WNT/β-catenin signaling pathway and germ cell differentiation in mammals

La préservation de la fertilité suscite une inquiétude croissante. Depuis les dernières décennies, la fertilité a diminué dans les pays industrialisés. En conséquence, un nombre croissant de couples a recours à la procréation médicalement assistée, sans grand succès par manque de connaissances sur les cellules reproductrices (cellules germinales). L’étude de ces cellules constitue un socle essentiel pour des applications médicales, mais jusqu’à présent, les mécanismes moléculaires qui régissent leur développement restent peu compris en raison de l’absence de modèles d'étude physiologiques.Les cellules germinales sont produites au cours du développement embryonnaire, se multiplient pour constituer un stock suffisant, se différencient dans la gonade en fonction du sexe de l'embryon, puis entrent en méiose pour devenir des gamètes fertiles, les ovocytes (femelles) et les spermatozoïdes (mâles). Mon laboratoire d’accueil a démontré que la voie de signalisation canonique WNT/β-catenin est nécessaire au développement de l'ovaire in vivo et qu'en absence d'activation de cette voie dans l'ovaire embryonnaire de souris, la prolifération et la différenciation des cellules germinales primordiales sont perturbées.Tirant partie de notre expertise dans l'analyse de modèles de différenciation gonadique, nous nous sommes intéressés au mécanisme d'action de la voie WNT/β-catenin dans la différenciation des cellules germinales, en analysant au moyen de modèles murins de perte et gain de fonction de β-catenin les conséquences de la modification de son expression dans les cellules de la gonade.Dans un premier temps, nous avons démontré que le maintien, après la naissance, de l'activité de la voie WNT/β-catenin dans les cellules souches spermatogoniales du testicule de souris stimule leur prolifération de manière massive, provoquant un défaut de différenciation en spermatozoïdes. Dans un deuxième temps, nous avons montré que l'ablation génétique du gène Ctnnb1 (codant pour β-catenin) dans les cellules germinales primordiales in vivo entraîne une perte précoce de leur pluripotence et une différenciation prématurée en ovogonies. Nous avons démontré, pour la première fois in vivo, que tandis que le développement ovarien progresse, β-catenin forme des complexes protéiques avec POU5F1 et CDH1 qui transitent du noyau des cellules germinales à leur membrane, permettant ainsi leur sortie de pluripotence et leur différenciation. Nos résultats indiquent que l'E3-ubiquitine ligase ZNRF3 régule la pluripotence des cellules germinales par une boucle de rétroaction négative.Ces données indiquent que dans l'ovaire comme dans le testicule, une régulation fine de l'activité de la voie de signalisation WNT/β-catenin est requise pour déterminer la fenêtre de différenciation des cellules germinales primordiales in vivo. De plus, la voie WNT/β-catenin contrôle la sortie de pluripotence des cellules germinales primordiales via une activité non-transcriptionnelle de β-catenin, permettant à terme de coordonner le développement des cellules somatiques et germinales de la gonade. Dans les années à venir, nos travaux pourraient aider à la génération des gamètes in vitro.Since the last decennials, fertility is decreasing in industrial countries, and nowadays, infertility reaches a prevalence of 9 to 18% of the general population, with a significant proportion of cases being due to defective gametogenesis. Accordingly, fertility preservation raises a growing concern. Nowadays, a growing number of couples undergo Assisted Reproductive Technology with little success due to the lack of knowledge on mechanisms orchestrating germ cell differentiation. Little is known about how primordial germ cells become gametogenesis-competent cells (gonocytes), because of the lack of suitable physiological models. Primordial germ cells give rise to the next generation by differentiating from pluripotent progenitors into highly specialized cells, the gametes, which in turn generate a totipotent zygote after fertilization. After specification in the early embryo, primordial germ cells colonize the gonad, lose pluripotency and gain their capacity for irreversible sexual differentiation. Gonocytes then become either female (oogonia) or male (spermatogonia), and eventually progress into meiotic divisions. So far, the mechanisms of germ cell changes in potency remain largely elusive.My host laboratory has demonstrated that activation of the canonical WNT/β-catenin signalling is required for ovarian development. Thus, absence of WNT/β-catenin activation eventually triggers defects in germ cell proliferation and sexual differentiation. However, genetic models of cell-specific ablation of Ctnnb1 (encoding β-catenin) were still missing to assess the contribution of WNT/β-catenin in the germ cells. We decided to analyse the role of this signalling pathway by generating specific mouse models for β-catenin loss or gain of function and by investigating the consequences of WNT/β-catenin deregulation in either the somatic or the germ cells of the developing gonad. We first demonstrated that WNT/β-catenin regulates spermatogonial stem cell proliferation and differentiation in the post-natal testis, demonstrating that this signalling activity must be finely tuned over time to ensure spermatogenesis.Secondly, we showed that genetic elimination of Ctnnb1 in mouse primordial germ cells in vivo leads to a precocious loss of pluripotency and to premature germ cell differentiation into gonocytes. We demonstrated, for the first time in vivo, that while ovarian development is getting forward, β-catenin forms proteic complexes containing POU5F1 and CDH1 that transit from the nucleus of the germ cells to their membrane, thus allowing primordial germ cells to exit pluripotency and eventually differentiate. Our results also reveal that the ZNRF3 E3-ubiquitine ligase negatively regulates germ cell exit from pluripotency through a negative feedback loop.Collectively, our results show that the WNT/β-catenin signalling is necessary for determining the proper window of differentiation in both somatic and germ cells. Moreover, WNT/β-catenin controls the germ cell exit from pluripotency through β-catenin non-transcriptional activity, eventually coordinating the development of the different cell types of the fetal ovary. In the future, our results might help to recapitulate gametogenesis in vitro

