Gonadotropins regulate cell adhesion and spermiation through a cross-talk between oxytocin and relaxin pathways in Senegalese sole

Our understanding of semi-cystic spermatogenesis in the Senegalese sole (Solea senegalensis), specifically the process where spermatids are released and undergo differentiation in the lumen, remains limited. Previously, we demonstrated that Luteinizing hormone (Lh) has the ability to penetrate the blood-testis barrier and reach released spermatids expressing the Lh receptor, thereby stimulating spermiogenesis. To deepen our knowledge, the current study aims to investigate how gonadotropins regulate the release of spermatids from Sertoli cells, known as spermiation. We conducted a six-week experiment exposing male Senegalese sole to recombinant gonadotropins (Follicle-stimulating hormone, rFsh, or rLh) via weekly injections at a dose of 6 μg/kg. Following treatment, we performed transcriptomic analysis using RNA-seq to identify differentially expressed genes (DEGs). We found that rLh significantly regulated 1316 genes, while rFsh regulated 717 genes, with 357 DEGs common to both. Pathway analysis revealed that both gonadotropins influenced the expression of cell-cell junctions and adhesion proteins. Notably, other affected pathways included those involving oxytocin (OXT) and relaxin (RLN), suggesting their potential roles in regulating spermiation. Immunohistochemistry (IHC) and in situ hybridization (ISH) were employed to investigate the cellular localization of OXT and RLN receptors in the testicular cortex and medulla, primarily finding them in Sertoli cells and spermatids. Proteins associated with cell adhesion identified through RNA-seq, such as CTNNB1, PARD3, GJA3, TJP2, CLDN4, TMEM47, and ITBG1, were observed in either spermatids or Sertoli cells, with their expression mainly repressed by gonadotropins. This repression indicates a disruption in Sertoli cell-spermatid junctions, potentially responsible for active spermiation and the enhanced permeability of the blood-testis barrier. Furthermore, we conducted an in vitro assay where testes explants were incubated with OXT, RLN, OXT inhibitor (L-371), RLN inhibitor (AT001), in combination with rFsh and rLh. Subsequent qPCR analysis indicated a trend towards decreased expression of cell adhesion genes by OXT and RLN, most notably by Lh by acting through the OXT and RLN pathways. In summary, our findings suggest that gonadotropins regulate cell adhesion and spermiation through a cross-talk between OXT and RLN pathways. These insights lay the groundwork for further research aimed at unraveling the process of spermiation in fish species. Two-year-old male Senegalese sole, F1 generation offspring from wild-caught parents, were acquired from the commercial supplier Stolt Sea Farm S.A. (Spain) and transferred to the IRTA fish research center in Sant Carles de la Ràpita (Spain). The fish were housed in 10 m³ fiberglass tanks with a recirculating system (IRTAmar1) and were fed five times a week with 0.75% of wet feed (mussels and polychaetes) and 0.55% of dry feed (balanced diet) based on total biomass. They were maintained for three months before the experiments under natural light cycles and regulated simulated natural temperatures. The experiment was conducted over six weeks from November to January to examine the specific impact of recombinant Fsh and Lh (rFsh and rLh) on testicular development. Males (347±9 gr) received intramuscular injections once a week with saline (control, n=8), rFsh (18 μg/kg, n=8) or rLh (18 μg/kg, n=8). The animal care and sample collection procedures were conducted in compliance with protocols approved by the Ethics Committee (EC) of the Institut de Recerca i Tecnologìa Agroalimentàries (IRTA) following the European Union Council Guidelines (86/609/EU). The study received specific approval from the IRTA EC.