Search

Pejerrey is a freshwater fish from South America with high potential for aquaculture. This study was designed to determine the effects of different dietary protein:lipid ratio on growth rate and the expression of growth, lipid metabolism and feeding-related genes of this species during early developmental stages. Pejerrey fry were fed for 60 days with four experimental diets containing low (400 g Kg) or high (500 g Kg) protein (LP or HP, respectively) and low (120 g Kg) or high (200 g Kg) lipid (LL or HL, respectively), in the combinations: LP-LL; LP-HL; HP-LL and HP-HL. Measurements of growth, lipid and fatty acid content of fry, expression of genes from the endocrine axis (gh, ghrs, igfs), fatty acid metabolism (∆6-desaturase), and food intake behavior (nucb2/nesfatin-1) were collected. Fry fed with diets LP-LL and HP-LL showed the highest growth rate and growth hormone (gh) mRNA expression levels. The gene expression of ∆6-desaturase was high in head of fry fed with diet LP-HL. The mRNA expression of nucb2/nesfatin-1 and gh followed the same patterns in head, and the inverse pattern in body. In conclusion, diets with LL ensure a higher growth of pejerrey fry compared to those that contain HL, without altering the final lipid amount nor the fatty acid profile on fry. In LL groups, the expression of genes from the GH-IGF axis is associated with the observed promotion of somatic growth. The expression of nucb2/nesfatin-1 indicates an effect of this peptide not related to food intake regulation, e.g., a negative regulatory role on GH expression, that would warrant future research., This work was partially funded by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina; PICT 2010–1493) and Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET; PIP 2014/0595) to LFC, and a Discovery grant, and Discovery Accelerator Supplement award from the Natural Sciences and Engineering Research Council (NSERC) of Canada to SU. SU received a new investigator salary award from the Canadian Institutes of Health Research (CIHR). JIB was recipient of a doctoral fellowship from ANPCyT (Argentina), and from the Emerging Leaders of the Americas Program (ELAP) scholarship from the Government of Canada and doctoral fellow from CONICET (Argentina).

In vertebrates, the reproduction is controlled by the brain-pituitary-gonadal (BPG) axis and kisspeptin has emerged as a key player of this axis. In this study, we analyzed changes in the expression levels of kiss1, kiss2, and their receptors, kissr2 and kissr3 during gametogenesis in the BPG axis of feral Odontesthes bonariensis. In females, levels of brain kiss1 showed an increase at final maturation (Fm), while kiss2 levels were shown to be high at primary growth (Pg) stage, with no differences in the expression of their receptors. In the pituitary, kiss1 and kiss2 peaked at the cortical alveoli (Ca) stage, and kissr3 at initial vitellogenesis. In parallel, there was an increase of kiss1, kissr2 and kissr3 in the ovary during the Ca stage and both receptors again at Fm stage. In males, the four genes were highly expressed in the brain at the arrested (A) stage. In the pituitary, kiss2 peaked at spermatogonial (SG) and spermatocytary (SC) stages; while kissr3 reached a peak at the spermiogenic stage (SP). In testes, kiss1 and kiss2 significantly increased during the SG and SC stages; meanwhile, kissr2 increased at SG and SC, whereas kissr3 levels were significantly high at SC and SP stages. Taken together these results showed that the kisspeptin system in pejerrey is expressed in the three levels of the BPG axis with different expression profiles during the gonadal cycle. These findings pointed that kisspeptins have different roles in gametogenesis in this species. Fil: Tovar Bohorquez, Mario Oswaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Mechaly, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Elisio, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Chalde, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Miranda, Leandro Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Pejerrey, Odontesthes bonariensis, is an euryhaline fish of commercial importance in Argentina. This work aimed to determine if water salinity affects the expression of genes involved in somatic growth (gh; ghr-I; ghr-II; igf-I), lipid metabolism (Δ6-desaturase) and food intake (nucb2/nesfatin-1). First, we identified the full-length cDNA sequences of Δ6-desaturase (involved in lipid metabolism) and nesfatin-1 (an anorexigen). Then, pejerrey juveniles were reared during 8 weeks in three different water salinity conditions: 2.5 g/L (S2.5), 15 g/L (S15) and 30 g/L (S30) of NaCl. Brain, pituitary, liver and muscle samples were collected in order to analyze mRNA expression. The expression of gh and ghr-II mRNAs increased in the pituitary of fish reared at S2.5 and S30 compared with the S15 group. The expression of ghr-I was higher in the liver of S30 group compared to S2.5 and S15. Igf-I mRNA expression in liver increased with the increment of water salinity, while it decreased in the muscle of S15 and S30 groups. Δ6-desaturase expression increased in S2.5 group compared to S15 in both liver and muscle. S30 caused a decrease in the Δ6-desaturase expression in liver compared to S15. The S30 treatment produced an increase in nucb2/nesfatin-1 mRNA expression in the brain and liver compared to S2.5 and S15. The changes in gene expression observed could help pejerrey perform better during salinity challenges. The S30 condition would likely promote pejerrey somatic growth in the long term. Fil: Bertucci, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Tovar Bohorquez, Mario Oswaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; España Fil: Gomez Requeni, Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Unniappan, Suraj. University of Saskatchewan; Canadá Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount ofdietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfishin vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose,l-tryptophan,oleic acid,linolenic acid(LNA),eicosapentaenoic acid(EPA) anddocosahexaenoic acid(DHA) on ghrelin and nesfatin-1 gene andprotein expressionwere studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation ofnucb2/nesfatin-1in the intestine.l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highlyunsaturated fatty acidsinhibitedpreproghrelin. EPA exposure led to a decrease inpreproghrelin, and an increase innucb2/nesfatin-1expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.

