Purification of luteinizing hormone (LH) in the sea bass (Dicentrarchus labrax) and development of a specific immunoassay (original) (raw)
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General and Comparative Endocrinology, 1991
Growth hormone (GH) was isolated from sea bass (Dicentrarchus labrax) pituitary extract by a simple one-step procedure involving immunoaffinity chromatography. A monoclonal antibody raised against chicken GH and found to immunostain very specifically the GH cells in the pituitary of the sea bass was coupled to CNBr-activated Sepharose 4B. Sea bass pituitary extracts were run on the affinity column, and the eluted material was analyzed on reversed-phase HPLC and found to consist of one single peak. The yield of purified hormone was 2.4 mg/g pituitary. Two monomeric forms (MW = 20,000 and 22,000 Da) of sea bass GH were identified by gel electrophoresis. Gel electrofocusing revealed apparent isoelectric points of 6.15, 6.50, and 6.95. Amino acid composition is consistent with other vertebrate GHs. The immunological relatedness was tested by immunoblotting using antisera raised against GH of different species. Polyclonal antisera raised against the isolated hormone exhibited a specific labeling of the GH cells in sea bass pituitary sections as well as of the immunoblotted purified GH.
Journal of Endocrinology, 2006
The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na C /K C -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1a (ef1a) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prl was 4 . 5-fold higher in FW than in SW, whereas pituitary sl was 1 . 8-fold higher in SW-compared with FW-adapted fish. Gill Na C /K C -ATPase specific activity was 2 . 3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6 . 5-fold lower in SW-than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FW euryhaline teleosts.
Biology of Reproduction, 2001
The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptinrelated peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10-8-10 Ϫ6 M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10 Ϫ6 M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.
Since the late 1980s, gonadotropins have been isolated and characterized in several fish species, but specific immunoassays for the follicle-stimulating hormone (FSH) have only been developed for a few. The present study reports the development and use of a specific and homologous competitive ELISA for measuring FSH in European sea bass (Dicentrarchus labrax) using a recombinant FSH and its specific antiserum. Recombinant European sea bass FSHb and FSH heterodimer were produced in the methylotrophic yeast Pichia pastoris and a baculovirus expression system, respectively. Specific polyclonal antibodies, generated by rabbit immunization against recombinant FSHb, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166 lg/ml). The sensitivity of the assay was 0.5 ng/ml [B 0 -2SD], and the intra-and inter-assay coefficients of variation were 2.12% (n = 10) and 5.44% (n = 16) (B i /B 0 $ 45%), respectively. A high degree of parallelism was observed between the standard curve and serially diluted plasma and pituitary samples of European sea bass.
Aquaculture Research, 2007
Mature black sea bass, Centropristis striata L. (2008 00 g), were captured in coastal South Carolina during the spawning season and administered hormones for ovulation induction and strip spawning. During both study years, control groups of females were incorporated into the study design and administered sham injections containing physiological saline solution. In 2004, females received a single intramuscular injection of human chorionic gonadotropin (hCG) (330 IU kg À1) (n 5 8) or two injections of hCG at 24-h intervals (n 5 8). In 2005, females received a single injection of hCG (n 510) or an analogue of luteinizing hormone releasing hormone (LHRHa) (n 510). In 2004, all ¢sh administered a single dose of hCG ovulated at least once. Six ¢sh ovulated on two consecutive days and one ¢sh ovulated on 3 days consecutively. In contrast, six of eight ¢sh receiving two doses of hCG ovulated once, ¢ve ovulated on 2 days successively and three ¢sh ovulated 3 days in succession. Of the ¢sh that spawned, no differences were found in any reproductive parameters. In 2005, all ¢sh administered hCG or LHRHa ovulated at least once. Three ¢sh administered hCG ovulated twice, four ¢sh ovulated on three consecutive days and one ¢sh 4 days successively. All ¢sh administered LHRHa spawned at least twice, six ¢sh ovulated thrice and three ¢sh ovulated 4 days, successively. A signi¢cant di¡erence in fertility was found between hCG (75.6 AE 11.4%) and LHRHa (55.6 AE 27.4%). The results of this study indicate that both hCG and LHRHa are e¡ective for ovulation induction in prespawning black sea bass.
General and Comparative Endocrinology, 2008
Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein a (GPa) and 13.6 kDa for FSHb subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPa and FSHb). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17b (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPa and FSHb), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.
Alvarifio, J.M.R., Zanuy, S., Prat, F., Carrillo, M. and Maiianos, E., 1992. Stimulation of ovulation and steroid secretion by LHRHa injection in the sea bass (Dicentrarchus labrax): effect of time of day. Aquuculfure, 102: 177-186.