The goal of this research is to understand the molecular physiology of gonadotropin-releasing hormone (GnRH) and pro-opiomelanocortin (POMC) neurons involved in male reproduction. An array of cells in the hypothalamus and forebrain secretes GnRH, which coordinates the release of the pituitary gonadotropins and ultimately governs testicular function. Other neurotransmitter systems in the brain, including members of the endogenous opioid family (e.g., beta-endorphin, from POMC) and the excitatory amino acid glutamate, process hormonal signals (e.g., testosterone) from the testis which, in turn, modulate the secretory activity of GnRH cells. Our first objective is to learn how testosterone regulates the synthesis of gene products within the GnRH cell. Here, we will focus on the proGnRH processing enzymes [carboxypeptidase H (CPH) and alpha-amidating enzyme (PAM)] and the glutamate receptor (GluR-K1 type). Using double in situ hybridization, we will map the distribution of the genes coding for CPH, PAM, and GluR-K1 within anatomically defined subsets of GnRH neurons and examine their regulation by testosterone. If we find that testosterone influences the expression of one or more of these genes, we will proceed to test the hypothesis that the effect of testosterone on gonadotropin secretion can be attributed to changes in pro-GnRH processing or alterations in receptor-mediated sensitivity to glutamate. We will approach this by coupling the GnRH promoter to the structural gene for CPH, PAM or GluR-K1 and use these constructs to create and study transgenic mice expressing multiple copies of one of these chimeric genes within GnRH neurons. Our second objective is learn more about the molecular physiology of testosterone-sensitive POMC neurons in the hypothalamus. We and others have previously shown that POMC neurons in the arcuate nucleus are functionally heterogeneous, but what molecular characteristics determine this heterogeneity and how gene products, in addition to POMC itself, are regulated within these neurons are unknown. We will use double in situ hybridization to measure POMC mRNA and aromatase or estrogen receptor mRNAs for the purpose of the studying how these genes are regulated by steroids within anatomically defined subsets of POMC neurons. We believe this work will foster improved knowledge of cellular neuroendocrinology and may offer clues to understanding clinical problems related to human reproduction.
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