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Evolution of germline gene expression profiles during rainbow trout ontogenesis and puberty onset. 7. International Workshop on the Biology of Fish Gametes

Le comportement animal nous fascine, nous étonne !En donnant de la voix, les porcs expriment un grandnombre d’émotions et communiquent avec leurssemblables et avec les hommes qui les côtoient. Les poissons ont des capacités d’apprentissage qui necessent de nous surprendre. Des jeux, des ateliers etanimations pour mieux comprendre le comportementdes animaux et évaluer objectivement le bien-êtreanimal.

Table S1. Influence of the feed ration, or the maturational status, on selected morphometric parameters, pituitary gene expression, and plasma hormone levels. RF, restricted ration; NF, normal ration; GSI, gonadosomatic index; K, condition factor; RU, relative units; nd, not determined. In the two different comparisons (ration effect and maturation effect; highlighted by different background color), asterisks indicate significant differences (N = 5–16; Student’s t-test, * P

Introduction: Fsh is a pituitary gonadotropin that regulates both gonadal functions, gametogenesis and steroidogenesis, in fish. The biological actions of Fsh are mediated through its binding to a membrane receptor (Fshr) that belongs to the superfamily of the G protein-coupled receptor (GPCR). Previous reports on in vivo disruption of the Fshr gene showed that Fsh signaling pathway was dispensable for male fertility but was required for oogenesis. In the present study we generated new mutant zebrafish lines to further investigate the role of fshr in reproductive and growth performances. Methods: We disrupted the fshr gene in zebrafish using the CRISPR/Cas9 method. We selected three CRISPR sites in the 10th exon of the gene to cause a large deletion easily detectable by a PCR-based genotyping method. Two mutant lines were generated from independent founders mated with transgenic vasa:eGFP females. The mutations were characterized by DNA sequencing in both lines. The deletions led to predicted truncated proteins enable to anchor to the membrane and to transduce Fsh signal. Results and Discussion: Heterozygous mutant males and females developed an apparently normal gametogenesis and were fertile. However heterozygous zebrafish population (fshr+/-) showed a female biased sexratio compared to the non mutated siblings. At 27 days post fertilization heterozygous mutants showed higher relative abundance of the gonadal aromatase gene (cyp19a1a) and increased levels of transcripts involved in granulosa cell differentiation (foxl2a2, gsdf, fshr, sox9a). In addition, transcripts accumulated during late oocyte growth (vasa, sox9b and nanos2) were also detected at higher levels. The relative abundance of sycp1 was unchanged indicating that meiosis initiation was normal. In contrast, in homozygous mutant meiosis was altered as revealed by decreased expression levels of sycp1. In addition, oocytes did not progress beyond the pre-follicle phase of primary growth. All adult homozygous mutants became males suggesting that impaired oogenesis during early ontogenesis caused sex reversal. Additional new phenotypes were observed in the homozygous males including lower sperm count and viability. This could result from 11-KT deficiency as revealed by decreased expression levels of cyp11b2. Perturbation of steroidogenesis could also explain the increased body weight and length observed in homozygous mutants. Conclusion: The marked phenotypic perturbations induced by the Fshr mutation confirm that Fsh and Lh have distinct functions. We showed that the loss of one fshr gene copy favours the differentiation of granulosa cells and oocyte growth. Fsh signalling pathway is dispensable for Sertoli cell differentiation but remains important for sperm quality and excretion.

Mise en évidence d'étapes de transition successives des cellules souches germinales qui conduisent à l'apparition de progéniteurs au cours de l’ontogénèse de la truite arc-en-ciel (Oncorhynchus mykiss). Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

Gene expression profiling during rainbow trout ontogenesis reveals successive changes gated in teleosts in germ stem cell molecular signature and regulatory pathways

un poster présenté; Expression profiling of new germline markers suggests successive waves of different spermatogonial stem cell subpopulations during rainbow trout ontogenesis. 6. International Workshop on the Biology of Fish Gametes

The invalidation of the FSHR gene in zebrafish leads to a gain of body weight, a biased sex-steroid and a decrease in sperm quality. 6. International Workshop on the Biology of Fish Gametes

Récolte de sperme sur des poissons zèbres mutés pour analyse en cytométrie de flux. 2. Journées des Installations Expérimentales LPGP

