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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.

reproduction, fertility, and development/reproduction, fertility and development

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