Germinal Vesicle stage (GV) oocytes were harvested at 39 hrs post-PMSG. Ten pL siRNA solution (Ezrin, Radixin, Moesin or negative siRNA) were injected into the cytoplasm of denuded GV oocytes one hour after their retrieval. Oocytes were matured in vitro and were then assessed after 17 hrs maturation. Only Metaphase II (MII) oocytes were selected on the basis of the presence of the first polar body. The zona pellucida (ZP) was subsequently removed with acidic Tyrode’s solution. ZP-free MII oocytes were inseminated with capacitated sperm for 1 hr at a final concentration of 105/mL, then washed and mounted in Vectashield/DAPI for observation under UV light. The fertilization index was evaluated: the total number of fused sperm/total number of eggs.

OOCYTE ERM PROTEINS PLAY A CRUCIAL ROLE IN GAMETE FUSION J. Cohen*, B. Lefevre*, C. Ialy Radio*, J.P. Wolf *, A. Ziyyat * *Université Paris Descartes. Institut Cochin. INSERM unité U1016, Paris, France

INTRODUCTION Fertilization culminates by the encounter of highly differentiated gametes. This interaction follows specific steps including recognition, adhesion and fusion. On the mammalian oocyte, Cd9 is the only molecule known at the moment to be essential for fertilization and to which is assigned a role in gamete membrane fusion. However, Cd9 has no fusion peptide. This assignment comes very likely from the fact that the gamete adhesion persists in the absence of Cd9 while the fusion cannot occur. Recently, we have shown that Cd9 generates fusion competent sites on the egg membrane for mammalian fertilization. These Cd9-induced adhesion sites, named pro-fusional, with strong adhesion, would be the actual location where fusion occurs. Indeed, in the absence of Cd9, the adhesion between the two gametes exists but remains abortive. Regarding the adhesive behavior experimentally measured, the crucial difference between WT and Cd9 null eggs comes from the strong interaction events which are absent in Cd9 null eggs (1). These adhesions are stronger because of the strong connection between the membrane and the cytoskeleton, which is not realized directly but through other molecules. The link would likely occur via Cd9-indirect partners such as Ezrin-Radixin-Moesin (ERM) that connect the actin cytoskeleton to the plasma membrane (2,3). ERM proteins are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. The purpose of our work was to confirm the hypothesis that Cd9 plays indirectly its role in gamete fusion thanks to its strong link to the cytoskeleton and that this link is mediated by ERM proteins. In order to demonstrate the role of these proteins, we have injected in oocytes cytoplasm small interfering RNA (siRNA) for each gene, following various combinations. We have then assessed functional and morphological effects of the siRNA. We verified their effectiveness in inhibition of target proteins expression, we measured the Fertilization Index (FI) and we studied oocyte microvilli morphology.

The main goal of our study was to improve the knowledge of the mechanisms that allow Cd9 protein to play its fundamental role in gamete fusion. Recently, Jegou et al. (1) measured adhesion forces between oocyte and sperm. When fusion occured with wild-type eggs, strong adhesion forces existed that reflected a potentially strong link between oocyte membrane and the underneath cytoskeleton. Interestingly this strong link was not observed in Cd9 -/- eggs and only « weak » forces remained. This discrepancy may reflect the fact that there is a stronger link between the two gametes than between the oocyte membrane and the cytoskeleton. This hypothesis is consistent with the fact that various proteins in somatic cells are known to make this link, such as EWI-2, EWI-F and ERM proteins. Whether this interaction also occurs with the cytoskeleton is unknown. ERM proteins are concentrated in actin-rich surface structures such as microvilli, filopodia and membrane ruffles (2). Two phosphorylation-dependent activation levels have been described for ERMs: when phosporylated, ERM proteins are active and located at the plasma membrane, when dephosphoshorylated, they are inactive and soluble in the cytoplasm (3). Our results showed that Fertilization Index was not decreased after isolated inhibition of Ezrin, Radixin or Moesin. This might be explained by redundancy mechanisms.

Moesin-deficient mice are perfectly normal and fertile without any Ezrin or Radixin hyper-regulation. Ezrin-deficient mice died at three weeks and their fertility was not explored. However, they showed abnormalities in their intestinal epithelium morphogenesis with microvilli disorganization. Ezrin is the only ERM protein detected in the developing small intestinal epithelium, and this is why no redudancy mechanism may occur in this case. Radixin-deficient mice are viable and fertile, but develop hyperbilirubinemia . In fact, combined injection of Ezrin, Radixin and Moesin siRNAs strongly affected fertilization, decreasing the FI. Given that ERM proteins promote the Cd9-cytoskeleton interaction in somatic cells, we can hypothesize that ERM are also critical in gamete adhesion/fusion mechanism. To explain their involvement, it is important to notice that their absence induced a disorganization of the oocyte microvilli, radius of curvature of which was larger, as in the experiments of Runge et al. (4) on Cd9 -/- oocytes. Indeed, a small radius of curvature at the tip of the microvilli maximizes the efficiency of membrane fusion and a dynamic role of the microvilli participates in sperm-oocyte contact.

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Combined injection of Ezrin, Radixin and Moesin siRNAs descreased Fertilization

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Injection of Ezrin, Radixin or Moesin siRNA did not affect fertilization. As these results were predictable because of the likely redundancy between the three genes, we made a mix of the 3 siRNAs (ERM siRNA) . After ERM siRNAs injection, FI was 1.2 (n=67) compared to 2.8 after Control siRNA injection (n=82), which means a 57% decrease (p<0.001).

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1.  Jegou A, et al. (2011) CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization. Proc Natl Acad Sci U S A 108(27):10946-10951. 2.  Sala-Valdes M, et al. (2006) EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins. J Biol Chem 281(28):

19665-19675 3.  Louvet-Vallee S (2000) ERM proteins: from cellular architecture to cell signaling. Biol Cell 92(5):305-316. 4.  Runge KE, et al. (2007) Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution. Dev Biol 304(1):317-325.

Decrease of Metaphase II oocyte pERM immunostaining after GV siRNA injection

ERM silencing encreased oocyte microvilli’s radius of curvature

When we analyzed oocytes by electronic microscopy, we found that microvilli had a larger radius of curvature after ERM RNA silencing. Radius of curvature of control oocytes (n oocytes = 5; n microvilli = 350) was 35 nm versus 48 nm in siRNA injected oocytes (+ 37.8 %, p<0.001; n ooocytes = 5; n microvilli = 521).

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