Ghrelin, and nesfatin-1 (encoded by nucleobindin2/nucb2) are two metabolic peptides with multiple biological effects in vertebrates. While sex steroids are known to regulate endogenous ghrelin and NUCB2 in mammals, such actions by steroids in fish remain unknown. This study aimed to determine whether estradiol (E2) and testosterone (T) affects the expression of preproghrelin, ghrelin/growth hormone secretagogue receptor (GHS-R), ghrelin O-acyl transferase (GOAT) and NUCB2 in goldfish (Carassius auratus). First, a dose-response assay was performed in which fish were intraperitoneally (ip) implanted with pellets containing 25, 50 or 100 μg/g body weight (BW) of E2 or T. It was found that sex steroids (100 μg/g BW) administered for 2.5 days achieved the highest E2 or T in circulation. In a second experiment, fish were ip implanted with pellets containing 100. μg/g BW of E2, T or without hormone (control). RT-qPCR analyses at 2.5 days post-administration show that gut preproghrelin and GOAT expression was upregulated by both E2 and T treatments, while the same effect was observed for GHS-R only in the pituitary. Both treatments also reduced hypothalamic preproghrelin mRNA expression. NUCB2 expression was increased in the forebrain of T treated group and reduced in the gut and pituitary under both treatments. These results show for the first time a modulation of preproghrelin and nucb2/nesfatin-1 by sex steroids in fish. The interaction between sex steroids and genes implicated in both metabolism and reproduction might help meeting the reproduction dependent energy demands in fish. Fil: Bertucci, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; España Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Unniappan, Suraj. University of Saskatchewan; Canadá

This review covers the current knowledge on the regulation of the somatic growth axis and its interaction with metabolism and feeding regulation. The main endocrine and neuroendocrine factors regulating both the growth axis and feeding behavior will be briefly summarized. Recently discovered neuropeptides and peptide hormones will be mentioned in relation to feeding control as well as growth hormone regulation. In addition, the influence of nutrient and nutrient sensing mechanisms on growth axis will be highlighted. We expect that in this process gaps of knowledge will be exposed, stimulating future research in those areas.

