Chez les mammifères, la détermination du sexe permet à un organe bipotentiel, la gonade, de se différencier en ovaire ou en testicule. Nrg1 (Neuregulin 1) est un facteur de croissance qui agit par phosphorylation de protéines cibles. Chez la souris, Nrg1 est nécessaire à la fertilité des adultes. Les mutants perte de fonction présente une hypoplasie testiculaire. Cependant, Nrg1 est exprimé dans la gonade embryonnaire, ce qui suggère son rôle précoce dans le développement de cet organe. Son rôle durant la gonadogenèse restait encore à établir. Mes travaux ont montré que Nrg1 est impliqué dans la prolifération des précurseurs des cellules de Sertoli chez le mâle, dans l’établissement de la vascularisation et par conséquent l’organisation des cordons testiculaires. Ce phénotype est aggravé en l’absence d’expression du gène Rspo1, un facteur qui stimule également la prolifération des précurseurs des cellules de Sertoli. Cela suggère une fonction additive entre les voies de signalisation contrôlées par Nrg1 et Rspo1 au cours du développement testiculaire. Chez les femelles, les mutants Nrg1 présentent une hypoplasie ovarienne et une réduction de la fertilité. Cela est associé à des cellules germinales atypiques. L’impact de ce phénotype sur la fertilité reste à déterminer. Mmd2 est spécifiquement exprimé dans le testicule mais son rôle est encore inconnu. Pour le déterminer durant la gonadogenèse, j’ai généré des modèles murins de perte de fonction par le système CRISPR-Cas9. Les premiers résultats montrent des cellules somatiques apoptotiques dans les testicules de ce modèle murin. Ces études restent cependant préliminaires et les causes de ce phénotype à élucider. / In mammals, sex determination allows a bipotential organ, the gonad, to differentiate into either an ovary or a testis. NRG1 (Neuregulin 1) is a growth factor that promotes phosphorylation of target proteins. In mice, Nrg1 is required for adult fertility. Loss-of-function mutants exhibit testicular hypoplasia. Nrg1 is expressed in embryonic gonads, suggesting an early role in the formation of this organ. However this role remained to be analysed. Here I show that Nrg1 is involved in the proliferation of precursors of Sertoli cells in male. It is also involved in the establishment of the vasculature and in turn partitioning of testis cords. This phenotype is more severe in the absence of Rspo1 expression. RSPO1 signalling also enhances proliferation of precursors of Sertoli cells. This suggests an additive function between the signalling pathways controlled by Nrg1 and Rspo1 during testicular development. In female, Nrg1 mutant ovaries exhibit hypoplasia and reduced fertility. This is associated with atypical germ cells. The relevance of this phenotype on fertility remains to be determined. Mmd2 is specifically expressed in testis but its role is still unknown. To investigate Mmd2 function, I have generated loss-of-function mouse models using the CRISPR-cas9 system. Preliminary results show apoptotic somatic cells in the testes of these mice. However, the causes of this phenotype remain to be clarified.

Attaining an understanding of the mechanisms underpinning development has been amongst the cardinal scientific challenges of our age. The transition from a single cell organism to the level of complexity evidenced in higher eukaryotes has been facilitated by the advent of intricate developmental networks involving a plethora of factors that synergise to allow for precise spatio-temporal expression of the proteins present in higher organisms. Development is often portrayed as a domino like cascade of events stemming from relatively uncomplicated origins that go on to branch out and form associations and interactions amongst multitudinous actors that will inexorably lead towards a higher state of order. Transcription factors occupy a central position within this tapestry of interactions. They regulate expression of the various required proteins and they provide the cues for the developmental events that will eventually shape an organism. These factors frequently remain unknown until some occurrence causes developmental processes to fail and inadvertently focus attention on the factors that facilitate development. Myotonic dystrophy is a useful paradigm of such a developmental dysfunction that has led to the discovery of a transcription factor integral to both muscle development and gonadogenesis in both Drosophila and higher eukaryotes.

Components of the molecular pathway underlying gonadogenesis in organisms with temperature-dependent sex determination (TSD) have been retained from genetic sex determination. Furthermore, although much of this network has been conserved, new functions for these genes have evolved in this different mode of sex determination. We find that the transcription factors Sox9 and Dmrt1 and the hormone Mis are involved in the formation of a testis and/or the repression of an ovary at a male-producing temperature. While Mis expression may be maintained by Sox9, the initial upregulation of Mis in the developing testis is most likely modulated by some other upstream factor. Dmrt1 appears to play an upstream role in testis sex determination. We provide evidence that the transcription factor Dax1 and the signaling molecule Wnt4, cloned for the first time in an organism with TSD, play roles in gonadogenesis in both sexes. Finally, we show that the transcription factor FoxL2 and the signaling molecule Rspo1 are involved in the formation of an ovary and/or the repression of a testis at a female-producing temperature. In the first investigation of Rspo1 in any organism exhibiting TSD, we demonstrate it is involved upstream in ovarian sex determination. Complementary to descriptive studies, we optimize a whole organ culture system in which gonad explants develop in vitro for up to three weeks. We show that expression of the sex-determining network in isolated gonads mimics in ovo patterns, revealing an endogenous temperature-sensing mechanism that does not require other embryonic tissues. Ectopic expression of Sox9 reveals a possible positive feedback regulation of Dmrt1. The use of this culture system opens the door to functional manipulation of the gonad at the molecular level and is suitable for a myriad of future studies. This work makes strides in elucidating the molecular network underlying gonadogenesis in an organism exhibiting TSD, and invites investigation of the evolution of gene function. The data lend insight into the changing roles of molecules in sex determination across diverse taxa, and into the evolution of developmental pathways in general. / text

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Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning.