Homologous desensitization and visualization of the tilapia GnRH type 3 receptor.

Two types of gonadotropin-releasing hormone (GnRH) receptors were found in the pituitary of tilapia (t), named GnRHR type 3 (tGnRHR3) and GnRHR type 1, according to phylogenetic analysis. tGnRHR3 is highly expressed in the posterior part of the pituitary which contains LH and FSH cells. We characterized tGnRHR3 in terms of both LH release rate and receptor internalization rate in response to continuous exposure to GnRH. Constant exposure of tilapia pituitary fragments to salmon GnRH analog (sGnRHa) resulted in an increased secretion rate for 3h, followed by a gradual decline, taking 17-19h, to the basal secretion rate. A chimera between tGnRHR3 and green fluorescent protein (GFP) was created and used to observe the changes in receptor distribution and translocation, activated by agonist with time. The results suggested that the receptor is initially localized at the plasma membrane and upon activation by a homologous ligand (e.g. sGnRHa) undergoes relatively rapid endocytosis. In summary, the present work demonstrates that tGnRHR3 has already undergone endocytosis after 30min, while desensitization of LH release occurs only after 17-19h. It is concluded that for tGnRHR3, internalization of the receptor is not exclusively responsible for the desensitization of LH release.

Cytochrome P450 aromatase in grey mullet: cDNA and promoter isolation; brain, pituitary and ovarian expression during puberty.

In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485 bp and encoded 495 predicted amino acid residues. The expression level of muCyp19b significantly increased in the brain as puberty advanced; however, its expression level in the pituitary increased only slightly with pubertal development. In the ovary, the muCyp19a expression level markedly increased as puberty progressed. The promoter regions of the two genes were also isolated and their functionality evaluated in vitro using luciferase as the reporter gene. The muCyp19a promoter sequence (650 bp) contained a consensus TATA box and putative transcription factor binding sites, including two half EREs, an SF-1, an AhR/Arnt, a PR and two GATA-3 s. The muCyp19b promoter sequence (2500 bp) showed consensus TATA and CCAAT boxes and putative transcription binding sites, namely: a PR, an ERE, a half ERE, a SP-1, two GATA-binding factor, one half GATA-1, two C/EBPs, a GRE, a NFkappaB, three STATs, a PPAR/RXR, an Ahr/Arnt and a CRE. Basal activity of serially deleted promoter constructs transiently transfected into COS-7, alphaT3 and TE671 cells demonstrated the enhancing and silencing roles of the putative transcription factor binding sites. Quinpirole, a dopamine agonist, significantly reduced the promoter activity of muCyp19b in TE671. The results suggest tissue-specific regulation of the muCyp19 genes and a putative alternative promoter for muCyp19b.

Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary.

A full-length cDNA encoding a dopamine receptor (DA-R) was obtained from the pituitary of tilapia (ta). This cDNA encodes a protein of 469 amino acids that exhibits the typical arrangement of GPCR. The taDA-R shows high similarity to the DA-Rs of mullet and fugu, and over 70% similarity to Xenopus, mouse and turkey D2 DA-Rs. Northern blot analysis revealed transcript for a single transcript in the pituitary, of approximately 3 kb. In a Southern analysis, the tilapia probe recognized specific bands in the genomic DNA of both mullet and catfish, suggesting high similarity between the corresponding genes. Phylogenetic analysis clearly aligned the taDA-D2-R with all vertebrate D2-like receptor sequences cloned to date, and it was therefore designated taDA-D2-R. taDA-D2-R was transiently expressed in COS-7 cells together with the reporter construct CRE-luciferase. Addition of the specific D2 dopamine agonists quinpirole or bromocriptine, in the presence of forskolin, led to a dose-dependent decrease in forskolin-induced cAMP levels. Both agonists yielded the same maximal inhibition (around 40%). However, the potency of taDA-D2-R for bromocriptine was higher than for quinpirole. As established for mammalian D2-like receptors, stimulation of the taDA-D2-R with quinpirole triggers pertussis-toxin-sensitive Gi/o-mediated, but not Gs-mediated signaling. In contrast to mammals, PCR analysis gave no evidence of alternative splicing in taDA-D2-R. Pharmacological and genetic manipulation of the taDA-D2-R should enable us to better define its physiological role and to further explore the usefulness of fish as a model system for understanding dopaminergic function in higher organisms.

Sequence analysis, endocrine regulation, and signal transduction of GnRH receptors in teleost fish.

Three gonadotropin-releasing hormones (GnRHs) and three cognate receptors have been identified in vertebrates, with distinct distributions and functions. According to their sequences, the receptors can be grouped into distinct classes: types I, II, and III. One branch contains all type-I GnRH receptors (GnRH-R-I) from mammals and fish; another branch clusters mainly amphibian and human type-II GnRH receptors; and a third branch includes evolved fish, mainly perciform species, type-III GnRH receptors. Taken tilapia GnRH receptors as a model, the present study summarizes the information regarding the amino-acid residues assumed to be involved in the receptors' structure, binding, activation, and intracellular signal transduction, including arrangement of the disulfide bonds, glycosylation sites, coupling to G proteins, and protein kinase A or protein kinase C phosphorylation sites.

Regulation of gonadotropin-releasing hormone (GnRH)-receptor gene expression in tilapia: effect of GnRH and dopamine.

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5-50 microg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 microg/kg and at 36 h. At the higher dose of sGnRHa (50 microg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1-100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Effects of long term feeding of Quillaja saponins on sex ratio, muscle and serum cholesterol and LH levels in Nile tilapia (Oreochromis niloticus (L)).

Seventeen-day-old Nile tilapia fry were fed a standard diet (C) or diets containing 50-700 mg kg(-1) Quillaja saponin (QS) extract (groups S50, S150, S300, S500 and S700). After the first 8 weeks, 30 randomly selected tilapia from each of the treatments were placed in separate aquaria and fed the standard diet without saponins from then on (these were designated S50/C, S150/C, S300/C, S500/C and S700/C). The fish grew from an initial average weight of approximately 30 mg to a final average weight of 79 g during the 6-month feeding period. The difference between the average weight of C-fed tilapia and the treatment with the highest average weight after 6 months was 53.5%. The sex ratio of tilapia in the saponin-fed groups deviated from the normal 50:50 male:female ratio, with the S700 group showing a significantly higher number of males. Quillaja saponin stimulated LH release from dispersed tilapia pituitary cells in vitro. This effect was abolished in the presence of dilute calf serum. Serum LH values did not show any diet-dependent trend in either male or female tilapia in vivo. In both continuously saponin-fed and only-initially saponin-fed groups, the average serum (but not muscle) cholesterol levels in males showed an increasing trend (R(2) values of 0.62 and 0.69) with increasing dietary saponin level. It was concluded that dietary QS has the potential to change the sex-ratio in favour of males. More investigations are required to determine the mechanism of action and the optimum dietary level of QS for maximum effects.