Gene expression of growth hormone receptors (GHRs), insulin-like growth factors (IGFs), myostatin (MSTN) and myogenin (MyoG) was analyzed in juveniles pejerrey fed with graded levels of lipids (L): 6% (L6), 10% (L10), 25% (L25). After 14 weeks, no changes were found in liver GHR-I GHR-II and IGF-II mRNA levels whereas IGF-I decreased in L10 and L25. Muscle GHR-I gene expression increased in L25 whereas GHR-II, IGF-II and MyoG were higher in L6. IGF-I and MSTN expression was not affected by the different diets. Adipose IGF-I mRNA levels decreased in L10. Correlations between body weight and members of GH/IGF system in liver and skeletal muscle were found only in L10 group. Correlations found in L10 group between both liver and skeletal muscle GHR-I and IGF-I were lost in either L6 or L25 groups. Thus, fish fed with apparently unbalanced dietary lipid contents (6% and 25%) exhibit a compensatory regulation of systemic and local components of the GH/IGF axis. Furthermore, the marked inhibition of muscle MyoG gene expression in L25 might limit excessive lipid deposition and fish growth. Our data suggest that a dietary lipid contents of 10% would promote a particular adjustment of the endocrine and autocrine/paracrine GH/IGF system, stimulating body growth and perhaps muscle hyperplasia. On the other hand, a higher dietary lipid content would uncouple the GH/IGF system, reducing hepatic IGF-I, while slightly increasing hepatic GHR-I, probably to prompt lipolysis., Centro de Endocrinología Experimental y Aplicada

Aquaculture fish diets usually contain an addition of fish oil to improve their nutritional value. The effect of the replacement of dietary fish oil (FO) by sunflower oil (SfO) on growth, fatty acid composition and expression of genes implicated in somatic growth, feed intake and fatty acid metabolism was studied in pejerrey fry. Fry were fed per 45 days with diets containing FO/SfO ratios of 100% FO; 50% FO:50% SfO; 20% FO:80% SfO; and 100% SfO. No differences were detected in growth and in the total per cent of saturated and monounsaturated fatty acids. Gh, ghr‐I and ghr‐II showed a higher mRNA expression in head and trunk of fry fed with 100% SfO diet. Expression of igf‐II was higher in trunk of fry fed with 100% SfO diet compared with 100% FO diet. The Δ6‐desaturase gene expression was upregulated in head and trunk of fry fed with 100% SfO diet. The nucb2/nesfatin‐1 gene expression decreased in the trunk of fry with increasing dietary SfO. We conclude that the replacement of fish oil by sunflower oil in pejerrey fry feed does not affect growth and is a viable strategy to reduce production costs of this fish, This work was partially funded by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina; PICT 2010‐1493) and Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET; PIP 2014/0595) to LFC. Lipid analyses were carried out under the framework of projects AGL2013‐40986‐R from MINECO, and PROMETEO 2014/085. Part of this work conducted at the laboratory of SU was supported by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada, to SU.

In mammals and fish, somatic growth and metabolism are coordinated by the GH-IGF axis, composed of growth hormone (GH), growth hormone receptors I and II (GHR-I and GHR-II), and the insulin-like growth factors I and II (IGF-I and IGF-II). In order to determine if dietary macronutrients regulate the hepatopancreatic expression of ghr-I, ghr-II, igf-I and igf-II independently of circulating GH, organ culture experiments were conducted. Briefly, goldfish hepatopancreas sections were incubated with different doses of glucose; L-tryptophan; oleic acid; linolenic acid (LNA); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). After two and four hours of treatment, the expression of ghr-I, ghr-II, igf-I and igf-II mRNAs was quantified. We found that glucose and L-tryptophan globally upregulate the mRNA expression of ghr-I; ghr-II; igf-I and igf-II. Duration of exposure, and unsaturation level of fatty acids differentially modulate ghr-I, ghr-II and igf-II mRNA expression. Additionally, we found that fatty acids increase the expression of igf-I depending on incubation time and fatty acid class. In conclusion, here we present evidence for GH-independent, direct effects exerted by dietary macronutrients on GHR and IGF in goldfish hepatopancreas. Fil: Bertucci, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; España Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Unniappan, Suraj. University of Saskatchewan; Canadá