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Supplemental Figures S1 - S2 - S3 - S4; What makes the spermatogonial stem cells (SSCs) self-renew or differentiate to produce spermatozoa is barely understood, in particular in nonmammalian species. Our research explores possible regulations of the SSC niche in teleost, locally by paracrine factors and peripherally by hormonal regulation. In the present study, we focus on the Gdnf-Gfra1 pathway that plays a major role in the regulation of SSC self-renewal in mammals. We describe a complex evolution of the genes encoding for Gdnf and Gfra1 proteins in trout with the emergence of three gdnf and two gfra1 paralogs. Using quantitative PCR measurements in isolated testicular cell populations, the gdnfb paralog was found expressed in A-spermatogonia and probably in another testicular cell type. In contrast, the transcript of gfra1a, the Gdnf receptor, was preferentially expressed in a population of undifferentiated A-spermatogonia (und A-Spg) separated by centrifugal elutriation. These und A-Spg also demonstrated high stemness potential in transplantation studies and preferentially expressed nanos2, a putative SSC marker in trout (Bellaiche et al., Biol Reprod 2014; 90:79). Flow cytometer experiments demonstrate that only a subfraction of und A-Spg express Gfra1. In trout, spermatogenesis develops along a strict annual cycle, and gdnfb and its receptor were expressed in a spermatogenetic activity-dependent manner. In particular, a dramatic increase of the gdnfb transcript coincided with the progressive cessation of rapid spermatogonial proliferation and of meiosis toward the end of the reproductive cycle. Together these results suggest that, in trout, Gdnfb is involved in the repression of und A-Spg differentiation. Fsh is an endocrine regulator of SSCs self-renewal through the up-regulation of Gdnf in rodents. We demonstrate that in trout, in vitro Fsh treatment stimulated the expression of the gfra1a1 but not of its ligand, gdnfb. Fsh treatment also stimulated the proliferation of und A-Spg cocultured with testicular somatic cells. Based on those results, the Gfra1-positive cells could correspond to the putative SSCs in rainbow trout, and we propose that the balance between SSC self-renewal and differentiation during the trout spermatogenetic cycle is under paracrine regulation by Gdnfb, which represses, and under peripheral regulation by Fsh via the control of gfra1a1 expression.

In vertebrates, spermatogenesis is under the direct control of two pituitary gonadotropic hormones named Fsh and Lh. In salmonids, the 2 hormones are differentially secreted in the plasma through the reproductive cycle, suggesting that Fsh and Lh are involved in the regulation of different steps of the process. Unlike in mammals, both fish gonadotropins exert a strong steroidogenic activity and, besides, some authors reported for their receptors a much lower selectivity towards the two ligands. Yet, their respective roles are not established in fish. Furthermore, the mechanisms of Fsh action that would not be mediated by steroids are poorly investigated. In trout, we showed that each gonadotropin mainly acts through its cognate receptor. The analysis of the variations of testicular transcriptome following an in vitro treatment with reproductive hormones (Fsh, Lh and androgens) permitted 1- to reveal that Fsh and Lh have distinct effects on gene expression, 2- to highlight two mechanisms of Fsh action, one dependent on the steroid production and the second one independent of that production, 3- to identify several Fsh regulated factors involved in cellular interactions and particularly in the control of germ cell proliferation / differentiation (anti mullerian hormone, midkine, insulin-like growth factor 1b, follistatin-like 3 and activin), 4- to propose an involvement of Fsh in late events of sperm maturation and release. In addition to knowledge on endocrine and molecular regulations in trout, this work provides a fund of knowledge useful in other teleosts to decipher the action of Fsh in triggering puberty onset. Finally, we showed that an immunization against the gonadotropin receptors is a potential method to delay sexual maturation in farmed fish.; Chez les vertébrés, le processus de la spermatogénèse est directement contrôlé par deux hormones gonadotropes hypophysaires, Fsh et Lh. Chez les salmonidés, les profils de sécrétion des 2 hormones diffèrent et présentent des variations significatives au cours du cycle de développement spermatogénétique, suggérant que Fsh et Lh interviennent à des étapes différentes du processus. A la différence des mammifères, chez les poissons les 2 gonadotropines exercent une forte activité stéroïdogène, et par ailleurs il a été rapporté par plusieurs auteurs que leurs récepteurs seraient moins sélectifs vis-à-vis des 2 ligands. Ainsi, le périmètre des actions respectives de Fsh et de Lh n'est pas défini chez les poissons. D'autre part, les mécanismes de l'action de Fsh qui ne seraient pas relayés par les stéroïdes sont très mal connus. Chez la truite, nous avons déterminé que chaque gonadotropine agit essentiellement par l'intermédiaire de son récepteur respectif. L'analyse des variations du transcriptome testiculaire après un traitement in vitro par les hormones de la reproduction (Fsh, Lh, androgènes) a permis 1- de révéler des actions distinctes de Fsh et de Lh sur l'expression des gènes, 2- de mettre en évidence deux mécanismes d'action de la Fsh, l'un dépendant et l'autre indépendant de la production de stéroïdes et 3- d'identifier plusieurs acteurs d'interaction cellulaire régulés par Fsh, et probablement impliqués dans les étapes précoces de prolifération ou de différenciation des cellules germinales, tels que l'hormone antimüllérienne, la midkine, l'insulin-like growth factor1b, la follistatine-like 3 et l'activine, 4-de proposer une implication de Fsh dans les évènements tardifs de maturation et d'excrétion du sperme. Au-delà des acquis concernant les régulations endocriniennes et moléculaires chez la truite, ces travaux constituent un apport de connaissances qui peut être étendu à d'autres téléostéens pour décrypter l'action propre à Fsh dans le déclenchement de la maturation pubertaire. Enfin, nous montrons qu'une vaccination contre les récepteurs des gonadotropines constitue une voie potentielle de contrôle des maturations précoces en élevage.

