Generation and use of recombinant gonadotropins in fish.

Understanding the differential roles of the pituitary gonadotropins Fsh and Lh in gonad maturation is crucial for a successful manipulation of the reproductive process in fish, and requires species-specific tools and appropriate active hormones. With the increasing availability of fish cDNAs coding for gonadotropin subunits, the production of recombinant hormones in heterologous systems has gradually substituted the approach of isolating native hormones. These recombinant hormones can be continually produced without depending on the fish as starting material and no cross-contamination with other pituitary glycoproteins is assured. Recombinant gonadotropins should be produced in eukaryotic cells, which have glycosylation capacity, but this post-translational modification varies greatly depending on the cell system, influencing hormone activity and stability. The production of recombinant gonadotropin beta-subunits to be used as antigens for antibody production has allowed the development of immunoassays for quantification of gonadotropins in some fish species. The administration in vivo of dimeric homologous recombinant gonadotropins has been used in basic studies and as a biotechnological approach to induce gametogenesis. In addition, gene-based therapies using somatic transfer of the gonadotropin genes have been tested as an alternative for hormone delivery in vivo. In summary, the use of homologous hormonal treatments can open new strategies in aquaculture to solve reproductive problems or develop out-of-season breeding programs.

The Kiss2 receptor (Kiss2r) gene in Southern Bluefin Tuna, Thunnus maccoyii and in Yellowtail Kingfish, Seriola lalandi - functional analysis and isolation of transcript variants.

The kisspeptin system plays an essential role in reproductive function in vertebrates, particularly in the onset of puberty. We investigated the kisspeptin system in two Perciform teleosts, the Southern Bluefin Tuna (SBT; Thunnus maccoyii), and the Yellowtail Kingfish (YTK; Seriola lalandi), by characterising their kisspeptin 2 receptor (Kiss2r) genes. In addition to the full length Kiss2r cDNA sequences, we have isolated from SBT and YTK a transcript variant that retained an intron. We have further obtained three ytkKiss2r transcript variants that contained deletions. In vitro functional analysis of the full length SBT and YTK Kiss2r showed higher response to Kiss2-10 than to Kiss1-10, with stronger transduction via PKC than PKA. The full length ytkKiss2r and two deletion variants were differentially expressed in the brain of male, but not in female, juvenile YTK treated with increasing doses of Kiss2-10 peptide. In the gonads, the expression level of the ytkKiss2r transcripts did not vary significantly either in the male or female fish. This is the first time that transcript variants of the Kiss2r gene that contain deletions and show responsiveness to treatments with kisspeptin have been reported in any teleost.

Perspectives on fish gonadotropins and their receptors.

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.

Sexual development in fish, practical applications for aquaculture.

Aquaculture is one of the fastest rising sectors of world food production. Hundreds of fish species are cultured, providing an affordable, high quality food source. Two aspects of sexual development are critically important for the continued improvement of cultured fish stocks: sexual dimorphism and control of reproduction. In this paper, we review the main methods used to control sex determination in fish and their application in some of the most widely cultured species. Specifically, we review the techniques available for the production of all-male, all-female, and sterile populations. Techniques for endocrinological control of reproduction are also discussed.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Purification and characterization of recombinant pufferfish (Takifugu rubripes) leptin.

Synthetic cDNA encoding pufferfish (Takifugu rubripes) leptin (pfLEP) was prepared according to the published sequence. The pfLEP, transformed into Escherichia coli and expressed upon induction with nalidixic acid, was found almost entirely in the insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography and gel-filtration. The respective yield of dimers and monomers was 50-100mg from 5L of fermentation culture. Circular dichroism analyses revealed similarity of the purified pfLEP secondary structure to that of mammalian leptins. The purified monomers and dimers showed a single band of approximately 15 kDa following SDS-PAGE in the presence of reducing agent, whereas the dimer showed one band of approximately 30 kDa in the absence of reducing agent, indicating its formation by S-S bonds. The purified product also showed a single peak following gel-filtration under nondenaturating conditions and reverse-phase chromatography. Monomeric and dimeric pfLEPs were stable for at least 6 months in sterile solution frozen at -20 degrees C or as lyophilized powder. Both pfLEPs were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin (hLEP) receptor, but their activity was four to five orders of magnitude lower than that of hLEP. The specificity of this activity was further evidenced by its complete inhibition by hLEP antagonist. In contrast to mammalian leptins, neither form of pfLEP bound to or formed 1:1 complex with chicken leptin-binding domain, likely due to low affinity. No specific binding of either ovine or pufferfish leptins to tilapia liver membranes was detected. This work is the first report on the purification of leptin from any fish species.

