SIGNAL TRANSDUCERS IN SPERM

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DESCRIPTION: (Scanned from the applicant's description) Fertilization in mammals depends upon the physiological priming of sperm during a process known as capacitation. The long range goal of this project is to determine the molecular and cellular mechanisms of capacitation. TRP2 is a sperm Ca2+ channel that is activated by sperm contact with the egg zona pellucida and drives the acrosome reaction. During differentiation and maturation of sperm, TRP2 assembles into heteromerc complexes that are posttranslationally modified during capacitation, thereby modulating ion channel function. As this channel plays a defined role in the fertilization process, and undergoes defined structural and functional regulation during sperm capacitation, it provides a unique model system in which to determine the underlying molecular processes. The mechanisms of TRP2 regulation during capacitation will be determined in this funding period by combining state-of-the-art electrophysiological methods and genomic/proteomic approaches. There are four Specific Aims. First, the proteins components of TRP2 complexes will be isolated by co-immunoprecipitation and identified by proteomic mass spectroscopy. Second, the phosphorylation state of TRP2 and of TRP2-associated proteins will be determined. Third, the effects of capacitation on TRP2 ion channel activity will be determined by electrophysiological studies. Finally, the role of TRP2-associated proteins and of protein phosphorylation in the control of ion channel activity will be determined by electrophysiological methods. These studies may be significant at several levels. First, an understanding of capacitation is central to our basic models of fertilization. Second, the inability to capacitate human sperm efficiently may account for aspects of infertility, as well as for part of failure rates during in vitro fertilization. Moreover, sperm Ca2+ channel antagonists are reported to have male contraceptive action and hence an understanding of the regulation of channel function is necessary for design of channel-based antifertility agents. Finally, TRP proteins are implicated in signal transduction processes in a wide range of cells, but our knowledge of these important channels has been limited by the lack of specific antagonists. A function-blocking antibody was generated and is utilized here to study the functions of the endogenous TRP2 channel of mouse sperm. Consequently, these studies may also provide a model for the functions of TRP channels in other tissues.