Gene expression of thyrotropin- and corticotrophin-releasing hormones is regulated by environmental salinity in the euryhaline teleost Sparus aurata.

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65-81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments.

Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinities.

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55ppt) for 14days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na+/K+-ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit ß (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15ppt). The high salinity (55ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity.

Pituitary gene and protein expression under experimental variation on salinity and temperature in gilthead sea bream Sparus aurata.

Temperature and salinity are important factors that affect several physiological processes in aquatic organisms, which could be produced by variation of certain hormones. In this study, the expression of pituitary hormones involved in the acclimation to different temperatures and salinities was examined in Sparus aurata, a euryhaline and eurytherm species, by Q-Real Time RT-PCR and Western blot analyses for mRNA and protein expression, respectively. Three different experimental conditions were designed with specimens (10 per treatment) acclimated to: a) low salinity water; b) sea water; and c) high salinity water. Additionally, fish under different salinities were acclimated to three different temperatures: 12, 19 and 26 degrees C. Animals were maintained seven weeks before sampling pituitary glands. Our results provided enough evidence for a differential expression of PRL, GH and SL in the pituitary of gilthead sea bream, under different temperature and salinity regimes.

Gene and protein expression for prolactin, growth hormone and somatolactin in Sparus aurata: seasonal variations.

The seasonal variation of PRL, GH and SL gene and protein expression has been analyzed in gilthead sea bream (Sparus aurata) pituitaries using Real-Time Q-PCR and Western Blots, respectively. Animals were cultured in earthen ponds under natural photoperiod, temperature and salinity conditions. Samples were taken during winter 2005 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Beta-actin, used as the housekeeping gene both for Q-RT-PCR and Western analysis, did not present significant differences among seasons. Higher expression was observed during spring and autumn for PRL, summer and winter for GH, and spring for SL. Expression of PRI, GH and SL, presented seasonal variation, suggesting that these hormones could play a role in the molecular signal transduction of environmental factors (especially of photoperiod and temperature) in eurythermal fish.

Sex steroids and metabolic parameter levels in a seasonal breeding fish (Sparus aurata L.).

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the two first reproductive cycles (RCs), in which the specimens develops as males. During the first RC (RC1), 11-ketotestosterone (11-KT) and testosterone (T), the main androgens in fish, play different and specific roles in the testicular physiology as they peak at different stages of RC1. Moreover, the profiles of T serum levels during the second RC (RC2) demonstrated that this androgen is not essential in the testicular regression process that occurs during this cycle. However, changes in serum levels suggest that 17beta-estradiol (E2) orchestrates this process during both RCs. Moreover, the E2 serum levels recorded during RC2 indicate that there is a threshold level that determines the initiation of ovarian development during this cycle without promoting complete feminization. We analysed triglyceride, protein, glucose and lactate serum levels in order to establish a relationship between the mobilization and transfer of nutrients, the hormonal changes that take place during the RC and body composition, finding that in vivo serum levels of metabolites change significantly throughout the first two RCs, although the physiological relevance of such changes is still unknown. Triglyceride levels seem to be affected by the beginning of ovary development during RC2 but not by testicular recrudescence during RC1. Moreover, glucose and lactate might be important sources of energy during the resting and testicular involution stages.

The involvement of thyroid hormones and cortisol in the osmotic acclimation of Solea senegalensis.

The peripheral conversion of the prohormone 3,5,3',5'-tetraiodothyronine (T4) to the biologically active 3,5,3'-triiodothyronine (T3), via enzymatic deiodination by deiodinases, is an important pathway in thyroid hormone metabolism. The aim of this study was to test if thyroid hormones and cortisol, as well as the outer ring deiodination (ORD) metabolic pathway, are involved in the osmoregulatory response of Senegalese sole (Solea senegalensis, Kaup 1858). We measured osmoregulatory and endocrine parameters in immature juveniles S. senegalensis acclimated to seawater (SW, 38 per thousand) and that were transferred and allowed to acclimate to different salinities (5 per thousand, 15 per thousand, 38 per thousand and 55 per thousand) for 17 days. An adjustment and a chronic regulatory period were identified following acclimation. The adjustment period immediately follows the transfer, and is characterized by altered plasma osmolalities. During this period, plasma cortisol levels increased while plasma free T4 (fT4) levels decreased. Both hormones levels returned to normal values on day 3 post-transfer. In the adjustment period, renal and hepatic ORD activities had increased concomitantly with the decrease in plasma fT4 levels in fishes transferred to extreme salinities (5 per thousand and 55 per thousand). In the chronic regulatory period, where plasma osmolality returned to normal values, plasma cortisol had increased, whereas plasma fT4 levels decreased in animals that were transferred to salinities other than SW. No major changes were observed in branchial ORD activity throughout the experiment. The inverse relationship between plasma cortisol and fT4 suggests an interaction between these hormones during both osmoregulatory periods while ORD pathway can be important in the short-term adjustment period.

Effect of sex-steroid hormones, testosterone and estradiol, on humoral immune parameters of gilthead seabream.

The role of sex-steroid hormones, testosterone (T) and 17beta-estradiol (E2), on the humoral immune parameters of the teleost gilthead seabream Sparus aurata was studied attempting to deepen on the knowledge of the immune-reproductive system interactions. Fish were injected intraperitoneally with coconut oil containing different dosages of T (0, 2, or 5 microg g(-1) body weight [bw]) or E2 (0, 1, or 2 microg g(-1) bw) and sampled 1, 3, and 7 days later. Hormonal levels and immune parameters (complement, peroxidase and antiprotease activities and IgM levels) were determined in plasma. Plasma hormone levels peaked at 1 day post-injection decreasing thereafter. Treatment with T significantly increased both complement and peroxidase activities after 3 days of injection but antiprotease activity and IgM levels remained unchanged. Treatment with E2 enhanced complement activity 1 day post-injection while decreased it after 3 and 7 days. However, peroxidase activity increased at 3 and 7 days post-injection while total IgM levels decreased. Implications of T and E2 in the immune-reproductive system interactions were discussed.