Eddie E. Deane

Modulation of fish growth hormone levels by salinity, temperature, pollutants and aquaculture related stress: a review

The focus of this review is on the importance and regulation of fish growth hormone (GH), during exposure to stress. Alterations in environmental salinity impose osmoregulatory stress on fish and upon exposure to increased salinities GH has been shown to be important in maintaining hypoosmoregulatory function. Whilst studies mainly on salmonids, demonstrate that GH essentially performs a role as a seawater adapting hormone a clear correlation of elevated GH with growth and isoosmotic salinity exposure has been identified from studies on sparids. Variations in water temperature have been shown to modulate fish GH with the overall consensus of highest levels of GH during the warmer seasons of the year, suggesting an important role for GH during the temperature acclimatization process, but whether this relates to growth is unclear. Environmentally important pollutants, including xenoestrogens and heavy metals have been shown to affect GH mediated mechanisms, in fish, possibly via interference with the GH receptor and/or GH transcription, whereas aquacultural related stressors such as handling, confinement/overcrowding and nutritional stress have also been shown to affect GH levels. In addition the impact of aquacultural related stressors can also pre-dispose fish to disease leading to chronic suppression of GH. Finally, GH has been recently demonstrated to exert an anti-apoptotic effect in fish cells, when exposed to chemical stress, providing evidence that GH can also serve as a protective agent.

Chronic Salinity Adaptation Modulates Hepatic Heat Shock Protein and Insulin-like Growth Factor I Expression in Black Sea Bream

Abstract: Black sea bream (Mylio macrocephalus) hepatic heat shock proteins hsp90, hsp70, and hsp60 were found to be thermally and reversibly inducible as they were elevated 2.0, 3.2, and 2.1 fold, respectively, on acute heat shock and returned to pre-heat-shock levels after a 40-hour recovery period. To establish whether salinity plays a role in regulating heat shock protein (hsp) and insulin-like growth factor-I (IGF-I) expression in a euryhaline marine fish, we adapted groups of juvenile black sea bream to salinities of 50 ppt (hypersaline), 33 ppt (seawater), 12 ppt (isoosmotic), and 6 ppt (hypoosmotic) for 8 months. The lowest levels of hsps were found in fish reared in an isoosmotic salinity and the highest in those adapted to hypersaline and hypoosmotic salinities. Hepatic β-actin messenger RNA abundance remained unchanged in all groups during salinity adaptation, whereas IGF-I mRNA abundance was highest in isoosmotic adapted black sea bream. This study is the first report of an effect of salinity ranging from hypersaline to hypoosmotic on the expression of different hsp forms and IGF-I in fish, and the possible relationship between environmental salinity, hepatic IGF-I expression, and hsp regulation is discussed.

Ontogeny of thyroid hormones, cortisol, hsp70 and hsp90 during silver sea bream larval development

We studied the profiles of silver sea bream (Sparus sarba) thyroxine (T(4)), triiodothyronine (T(3)), cortisol and the heat shock protein (hsp) families hsp70 and hsp90 during larval development. Eggs from sexually mature female sea bream were fertilized and larvae were collected at incremental time intervals between 1-46 days post hatch (dph). Both T(4) and T(3) were detected in 1 dph larvae and it was found that both increased as development progressed with a distinct surge in amounts between 21-35 dph, a time associated with direct development of larvae to juveniles. Cortisol increased from 1 dph reaching a maximum and constant level from 35 dph onwards. Using RT-PCR coupled with radioisotope hybridization of immobilized cDNA we assessed the transcript levels of hsp70 and it was found that transcript remained unaltered between 1-14 dph before progressively increasing. Immunoblotting was used to study the larval concentrations of hsp70 and hsp90 and it was found that hsp70 was not significantly changed between 1-14 dph whereas hsp90 increased from 1 dph onwards. These findings suggest an important role for hsp90 in the corticosteroid receptor complex during silver sea bream larval development.