In vertebrates, spermatogenesis is under the direct control of two pituitary gonadotropic hormones named Fsh and Lh. In salmonids, the 2 hormones are differentially secreted in the plasma through the reproductive cycle, suggesting that Fsh and Lh are involved in the regulation of different steps of the process. Unlike in mammals, both fish gonadotropins exert a strong steroidogenic activity and, besides, some authors reported for their receptors a much lower selectivity towards the two ligands. Yet, their respective roles are not established in fish. Furthermore, the mechanisms of Fsh action that would not be mediated by steroids are poorly investigated. In trout, we showed that each gonadotropin mainly acts through its cognate receptor. The analysis of the variations of testicular transcriptome following an in vitro treatment with reproductive hormones (Fsh, Lh and androgens) permitted 1- to reveal that Fsh and Lh have distinct effects on gene expression, 2- to highlight two mechanisms of Fsh action, one dependent on the steroid production and the second one independent of that production, 3- to identify several Fsh regulated factors involved in cellular interactions and particularly in the control of germ cell proliferation / differentiation (anti mullerian hormone, midkine, insulin-like growth factor 1b, follistatin-like 3 and activin), 4- to propose an involvement of Fsh in late events of sperm maturation and release. In addition to knowledge on endocrine and molecular regulations in trout, this work provides a fund of knowledge useful in other teleosts to decipher the action of Fsh in triggering puberty onset. Finally, we showed that an immunization against the gonadotropin receptors is a potential method to delay sexual maturation in farmed fish.; Chez les vertébrés, le processus de la spermatogénèse est directement contrôlé par deux hormones gonadotropes hypophysaires, Fsh et Lh. Chez les salmonidés, les profils de sécrétion des 2 hormones diffèrent et présentent des variations significatives au cours du cycle de développement spermatogénétique, suggérant que Fsh et Lh interviennent à des étapes différentes du processus. A la différence des mammifères, chez les poissons les 2 gonadotropines exercent une forte activité stéroïdogène, et par ailleurs il a été rapporté par plusieurs auteurs que leurs récepteurs seraient moins sélectifs vis-à-vis des 2 ligands. Ainsi, le périmètre des actions respectives de Fsh et de Lh n'est pas défini chez les poissons. D'autre part, les mécanismes de l'action de Fsh qui ne seraient pas relayés par les stéroïdes sont très mal connus. Chez la truite, nous avons déterminé que chaque gonadotropine agit essentiellement par l'intermédiaire de son récepteur respectif. L'analyse des variations du transcriptome testiculaire après un traitement in vitro par les hormones de la reproduction (Fsh, Lh, androgènes) a permis 1- de révéler des actions distinctes de Fsh et de Lh sur l'expression des gènes, 2- de mettre en évidence deux mécanismes d'action de la Fsh, l'un dépendant et l'autre indépendant de la production de stéroïdes et 3- d'identifier plusieurs acteurs d'interaction cellulaire régulés par Fsh, et probablement impliqués dans les étapes précoces de prolifération ou de différenciation des cellules germinales, tels que l'hormone antimüllérienne, la midkine, l'insulin-like growth factor1b, la follistatine-like 3 et l'activine, 4-de proposer une implication de Fsh dans les évènements tardifs de maturation et d'excrétion du sperme. Au-delà des acquis concernant les régulations endocriniennes et moléculaires chez la truite, ces travaux constituent un apport de connaissances qui peut être étendu à d'autres téléostéens pour décrypter l'action propre à Fsh dans le déclenchement de la maturation pubertaire. Enfin, nous montrons qu'une vaccination contre les récepteurs des gonadotropines constitue une voie potentielle de contrôle des maturations précoces en élevage.

