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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
Published on: February 22, 2016
Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida
M J Ringuette1, M E Chamberlin, A W Baur
1Laboratory of Cellular and Developmental Biology, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
At fertilization, mammalian sperm bind is a species-specific manner to the extracellular zona pellucida that surrounds ovulated eggs. ZP3, an 83,000-85,000 Da glycoprotein of the murine zona pellucida, has been shown to inhibit sperm binding via its O-linked oligosaccharide side chains. We have recently isolated cDNA clones coding for ZP3 and have demonstrated that ZP3 transcripts are accumulated in oocytes where their expression is developmentally regulated during oogenesis. We now report that ZP3 mRNA is 1317 nt long with an estimated poly(A) tail of 200-300 nt. The short 29-nt 5' untranslated region is followed by a single open reading frame coding for a polypeptide chain of 46,307 Da which includes six possible sites for N-linked oligosaccharides. The N-terminus of ZP3 contains a potential 22-amino acid signal peptide which upon cleavage would result in a secreted core protein of 43,943 Da. The termination codon is a part of the AATAAA polyadenylation signal and is contained in an unusually short 16-nt 3' untranslated region. Sequences homologous to ZP3 are conserved among mammals and are expressed in ovarian tissue as mature transcripts with indistinguishable molecular weights.
Insights
Zona Pellucida 3 (ZP3) glycoprotein inhibits sperm binding through its oligosaccharide chains. This study details the ZP3 mRNA structure and its conserved expression in mammalian ovarian tissue, crucial for fertilization.
Area of Science:
- Reproductive Biology
- Molecular Genetics
- Glycobiology
Background:
- Mammalian sperm-zona pellucida binding is species-specific, mediated by the zona pellucida (ZP) surrounding ovulated eggs.
- Murine ZP3 glycoprotein, a key component of the ZP, inhibits sperm binding via its O-linked oligosaccharide side chains.
- Previous work identified ZP3 cDNA clones and demonstrated developmentally regulated expression in oocytes.
Purpose of the Study:
- To characterize the molecular structure of ZP3 mRNA.
- To investigate the conserved nature and expression patterns of ZP3 in mammalian ovarian tissue.
Main Methods:
- Isolation of cDNA clones coding for ZP3.
- Analysis of ZP3 mRNA length, including 5' and 3' untranslated regions and poly(A) tail.
- Prediction of polypeptide chain characteristics, including signal peptide and N-linked glycosylation sites.
- Comparative analysis of ZP3 sequences and transcript expression in ovarian tissues across mammalian species.
Main Results:
- ZP3 mRNA is 1317 nucleotides long with a 200-300 nt poly(A) tail.
- The coding region specifies a 46,307 Da polypeptide with a potential 22-amino acid signal peptide, yielding a 43,943 Da secreted core protein.
- ZP3 mRNA features a short 5' UTR (29 nt) and a very short 3' UTR (16 nt) containing the polyadenylation signal.
- Homologous ZP3 sequences are conserved among mammals, with mature transcripts of similar molecular weights found in ovarian tissue.
Conclusions:
- The detailed molecular structure of ZP3 mRNA provides insights into its regulation and function.
- Conserved ZP3 sequences and expression patterns suggest a critical, conserved role in mammalian fertilization.
- Understanding ZP3 structure and regulation is vital for reproductive biology research.