Advances and perspectives on the regulation and expression of piscine heat shock proteins

The focus of this review is on the regulation and expression of heat shock proteins (HSPs), in fish. Within the past decade, molecular studies pertaining to the isolation and characterization of fish HSP genes have been rapidly expanding with many gene sequences from many fish species being reported. Most of the gene sequences presently available belong to the HSP70 family but genes encoding constitutive and inducible members of the HSP90 family have also been identified as well as genes encoding HSP60, HSP47 and small HSP families. Environmental stressors such as alterations in environmental salinity, disease and chemical exposure are known to alter HSP expression and the regulation of HSPs by hormones has received much attention recently. Heat shock proteins are known to play key roles during embryonic development and recent findings have defined their ontogenetic profiles following hatching. Finally, we are gradually starting to add to our understanding as to the transcriptional regulation of HSPs especially the role and importance of the heat shock factor (HSF).

Hormonal status and phagocytic activity in sea bream infected with vibriosis.

Serum taken from female, sexually mature, silver sea bream (Sparus sarba) displaying either no symptoms of vibriosis, mild infection, severe infection or moribundity were assayed for a number of key hormones. Serum cortisol levels were not significantly different among symptomless, mildly- and severely-infected groups, whereas moribund fish displayed hypercortisolemia with a 14-fold increase in serum cortisol in comparison to symptomless fish. Serum estradiol levels were significantly reduced 19-fold in mildly-infected fish and remained at a low level as infection progressed, whereas serum testosterone increased gradually during vibriosis with a 1.8-fold increase in moribund groups in comparison to symptomless groups. Both serum thyroxine (T(4)) and triiodothyronine (T(3)) gradually decreased during vibriosis being 26- and 2.8-fold lower, respectively, in moribund fish in comparison to symptomless fish. The non-specific immune response, as determined by phagocytic activity, was also assessed using macrophages isolated from the pronephros and spleen of infected fish. Phagocytic indices significantly increased in mildly- and severely-infected fish and then decreased from these stimulated levels in moribund fish.

Larval development of silver sea bream (Sparus sarba): ontogeny of RNA-DNA ratio, GH, IGF-I, and Na(+)-K(+)-ATPase.

To ascertain some of the important biochemical and molecular events that take place during early larval development of silver sea bream (Sparus sarba), we undertook a study of changes in the morphology as well as the ontogeny of the RNA-DNA ratio, growth hormone (GH), insulin-like growth factor I (IGF-I) messenger RNA abundance, Na+-K+-ATPase subunit mRNA abundance, and Na+-K+-ATPase enzyme activity. Larvae samples were collected at 1 to 46 days posthatch (dph). At 7 dph the yolk sac was fully absorbed, and from 28 dph onward larvae underwent rapid developmental changes to the juvenile stage. The RNA-DNA ratio was highest at 1 dph, decreased to low levels between 7 and 21 dph, then increased by 28 dph, and then again by 46 dph. The ontogenetic profiles of GH, IGF-I, and Na+-K+-ATPase α1 and β1 subunits were studied using reverse transcriptase polymerase chain reaction, coupled with radioisotope hybridization of immobilized DNA. Growth hormone abundance reached a constant and high level from 35 dph onward, whereas the IGF-I level reached a peak at 35 dph and then significantly decreased. Both Na+-K+-ATPase α1 and β1 subunit mRNAs increased up to 35 dph, however, at 46 dph the α1 subunit remained high whereas the β1 subunit decreased. Na+-K+-ATPase activity was low in 1-dph larvae but increased rapidly as development progressed. The importance of these findings is discussed within the context of larval development.

Hormonal modulation of branchial Na+-K+-ATPase subunit mRNA in a marine teleost Sparus sarba.

