Generation of a mouse sperm membrane fraction with zona receptor activity.
To identify mouse sperm components involved in primary sperm binding to zonae pellucidae, a mouse sperm plasma membrane-enriched fraction was generated using a vortex method. The crude membrane fraction recovered after vortexing was resolved into three bands and a pellet by centrifugation on a discontinuous sucrose gradient. Ultrastructural analysis indicated that Bands 2 and 3 were composed predominantly of membranes, although Band 3 was contaminated with mitochondria; Band 1 and the gradient pellet contained insufficient material and were unsuitable for ultrastructural analysis. To determine where plasma membranes migrate in the gradient, sperm were labeled vectorially with 125I; subsequently, membrane fractions were analyzed by SDS-PAGE and autoradiography. Band 2 was enriched threefold in radiolabel when compared with Band 3. Examination of intact and vortexed sperm stained with regionally distributed anti-mouse sperm monoclonal antibodies revealed that vortexing removed anterior head plasma membrane preferentially. Bioactivity, defined as the ability to inhibit primary sperm binding to the zona pellucida in a concentration-dependent manner, was contained in the crude membrane fraction and Band 2 exclusively, with inhibition of 53% and 44%, respectively, at the maximum concentration tested. Band 3 exhibited no significant bioactivity. We conclude from these results that a plasma membrane-enriched fraction, Band 2, isolated from mouse cauda epididymal sperm, exhibits zona pellucida receptor activity.
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
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D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004527:Egg Proteins; D005260:Female; D006023:Glycoproteins / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D011956:Receptors, Cell Surface / Q000378:metabolism; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins