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

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.

Figure 1. IgSF8 expression and association with CD9 in mouse eggs

Panel A: ZP-free eggs (150 per lane) were surface-biotinylated and then lysed in buffer containing 1% Triton X-100; immmunoprecipitations were performed with an anti-CD9 antibody (lane 1) or nonimmune rat IgG (lane 2). Detection was performed with avidin-HRP; CD9 is present in immunoprecipitations using the anti-CD9 antibody but not the nonimmune IgG. Panel B: Immunoblot analysis shows IgSF8 protein expression in mouse eggs. Lane 1: Lysate of 75 eggs (1.88 μg). Lane 2: Lysate of mouse brain (0.5 μg). Panels C and D: ZP-free mouse eggs (200 per lane) were lysed in 1% Triton X-100 (lane 1) or CHAPS (lane 2) and immunoprecipitated with an anti-CD9 antibody, and then immunoblotted with an anti-CD9 antibody (Panel C) or an anti-IgSF8 antibody (Panel D). These immunoprecipitations show co-immunoprecipitation of IgSF8 with CD9.

Figure 2

Indirect immunofluorescence of mouse oocytes and eggs show colocalization of IgSF8 with CD9. A-F: Metaphase II egg labeled with anti-IgSF8 antibody (A), anti-CD9 antibody (B), non-immune goat IgG (C), with corresponding DAPI images showing the localization of the metaphase II DNA (D-F). G-L: Germinal vesicle-intact oocyte labeled with anti-IgSF8 antibody (G), anti-CD9 antibody (H), non-immune goat IgG (I), with corresponding DAPI images showing the germinal vesicle (J-L).

Figure 3. IVF outcomes from inseminations with sperm:egg ratios of 25:1, 100:1, and 500:1

Graphs show the percentages of ZP-free eggs (y-axes) with the indicated numbers of sperm bound (left panels) and fused (right panels) on the x-axes. In these studies, ZP-free eggs were inseminated for 60 min with sperm:egg ratios of 500:1 (Panels A,D; n=75 eggs), 100:1 (Panels B,E; n=200 eggs), or 25:1 (Panels C,F; n=330 eggs).

Figure 4. Effects of anti-IgSF8 or anti-CD9 antibodies on sperm-egg binding and fusion in inseminations with sperm:egg ratios of 25:1 or 100:1

ZP-free eggs were pre-treated with either control IgG (nonimmune goat IgG), anti-CD9, or anti-IgSF8 for 60 min, then inseminated for 60 min with the indicated sperm:egg ratio; the average numbers of sperm bound (Panels A,C) or fused (Panels B,D) per egg were examined. Panels A, B: Eggs inseminated with a sperm:egg ratio of 25:1. Panels C, D: Eggs inseminated with a sperm:egg ratio of 100:1. Data are from 3-6 experiments with 71-220 eggs per treatment group and insemination condition. Asterisks indicate statistically significantly differences (p < 0.05) from the control IgG group.

Figure 5. Inhibition of sperm-egg fusion by the anti-CD9 antibody in inseminations with sperm:egg ratio of 500:1

ZP-free eggs were pre-treated with either control IgG (nonimmune rat IgG) or anti-CD9 for 60 min, then inseminated for 60 min with a sperm:egg ratio of 500:1; the average numbers of sperm bound (Panel A) or fused (Panel B) per egg were examined. Data are from three experiments with 75-83 eggs per treatment group. Asterisk indicates statistically significantly differences (p < 0.05) from the control IgG group.

Figure 6. Effects of anti-IgSF8 and anti-CD9 antibodies on sperm-egg binding in 15 min inseminations

ZP-free eggs were pre-treated with either control IgG (nonimmune goat IgG), anti-CD9, or anti-IgSF8 for 60 min, then inseminated for 15 min with a sperm:egg ratio of 500:1. The average number of sperm bound per egg was examined. Data from three experiments with 86-109 eggs per treatment group. Asterisks indicate statistically significantly differences (p < 0.05) from the control IgG sample.