Abstract

The long range objective of the proposed research is the isolation and characterization of glycoproteins comprising the mammalian egg envelope, the zona pellucida, with the goal of correlating structures of specific zona macromolecules with reproductive function. Sperm-egg interactions in mammalian species are known to involve recognition of carbohydrate ligands in the zona pellucida by complementary receptor molecules on the sperm surface. The scientific goal of this application is to identify and characterize the biologically active carbohydrate chains of a sperm adhesive glycoprotein, ZP3alpha, of the pig zona pellucida. Biochemical studies will profile oligosaccharide populations at individual glycosylation sites. Oligosaccharides released from purified glycopeptides by chemical or enzymatic cleavage will be separated by high pH anion exchange chromatography and structurally characterized by mass spectrometric methodologies to define site-specific carbohydrate populations. The contribution of saccharides to the sperm adhesive properties of ZP3alpha will be probed by assaying the ligand activity of ZP3alpha glycopeptides as well as ZP3alpha preparations subjected to stepwise enzymatic deglycosylation. Ligand activity will be assessed by competitive sperm-zona attachment assays as well as by a competitive ligand binding assay employing immobilized boar sperm membrane vesicles as the source of receptors and biotinylated ZP3alpha as tracer. Collectively, the data obtained will yield new and important information on the structure and function of mammalian zona glycoproteins, particularly in regard to their carbohydrate moieties. Considering common mechanisms of gamete interactions across species and the immunological and structural similarities of human and pig zona proteins, such knowledge will contribute to a better understanding of human reproductive biology as well as the eventual development of novel approaches to prefertilization contraception and the diagnosis and treatment of infertility.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD023163-05
Application #
2198784
Study Section
Reproductive Biology Study Section (REB)
Project Start
1987-09-01
Project End
1997-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
Wayne State University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202

  Publications

O'Leary, Valerie B; Mills, James L; Parle-McDermott, Anne et al. (2005) Screening for new MTHFR polymorphisms and NTD risk. Am J Med Genet A 138A:99-106
O'Leary, Valerie B; Mills, James L; Pangilinan, Faith et al. (2005) Analysis of methionine synthase reductase polymorphisms for neural tube defects risk association. Mol Genet Metab 85:220-7
Kirke, Peadar N; Mills, James L; Molloy, Anne M et al. (2004) Impact of the MTHFR C677T polymorphism on risk of neural tube defects: case-control study. BMJ 328:1535-6
O'Leary, Valerie B; Mills, James L; Kirke, Peadar N et al. (2003) Analysis of the human folate receptor beta gene for an association with neural tube defects. Mol Genet Metab 79:129-33
Molloy, Anne M; Mills, James L; McPartlin, Joseph et al. (2002) Maternal and fetal plasma homocysteine concentrations at birth: the influence of folate, vitamin B12, and the 5,10-methylenetetrahydrofolate reductase 677C-->T variant. Am J Obstet Gynecol 186:499-503
Mills, J L; Kirke, P N; Molloy, A M et al. (1999) Methylenetetrahydrofolate reductase thermolabile variant and oral clefts. Am J Med Genet 86:71-4
Yurewicz, E C; Sacco, A G; Gupta, S K et al. (1998) Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles. J Biol Chem 273:7488-94
Yurewicz, E C; Pack, B A; Armant, D R et al. (1993) Porcine zona pellucida ZP3 alpha glycoprotein mediates binding of the biotin-labeled M(r) 55,000 family (ZP3) to boar sperm membrane vesicles. Mol Reprod Dev 36:382-9
Yurewicz, E C; Zhang, S; Sacco, A G (1993) Generation and characterization of site-directed antisera against an amino-terminal segment of a 55 kDa sperm adhesive glycoprotein from zona pellucida of pig oocytes. J Reprod Fertil 98:147-52
Dabich, D; Yurewicz, E C; Battel, V A (1993) Deglycosylation with trifluoromethanesulfonic acid differentially affects inhibitor activities of turkey ovomucoid. Biochim Biophys Acta 1164:47-53

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