In vertebrates, kisspeptins and their receptors are known to be related to puberty onset and gonadal maturation, however, there are few studies concerning their role in early development. Here, we characterize the kisspeptin system in the pejerrey, Odontesthes bonariensis, a fish with strong temperature-dependent sex determination. We reconstructed the phylogenetic history of the two ligands (kiss1 and kiss 2) and two receptors (kissr2 and kissr3) in pejerrey in the context of recent classifications of bony fishes, determined their tissue distribution and documented the early expression pattern of these ligands and receptors. Phylogenetic analysis of these gene families clearly resolved the percomorph clade and grouped pejerrey with Beloniformes. Paralogous sets of genes putatively arising from the teleost-specific genome duplication event (3R) were not detected. Kisspeptins and their receptors showed a wide tissue distribution in adult pejerrey, including tissues not related to reproduction. In larvae reared at 24 °C, the four kisspeptin elements were expressed in the head from week 1 to week 8 of life, with no differences in transcript levels. Larvae kept at a female-producing temperature (17 °C) did not show statistically significant differences in the transcript levels of all analyzed genes during the sex determination/differentiation period; however, in those larvae raised at male producing temperature (29 °C), kiss2 levels were increased at week 4 after hatching. These results showed that all members of the kisspeptin system are expressed at this early period, and the increase of kiss2 transcripts at week 4 could be interpreted as it would be related to the differentiation of the brain-pituitary axis in male development Fil: Tovar Bohorquez, Mario Oswaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Mechaly, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Hughes, Lily C.. The George Washington University; Estados Unidos Fil: Campanella, Daniela. The George Washington University; Estados Unidos Fil: Orti, Guillermo. The George Washington University; Estados Unidos Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT-qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1. Fil: Sundarrajan, Lakshminarasimhan. University of Saskatchewan; Canadá Fil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; España Fil: Bertucci, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Ramesh, Naresh. University of Saskatchewan; Canadá Fil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Unniappan, Suraj. University of Saskatchewan; Canadá

The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [(3)H]corticosterone or [(3)H]testosterone as substrate. The enzyme localizes in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither K(m) nor V(max) for both corticosterone and testosterone were significantly different among reproductive periods. The K(m) value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [(3)H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9 nM and 35.24±2.3 nM, respectively. In vitro experiments suggest that 5α-DHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.

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.

Growth and mRNA levels of the pituitary adenylate cyclase-activating polypeptide (PACAP) and its related peptide (PRP), and the system controlled by the growth hormone (GH) and insulin-like growth factors (IGFs) were analyzed in pejerrey fry fed with graded levels of dietary lipids: 10% (L10), 13% (L13) and 21% (L21). First, the full sequence of pejerrey PRP-PACAP was obtained by RT-PCR, using primers based on conserved fragments of teleosts PACAP sequences. The growth of the fish at 83 days after hatching (dah) and the GH mRNA levels were not significantly affected by the dietary treatment. Conversely, PRP-PACAP expression significantly decreased with increasing dietary lipids (L10 > L21). While GH receptor (GHR)-I and IGF-I transcripts did not differ among groups, GHR-II transcripts decreased in group L21. IGF-II expression apparently followed the same trend. These results in combination with the lower expression of the anorexigenic PRP-PACAP in fish fed diet L21 and the correlation analysis evidencing a particularly fine tuning of the GH–IGF system in group L13, suggest that this diet may cover the energy demands for growing pejerrey from 27 dah onwards. Our results show for first time in fish a differential response of PRP-PACAP transcripts to dietary manipulations, and confirm the sensitivity of the pejerrey GH–IGF system to changes in diet composition despite the lack of (or in advance to) a clear response of somatic growth.

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 Ca2+ 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.

In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2α treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain.

