The objective of our research is to understand how a specific gene is expressed and regulated in male reproductive tissues. In this proposal, proopiomelanocortin (POMC) will be used as a model system. POMC, which is a precursor protein for ACTH, beta-endorphin and melanocyte-stimulating hormones, has been well characterized in many tissues. Our recent demonstration of the existence of POMC-like mRNA in testis and epididymis suggests the expression of a POMC gene and synthesis of POMC peptides in these tissues. Since the regulation of a specific gene expression in Leydig cells or epididymis has not yet been well understood, we have chosen to investigate the regulation of POMC gene expression in these tissues. Our first specific aim is to investigate both in vivo and in vitro the regulation of POMC mRNA level and POMC peptide synthesis, processing, and secretion in testis and epididymis by using factors known to affect functions of Leydig cells (gonadotropins, prolactin LHRH analogs, etc.) or epididymis (androgen, testis fluid). The regulation of POMC mRNA and its peptide levels will also be examined at cellular levels by using in situ cDNA hybridization histochemistry technique. Our second specific aim is to understand the molecular mechanisms involved in the change of POMC mRNA level by modulators. To answer this question, we will determine whether the rate of synthesis of POMC mRNA as measured by nuclear transcriptional activity of the POMC gene or the stability of POMC mRNA will be altered by modulators. Finally, the structure of heterogeneous POMC mRNAs expressed in the testis and epididymis will be characterized. The POMC-like mRNAs found in the testis, epididymis, or Leydig cell lines are approximately 150-200 bases shorter than those found in the pituitary or hypothalamus. To understand the mechanism involved in the production of heterogeneous POMC mRNA in different tissues, we will determine the POMC mRNA structure by cDNA cloning and DNA sequencing analysis, characterize POMC gene(s) isolated from rat genomic libraries, and identify the transcription unit of POMC gene and the pathway of processing nuclear precursor of POMC mRNA. The proposed research will provide useful information in studying the regulation of a specific gene expression in Leydig cells and epididymis. In addition, since POMC is a precursor for several peptide hormones and neurotransmitters, the proposed research will facilitate our understanding of the biological role of these individual peptides derived from POMC in the male reproductive physiology.
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