Controlo da secreção e síntese da hormona de crescimento e da prolactina em pituitárias de dourada (Sparus aurata L.) incubadas in vitro

Dissertação de Mestrado, Estudos Marinhos e Costeiros, Unidade de Ciências do Mar e do Ambiente, Universidade do Algarve, 1994O peixe utilizado no presente trabalho, a dourada (Spcnvs aura,a L), é uma espécie importante para a aquacultura ao longo de toda a costa mediterrânea. A hormona de crescimento (GH) e a prolactina (PRL) libertadas pela glandula pituitária deste teleósteo, regulam dum modo fundamental alguns processos fisiológicos particularmente o crescimento.The fish used in tlie present study, the sea bream {Sparus aura,a L.), is an important aquaculture species along the Mediterranean coast. The growth hormone (GH) and prolactin (PRL), released by the pituitary gland of this telcost fish, play a central role in controlling several aspects of its physiology, particularly growth. In this study, we examined the effect of several peptides on the activity ol somatotrophs and prolactin cells in cultured pituitary gland

Characterization of sea bream (Sparus aurata L.) prolactin and control of release from pituitary gland

Tese de Doutoramento, Ciência Biológicas, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2004The sea bream {Sparus aurata L.) is an important aquaculture species along the Mediterranean coast. Prolactin (PRL) released by the pituitary gland play a central role in controlling several aspects of sea bream physiology. In this study, we examined the effect of several internai and externai factors on the activity of PRL cells in cultured pituitary glands. Sea bream pituitaries were cultured during 18hrs at 21ºC and the hormones present in the culture médium separated by SDS-PAGE. PRL was identified by Western blotting using antiserum directed against chum salmon PRL and quantified by optical densitometry.A dourada {Sparus aurata L.) é uma espécie importante para a aquacultura ao longo de toda a costa mediterrânea. A prolactina (PRL) é uma hormona libertada pela glândula pituitária e regula de um modo fundamental alguns aspectos da fisiologia deste teleósteo. No presente trabalho foi estudado o efeito que diferentes factores, externos e internos, exercem na actividade in vitro das células de PRL. Para este estudo foram feitas culturas de pituitárias de dourada com uma duração de 18 horas a uma temperatura média de 21ºC. A PRL, após separação por SDSPAGE, foi identificada por "Western blotting" utilizando anticorpos da PRL do salmão e, posteriormente, quantificada por densidade óptica

The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata, L.)

The effect of estradiol-17b (E2) implants on the in vitro secretion of prolactin (PRL) and its modulation by vasoactive intestinal peptide (VIP) in a marine teleost, sea bream (Sparus aurata L.), was determined. Experiments were conducted during winter and spring. During winter, fish (n¼130, body weight 50–70 g) were randomly divided into 2 groups; control and E2 treated (10 mg/kg, wet weight). Fish were sacrificed after 7 days treatment and in vitro pituitary cultures in Ringer bicarbonate supplemented with increasing doses (0–200 nM) of VIP were carried out for 18 h. Culture medium was analysed by PAGE and secreted PRL quantified by densitometry. Fish treated with E2 secreted significantly more PRL ðP < 0:05Þ in vitro than control fish. In E2 primed fish VIP caused a dose-dependent inhibition of PRL secretion in vitro. VIP had no detectable effect on the secretion of PRL from control pituitaries. Treatment with E2 had a different effect during spring; PRL secretion was significantly decreased ðP < 0:01Þ compared with the control fish. Anatomical evidence of abundant VIPimmunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIPin the regulation of PRL cells. In conclusion, the responsiveness of PRL in the pituitary gland varied with season. Moreover, in the sea bream VIP appears to modulate PRL secretion from E2 primed pituitary glands.The present work was funded by the Fundac ~ao para a Ci^encia e a Tecnologia, Portugal (PraxisXXI 2/2.1/ BIA/211/94). J.F. and L.B. were funded by FCT Grants BPD/22033 and 4/5.3/PRODEP/2000, respectively. The authors thank the anonymous reviewers for their helpful critique

Cloning, expression, and tissue localisation of prolactin in adult sea bream (Sparus aurata)

A major action of prolactin (PRL) in teleost fish is the maintenance of hydromineral balance in euryhaline species in fresh water. The function of PRL in marine teleosts is less certain and unlike euryhaline teleosts, such as tilapia and salmon, there is relatively little information about protein or gene structure. Associated with studies to determine potential functions of PRL, pituitary prolactin cDNA has been cloned and sequenced from sea bream (Sparus aurata), a marine teleost. The sequence obtained spanned 1349 bp and contained an open reading frame encoding a protein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids. N-terminal sequencing of the native protein confirmed unambiguously the cleavage site, Ala24, Val25, predicted from alignments of the sea bream PRL cDNA with that of other teleosts. The presence of only one form of PRL in sea bream was supported by identification using Northern blots of only a single transcript of 1.35 kb. Reverse transcription and polymerase chain reaction techniques coupled with Southern blot analysis resulted in the detection of PRL in the pituitary but also in the intestine, liver, ovary, and testes.This work was supported by a grant from the Portuguese Fundacao para Ciencia e Tecnologia (2/2.1/BIA/211/94). Cecilia R. A. Santos was in receipt of a JNICT grant (BD/2632/93/IG). We are grateful toMs Nair Enxerto for assistance in the study. Note added in proof. In a recent publication Bole-Feysot et al., 1998, refer to over 300 separate functions of PRL in vertebrates. (Bole- Feysot, C., Goffin, V., Edery, M., Binart, N., and Kelly, P. A. (1998). Prolactin (PRL) and its receptor: Actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19, 225–268)

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p < 0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p < 0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p < 0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na+/K+-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.The present work was funded by the Ministry of Science and Technology (FCT), Portugal, project PRAXIS/P/BIA/13174/98 and pluriannual funding to the Centre of Marine Sciences (CCMar). L. Brinca and P.M. Guerreiro were funded by the Ministry of Science (FCT), Portugal through Grants, 4/5.3/PRODEP/2000 and BPD/9464/ 2002, respectively. The authors express their appreciation to Dr. Patricia M. Ingleton for helpful discussion about the study. The constructive critique and helpful comments of two anonymous reviewers’ contributed to improve the manuscript