Sonication of mouse sperm membranes reveals distinct protein domains

    Authors

    S. S. Baker; R. A. Cardullo;C. D. Thaler

    Comments

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    Abbreviated Journal Title

    Biol. Reprod.

    Keywords

    acrosome reaction; fertilization; gamete biology; signal transduction; sperm; ZONA-PELLUCIDA; EXTRACELLULAR-MATRIX; PLASMA-MEMBRANE; MAMMALIAN; FERTILIZATION; BOAR SPERMATOZOA; IN-VITRO; BINDING; CAPACITATION; HYALURONIDASE; ACTIVATION; Reproductive Biology

    Abstract

    Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1-3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with I-125 demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions.

    Journal Title

    Biology of Reproduction

    Volume

    66

    Issue/Number

    1

    Publication Date

    1-1-2002

    Document Type

    Article

    Language

    English

    First Page

    57

    Last Page

    64

    WOS Identifier

    WOS:000172912800009

    ISSN

    0006-3363

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