This applicant proposes a program of research to prepare her for a career in academic medicine and basic science research studying male reproductive/urologic tract function, addressing the regulation of epididymal luminal acidification under physiological and pathophysiological conditions, during adulthood and postnatal development. The research will be conducted in the laboratory of Dr. Sylvie Breton at the Program in Membrane Biology and Renal Unit, Massachusetts General Hospital. The epididymis and vas deferens (VD) are the sites where spermatozoa mature and are stored in an immotile state. The luminal environment of these organs is tightly regulated and maintained at an acidic pH in order to keep spermatozoa from achieving motility. In particular, the applicant will study the mechanism by which an alkaline epididymal/VD luminal pH stimulates H+ATPase trafficking from subapical vesicles to the apical plasma membrane in clear cells. In addition, she will further investigate the role of the bicarbonate-activated """"""""soluble"""""""" adenylyl cyclase (sAC), cAMP, and downstream effectors (such as PKA and Epac) on this trafficking phenomenon. Other sources of cAMP, such as G protein-regulated transmembrane adenylyl cyclases, and other second messenger molecules (such as cGMP) in clear cells will also be investigated. These studies will also evaluate the effects of steroid hormone agonists and antagonists (DES, ethinyl estradiol, GnRH antagonist and flutamide) and toxic compounds (Cadmium and Lead) have on the epididymal cellular profile, protein expression, H+ATPase trafficking and function, and on the cellular profile of the epididymis/VD during adulthood. The work proposed in this application will be carried out on rat and/or mouse epididymal/VD epithelial cells during adulthood and postnatal development, in vivo, in situ, and in vitro, using a multidisciplinary approach to evaluate protein expression and vesicle recycling mechanisms. The techniques proposed include microspectrofluorescence, cell biological, molecular biological techniques, and the use of a proton-selective self-referencing electrode. Newly available transgenic mice expressing enhanced green fluorescence protein (EGFP) specifically in clear cells (B1-EGFP mice) will provide a unique model to examine the development and modulation of epithelial cell phenotypes in response to various manipulation.