Isolation of dopamine D(2) receptor (D (2)R) promoters in Mugil cephalus.

This paper reports the isolation of two putative D(2)R promoters from grey mullet, one 5'flanking and the other an intronic sequence immediately upstream of the first coding exon. Promoter activity of the intronic sequence was confirmed in vitro through functional analysis using luciferase as reporter gene. The functional characteristics of the region flanking the 5'UTR is currently under investigation.

Carotenoid and retinoid transport to fish oocytes and eggs: what is the role of retinol binding protein?

Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehydroretinol and retinyl-esters) that are utilized during embryonic development, after fertilization. The carotenoids (mainly astaxanthins) are transported in the plasma by the low density lipoproteins, high density lipoproteins, and very high density lipoproteins (VHDL) and were found to be associated also with serum albumin. Retinals were found to be associated vitellogenin (VTG), a component of the plasma VHDL fraction that is internalized by oocytes during vitellogenesis. However, the transport of retinols and retinyl-esters that were located in the oil droplet fraction of homogenized eggs, has yet to be elucidated. Retinols are more abundant in freshwater fish eggs than in eggs of marine fish species. Since retinol is transported in the plasma of vertebrates in association with retinol binding protein (RBP), recent studies on the molecular characterization and expression sites of RBP, could contribute to determining the involvement of RBP in transporting retinol to developing oocytes in vertebrates.Recently, results from our laboratory show that RBP mRNA levels in the liver and RBP plasma levels did not significantly change with the onset and during vitellogenesis in the Rainbow trout. These results were in contrast with a dramatic elevation in the mRNA levels of VTG in the liver and an increase in VTG plasma levels that was observed in the same females. Moreover, 17beta-estradiol treatment of immature fish, resulted in relatively lower mRNA levels of RBP in the liver, concomitantly with an increase in the level of VTG transcripts and the appearance of VTG in the plasma of treated fish. In addition, RBP was localized in the cytosol of ovulated oocytes. These results for Rainbow trout are similar to those reported for the chicken but differ from those of Xenopus, where an increase in RBP mRNA was reported in the liver and higher levels of retinal and retinol were found in the plasma of 17beta-estradiol treated animals. The results, reported here for the first time in Rainbow trout, showing RBP transcripts in the ovary, oviduct (the ovarian tissue adjacent to the gonopore) and oocytes, suggest a modulating role for RBP in follicular development, as has been suggested for the bovine ovary.

Characterization of tilapia FSHbeta gene and analysis of its 5' flanking region.

The objective of the current study was to unveil molecular mechanisms underlying transcriptional regulation of the FSHbeta gene expression in the pituitary of tilapia (Oreochromis mossambicus). The full-length sequence of tilapia FSHbeta (tFSHbeta) gene was determined. Its transcriptional unit (2.7 kb) exhibits the conserved genomic organization, i.e. three exons and two introns. Primer extension and RT-PCR analysis revealed heterogeneity of the tFSHbeta transcripts, due to alternate mRNA splicing and multiple initiation sites for transcription. Examination of the 5' flanking region (5'FR) of the tFSHbeta gene identified potential CAAT and TATA promoter proximal elements as well as several sequences of cis-acting motifs known to dictate inducible and tissue-specific transcriptional regulation in other gonadotropin genes. Chimeric constructs containing 1.7 kb of the tFSHbeta 5'FR fused to a luciferase (LUC) reporter gene were transiently transfected into primary culture of tilapia pituitary cells. The tFSHbeta-LUC construct was efficiently expressed under basal conditions and was rapidly induced by GnRH stimulation. Our data indicate that the 5'FR contains a functional promoter, which is responsive to GnRH treatment. In addition, 5' deletion analysis showed that the 1.7 kb, DNA sequence of the FSHbeta 5'FR encompasses both positive and negative regulatory elements.

