Hetero-oligomerization-dependent Binding of Pig Oocyte Zona Pellucida Glycoproteins ZPB and ZPC to Boar Sperm Membrane Vesicles*
- From the ‡Department of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan 48201, the ¶Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India, and the ‖Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
Abstract
The zona pellucida surrounding the pig oocyte contains two M r 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB+pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.
Footnotes
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↵* This work was supported by National Institutes of Health Grant HD23163 (to E. C. Y.). Matrix assisted laser desorption/ionization mass spectrometry analysis was performed at the Michigan State University Mass Spectrometry Facility, which is supported in part by National Institutes of Health Grant RR00480 (to D. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Wayne State University, Dept. of Ob/Gyn, 275 East Hancock, Detroit, MI 48201. Tel.: 313-577-1228; Fax: 313-577-8554; E-mail: e.yurewicz{at}wayne.edu.
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↵1 The abbreviations used are: HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; TBS, Tris-buffered saline; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethane sulfonic acid.
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↵2 E. C. Yurewicz, unpublished results.
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↵3 E. C. Yurewicz and G. K. Fontenot, unpublished results.
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- Received September 9, 1997.
- Revision received January 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