The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. Accordingly, the regulatory network for GH is complex and includes many endocrine and environmental factors. In fish, the neuroendocrine control of GH is multifactorial with multiple inhibitors and stimulators of pituitary GH secretion. In fish, GH release is under a tonic negative control exerted mainly by somatostatin. Sex steroid hormones and nutritional status influence the level of brain expression and effectiveness of some of these GH neuroendocrine regulatory factors, suggesting that their relative importance differs under different physiological conditions. At the pituitary level, some, if not all, somatotropes can respond to multiple regulators. Therefore, ligand- and function-specificity, as well as the integrative responses to multiple signals must be achieved at the level of signal transduction mechanisms. Results from investigations on a limited number of stimulatory and inhibitory GH-release regulators indicate that activation of different but convergent intracellular pathways and the utilization of specific intracellular Ca(2+) stores are some of the strategies utilized. However, more work remains to be done in order to better understand the integrative mechanisms of signal transduction at the somatotrope level and the relevance of various GH regulators in different physiological circumstances.

In teleost fish, melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide released from the pituitary during white background adaptation. In the periphery MCH concentrates melanin granules in melanophores thus lightening the body color of fish. Evidence from mammalian studies has demonstrated the involvement of MCH in the control of energy balance and the reproductive axis. Information about the hormonal regulation of MCH neurons in non-mammalian systems is scarce and nothing is known about its role in the regulation of the reproductive axis. We here report the molecular characterization of two MCH precursors in the goldfish. Both precursors are peripherally expressed and the expression in the central nervous system is restricted to the mediobasal hypothalamus. Hypothalamic MCH-mRNA production is upregulated during white background adaptation. Both testosterone and estradiol stimulate MCH mRNA expression in the hypothalamus in a sex-dependent manner, with females showing the greatest responsiveness. In addition, in vitroexperiments demonstrated that graded doses of salmon MCH stimulate LH, but not GH, secretion from dispersed pituitary cells. Results suggest that hypothalamic MCH may participate in the steroid positive feedback loop on pituitary LH secretion.

In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu1,Tyr7,Gly10]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro2]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.

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.

This study analyzes daily changes in the expression of somatostatin precursors PSS-I and PSS-III (structurally related to cortistatin) in the goldfish brain. The results indicate that PSS-I expression correlates with the light cycle only in optic tectum-thalamus (OT-Tha). PSS-III expression correlates with the light cycle in telencephalon-preoptic area (Tel-POA) and OT-Tha. In Tel-POA, PSS-III reaches a minimum level at the beginning of the active phase and a maximum level late in this phase. These results suggest that PSS-I in OT-Tha and PSS-III in Tel-POA and OT-Tha could be involved in the control of the activity cycles in goldfish.

In the present study the brain distribution of three somatostatin (SRIF)-encoding genes, PSS-I, PSS-II, and PSS-III, was analyzed by in situ hybridization (ISH) in the goldfish. The PSS-I mRNA showed the widest distribution throughout the brain, whereas PSS-II transcripts were restricted to some hypothalamic nuclei. On the other hand, PSS-III presents an intermediate distribution pattern. All SRIF encoding genes are expressed in hypophysiotropic nuclei supporting the idea that, in addition to SRIF-14, [Pro(2)] SRIF-14, and gSRIF-28 have pituitary-controlling functions. Moreover, each of the genes is expressed in nuclei directly associated with feeding behavior, suggesting a role for SRIF peptides in the central control of food intake and energy balance. Alternatively, they might have a role in processing sensory information related with feeding behavior, since PSS genes are expressed in the main gustatory, olfactory, and visual centers, which project to the hypothalamic feeding center in teleost fish.

In the present paper the effects of estradiol and testosterone on the expression of the types 1 and 5 somatostatin receptors (sst1 and sst5) in the goldfish forebrain and pituitary were investigated. Estradiol increased the sst1 expression in both the forebrain and pituitary in a dose- and time-dependent manner. In addition, estradiol also increased the pituitary expression of sst5. On the other hand, testosterone had no effects on the expression of these receptor subtypes. Mature female goldfish were found to have higher sst1 and sst5 expression in the pituitary, as well as a higher expression of sst1 in the forebrain compared to sexually regressed animals. As estradiol treatment increases serum growth hormone levels in goldfish, these data suggest that sst1 and sst5 receptors are likely not directly involved in this aspect of growth hormone release.

