CHARACTERIZATION OF SPERM MEMBRANES

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DESCRIPTION (Adapted from the applicant's abstract): The long term objective is to characterize structure-function relationships of ram sperm surface membrane domains essential to fertilization. The investigator has shown that changes in lipid composition and properties of membrane domains occur during epididymal maturation and that: (a) unique cholesterol transfer protein(s) (CTP) and lipoprotein particles (LPP) exist in male tract fluids; (b) lipid modifications alter sperm function (induction of the acrosome reaction (AR) and motility); and (c) membranes from two domains (the plasma membrane (PM) overlying the acrosome and the PM overlying the tail) can be isolated.Two questions are addressed. "How are unique PM domains developed and maintained?"and "How do layers of membranes (e.g., "onion-like" arrangements of PM and inner membranes) interact during development?" A hypothesis links progressive modifications of ram sperm PM lipids by luminal/internal lipid transfer proteins and luminal LPP to acquisition of membrane function. Experimental goals and questions are: Goal 1 (determination of lipid asymmetry of PM overlying the acrosome) answers the question: Is PM topography distinct in mature vs immature sperm?Two experiments characterize phospholipid flipase activities and lipid asymmetry of isolated membrane vesicles. Goal 2 (consequences of lipid asymmetry of the PM overlying the acrosome) answers the question: Does transbilayer movement of cholesterol (chol) permit fusion between two surfaces (IPM and outer acrosomal membrane) uniquely enriched in unsaturated phospholipid? Three experiments assess the extent of lipid asymmetry in the PM of immature sperm, test a model system to determine the block to the AR in immature sperm, and examine the role of transbilayer chol movement in membrane destabilization. Goal 3 (determination of lipid composition/asymmetry of PM overlying the tail) answers the question: Has the lipid composition and asymmetry of the PM overlying the tail been altered during epididymal transit? Two experiments determine the lipid composition of the tail membrane domain and assess lipid asymmetry of these membrane vesicles. Goal 4 (determination of the mechanism by which lipids are altered in the PM) answers the question: Given that the sperm cell exists in a sea of LPPs and CTPs, how are individual surface domains selectively modified during epididymal transit? Four experiments test for membrane domain specific binding of CTP)(s), determine if CTP can transfer chol into specific membrane domains, localize a PC transfer protein in the acrosomal region, and test for a PE transfer protein in epididymal fluids.