Project description:In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the endocrine mechanisms involved in the regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood.This study aimed at describing the transcriptomic changes taking place in the Senegalese sole testis in response to hCG stimulation in vivo. Gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results provide evidence for the first time that key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis are under gonadotropic control.
Project description:The kiss system is known to regulate puberty and to be involved in the onset of reproduction, acting upstream of the Brain-Pituitary-Gonad axis in gonadotrophin releasing hormone neurons and stimulating secretion of GnRH. Kisspeptin has been proven to be a novel therapeutic for fertility disorders in humans. In fish, positive results have also observed the prospective of this hormone to induce gamete development. During the last two decades, support has been obtained describing the key role of microRNAs (miRNA) in regulating gene expression at the post-transcriptional level and they have been recommended as relevant biomarkers in reproductive studies. Senegalese sole is a marine flatfish species with high importance in the European aquaculture industry, due to its good growth rate and high market value. However, this species presents a bottleneck in terms of reproduction, which does not allow to close the cycle in captive conditions. Hence, this study aimed to observe the effect of a novel hormonal treatment using kisspeptin in miRNA expression related to reproductive performance in cultured Senegalese sole breeders. For this purpose, Senegalese sole F1 broodstock were held at CCMAR facilities in 4 tanks of 3m3 (n = 12/per tank, sex ratio 1:1) under natural photoperiod and temperature conditions. During the reproductive season, the animals from each 2 of the tanks were treated either with SSkiss2_v2 decapeptide in a single intramuscular injection, or with a PBS injection for control groups. After the treatment (2 and 4 days), all fish were anesthetized and sampled for blood collection. Afterwards, plasma was obtained to determine several parameters related to reproductive physiology (Fsh, Lh and sexual esteroids (T, 11-KT and E2)) and miRNAs regulation. After observing the high implication of this treatment in the gonadotrofins, above all in Fsh and sexual esteroids, above all in T, the RNA was extracted from blood plasma using miRNeasy Serum/Plasma Kit (Qiagen, Germany) and libraries were prepared using NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA). Small RNA sequencing (sRNA-seq) (NextSeq 500) was performed on sole treated with kisspeptin (n = 16) and non-treated control fish (n = 14). A total of 1,467 unique miRNAs were expressed in Senegalese sole plasma. A multidimensional scaling (MDS) plot showed no clear clustering of the samples with the kisspeptin treatment. However, the differential expression analysis revealed one miRNA, miR-1-3p, to be highly up-regulated in both males and females treated with kisspeptin compared to control sole. This miRNA is linked with myogenesis and angiogenesis, and skeletal muscle development in fish. Three isomirs (let 7e, miR-199a-3p, miR-100-5p) were differentially expressed between females treated and control fish. MicroRNA let-7e is an important regulator of endothelial function and inflammation, related to testosterone pathway release; miR-199a-3p plays an important role in inflammation and women fertility and miR-100-5p might be implicated in the regulation of cell growth and oocytes maturation. In conclusion, kisspeptin treatment may affect gonad maturation in females and muscle growth in males. Our data support the possibility of using circulating miRNAs as less invasive physiological biomarkers in fish reproduction.
Project description:Betanodaviruses (VNNV) are the causative agents of the viral nervous necrosis, a disease that affects cultured Senegalese sole (Solea senegalensis). VNNV reassortant strains, combining genomic segments from the RGNNV and SJNNV genotypes, have previously been isolated from wild and farmed several fish species. The wild reassortant wSs160.03 isolated from sole has been proved to be more virulent for sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations of wSs160.03 strain performed by reverse genetic, have allow to obtain a mutant reassortant strain (rSs160.03247+270) with mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine), which provoke a 40% decreased mortality. In the current study, the RNA-Seq technology has been used to compare the Senegalese sole transcriptomes in two organs (head-kidney and eye/brain) after infection with the wild reassortant strain and the mutant reassortant strain. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wSs160.03 strain, whereas only 393 genes were differentially expressed in animals infected with the rSs160.03247+270 strain, indicating a 37.9% decrease in the number of DEGs after infection with the mutated reassortant strain. An inversion in the proportion of genes up/down-regulated in nervous tissue of these animals was also obtained for both reassortants. To understand the biological functions of identified DEGs involved in VNNV infection, a gene ontology (GO) enrichment analysis was performed with fold change > 1.5 (up- or down-regulated) and p-value < 0.05. Different profiles of GO were obtained in the subclasses biological process, cellular component, and molecular function for each reassortant strains. Regarding the immune response, genes coding for proteins acting as mediators of IFN type I expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were up-regulated in animals infected with the wild type reassortant, whereas no-differential expression of these genes was observed in animals infected with the mutant reassortant. The different transcriptomic profiles obtained could help to better understand VNNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.
Project description:We report the effect of incubation temperature on embryos and larvae of Solea senegalensis subjected to two different incubation temperatures (15 °C or 21 °C). We found that at some stages, a higher incubation temperature was associated with the expression of miRNAs positively related with growth Examination of miRNA expression in Senegalese sole whole embryos and larvae subjected to two different incubation temperatures, using the SOLiD platform and validation by qPCR
Project description:In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the endocrine mechanisms involved in the regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood.This study aimed at describing the transcriptomic changes taking place in the Senegalese sole testis in response to hCG stimulation in vivo. Gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results provide evidence for the first time that key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis are under gonadotropic control. Testes samples from saline- and hCG-injected male Senegalese sole (8 fish per group) were pooled separatedly. Pooled testicular RNAs from each group were amplified and the resulting cRNAs labelled with Cy3 and Cy5, respectively, mixed in equal amounts and hybridized to the microarray for 17 h at 60 ºC. Each hybridization was performed in duplicate.
Project description:Senegalese sole (Solea senegalensis) were treated with CuSO4 or lipopolysaccharide (LPS), an immune-system stimulant. Controls were treated with phosphate-buffered saline (PBS). Fish were sampled after 6 hours or 24 hours. Hepatic RNA from each treatment group was combined into pools, reverse transcribed to cDNA and labeled with Cy3-dCTP or Cy5-dCTP. Hybridisations consisted of a labeled pooled sample versus a reference mixture made from all PBS-treated samples. The microarray used was made from amplicons of European flounder (Platichthys flesus) hepatic cDNA.
Project description:Senegalese sole males born and raised in captivity (F1 males) do not spawn naturally and typically display lower gamete quality when compared with wild individuals. Broodstock nutrition is an important aspect when dealing with reproduction because it influences not only fish health, but also gamete and progeny quality. The usage of dietary algae antioxidants to improve fish reproduction is under-explored, especially in terms of the male reproductive system. In this experiment, 6 % of a blended meal of Phaeodactylum tricornutum and Gracilaria gracilis was incorporated in Senegalese sole broodstock feed, to evaluate the effects on sperm quality of F1 males throughout the breeding season. RNA-seq was employed to assess differences in the gonadal tissue of fish fed the algae supplemented diets.
Project description:Pseudo-albinism is a complex condition, caused by a multitude of factors and negatively affecting flatfish aquaculture. The majority of studies have targeted genes involved in pigmentation, the most notable change in these animals. In this study, a global transcriptome approach was taken to understand the underlying basis of Senegalese sole pseudo-albinism, using as targets two important innate immune barriers, the skin and intestine. RNA-seq transcriptome analysis identified 573 differentially expressed transcripts (DETs) between pseudo-albino and pigmented soles in the skin and/or the gut. DETs were mainly linked to pigment production, skin structure and regeneration, mucus production and smooth muscle contraction.