Evolution of GnRH Systems and Their Regulatory Neuropeptides in Endocrine and Paracrine Control of Spermatogenesis in the Lesser Spotted Dogfish, Scyliorhinus canicula

Main Objective

The goal of this project is to identify peptides involved in the regulation of spermatogenesis in a shark, the lesser spotted dogfish. The proposed program involves identifying the sequences of GnRH system regulatory peptide precursors (GnRHs, GnIH, Kisspeptin/Neurokinin/Dynorphin) from available genomic data and studying their biological activities on pituitary and testicular cultures. The study of the testicular paracrine function of these peptides is an emerging topic for understanding the influence of environmental factors on reproductive functions. By focusing on a Chondrichthyan, this project will help place the results within an evolutionary framework of the Gnathostome hypothalamo-pituitary-gonadal axis and improve our knowledge of Elasmobranch reproduction, a group with significant conservation issues.

Study Material, Program, and Methods

The lesser spotted dogfish, Scyliorhinus canicula (L.), is a shark abundant on our coasts (IUCN Status: least concern). The animals will be sourced from the Lycée Maritime et Aquacole and the Cité de la Mer in Cherbourg. They will be housed at the Coastal Environment Research Center of the University of Caen’s marine station.

The proposed program and methods are:

A) In silico analyses. Complete the search for peptide precursors and their receptors by querying the genome and transcriptome of S. canicula and other Chondrichthyan genomes. Conduct phylogenetic and synteny analyses to determine gene gains and losses and propose an evolutionary history for the studied genes.

B) Expression analyses. Clone cDNAs and analyze expression by RT-PCR (tissue expression and during spermatogenesis) and by in situ hybridization of mRNAs for peptide precursors and their receptors (e.g., GnRH1 & GNRHR1b, GnRHR2b, oxytocin, neurophysin, somatostatins 1 and 7, Orexin 1, Kiss1 & KissR3, Leptin & LEPR), as well as protein hormones and their receptors (e.g., GCα, LHβ, FSHβ, FSHR, LHR) whose expressions are anticipated based on the work of Hara et al. [5].

C) In vitro analyses of endocrine role. Cultures of whole pituitaries and ventral lobes (VPD) will be developed in close collaboration with S. Dufour and K. Rousseau (BOREA, MNHN, Paris) to test the ability of neuropeptides (GnRHs, GnIH, Kiss, Leptin) to control the expression of pituitary hormones (GCα, LHβ, FSHβ, TSHβ2, PRL1, GH) and GnRHRs.

D) In vitro analyses of paracrine role. Four stages of spermatogenesis (spermatogonia, spermatocytes, young spermatids, old spermatids) will be cultured as explants in the presence of gonadotropic hormones (LH, FSH) to determine the cellular dynamics (proliferation, apoptosis) in response to the treatment. The homologous gonadotropic hormones, already obtained, are produced by expression of fused subunits [20]. The expression of genes encoding key players in steroidogenesis will be analyzed (STAR protein, 3bHSD, and 17bHSD) as well as the expression of various receptors for peptide or protein ligands (e.g., GNRHR, KissR, LRPR, GHR, LHR, FSHR, ORγ). In a second step, the different forms of GnRH will be tested for their ability to modulate the previously observed responses.

Outlook

This project is a continuation of a Master 2 project (F. Jeanne, M2 Marine Sciences UniCaen, on the characterization of gonadotropic hormones and their receptors) and a unifying project of the MNHN (Neuropeptides and regulation of spermatogenesis in an Elasmobranch, 2020-2021). In terms of outlook, it is part of the understanding of reproductive functions in a marine organism, the dogfish, which is of phylogenetic and socio-economic interest (fisheries) and belongs to the Elasmobranchs, of which 40% of species are threatened. This work will also be conducted within a network of partners with scientific complementarity (H. Tostivint (UMR 7221): evolutionary history of peptides; S. Dufour & K. Rousseau: evolution of neuroendocrine control of reproduction in Actinopterygians; P. Sourdaine: reproductive physiology of Elasmobranchs) and will allow for significant advances in the knowledge of Chondrichthyans, whose physiology is too little studied internationally, even though they are the living representatives of the first Gnathostomes. This thesis project also integrates, through its comparative and evolutionary studies of neuropeptides, into the ANR pre-project CandyFISH (APG 2021 coordinated by H. Tostivint) on the caudal neurosecretory system of fish to understand, in particular, the influence of the environment on fish fitness and, consequently, with applications in aquaculture.

This project will provide a knowledge base on the reproductive physiology of Elasmobranchs, which in the longer term can allow for mastery of their reproduction, one of the levers for preserving the resource.