Question

2. You have isolated a protein from sperm that you suspect is involved in binding to the zona pellucida and triggering the acrosome reaction. You generate an antibody to this protein and use the antibody to isolate the protein. You have obtained peptide information and used this peptide data to identify the DNA sequence that encodes your protein of interest.

a. What zona protein do you suspect this sperm protein binds to?

b. What expression pattern is consistent with your hypothesized role?

c. Describe one experiment to show that this protein is necessary for sperm-egg binding. What results would be consistent with your hypothesis?

Answer 1

a.In vitro studies suggest that sperm-egg binding is mediated by protein-carbohydrate complementarity

b.zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian

oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the

study was to determine if all four ZP proteins are expressed and present in the human. METHODS: cDNA derived

from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were

heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). RESULTS: All four genes

were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed

that the mouse ZPB gene, although present, is likely to encode a non-functional protein. CONCLUSIONS: Four ZP

genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corres-

ponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse

model

Key words: human/mouse ZPB/oocyte/proteomics/zona pe

c.The zona pellucida of the egg has been removed, exposing the plasma membrane, which contains numerous microvilli. The ability of an individual's sperm to penetrate hamster eggs is used as an assay of male fertility; penetration of more than 10–25% of the eggs is considered to be normal.

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