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Loss of surface EWI-2 on CD9 null oocytes
Zhi-Yong He1, Surabhi Gupta, Diana Myles
1Department of Molecular and Cellular Biology, College of Biological Sciences, University of California Davis, Davis, California 95616, USA.
Abstract:
CD9, a member of the tetraspanin family, associates with a variety of other proteins to form the tetraspanin web. CD9 forms direct and relatively stable associations with the immunoglobulin superfamily proteins EWI-2 and EWI-F. Deletion of the Cd9 gene results in female infertility since Cd9 null mice produce oocytes that fail to fuse. It is thought that the absence of CD9 causes the inability of the oocytes to fuse. In this study, we report that the expression level of EWI-2 on the Cd9(-/-) oocyte surface is <10% of the wild-type level. Hence, the severe reduction in EWI-2 activity may be responsible for the loss of fusion ability. An entirely different mutant of CD9, not a deletion but a depalmitoylated construct, does not affect in vivo female fertility suggesting that the palmitate modification of CD9 is not essential for its putative fusion function. Additionally, the level of EWI-2 on the surface of the oocytes from these females was comparable to the EWI-2 level on wild-type oocytes. We also found that soluble, recombinant EWI-2 binds preferentially to acrosome-reacted sperm but the bound EWI-2 does not inhibit sperm-oocyte fusion. Overall, the results indicate that deletion of CD9, which is known to have multiple associations, may have pleiotropic effects on function that will require further dissection.
Insights
The CD9 protein is crucial for female fertility, as its absence in mice leads to oocyte fusion failure. Reduced EWI-2 expression on CD9-deficient oocytes likely causes this infertility, suggesting CD9
Area of Science:
- Reproductive biology
- Cell adhesion molecules
- Molecular genetics
Background:
- CD9, a tetraspanin protein, forms complexes with other proteins, known as the tetraspanin web.
- CD9 directly associates with immunoglobulin superfamily proteins EWI-2 and EWI-F.
- CD9 deletion in mice causes female infertility due to oocyte fusion defects.
Purpose of the Study:
- To investigate the role of CD9 in oocyte fusion and female fertility.
- To determine the impact of CD9 deletion on EWI-2 expression and function.
- To explore the necessity of CD9 palmitoylation for its role in fertility.
Main Methods:
- Analysis of oocyte fusion in CD9 knockout (Cd9(-/-)) mice.
- Quantification of EWI-2 surface expression on wild-type and Cd9(-/-) oocytes.
- Assessment of female fertility in mice with a depalmitoylated CD9 mutant.
- In vitro binding assays with recombinant EWI-2 and sperm.
Main Results:
- CD9 deletion significantly reduces EWI-2 surface expression on oocytes (<10% of wild-type).
- A depalmitoylated CD9 mutant retains in vivo female fertility, indicating palmitoylation is not essential for fusion.
- Soluble EWI-2 binds to acrosome-reacted sperm but does not inhibit sperm-oocyte fusion.
Conclusions:
- The severe reduction of EWI-2 on Cd9(-/-) oocytes is a likely cause of the observed female infertility.
- CD9's palmitoylation is not essential for its function in female fertility.
- CD9 deletion may have pleiotropic effects beyond EWI-2 regulation, requiring further investigation.
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