Functional analysis and regulation of epididymal OCTN2

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DESCRIPTION (provided by applicant): Protection of cells from osmotic stress is critical for their survival as cells will undergo apoptosis if not protected. Epididymal luminal fluid is hypertonic and therefore epididymal cells need to adapt to the higher osmolality to limit the effects of osmotic stress. Like the kidney, the epididymis accumulates osmolytes to adapt to changes in osmolality. Movement of osmolytes is achieved by specific transporters, e.g. sodium-myo inositol cotransporter which transports the osmolyte myo-inositol. L-carnitine is another osmolyte that plays a key role in the adaption of hyperosmolality and is found in high concentrations in epididymal luminal fluid. Therefore, this study will examine how the transporter for L-carnitine, Novel Organic Cation Transporter 2 (OCTN2), plays a critical role in allowing epididymal cells to adapt to a hyperosmotic environment and ultimately protecting themselves from osmotic stress. Our working hypothesis is that osmotic stress of epididymal cells results in an influx of ions which increases the intracellular ionic strength. This increase activates the MAPK pathway, which in turn induces transcription of hypertonicity responsive transcription factors, e.g. TonEBP. These transcription factors then bind to their cognate binding sites on the OCTN2 promoter and allow transcription of OCTN2 to proceed. The transport protein is then translated, trafficked to the basolateral membrane and L-carnitine transported into the cell. Accumulation of the osmolyte, L-carnitine, will counteract the effects of ions by stabilizing key proteins and DNA. At this stage the epididymal cells have adapted to the new osmotic enviroment and afforded themselves protection from osmotic stress. Specifically, the following hypotheses will be tested: (1) OCTN2 is responsible for transport of L-carnitine into the epididymis; (2) The OCTN2 promoter is regulated by tonicity responsive transcription factors; (3) OCTN2 transcription and transporting activity are regulated by changes in tonicity via specific signal transduction pathways; (4) Loss of OCTN2 results in the loss of the ability of the epididymal epithelium to adapt to a hyperosmotic enviroment resulting in susceptibility to osmotic stress leading to epididymal dysfunction and male infertility. The findings from this proposal will provide fundamental information for the treatment of certain forms of male infertility and for the development of a male contraceptive.