1. The mutation of FSHR affects reproductive physiology and growth in zebrafish
- Author
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Sambroni, Elisabeth, Patinote, Amélie, Goupil, Anne-Sophie, Le Gac, Florence, Lareyre, Jean-Jacques, Laboratoire de Physiologie et Génomique des Poissons (LPGP), Institut National de la Recherche Agronomique (INRA)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Laboratoire de Physiologie et Génomique des Poissons ( LPGP ), Institut National de la Recherche Agronomique ( INRA ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Instituto Nacional de Pesquisas da Amazônia (INPA). Manaus, BRA.
- Subjects
fish ,endocrine system ,[ SDV ] Life Sciences [q-bio] ,fsh receptor ,animal fertility ,[SDV]Life Sciences [q-bio] ,fertilité animale ,poisson zèbre ,physiologie de la reproduction ,récepteur fsh ,performance de croissance ,reproduction ,poisson ,danio rerio ,cyprinidae ,danio devario - Abstract
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.
- Published
- 2018