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Fiche Aquaculture-Recherche; National audience; Au cours de l'incubation artificielle des oeufs de salmonidés, certains blanchissent plus ou moins rapidement, et dans des proportions variables selon la qualité initiale des pontes.

National audience; Female rainbow trout were induced to ovulate by GnRHa. Half of the eggs of each female was collected and inseminated on ovulation day (I-D0) and the other half a week later (I-D7). Fry survival was significantly better in I-D7 than in I-D0 offspring. Moreover, a high percentage of triploid fry (65 to 90%) was observed in I-D0 offspring from some females, whereas no triploid fry was observed anymore. in I-D7 offspring from the same females.

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National audience; To better improve understanding of the molecular mechanisms involved in the actions of gonadotropins in fish, a structural and functional characterization of trout gonadotropin receptors has been undertaken. The present study describes two distinct gonadotropin receptors in trout showing similarities with those of other vertebrates but also differences in structural determinants of the FSHR. As observed in their mammalian counterparts, trout LH receptor (LHR) harbours a large N-terminal hormone binding domain with 9 leucine reach repeats (LRR) flanked by a proximal and distal cysteine rich region (CRR). A similar sequential organization of 9 clustered LRR and 2 CRR motifs is also observed on mammalian FSH receptors however this is absent in the trout with only one out of four conserved cysteine residues being present upstream of the first LRR motif. The functional specificity of trout gonadotropin receptors was investigated in the heterologous mammalian COS-7 cells using homologous (trout) and heterologous (chinook salmon) purified gonadotropins. Contrary to published data in catfish and zebrafish, trout gonadotropin receptors show a high selectivity for their respective cognate ligand.

National audience; The consequences of immunization against specific regions of gonadotropin receptors were analysed in rainbow trout, on gonadal development and plasma testosterone levels. Males immunized against LHR alone or in combination with FSHR, showed a decrease in mean GSI. related to an inhibition of the spermatogenetic process; females immunized against FSHR or LHR exhibited delayed vitellogenesis. Vaccination against FSHR, and to a less extent against LHR, had a global inhibitory effect on plasma testosterone levels.

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International audience; The aim of the present paper was to obtain somatotroph- and gonadotroph-enriched populations from collagenase dispersed pituitaries of male rainbow trout. Inasmuch as the percentage of immunoreactive gonadotrophs and somatotrophs present in pituitaries was higher at spermiation than at the beginning of spermatogenesis, we tried such a cell separation with fish at this stage of spermatogenesis. Cells were fractionated using their differences in buoyant density with centrifugation in Percoll solutions. The use of Percoll linear gradients (1.110 to 1.027 g/ml) showed that somatotroph cells have a density of between 1.102 and 1.064 g/ml whereas gonadotrophs are spread over the range of the gradient. It was thus possible, by using linear or discontinuous Percoll gradients, to obtain 95 to 67% (mean 80%) enriched somatotropic cell fractions while no enriched gonadotropic cell fractions were collected. The fractionated cells kept their ability to be cultured and to be responsive to specific secretagogues. Somatostatine induced a 80 to 85% decrease in growth hormone release per somatotroph in the initial cell suspension as well as in the different cell fractions. On the other hand, the basal growth hormone release per cell was lower in the fractions containing cells with a density lower than 1.062 g/ml. Inversely, the gonadotrophs have a basal release per cell independent of their density, and this is also available for their responsiveness to salmon gonadotropin-releasing hormone.

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International audience; In vitro potencies of native and modified forms of salmon and mammalian gonadotropinreleasing hormone (GnRH) were studied in relation with their susceptibility to degradation by intact pituitary cells maintained in culture. The kinetics of degradation and the origin of the proteases involved in this process were examined. All the molecules tested (native and modified forms) were equipotent at doses between 1Om6 and lo-’ M in inducing GtH release by cultured pituitary cells. On the other hand, their effectiveness differed at lo-’ and IO-’ M leading to the establishment of the following hierarchy of bioactivity: the native forms, LHRH and sGnRH, were the less potent, the fish analogues (DAla6Pro9Net)sGnRH and (DArg&Pro’Net)sGnRH were the more potent, and mammalian analogues with substitutions at position 6 and/or 10 were intermediate in potency. The native form sGnRH was weakly degraded while no degradation of the mod&d molecules was observed. The degradation of the native sGnRH occurred after 12 and 24 hr of incubation and the results indicate that the peptidases involved are released from the cells into the incubation medium.

International audience; Modified forms of mammalian and fish GnRH have higher in vivo and in vitro bioactivities than native forms. Using pituitary cultured cells, native salmon GnRH is the only one to be submitted to a very low degradation.