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This Article Full Text Full Text (PDF) All Versions of this Article: 73/4/721    most recent biolreprod.105.041533v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sleight, S. B. Articles by Visconti, P. E. Search for Related Content PubMed PubMed Citation Articles by Sleight, S. B. Articles by Visconti, P. E. Agricola Articles by Sleight, S. B. Articles by Visconti, P. E.

Isolation and Proteomic Analysis of Mouse Sperm Detergent-Resistant Membrane Fractions: Evidence for Dissociation of Lipid Rafts During Capacitation¹

Center for Research in Contraceptive and Reproductive Health (CRCRH),³ Department of Cell Biology, Department of Neurosciences,⁴ Department of Urology,⁵ University of Virginia, Charlottesville, Virginia 22908 Department of Veterinary and Animal Sciences,⁶ University of Massachusetts, Amherst, Massachusetts 01003

Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.

fertilization, signal transduction, sperm, sperm capacitation, testis

² Correspondence: Pablo E. Visconti, Department of Veterinary and Animal Sciences, University of Massachusetts, 208 Paige Laboratories, Amherst, MA 01003. FAX: 413 545 6326; pvisconti{at}vasci.umass.edu

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