Transport du CO2 et échanges ioniques chez un animal autotrophe, le vestimentifère Riftia pachyptila : étude de l'anhydrase carbonique et des transporteurs impliqués

The hydrothermal vent vestimentiferan tubeworm Riftia pachytila consists on a very particular symbiosis model. Indeed, vestimentiferan are the only animals known to rely entirely on intracellular CO2-assimiliating and sulfato-oxidizing symbionts, remotely located from the surrounding medium, to sustain their metabolism. This suggest a complete subordination of the symbionts through their host regarding oxygen, sulfide, nitrogen and carbon, and at the opposite, the host loses its entire digestive tract and any way to ingest food, once the symbiosis becomes effective. We investigated specifically the cellular mechanisms responsible for carbon dioxide transport and conversion needed for bacterial metabolism, at the level of the plume and the trophosome tissues in Riftia. Our work combined physiological and pharmacological approaches on isolated bacteriocyte suspensions with in situ hybridization, immunlocalization, biochemical and molecular characterization.We showed that two isoforms of carbonic androhydrases (CA), related to the α-CA multigenic family, are expressed in Riftia. In the trophosome, both the cytosolic and the membrane-bound isoforms have been evidenced. They were co-localized with a V-H+-ATPase in all the symbiotic cells whereas Na+K+-ATPase occurrence were restricted to peripheric cells of the lobules. We also evidenced the responsiveness of bacteriocytes to amiloride and NPPB, inhibitors specific for sodium and chloride exchanges respectively. In the branchial plume, epithelial cells expressed only the cytolosic CA isoform, the cDNA and corresponding protein exhibiting characteristics slightly different from the trophosome cytosolic CA. The epithelial cells appeared to drive orientated ion fluxes, with a V-H+-ATPase on their apical side, in contact with the surrounding environment, and a Na+K+-ATPase on their basolateral side. These results are of interest in terms of understanding the carbon-related metabolism in Riftia pachytila toward the carbonic anhydrase key enzyme. They also point out the multiple interactions between ion exchanges and carbonic anhydrase activity, and provide further insights in characterization of peculiar transporters, that could be modified from those generally described in heterotrophic systems, because of the symbiotic mode of life of Vestimentiferan and their specific needs for nitrate, sulfide and carbon dioxide.Le vestimentifère des sources hydrothermales Riftia pachytila constitue un modèle de symbiose particulièrement original et quasiment unique dans le monde animal. Riftia est en effet à ce jour le seul organisme connu ayant développé une symbiose obligatoire avec une bactérie intracellulaire, sulfato-oxydante utilisant le CO2 moléculaire comme source de carbone, et localisée dans un organe interne isolé de tout contact direct avec le milieu environnant. Cet état de fait suggère la dépendance totale de la bactérie symbiotique vis-à-vis de son hôte, que ce soit pour une source d'oxygène, d'énergie (H2S), d'azote (NO3-) ou de carbone (CO2), et l'inverse est également vrai puisqu'une fois la symbiose établie, l'hôte perd toute capacité d'ingérer quelque nourriture que ce soit. Au cours de cette thèse, nous avons cherché à approfondir spécifiquement les mécanismes cellulaires liés au transport et à la conversion du CO2 mis en place dans la branchie et le trophosome de Riftia, et nécessaires au métabolisme des symbiontes. Pour cela, nous avons combiné une approche cellulaire et pharmacologique sur des suspensions de bactériocytes isolés à des techniques d'hybridation in-situ, d'immunolocalisation, de biochimie et de biologie moléculaire. Nous avons pu déterminer que Riftia exprime certainement deux isoformes d'anhydrases carboniques (CA) apparentées aux α-CA. Dans les lobules du trophosome, une isoforme cytoplasmique et une isoforme membranaire ont pu être mises en évidence. Un transporteur de type V-H+-ATPase a été co-localisé avec l'anhydrase carbonique dans toutes les cellules symbiotiques, alors que la présence de Na+-K-ATPase apparaît être restreinte aux cellules les plus périphériques du lobule. Nous avons également pu mettre en évidence la sensibilité des bactériocytes à l'amiloride et au NPPB, deux inhibiteurs des échanges d'ions sodiums et chlorures respectivement. Dans l'épithélium branchial, une seule isoforme de CA cytoplasmique a pu être mise en évidence, présentant des caractéristiques légèrement différentes de celles du trophosome. L'épithélium branchial joue un rôle manifeste dans le transport orienté des ions : une V-H+-ATPase a été immunolocalisée au niveau de la membrane apicale des cellules épithéliales, en contact avec le milieu environnant, et une Na+K+-ATPase au niveau basolatéral. Nos travaux ont permis d'apporter de nouvelles données sur le métabolisme du carbone chez Riftia pachytila au travers d'une enzyme clé, l'anhydrase carbonique, mais également d'élargir les perspectives d'étude à la caractérisation potentielle de nouveaux transporteurs membranaires, impliqués dans des échanges ioniques particuliers liés au mode de vie symbiotique de Riftia

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals

International audienceIn mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/b-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals

In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/β-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

International audienceAbstract R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals

In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/β-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal

The −KTS splice variant of WT1 is essential for ovarian determination in mice

IMPORTANT , l'article est en accès libre, le lien est sur le site http://ibv.unice.fr/research-team/chaboissier/ , il est surligné si on utilise Google Chrome http://ibv.unice.fr/research-team/chaboissier/#:~:text=https%3A//www.science.org/stoken/author%2Dtokens/ST%2D1527/fullInternational audienceSex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified −KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of − KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify −KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation