sex steroids, genetic regulation of GnRH synthesis in the normal menstrual cycle, and changes in hypothalamic function during puberty and menopause. GnRH synthesis and regulation in gonadal and placental cells will be investigated. Rat GnRH cDNA will be cloned in the expression vector Lambdagt11. Western blot analysis of expressed proteins and screening by hybridization with specific oligonucleotides will be used to identify clones containing GnRH sequences. The GnRH cDNA will be used as a specific probe to study regulation of GnRH mRNA levels in different physiological conditions by Northern and dot-blot hybridization to hypothalamic RNA. GnRH synthesizing cells in the hypothalamus and elsewhere will be identified by in situ hybridization using biotinylated cDNAs. The cDNA will be sequenced by M13 and/or Maxam-Gilbert techniques and used to derive the amino acid sequence of the GnRH precursor. It will then be used to isolate GnRH containing clones from preexisting Charon genomic libraries. Relevant parts of the genomic clone will be sequenced and used in expression studies with DNA mediated gene transfer. These studies will help to provide a rational basis for diagnosis and treatment of disorders affecting the GnRH-gonadotrope axis, including premature puberty and hypogonadotropic hypogonadism, as well as furthering understanding of the integration of neural and endocrine systems in genetic regulation of reproductive function.