Regulation of gonadotropin subunit genes in tilapia.

A steroidogenic tilapia gonadotropin (taGtH=LH) was purified from pituitaries of hybrid tilapia (Oreochromis niloticus x O. aureus) and a homologous RIA was established. This RIA enabled the study of the endocrine regulation of GtH release, the transduction pathways involved in its secretion and its profile during the spawning cycle. Discrepancies between steroid and taGtH peaks during the cycle led to the conclusion that an additional gonadotropin similar to salmonid FSH operates early in the cycle. In order to identify this hormone and to study the endocrine control of synthesis of all gonadotropin (GtH) subunits, a molecular approach was taken. The cDNA sequences and the entire gene sequences encoding the FSHbeta and LHbeta subunits, as well as an incomplete sequence of the glycoprotein hormone alpha subunit (GPalpha), were cloned. Salmon gonadotropin-releasing hormone (sGnRH) elevated mRNA steady-state levels of all three GtH subunits in cultured pituitary cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) also stimulated the expression of these subunits and potentiated the effect of GnRH, except that NPY did not affect FSHbeta. The GnRH and NPY effects were found to be mediated mainly through protein kinase C (PKC), while protein kinase A (PKA) cascade was involved to a lesser extent. Mitogen-activated protein kinase (MAPK) cascade takes part in mediating GnRH effects, possibly via PKC. Testosterone (T) and estradiol (E2), but not 11-ketotestosterone (KT), are able to elevate GPalpha and LHbeta mRNAs in pituitary cells of early maturing or regressing males. Low levels of T exposure are associated with elevated FSHbeta mRNA in cells of mature fish, while higher levels suppress it, but elevate LHbeta mRNA. In vivo observations also showed the association of low T levels with increased FSHbeta mRNA and high T levels with elevated LHbeta mRNA. In accordance with these findings, analysis of LHbeta and FSHbeta 5' gene-flanking regions revealed on both gene promoters a GtH-specific element (GSE), half site estrogen response elements (ERE), cAMP response element (CRE) and AP1. In vitro experiments showed that recombinant human activin-A leads to higher levels of GPalpha, FSHbeta and LHbeta mRNAs in pituitary cell culture. Porcine inhibin marginally decreased the mRNA levels of GPalpha and FSHbeta, but at a low level (1 ng/ml) it stimulated that of LHbeta. These results shed some light on certain hypothalamic and gonadal hormones regulating the expression of GtH subunit genes in tilapia. In addition, they provide evidence for their differential regulation, and insight into their mode of action.

Adenylyl cyclase interaction with the D2 dopamine receptor family; differential coupling to Gi, Gz, and Gs.

1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the alpha subunit of Gz. PTX treatment was employed to inactivate endogenous alpha i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively. 4. We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.

Human D3 dopamine receptor in the medulloblastoma TE671 cell line: cross-talk between D1 and D3 receptors.

In search of a cell line in which the D3 dopamine receptor is expressed endogenously, we found that the neuron-derived human medulloblastoma cell line TE671 expresses the human D3 (hD3) and D1 (hD1) receptor, but neither the D2 or D4 receptors. Exposure of TE671 cells to the D3 agonist 7-OH-DPAT (DPAT), or to the D1 agonist SKF-38393 (SKF) increased the expression of hD3 or hD1 mRNA, respectively. Moreover, whereas DPAT had no effect on hD1 mRNA levels, stimulating the cells with SKF caused an increase in both hD1 and hD3 transcript levels. These results suggest (i) that following ligand stimulation, hD3 and hD1 receptors are upregulated to enhance their own receptor expression, and (ii) that upregulation of hD1 receptor transcripts leads to a stimulation of the hD3 dopamine receptor transcripts.

Functional expression of the murine D2, D3, and D4 dopamine receptors in Xenopus laevis oocytes.

