Functional and evolutionary analysis of flatfish gonadotropin receptors reveals cladal- and lineage-level divergence of the teleost glycoprotein receptor family.

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

Effect of different glycaemic conditions on gene expression of neuropeptides involved in control of food intake in rainbow trout; interaction with stress.

To assess mechanisms relating to food intake and glucosensing in fish, and their interaction with stress, we evaluated changes in the expression of orexigenic (NPY) and anorexigenic (POMC, CART and CRF) peptides in central glucosensing areas (hypothalamus and hindbrain) of rainbow trout subjected to normoglycaemic (control), hypoglycaemic (4 mg insulin kg(-1)) or hyperglycaemic (500 mg glucose kg(-1)) conditions for 6 h under normal stocking density (NSD; 10 kg fish mass m(-3)) or under stress conditions induced by high stocking density (HSD; 70 kg fish mass m(-3)). Hyperglycaemic NSD conditions resulted in decreased mRNA levels of NPY and increased levels of CART and POMC in the hypothalamus as well as increased mRNA levels of CART and CRF in the hindbrain compared with hypo- and normoglycaemic conditions. HSD conditions in normoglycaemic fish induced marked changes in the expression of all peptides assessed: mRNA levels of NPY and CRF increased and mRNA levels of POMC and CART decreased in the hypothalamus, whereas the expression of all four peptides (NPY, POMC, CART and CRF) decreased in the hindbrain. Furthermore, HSD conditions altered the response to changes in glycaemia of NPY and POMC expression in the hypothalamus and CART expression in the hypothalamus and the hindbrain. The results are discussed in the context of food intake regulation by glucosensor systems and their interaction with stress in fish.

2-D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild-caught and hormone-treated F1 fish.

In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild-caught males. To gain insights into the molecular mechanisms that may be altered in the F1 testis, we used 2-D DIGE to compare the protein profiling of the testis of wild-caught males at the spermiation stage with that of F1 males showing different stages of germ cell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins by MS/MS revealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germ cell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males.

New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis.

BACKGROUND: The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. RESULTS: Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis (leukocyte cell-derived chemotaxin 2,), angiogenesis (thrombospondin), and prevention of apoptosis (S100a10 calcium binding protein). CONCLUSION: This study has identified a number of differentially expressed genes in the ovary that were not previously found to be regulated during ovarian development in marine fish. Specifically, we found evidence, for the first time in teleosts, of the activation of chemoattractant, angiogenic and antiapoptotic pathways in hypertrophied follicular cells at the onset of ovarian atresia.

Molecular cloning of Senegalese sole (Solea senegalensis) follicle-stimulating hormone and luteinizing hormone subunits and expression pattern during spermatogenesis.

Pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. However, in teleosts with testis of semi-cystic type and asynchronous spermatogenesis, as the flatfish Senegalese sole (Solea senegalensis), the physiological roles of FSH and LH are still not well understood. To gain insight into this mechanism, full-length complementary DNAs (cDNAs) encoding Senegalese sole FSH beta and LH beta subunits, and the common glycoprotein alpha subunit (CG alpha), were cloned and sequenced. The three cDNAs consisted of 550, 582 and 744 nucleotides encoding peptides of 120, 148 and 132 amino acids, respectively. Comparison of the deduced amino acid sequences of sole FSH beta, LH beta and CG alpha with those from other teleosts indicated that cysteine residues and potential N-linked glycosylation sites were fully conserved with respect to other percomorphs and salmonids. However, the primary structure of FSH beta and LH beta in pleuronectiforms appeared to be highly divergent. In situ hybridization of mature male pituitaries showed that fshb, lhb and cga mRNAs were localized in the proximal pars distalis and in the periphery of pars intermedia. Real-time quantitative reverse transcription-polymerase chain reaction indicated that the levels of all three transcripts in the pituitary of males increased during winter and spring, at the time when plasma levels of androgens raised and testicular germ cell development and spermatozoa production were stimulated. These results suggest that FSH and LH may regulate spermatogenesis in Senegalese sole similarly to that described for other teleosts with testis of cystic type and synchronous germ cell development.

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility.

The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.

High transcript level of fatty acid-binding protein 11 but not of very low-density lipoprotein receptor is correlated to ovarian follicle atresia in a teleost fish (Solea senegalensis).

Transcripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly defined teleost fish FABP subfamily that is a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9. RT-PCR revealed high levels of vldlr transcript splicing variants in the ovaries and, to a lesser extent, in somatic tissues, whereas fabp11 was highly expressed in the ovaries, liver, and adipose tissue. In situ hybridization analysis showed vldlr and fabp11 mRNAs in previtellogenic oocytes, whereas no hybridization signals were detected in the larger vitellogenic oocytes. Transcript expression of fabp11 was strongly upregulated in somatic cells surrounding atretic follicles. Real-time quantitative RT-PCR demonstrated that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively. These results suggest that the expression level of vldlr transcripts may be used as a precocious functional marker to quantify the number of oocytes recruited for vitellogenesis and that fabp11 mRNA may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia in fish.