Ahmed Ziyyat

H19 acts as a trans regulator of the imprinted gene network controlling growth in mice.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.

CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion

The process of gamete fusion has been largely studied in the mouse and has revealed the crucial role of the tetraspanin CD9. By contrast, human gamete fusion remains largely unknown. We now show that an anti-α6 integrin mAb (GoH3) strongly inhibited human sperm-egg fusion in human zona-free eggs. Furthermore, a mAb directed against CD151, a tetraspanin known to associate with α6β1, partially inhibited sperm-egg fusion. By contrast, the addition of an anti-CD9 mAb to zona free eggs had no effect. The integrin α6β1, CD151 and CD9 tetraspanins were evenly distributed on human zona-intact oocytes. On zona-free eggs, the integrin α6β1 and tetraspanin CD151 patched and co-localized but the tetraspanin CD9 remained unchanged. CD9 mAb prevented α6β1 integrin clustering and gamete fusion when added prior to, but not after, zona removal. Antibody-mediated aggregation of integrin α6β1 yielded patches that were bigger and more heterogeneous in mouse oocytes lacking CD9. Moreover, a strong labelling of α6β1 could be observed at the sperm entry point. Altogether, these data show that CD9 controls the redistribution of some membrane proteins including the α6β1 integrin into clusters that may be necessary for gamete fusion.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm–egg fusion is a direct consequence of CD9 controlled sperm–egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

Transfer of oocyte membrane fragments to fertilizing spermatozoa

Several families of molecules are implicated in the membrane fusion process between sperm and oocyte. Among these, CD9 tetraspanin, a membrane‐organizing molecule, plays a crucial role, since the fertilizing ability of CD9−/− oocytes is dramatically impaired. CD9 controls alpha6‐beta1 integrin relocation involved in the membrane reorganization that occurs on oocyte fertilization but is not expressed on sperm. We report here that, together with several other proteins, the CD9 tetraspanin is transferred from the oocyte to the fertilizing spermatozoa present in the perivitelline space before fertilization. Transfer of CD9 from oocyte to sperm from CD9−/− male mice still occurs. CD9 acquisition by sperm results from a transfer of membrane fragments from the plasma membrane of the oocyte, in a process similar to trogocytosis, the recently described mechanism of intercellular exchange of membrane patches. Acquisition of CD9 by the sperm may be crucial for the membrane reorganization in sperm required for fusion with the oocyte, a process that is similar to the role CD9 plays in oocyte membrane reorganization.—Barraud‐Lange, V., Naud‐Barriant, N., Bomsel, M., Wolf, J.‐P., Ziyyat, A. Transfer of oocyte membrane fragments to fertilizing spermatozoa. FASEB J. 21, 3446–3449 (2007)

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

BackgroundBased on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.ResultsUsing Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrins function in the adhesion/fusion process.ConclusionThese findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

Role of sperm αvβ3 integrin in mouse fertilization

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin α6β1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of αvβ3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti‐αv or anti‐β3 antibodies were performed before in vitro fertilization on cumulus‐intact and zona‐free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm αvβ3 integrin. Developmental Dynamics 239:773–783, 2010.

Refined Mapping of a Quantitative Trait Locus on Chromosome 1 Responsible for Mouse Embryonic Death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Sperm-egg interaction: is there a link between tetraspanin(s) and GPI-anchored protein(s)?

Both female mice deficient in CD9 tetraspanin‐ and oocyte‐specific glycosyl‐phosphatidylinositol‐anchored family proteins showed severely reduced fertility due to the failure of sperm‐egg fusion. This raises the question of a link between these two groups of proteins at the oocyte membrane. We propose two hypotheses to explain why the absence of one of these proteins from the oocyte membrane results in the same phenotype. The first hypothesis envisages different levels of control by these molecules of the common induced signaling cascade. The second relies on the known involvement of these molecules in the overall organization of the plasma membrane. Their disappearance could thus prevent sperm‐egg fusion either by disruption of the signaling cascade and/or by an important disorganization of the oocyte membrane. In this review, describing their structural and functional characteristics, and using published results on the oocyte, we try to analyze how these two protein families could interact.