Spexin (SPX) is a 14 amino acid peptide hormone that has pleiotropic functions across vertebrates, one of which is involvement in the brain-pituitary-gonad axis of fish. SPX(1) has been identified in each class of vertebrates, and a second SPX (named SPX2) has been found in some non-mammalian species. We have cloned two spexin paralogs, designated as Spx1a and Spx1b, from Nile tilapia (Oreochromis niloticus) that have varying tissue distribution patterns. Spx1b is a novel peptide only identified in cichlid fish, and is more closely related to Spx1 than Spx2 homologs as supported by phylogenetic, synteny, and functional analyses. Kisspeptin, Spx, and galanin (Gal) peptides and their corresponding kiss receptors and Gal receptors (Galrs), respectively, are evolutionarily related. Cloning of six tilapia Galrs (Galr1a, Galr1b, Galr2a, Galr2b, Galr type 1, and Galr type 2) and subsequent in vitro second-messenger reporter assays for Gα s , Gα q , and Gα i suggests that Gal and Spx activate Galr1a/Galr2a and Galr2b, respectively. A decrease in plasma follicle stimulating hormone and luteinizing hormone concentrations was observed with injections of Spx1a or Spx1b in vivo. Additionally, application of Spx1a and Spx1b to pituitary slices decreased the firing rate of LH cells, suggesting that the peptides can act directly at the level of the pituitary. These data collectively suggest an inhibitory mechanism of action against the secretion of gonadotropins for a traditional and a novel spexin paralog in cichlid species.
The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common α-subunit (GpA1) and unique β-subunits (Gpβ1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical β-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHβ were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHβ after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1μΜ) significantly increased the expression of lGpHβ and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHβ with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.
21Spexin (SPX) is a 14 amino acid peptide hormone that has pleiotropic functions 22 across vertebrates, one of which is involvement in the brain-pituitary-gonad axis of fish. 23 SPX(1) has been identified in each class of vertebrates, and a second SPX (named SPX2) 24 has been found in some non-mammalian species. We have cloned two spexin paralogs, 25 designated as Spx1a and Spx1b, from Nile tilapia (Oreochromis niloticus) that have 26 varying tissue distribution patterns. Spx1b is a novel peptide only identified in cichlid 27 fish, and is more closely related to Spx1 than Spx2 homologs as supported by 28 phylogenetic, synteny, and functional analyses. Kisspeptin, Spx, and galanin (Gal) 29 peptides and their corresponding kiss receptors and Gal receptors (Galrs), respectively, 30 are evolutionarily related. Cloning of six tilapia Galrs (Galr1a, Galr1b, Galr2a, Galr2b, 31 Galr type 1, and Galr type 2) and subsequent in vitro second-messenger reporter assays 32 for Gαs, Gαq, and Gαi suggests that Gal and Spx activate Galr1a/Galr2a and Galr2b, 33 respectively. A decrease in plasma follicle stimulating hormone and luteinizing hormone 34 concentrations was observed with injections of Spx1a or Spx1b in vivo. Additionally, 35 application of Spx1a to pituitary slices decreased the firing rate of LH cells, suggesting 36 direct inhibition at the pituitary level. These data collectively suggest an inhibitory 37 mechanism of action against the secretion of gonadotropins for a traditional and a novel 38 spexin paralog in cichlid species. 39 40 42 methods [1], and then also by chemical methods [2] in humans. These computational 43 methods have been attempted mostly on the basis of the characteristics of the 44 prohormones from which active neuropeptides are processed. The mature peptide 45 sequence contains 14 amino acids that are flanked by monobasic and dibasic proteolytic 46 cleavage sites. The mature spexin peptide was found to be identical in all tetrapods and 47 elephant shark, and differs in only one amino acid (A 13 T) in piscine species. A paralog of 48 spexin, termed Spx2, was later identified in non-mammalian species [3]. In mammals, 49 Spx was found to participate in inducing stomach contraction [1], inhibiting 50 adrenocortical cell proliferation[4], postnatal hypoxia response [5], cardiovascular and 51 renal modulation[6], nociceptive response [7], fatty acid absorption and weight regulation 52 [8], and diabetes [9]. In teleosts, functional studies of Spx1 mainly focused on its 53 inhibitory role in the regulation of reproduction [10] and food intake [2] [11]. However, a 54 recent study reported that spx1 knock-out zebrafish exhibited normal reproductive 55 capability but higher food intake than wild type fish, an effect mediated via increased 56 expression of the appetite stimulant, agouti-related peptide AgRP1 [12].57The galanergic neurotransmission system is one of the newest described signaling 58 systems. Today, the galanin family consists of galanin (Gal), galanin-like peptide (GalP), 59...
The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are the principle endocrine drivers of reproductive processes in the gonads of jawed vertebrates. Canonically, FSH recruits and maintains selected ovarian follicles for maturation and LH induces the stages of germinal vesicle breakdown and ovulation. In mammals, LH and FSH specifically activate cognate G-protein-coupled receptors that affect the proteins involved in steroidogenesis, protein hormone synthesis, and gametogenesis. This dual-gonadotropin model also exists in some fish species, but not in all. In fact, due to their diverse number of species, extended number of ecological niches, and remarkably flexible reproductive strategies, fish are appropriate as models to understand the co-evolution of gonadotropins and their receptors. In this study, we cloned and characterized the expression profile over the final stages of ovarian maturation of carp (Cyprinus carpio) LHCGR and FSHR. Expression of both gonadotropin receptors increased in the later stage of early vitellogenesis, suggesting that both LH and FSH play a role in the development of mature follicles. We additionally tested the activation of cLHCGR and cFSHR using homologous and heterologous recombinant gonadotropins in order to gain insight into an evolutionary model of permissive gonadotropin receptor function. These data suggest that carp (Cyprinus carpio) gonad development and maturation depends on a specific gonadotropin profile that does not reflect the temporally distinct dual-gonadotropin model observed in salmonids or mammals, and that permissive gonadotropin receptor activation is a specific feature of Ostariophysi, not all teleosts.
As the male reproductive organ, the main task of the testis is the production of fertile, haploid spermatozoa. This process, named spermatogenesis, starts with spermatogonial stem cells, which undergo a species-specific number of mitotic divisions until starting meiosis and further morphological maturation. The pituitary gonadotropins, luteinizing hormone, and follicle stimulating hormone, are indispensable for vertebrate spermatogenesis, but we are still far from fully understanding the complex regulatory networks involved in this process. Therefore, we developed an ex vivo testis cultivation system which allows evaluating the occurring changes in histology and gene expression. The experimental circulatory flow-through setup described in this work provides the possibility to study the function of the male tilapia gonads on a cellular and transcriptional level for at least 7 days. After 1 week of culture, tilapia testis slices kept their structure and all stages of spermatogenesis could be detected histologically. Without pituitary extract (tilPE) however, fibrotic structures appeared, whereas addition of tilPE preserved spermatogenic cysts and somatic interstitium completely. We could show that tilPE has a stimulatory effect on spermatogonia proliferation in our culture system. In the presence of tilPE or hCG, the gene expression of steroidogenesis related genes (cyp11b2 and stAR2) were notably increased. Other testicular genes like piwil1, amh, or dmrt1 were not expressed differentially in the presence or absence of gonadotropins or gonadotropin containing tilPE. We established a suitable system for studying tilapia spermatogenesis ex vivo with promise for future applications.
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