GnRH controls vertebrate reproduction in several ways. This hormone not only affects the secretion of gonadotropins from the pituitary gland but also has a direct influence on several gonadal functions such as steroidogenesis, spermatogenesis, and spermiation. In the present paper we have studied the in vitro effects of GnRH on the testicular steroidogenesis of Bufo arenarum to ascertain the role of this peptide in the control of the steroidogenic pathway previously described in this species. It was found that GnRH is able to reduce basal as well as hCG-stimulated testosterone release, having an inhibitory effect on P450 c17 activity. Thus, GnRH could be involved in the mechanism that regulates the metabolic change in the testicular steroidogenesis. Additionally, testicular GnRH binding site has been characterised, showing a K d of 34 nM and a maximum binding of 4.7 pmol/mg protein.

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.

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.

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.

International audience; 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.

Publisher Summary This chapter discusses the neuroendocrine territories in fish. It describes the anatomy of the main neuroendocrine territories of the teleost brains. Teleost fish lack a canonical median eminence, and the hypothalamic neurons terminate very close to the adenohypophysial cells or make synaptoid contact upon them. This anatomical characteristic allows the study of the hypothalamo–hypophysial system by retrograde tracing experiments. Tract‐tracing techniques have corroborated early studies showing the preoptic area and tuberal hypothalamus as loci for the neuronal cell bodies whose axons reach the neuro‐ and adenohypophysis along well‐defined fiber tracts. The chapter also discusses several neuronal systems that produce hypothalamic releasing or inhibitory peptides and neurotransmitters, and innervate the pituitary. The hypothalamus–pituitary complex in teleost species shows a particular specialization in which the median eminence is greatly reduced or absent. As a consequence, the hypothalamic control of the pituitary is exerted by an important pituitary innervation that penetrates to the adenohypophysis as interdigitation of neuronal tissue. This neuronal circuitry integrates incoming information from both external and internal environments by expressing the appropriate set of hormonal receptors and through interneuronal communication.

In the present study the brain distribution of three somatostatin (SRIF)-encoding genes, PSS-I, PSS-II, and PSS-III, was analyzed by in situ hybridization (ISH) in the goldfish. The PSS-I mRNA showed the widest distribution throughout the brain, whereas PSS-II transcripts were restricted to some hypothalamic nuclei. On the other hand, PSS-III presents an intermediate distribution pattern. All SRIF encoding genes are expressed in hypophysiotropic nuclei supporting the idea that, in addition to SRIF-14, [Pro(2)] SRIF-14, and gSRIF-28 have pituitary-controlling functions. Moreover, each of the genes is expressed in nuclei directly associated with feeding behavior, suggesting a role for SRIF peptides in the central control of food intake and energy balance. Alternatively, they might have a role in processing sensory information related with feeding behavior, since PSS genes are expressed in the main gustatory, olfactory, and visual centers, which project to the hypothalamic feeding center in teleost fish.

It was previously demonstrated that both cholecystokinin (CCK) and bombesin (BBS) stimulate growth hormone (GH) secretion in goldfish. Both peptides induce satiety and it was speculated that they integrate satiation and the postprandial increase in GH circulating levels. In the present paper we investigated the effects of CCK and BBS on the forebrain expression of the somatostatin gene family in goldfish to analyze if somatostatin peptides may be part of the effector mechanisms of CCK and BBS. We found that peripherally as well as centrally administered CCK decreases mRNA levels of preprosomatostatin (PSS)-I that encodes for SRIF-14, having no effects on PSS-II and PSS-III, which encode for gSRIF-28 and [Pro2] SRIF-14, respectively. In addition, a direct action on the pituitary to stimulate GH release, this inhibition of PSS-I expression provides a possible mechanism for CCK to increase postprandial GH levels. On the other hand, BBS inhibits the forebrain expression of PSS-I and PSS-II but does not affect PSS-III regardless of the route of administration. We conclude that this could be the most likely mechanism of action of BBS to increase GH secretion, since there are few BBS-immunoreactive (IR) fibers and BBS binding sites in the anterior pituitary of goldfish.