Voie de signalisation WNT/ β-catenin et différenciation des cellules germinales chez les mammifères

Since the last decennials, fertility is decreasing in industrial countries, and nowadays, infertility reaches a prevalence of 9 to 18% of the general population, with a significant proportion of cases being due to defective gametogenesis. Accordingly, fertility preservation raises a growing concern. Nowadays, a growing number of couples undergo Assisted Reproductive Technology with little success due to the lack of knowledge on mechanisms orchestrating germ cell differentiation. Little is known about how primordial germ cells become gametogenesis-competent cells (gonocytes), because of the lack of suitable physiological models. Primordial germ cells give rise to the next generation by differentiating from pluripotent progenitors into highly specialized cells, the gametes, which in turn generate a totipotent zygote after fertilization. After specification in the early embryo, primordial germ cells colonize the gonad, lose pluripotency and gain their capacity for irreversible sexual differentiation. Gonocytes then become either female (oogonia) or male (spermatogonia), and eventually progress into meiotic divisions. So far, the mechanisms of germ cell changes in potency remain largely elusive.My host laboratory has demonstrated that activation of the canonical WNT/β-catenin signalling is required for ovarian development. Thus, absence of WNT/β-catenin activation eventually triggers defects in germ cell proliferation and sexual differentiation. However, genetic models of cell-specific ablation of Ctnnb1 (encoding β-catenin) were still missing to assess the contribution of WNT/β-catenin in the germ cells. We decided to analyse the role of this signalling pathway by generating specific mouse models for β-catenin loss or gain of function and by investigating the consequences of WNT/β-catenin deregulation in either the somatic or the germ cells of the developing gonad. We first demonstrated that WNT/β-catenin regulates spermatogonial stem cell proliferation and differentiation in the post-natal testis, demonstrating that this signalling activity must be finely tuned over time to ensure spermatogenesis.Secondly, we showed that genetic elimination of Ctnnb1 in mouse primordial germ cells in vivo leads to a precocious loss of pluripotency and to premature germ cell differentiation into gonocytes. We demonstrated, for the first time in vivo, that while ovarian development is getting forward, β-catenin forms proteic complexes containing POU5F1 and CDH1 that transit from the nucleus of the germ cells to their membrane, thus allowing primordial germ cells to exit pluripotency and eventually differentiate. Our results also reveal that the ZNRF3 E3-ubiquitine ligase negatively regulates germ cell exit from pluripotency through a negative feedback loop.Collectively, our results show that the WNT/β-catenin signalling is necessary for determining the proper window of differentiation in both somatic and germ cells. Moreover, WNT/β-catenin controls the germ cell exit from pluripotency through β-catenin non-transcriptional activity, eventually coordinating the development of the different cell types of the fetal ovary. In the future, our results might help to recapitulate gametogenesis in vitro.La préservation de la fertilité suscite une inquiétude croissante. Depuis les dernières décennies, la fertilité a diminué dans les pays industrialisés. En conséquence, un nombre croissant de couples a recours à la procréation médicalement assistée, sans grand succès par manque de connaissances sur les cellules reproductrices (cellules germinales). L’étude de ces cellules constitue un socle essentiel pour des applications médicales, mais jusqu’à présent, les mécanismes moléculaires qui régissent leur développement restent peu compris en raison de l’absence de modèles d'étude physiologiques.Les cellules germinales sont produites au cours du développement