The effect of hormone treatment on the abundance of Na+-K+-ATPase alpha- and beta-subunit mRNA in Sparus sarba branchial tissue was investigated. Groups of seawater (33/1000) and hypo-osmotic (6/1000) acclimated fish were injected daily, with either saline, cortisol, recombinant bream growth hormone (rbGH) or ovine prolactin (oPRL). Total RNA from branchial tissue was analyzed by Northern blotting using PCR amplified Na+-K+-ATPase alpha- and beta-subunit cDNA clones. Na+-K+-ATPase alpha- and beta- subunit transcripts of 3.3kb and 2.4kb respectively, were detected and their abundance, after hormone treatment was assessed using RNA dot blots. The abundance of subunit mRNAs increased 1.4-1.9 fold, relative to controls, after cortisol treatment. The alpha:beta mRNA ratio also increased in cortisol treated seawater acclimated fish. Growth hormone treatment did not cause any significant changes in Na+-K+-ATPase subunit mRNA, whereas prolactin significantly reduced alpha-subunit mRNA levels by approximately 0.5 fold in both seawater and hypo-osmotic conditions. The data from this study add further support to the generally accepted roles that cortisol and prolactin have in the modulation of Na+-K+-ATPase activity. It can be concluded from this study that S. sarba branchial Na+-K+-ATPase subunit expression is multihormonally regulated.

Cortisol can be pro- or anti-apoptotic in sea bream cells: Potential role of HSP70 induction for cytoprotection

Cortisol, and heat shock protein 70 (HSP70) are known to perform key roles as part of the fish stress response. In the present study, two in vitro systems were used to investigate a possible cortisol-HSP70-apoptosis regulatory relationship. Using a developed silver sea bream fibroblast cell line (SSF), cortisol was found to induce HSP70 synthesis with a concomitant protection against camptothecin induced apoptosis. The induction of HSP70 synthesis using azetidine was also found to protect SSF against apoptosis. A primary culture of silver sea bream macrophages (SSM) displayed reduced HSP70, underwent apoptosis and displayed reduced phagocytic activity upon exposure to cortisol. The effect of cortisol on HSP70 expression in both SSF and SSM were blocked by the glucocorticoid antagonist, RU486. Treatment of SSM with azetidine protected against apoptosis and also enhanced phagocytic activity. The data from this study demonstrates for the first time that cortisol can be either anti- apoptotic or pro-apoptotic in different fish cells and such actions can be mediated via HSP70 induction or suppression respectively.

Growth Hormone Increases hsc70/hsp70 Expression and Protects against Apoptosis in Whole Blood Preparations from Silver Sea Bream

Abstract: Preparations of whole blood, from silver sea bream (Sparus sarba), were used for heat shock protein 70 (hsp70) and apoptosis studies. It was found that the expression of both gene members of the hsp70 family (hsc70 and hsp70) were upregulated during acute heat shock. The transcript abundance of both of these genes was increased when cells were exposed to growth hormone (GH) at concentrations of 10 and 100 ng/mL. It was also found that GH at concentrations of 10 and 100 ng/mL could protect whole blood from camptothecin‐induced apoptosis as determined by DNA fragmentation analysis.

Hypercortisolemia does not affect the branchial osmoregulatory responses of the marine teleost Sparus sarba

The effect of cortisol treatment on branchial Na(+)-K(+)-ATPase subunit mRNA abundance, enzyme activity, chloride cell number/morphometrics and serum electrolyte levels were investigated for the marine teleost Sparus sarba. Groups of fish received intraperitoneal injections of cortisol at a concentration of 4 micrograms/g body weight, daily, over a seven-day period. This dose of cortisol was sufficiently high enough to maintain a condition of hypercortisolemia as serum cortisol levels in treated fish were eleven fold higher than controls at time of sacrifice. By using branchial Na(+)-K(+)-ATPase alpha- and beta-subunit cDNA clones we were able to demonstrate that cortisol administration to S. sarba caused a significant elevation in the abundance of alpha-mRNA whereas the levels of beta-mRNA were unchanged. In addition Na(+)-K(+)-ATPase activity remained unaltered by cortisol administration. Branchial chloride cell number, exposure, apical area as well as serum Na+ and Cl- levels remained unchanged after cortisol administration. The results of this study suggest that elevated cortisol level may not necessarily translate into modulated branchial Na(+)-K(+)-ATPase activity and chloride cell function in hypo-osmoregulating marine fish.