Immunoglobulin superfamily member IgSF8 (EWI-2) and CD9 in fertilisation: evidence of distinct functions for CD9 and a CD9-associated protein in mammalian sperm–egg interaction
Amanda I. Glazar A and Janice P. Evans A BA Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolf Street, Baltimore, MD 21205, USA.
B Corresponding author. Email: jpevans@jhsph.edu
Reproduction, Fertility and Development 21(2) 293-303 https://doi.org/10.1071/RD08158
Submitted: 22 July 2008 Accepted: 29 September 2008 Published: 27 January 2009
Abstract
On the mouse egg, the tetraspanin CD9 is nearly essential for sperm–egg fusion, with another tetraspanin, CD81, playing a complementary role. Based on what is known about these proteins, egg tetraspanins are likely to be involved in regulation of membrane order through associations with other egg membrane proteins. Here, we identify a first-level interaction (stable in 1% Triton X-100) between CD9 and the immunoglobulin superfamily member IgSF8 (also known as EWI-2), the first evidence in eggs of such an interaction of CD9 with another protein. We also compared the effects of antibody-mediated perturbation of IgSF8 and CD9, evaluating the robustness of these perturbations in IVF conditions that heavily favour fertilisation and those in which fertilisation occurs less frequently. These studies demonstrate that IgSF8 participates in mouse gamete interactions and identify discrete effects of antibody-mediated perturbation of CD9 and IgSF8. An anti-IgSF8 antibody had moderate inhibitory effects on sperm–egg binding, whereas an anti-CD9 antibody significantly inhibited sperm–egg fusion and, in certain assays, had an inhibitory effect on binding as well. The present study highlights the critical importance of design of IVF experiments for the detection of different effects of experimental manipulations on gamete interactions.
Additional keywords: sperm–egg binding, sperm–egg fusion, tetraspanin.
Acknowledgements
This work was funded by grants from the National Institutes of Health to J.P.E. (HD037696, HD045671). The authors thank Chris Hung for critical reading of the manuscript.
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