embryonnaire, se multiplient pour constituer un stock suffisant, se différencient dans la gonade en fonction du sexe de l'embryon, puis entrent en méiose pour devenir des gamètes fertiles, les ovocytes (femelles) et les spermatozoïdes (mâles). Mon laboratoire d’accueil a démontré que la voie de signalisation canonique WNT/β-catenin est nécessaire au développement de l'ovaire in vivo et qu'en absence d'activation de cette voie dans l'ovaire embryonnaire de souris, la prolifération et la différenciation des cellules germinales primordiales sont perturbées.Tirant partie de notre expertise dans l'analyse de modèles de différenciation gonadique, nous nous sommes intéressés au mécanisme d'action de la voie WNT/β-catenin dans la différenciation des cellules germinales, en analysant au moyen de modèles murins de perte et gain de fonction de β-catenin les conséquences de la modification de son expression dans les cellules de la gonade.Dans un premier temps, nous avons démontré que le maintien, après la naissance, de l'activité de la voie WNT/β-catenin dans les cellules souches spermatogoniales du testicule de souris stimule leur prolifération de manière massive, provoquant un défaut de différenciation en spermatozoïdes. Dans un deuxième temps, nous avons montré que l'ablation génétique du gène Ctnnb1 (codant pour β-catenin) dans les cellules germinales primordiales in vivo entraîne une perte précoce de leur pluripotence et une différenciation prématurée en ovogonies. Nous avons démontré, pour la première fois in vivo, que tandis que le développement ovarien progresse, β-catenin forme des complexes protéiques avec POU5F1 et CDH1 qui transitent du noyau des cellules germinales à leur membrane, permettant ainsi leur sortie de pluripotence et leur différenciation. Nos résultats indiquent que l'E3-ubiquitine ligase ZNRF3 régule la pluripotence des cellules germinales par une boucle de rétroaction négative.Ces données indiquent que dans l'ovaire comme dans le testicule, une régulation fine de l'activité de la voie de signalisation WNT/β-catenin est requise pour déterminer la fenêtre de différenciation des cellules germinales primordiales in vivo. De plus, la voie WNT/β-catenin contrôle la sortie de pluripotence des cellules germinales primordiales via une activité non-transcriptionnelle de β-catenin, permettant à terme de coordonner le développement des cellules somatiques et germinales de la gonade. Dans les années à venir, nos travaux pourraient aider à la génération des gamètes in vitro

Constitutive WNT/CTNNB1 activation triggers spermatogonial stem cell proliferation and germ cell depletion

International audienceThe differentiation of germ cells into oogonia or spermatogonia is the first step that eventually gives rise to fully mature gametes. In the female fetal gonad, the RSPO1/WNT/CTNNB1 signalling pathway is involved in primordial germ cell proliferation and differentiation into female germ cells, which are able to enter meiosis. In the postnatal testis, the WNT/CTNNB1 pathway also mediates proliferation of spermatogonial stem cells and progenitor cells. Here we show that forced activation of the WNT/CTNNB1 pathway in fetal gonocytes using transgenic mice leads to deregulated spermatogonial proliferation, and exhaustion of the spermatocytes by apoptosis, resulting in a hypoplastic testis. These findings demonstrate that a finely tuned timing in WNT/CTNNB1 signalling activity is required for spermatogenesis

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

Arrest of WNT/β-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries

International audienceGerm cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell–intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3 , a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis

Retinoic acid synthesis by ALDH1A proteins is dispensable for meiosis initiation in the mouse fetal ovary

International audienceIn mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells