Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs.

Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1-3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5-/-) was produced. Assessment of sstr5-/- indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5-/- medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5-/- fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.

Melanin concentrating hormone (MCH) is involved in the regulation of growth hormone in Cichlasoma dimerus (Cichlidae, Teleostei).

Growth hormone (GH) is the main pituitary hormone involved in somatic growth. In fish, the neuroendocrine control of GH is multifactorial due to the interaction of multiple inhibitors and stimulators. Melanin-concentrating hormone (MCH) is a cyclic peptide involved in skin color regulation of fish. In addition, MCH has been related to the regulation of food intake in both mammals and fish. There is only one report presenting evidences on the GH release stimulation by MCH in mammals in experiments in vitro, but there are no data on non-mammals. In the present work, we report for the first time the sequence of MCH and GH cDNA in Cichlasoma dimerus, a freshwater South American cichlid fish. We detected contacts between MCH fibers and GH cells in the proximal pars distalis region of the pituitary gland by double label confocal immunofluorescence indicating a possible functional relationship. Besides, we found that MCH increased GH transcript levels and stimulated GH release in pituitary cultures. Additionally, C. dimerus exposed to a white background had a greater number of MCH neurons with a larger nuclear area and higher levels of MCH transcript than those fish exposed to a black background. Furthermore, fish reared for 3 months in a white background showed a greater body weight and total length compared to those from black background suggesting that MCH might be related to somatic growth in C. dimerus. Our results report for the first time, that MCH is involved in the regulation of the synthesis and release of GH in vitro in C. dimerus, and probably in the fish growth rate.

Forebrain mapping of secretoneurin-like immunoreactivity and its colocalization with isotocin in the preoptic nucleus and pituitary gland of goldfish.

Secretoneurin, a 33-34 amino acid neuropeptide derived from the proteolytic processing of the secretogranin-II precursor protein, is reasonably well conserved in evolution. Goldfish secretoneurin shares >75% similarity overall with other vertebrate secretoneurin sequences. The secretoneurin peptide has numerous functions that include neuroinflammation, neurotransmitter release, and neuroendocrine regulation. A detailed description of the central distribution of secretoneurin immunoreactivity is only known for the rat. Using our polyclonal antibody against the central, conserved core of the secretoneurin peptide we studied the distribution of secretoneurin-like immunoreactivity in the goldfish brain. Secretoneurin immunoreactivity was found in the olfactory bulb, entopeduncular nucleus, preoptic nucleus, lateral part of the lateral tuberal nucleus, posterior periventricular nucleus, nucleus of the posterior recess, the nucleus of the saccus vasculosus, and nucleus isthmi. Secretoneurin-immunoreactive fibers were found in the dorsal part of the dorsal telencephalon, ventral and lateral parts of the ventral telencephalon, periventricular preoptic nucleus, pituitary, and the ventrocaudal aspect of the nucleus of the lateral recess. The most conspicuous secretoneurin immunoreactivity was found in the magnocellular and parvocellular cells of the preoptic nucleus that project to the pituitary. Double-labeling studies indicated coexpression with isotocin, the fish homolog of mammalian oxytocin. Clear colabeling for secretoneurin and isotocin in fibers terminating in the neurointermediate lobe suggests that secretoneurin maybe coreleased with isotocin. Previous work indicates that secretoneurin stimulates the release of luteinizing hormone from the goldfish anterior pituitary. Our findings further support a reproductive role for secretoneurin and related peptides, given the importance of oxytocin family peptides in reproductive behavior in vertebrates.

The secretogranin II-derived peptide secretoneurin stimulates luteinizing hormone secretion from gonadotrophs.

Secretoneurin (SN) is a 33- to 34-amino acid neuropeptide derived from secretogranin-II, a member of the chromogranin family. We previously synthesized a putative goldfish (gf) SN and demonstrated its ability to stimulate LH release in vivo. However, it was not known whether goldfish actually produced the free SN peptide or whether SN directly stimulates LH release from isolated pituitary cells. Using a combination of reverse-phase HPLC and mass spectrometry analysis, we isolated for the first time a 34-amino acid free gfSN peptide from the whole brain. Moreover, Western blot analysis indicated the existence of this peptide in goldfish pituitary. Immunocytochemical localization studies revealed the presence of SN immunoreactivity in prolactin cells of rostral pars distalis of the anterior pituitary. Additionally, we found that magnocellular cells of the goldfish preoptic region are highly immunoreactive for SN. These neurons send heavily labeled projections that pass through the pituitary stalk and innervate the neurointermediate and anterior lobes. In static 12-h incubation of dispersed pituitary cells, application of SN antiserum reduced LH levels, whereas 1 and 10 nM gfSN, respectively, induced 2.5-fold (P < 0.001) and 1.9-fold (P < 0.01) increments of LH release into the medium, increases similar to those elicited by 100 nM concentrations of GnRH. Like GnRH, gfSN elevated intracellular Ca(2+) in identified gonadotrophs. Whereas we do not yet know the relative contribution of neural SN or pituitary SN to LH release, we propose that SN could act as a neuroendocrine and/or paracrine factor to regulate LH release from the anterior pituitary.

Neuropeptides and the control of food intake in fish.

The brain, particularly the hypothalamus, integrates input from factors that stimulate (orexigenic) and inhibit (anorexigenic) food intake. In fish, the identification of appetite regulators has been achieved by the use of both peptide injections followed by measurements of food intake, and by molecular cloning combined with gene expression studies. Neuropeptide Y (NPY) is the most potent orexigenic factor in fish. Other orexigenic peptides, orexin A and B and galanin, have been found to interact with NPY in the control of food intake in an interdependent and coordinated manner. On the other hand cholecystokinin (CCK), cocaine and amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF) are potent anorexigenic factors in fish, the latter being involved in stress-related anorexia. CCK and CART have synergistic effects on food intake and modulate the actions of NPY and orexins. Although leptin has not yet been identified in fish, administration of mammalian leptin inhibits food intake in goldfish. Moreover, leptin induces CCK gene expression in the hypothalamus and its actions are mediated at least in part by CCK. Other orexigenic factors have been identified in teleost fish, including the agouti-related protein (AgRP) and ghrelin. Additional anorexigenic factors include bombesin (or gastrin-releasing peptide), alpha-melanocyte-stimulating hormone (alpha-MSH), tachykinins, and urotensin I. In goldfish, nutritional status can modify the expression of mRNAs encoding a number of these peptides, which provides further evidence for their roles as appetite regulators: (1) brain mRNA expression of CCK, CART, tachykinins, galanin, ghrelin, and NPY undergo peri-prandial variations; and (2) fasting increases the brain mRNA expression of NPY, AgRP, and ghrelin as well as serum ghrelin levels, and decreases the brain mRNA expression of tachykinins, CART, and CCK. This review will provide an overview of recent findings in this field.

In vitro hCG and human recombinant FSH actions on testicular steroidogenesis in the toad Bufo arenarum.

In order to study the regulation of testicular steroidogenesis in the toad Bufo arenarum, the effect of gonadotropins (hCG and hrFSH) on steroidogenic enzymes was determined using an in vitro system. 3beta-Hydroxysteroid dehydrogenase/isomerase activity was not affected by any of the gonadotropins, at any of the concentrations used. In contrast, 5alpha-reductase activity was strongly reduced by both hCG and hrFSH. Human chorionic gonadotropin inhibited the activity of cytochrome P450 17alpha-hydroxylase-C(17-20) lyase (P450(c17)), only at the highest concentration used, while hrFSH strongly reduced P450(c17) activity at all the doses assayed. In conclusion, these data suggest that LH (hCG) and FSH regulate steroidogenic enzymes such as 5alphaRed and P450(c17). The results also suggest that FSH could be involved in the regulation of the change in steroidogenesis undergone by the testis during the breeding season. In turn, the inhibition of P450(c17) activity could result in a reduction of androgen production and an increment of C21 steroids.

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H.

The biosynthesis of androgens in Bufo arenarum takes place through the 5-ene pathway that includes 5-androstane-3beta,17beta-diol as intermediate in testosterone biosynthesis. Besides testosterone and 5alpha-dihydrotestosterone, testes are able to synthesize 5alpha-pregnan-3,20-dione and several 3alpha- and 20alpha-reduced derivatives. Steroid biosynthesis changes during the breeding period (spring and early summer), turning from androgen to C21 steroid production. During the reproductive season, the production of progesterone, 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one, and 5alpha-pregnan-3,20-dione increases significantly. The function of most of these steroids in amphibians remains unknown. However, 5alpha-androstan-3alpha,17beta-diol and 3alpha-hydroxy-5alpha-pregnan-20-one were shown to be neuroactive in mammals, modulating sexual behavior. Thus, 5alpha/3alpha-reduced steroids could be involved in the regulation of the reproductive behavior in B. arenarum, a species with a dissociated reproductive pattern. Percentage contribution of each enzymes to the total metabolism reveals that neither 3beta-hydroxysteroid dehydrogenase/isomerase nor 5alpha-reductase change throughout the reproductive cycle. However, a strong reduction in 17-hydroxylase-C(17-20) lyase activity occurs in the reproductive season, suggesting that this enzyme could represent a key enzyme in the regulation of the seasonal change of steroidogenesis. Also, 3alpha-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase activities increase during the reproductive period, implying that steroid metabolism is clearly focused on C21-reduced steroids.

Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum.

Testis fragments from Bufo arenarum were incubated with [7(n)-(3)H]pregnenolone (P5), [1,2-(3)H]dehydroepiandrosterone (DHEA) and [1,2,6.7-(3)H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3beta-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17chi-hydroxylase, C17-20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17beta-hydroxysteroid dehydrogenase (17betaHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17betaHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17betaHSD could be due to the reduction of C19-substrates. The activity of 5chi-reductase was inhibited by CNK only if results from P5 and DHEA were considered.

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum.

Sliced testis tissue from Bufo arenarum was incubated in the presence of [3H]pregnenolone. Testis fragments were also used for double isotope experiments using [3H]pregnenolone and [14C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C19 steroids such as dehydroepiandrosterone, 5-androsten-3 beta, 17 beta diol, testosterone, 5 alpha-dihydrotestosterone and a C21 steroid, 5 alpha-pregnan-3,20 dione. Progesterone mainly converts to 5 alpha-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3 beta, 17 beta diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis.

GnRH molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes). Immunological and chromatographic evidence for the presence of a novel molecular variant.

Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column.

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (cIGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.

Immunoreactive GnRH suggesting a third form of GnRH in addition to cIIGnRH and sGnRH in the brain and pituitary gland of Prochilodus lineatus (Characiformes).

Molecular variants of GnRH (gonadotropin-releasing hormone) in brain and pituitary extracts of the South American characiforme Prochilodus lineatus were studied using a combination of reverse-phase high-performance liquid chromatography and radioimmunoassay with different antisera. In brain extracts our study revealed that this fish has at least two different types of GnRH: cIIGnRH (chicken II) and sGnRH (salmon), and possibly a third variant of this molecule. In pituitary extracts we could find only two immunoreactive peaks corresponding to sGnRH and the possible third form.