Membrane Glycoproteins from Spermatozoa: Partial Characterization of an Integral Mr = ∿24,000 Molecule from Rat Spermatozoa that is Glycosylated during Epididymal Maturation1

Abstract

Spermatozoa from the rat cauda epididymidis were treated with either the galactose oxidase-NaB[3H]4 or the NaIO4-NaB [3H]4 technique to label cell surface moieties of galactose and sialic acid, respectively. Following extraction with 40 mM octyl-beta-D-glucopyranoside (OBG), electrophoresis in sodium dodecylsulfate-polyacrylamide gels (SDS-PAGE) revealed a single radioactive peak migrating at Mr = approximately 24,000 in 11.2%, 14% and 16.8% tube gels. SDS-PAGE of the same OBG extract on 5.6% and 14% gels showed that this molecule was the same as that reported elsewhere, having a molecular weight varying from 32,000 to 37,000. The amount of labeled molecule extracted with 8 M urea or with 6 M guanidine-HCl was 30 and 50%, respectively, of that achieved with OBG. However, when labeled sperm were treated under other conditions (at pH 8, pH 3, 0.1-3 M NaCl [at pH 7.2], 5 mM ethylenediaminetetraacetic acid [EDTA], 20-200 mM dithiothreitol or 20-200 mM betamercaptoethanol, at varying temperatures and extraction times), the amount of labeled molecule extracted was less than 1% of that obtained with OBG. The molecule aggregates in aqueous buffers, as shown by chromatography using Sephadex G-100 and by centrifugation through a sucrose density gradient. Analysis by charge-shift electrophoresis suggested that the molecule contains an exposed hydrophobic domain(s). Mild trypsin treatment released all the labeled carbohydrate with the majority of the label attached to a Mr = 10,000 fragment. These data support the hypothesis that the molecule is an integral membrane glycoprotein, and suggest that it is only partially buried in the lipid matrix of the plasma membrane.