In this study, we examined (i) the preprandial, postprandial and starvation-induced changes in the preproghrelin mRNA expression and serum ghrelin levels, and (ii) the effects of intracerebroventricular and intraperitoneal administration of ghrelin on food intake in goldfish. Slot blot analysis revealed a significant postprandial decrease in preproghrelin mRNA expression in the hypothalamus (1 and 3 h after feeding) and gut (3 h after feeding). A similar postprandial decrease (1 and 3 h after feeding) in serum ghrelin levels was also detected. In the fish that were unfed at the regular feeding time, the hypothalamic preproghrelin mRNA expression and the serum ghrelin levels remained unchanged, while the preproghrelin mRNA expression in the gut decreased 3 h after the regular feeding time. Starvation increased preproghrelin mRNA expression in the hypothalamus and gut on the 7th day. Serum ghrelin levels were significantly elevated on days 3 and 5 of starvation. Intracerebroventricular injections of n-octanoylated ghrelin-like peptides (gGRL[1–12]) (10 ng/g body weight) and human ghrelin (1 and 10 ng/g body weight) and intraperitoneal injections of n-octanoylated gGRL[1–12] (10 ng/g body weight), gGRL[1–19] (100 ng/g body weight) and human ghrelin (10 and 100 ng/g body weight) stimulated food intake in goldfish. The patterns of synthesis, secretion and actions indicate that ghrelin is an orexigen in goldfish.

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3beta,17beta-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C(21)-steroids such as 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C(21)-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C(17-20) lyase (CypP450(c17)) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3beta-hydroxysteroid dehydrogenase (3alphaHSD)/isomerase could also be involved. Moreover, the reduction in CypP450(c17) leading to a reduction in C(19)-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450(c17) possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450(c17) activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450(c17) activity.

Sex steroid hormones have been shown to regulate somatostatin (SRIF) gene expression in goldfish brain, which in turn influences the regulation of GH secretion. In this study, the influences of sex steroids on pituitary responsiveness to SRIF-14 and the pituitary expression of a type two SRIF receptor (sst(2)) were examined. Results from in vitro perifusion of pituitary fragments show that pituitaries from estradiol-primed sexually regressed female fish have significantly lower GH release responsiveness to pulse exposure to SRIF-14 than pituitaries from control or testosterone-treated sexually regressed females. Results from in vitro static culture show that pituitaries from sexually mature female fish have lower GH release responsiveness to SRIF-14 than those from sexually regressed females. In addition, the sst(2) receptor mRNA levels in pituitaries from mature and recrudescent female fish are significantly lower than in sexually regressed female fish. Our results indicate that estradiol acts at the level of the pituitary to regulate GH secretion by influencing the responsiveness to SRIF-14. The underlying mechanism includes, in part, reduction of the expression of sst(2) receptors, presumably leading to the lower number of the receptors available for SRIF binding.

Recently, our laboratory has identified three distinct pre-pro-somatostatin (PSS) genes in goldfish brain: PSS-I encodes for somatostatin (SRIH)-14, PSS-II encodes SRIH-28, which contains [Glu1, Tyr7, Gly10] SRIH-14 at its C-terminus, and PSS-III encodes [Pro2] SRIH-14. In goldfish, increasing levels of the sex steroid estradiol increase the plasma levels of growth hormone (GH). However, whether sex steroids act at the level of the brain to regulate GH release is unclear. In the present study, the effects of sex steroids on the expression of the three PSS genes in goldfish forebrain were examined. The results demonstrate that treatment with estradiol significantly increases the expression of PSS-I and PSS-III genes in both male and female fish. The effects of estradiol were evident after only 2.5 days of treatment. Testosterone treatment increased the expression of PSS-I and PSS-III genes in female but not male fish, and only at the highest dose used. In addition, the effects of testosterone were evident only after treatment for 5 or 10 days and were blocked by an aromatase inhibitor, suggesting that testosterone must be converted to estradiol to exhibit the effect. Neither estradiol nor testosterone treatment had effects on the expression of the PSS-II gene. These results suggest that sex steroids can act either directly or indirectly on the brain to regulate PSS-I and PSS-III gene expression, influencing in turn the regulation of GH secretion.

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.

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.

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.

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.