The long-term goal is to identify mechanisms regulating glycolysis druing spermatogenesis and sperm function. Isolated spermatids are unacble to maintain ATP levels when glucose is their oonly energy substrate, while sperm require glucose for in vitro fertilization, suggesting that unique mechanisms regulate glycolysis in male germ cells. Glyceraldehyde 3- phosphate dehydrogenase (GAPD) appears to be the rate limiting enzyme for glycolysis in spermatids. A gene (Gapd-s) encoding a unique form of this enzyme (GAPD-S) is expressed only in spermatids and may serve a pivotal role in regulating glycolysis in these cells. Competitve inhibitors of NAD coenzyme binding to this enzyme suppress glycolysisin spermatids, and competitive inhibitors or substrate(glyceraldhyde 3 phosphate) binding are male reproductive toxicants. GAPD-S also has unique sequence features that may be involved in regulating its activity. It is hypothesized that glycolysis is suppressed in late spermatids due to inhibition of GAPD-S, but that GAPD-S is activated in sperm where glycolysis provides energy required for fertilization.
The specific aims of this study are to: 1) Determine when GAPD-S is synthesized and where it is localized in spermatozoa. Specific antibody probes will be used to define when the GAPD-S protein is expressed during spermatid differentiation (spermiogenesis) and determine whether this enzyme binds to sperm structural components. 2) Define the role of GAPD-S during spermiogenesis, sperm maturaltion and fertilization. In vitro studies will determine when GAPD-S is activation during the development and function of sperm and define its role in providing energy required fertilization. Target disruption of the Gapd-s gene by homogous recombination will be used to determine whether GAPD-S is required for spermiogenesis and/or fertility in mice. 3) Identify the molecular mechanisms that regulate GAPD-S in spermatids and sperm. To determine how GAPD-S is regulated, activities of recombinant GAPD-S and GAPD will be compared in cell-free assays in the presence of competitive inhibitors of coenzyme and substrate binding. The studies will determine whether the regulation of energy production in spermatogenic cells is due to unique structural and functional features of GAPD-S, and will provide fundamental information on the role of GAPD-S in fertilization. Mutations in the Gapd-s gene or inhibition of the GAPD-S enzyme by environmental agents may cause infertility in men, and the GAPD-S enzyme may be a feasible target for a highly specific contraceptive agent.
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