DOI: 10.20350/digitalcsic/17675
title: Gonadotropins regulate cell adhesion and spermiation through a cross-talk between oxytocin and relaxin pathways in Senegalese sole [Dataset]

description: Dataset are from our own production and does not forms part of other datasets Two-year-old male Senegalese sole, F1 generation, 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. Total RNA from the testis was extracted using the RNeasy Plus Mini Kit (Qiagen). For library construction (n = 4 for each group: saline, rFsh and rLh), the SMARTseq2 protocol was employed, with some modifications. The libraries were sequenced on a NovaSeq 6000 S1 sequencing system (Illumina) in paired-end mode with 100 bp paired-end reads length. The resulting reads were processed for image analysis, base calling, and quality scoring using the manufacturer’s software, Real Time Analysis (RTA 1.18.66.3), followed by the generation of FASTQ sequence files by CASAVA 1.8.
description: 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
description: Raw Data (FASTQ files) from samples; Count and Normalized expression matrices; Platforms (1): GPL35005 Illumina NovaSeq 6000 (Solea senegalensis). Samples (15): GSM8578195 Testis_FSH_m61; GSM8578196 Testis_FSH_m62; GSM8578197 Testis_FSH_m64; GSM8578198 Testis_FSH_m68; GSM8578199 Testis_FSH_m69; GSM8578200 Testis_LH_m63; GSM8578201 Testis_LH_m64; GSM8578202 Testis_LH_m66; GSM8578203 Testis_LH_m67; GSM8578204 Testis_LH_m68; GSM8578205 Testis_Saline_m63; GSM8578206 Testis_Saline_m64; GSM8578207 Testis_Saline_m66; GSM8578208 Testis_Saline_m67; GSM8578209 Testis_Saline_m68. Supplementary file: GSE279689_raw_counts.csv.gz840.2