MOLECULAR MECHANISMS OF HFSH/B GENE REGULATION

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DESCRIPTION (Taken from the applicant's Abstract) One of the most common and difficult to manage disorders of the reproductive system is altered gonadotropin regulation related to overactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The first Specific Aim of this mentored project is directed towards understanding the normal regulation of the reproductive system, in particular the regulation of the hFSH-beta gene by gonadotropin specific factors, gonadotropin releasing hormone (GnRH) and gonadal steroids. This will be accomplished by l) comparison of the hFSH-beta gene to known cAMP-responsive, gonadotropin-specific, estrogen, and androgen response elements and GATA-binding sites known to regulate other gonadotropin genes, 2) electromobility shift and DNase protection assays to demonstrate DNA binding and mutation of any binding sites found to assess their importance for expression and 3) expression of 3' deleted gene constructs in transgenic mice to map candidate enhancers. In Specific Aim Two, the importance of beta-endorphin in mediating stress-induced anovulation will be evaluated in physiologic experiments using beta-endorphin knockout mice. The importance of glucocorticoids for this effect will be tested by demonstrating DNA binding of glucocorticoid receptor and in physiologic experiments of stress-induced anovulation in adrenalectomized beta-endorphin deficient mice. Work in this field has been hampered by the lack of a convenient expression system; therefore, continued efforts to develop an FSH-producing cell line from mouse gonadotropin cells immortalized with the SV40tsTAg linked to the hFSH-beta promoter and optimizing transient transfections is the third Specific Aim of this project. An improved understanding of the physiologic and molecular regulation of the reproductive system and the interaction of the HPA axis with that system will lead to improved treatment of the reproductive effects of disorders of the HPA axis. Through a mentored career development plan, the Principal Investigator will enhance knowledge of biochemistry and molecular biology through course work, enhance skills in application of basic science research to understanding endocrinology, gain expertise in research design and statistical analysis, and gain appreciation of principles of responsible scientific conduct. By the end of the five year mentorship period, this will result in achievance of independence in the design and implementation of research projects.