The different murine D2-type dopamine receptors (D2L, D2S, D3L, D3S, and D4) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [125I]sulpride. They were all shown to activate Cl- influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na+/K+/2Cl- cotransporter component of the Cl- influx from the total unidirectional Cl- influx. The D3L subtype was found to operate exclusively through the bumetanide-insensitive Cl- influx whereas the other D2-type receptors acted on the Na+/K+/2Cl- cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na+/K+/2Cl- cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.

The tilapia prolactin I gene: evolutionary conservation of the regulatory elements directing pituitary-specific expression.

To study the elements involved in the pituitary specific transcriptional regulation of the tilapia prolactin I gene (tiPRL I), we have cloned and entirely sequenced a 3.4-kb genomic fragment immediately upstream from the first exon. In footprinting experiments, three tilapia sequences are protected from DNase I digestion by rat pituitary extracts (base pair coordinates -643 to -593, -160 to -111, and -73 to -46). Computer analysis of the nucleotide sequence reveals significant homology to mammalian binding sites for Pit-1, a transcription factor that is known to mediate pituitary-specific expression of the PRL genes in mammals. The tiPRL I 5'-flanking sequences can direct transient expression of a linked luciferase reporter gene in transfected rat pituitary cell lines and tilapia pituitary primary cell cultures. Transient expression experiments with 5'-deletion mutants reveal three regulatory regions. Two have a stimulatory effect on transcription and one an inhibitory effect. Electrophoretic mobility-shift assays (EMSA) demonstrate that the rat Pit-1 factor specifically binds to tilapia DNA sequences. Several such tilapia Pit-1 binding sites mediate activation of a linked heterologous promoter in transfected rat and tilapia pituitary cells. As evidenced by EMSA, a Pit-1-like protein is present in tilapia pituitary extracts. All these data point to a high conservation of the molecular mechanisms involved in pituitary-specific expression of the PRL genes in vertebrates.

Possible sites of dopaminergic inhibition of gonadotropin release from the pituitary of a teleost fish, tilapia.

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 microM) totally abolished this stimulation. Dopamine (DA; 1 microM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 microM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D2 type mode of action. The inhibition of GtH release in culture, caused by 1 microM APO, the specific DA D2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 microM) in perifusion was not reduced by DA (1 microM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca2+ ionophore A23187, however, their inhibitory effect was reversed by 10 microM ionomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic and thyroidal regulation of growth hormone in tilapia.

A radioimmunoassay (RIA) for recombinant tilapia growth hormone (GH) was established and validated. The ability of various hypothalamic factors to regulate GH secretion in the tilapia hybrid (Oreochromis niloticus x Oreochromis aureus) was studied. Somatostatin1-14 (SRIF1-14; 10-100 micrograms/kg) was found to reduce circulating GH levels in a dose-dependent manner. SRIF1-14 (0.1-1000 nM) inhibited GH release from perifused pituitary fragments (ED50 0.83 nM). Human growth hormone-releasing hormone fragment 1-29 (hGHRH1-29; 100 micrograms/kg) doubled circulating GH levels and modestly stimulated GH secretion in vitro. Carp growth hormone-releasing hormone (cGHRH) stimulated GH secretion in vitro to a similar degree at the same dose (1 microM). Injection of salmon gonadotropin-releasing hormone (sGnRH) superactive analog (10-100 micrograms/kg) increased plasma GH levels sixfold. sGnRH also stimulated GH release in vitro (ED50 142.56 nM). Dopamine (0.1-10 microM) and the D1 DA receptor agonist SKF 38393 increased GH secretion from perifused pituitary fragments dose-relatedly. Thyrotropin-releasing hormone (TRH) had no effect on GH secretion from perifused pituitary fragments, but increased plasma GH levels, as did bovine thyroid stimulating hormone (bTSH). The increased plasma GH in the bTSH-treated fish coincided with a dramatic increase in T4; however, TRH increased GH without changing T4 levels. T3 increased the synthesis of GH by isolated pituitaries (incorporation of [3H]leucine). SRIF1-14 seems to be a most potent hypothalamic regulator of GH secretion in tilapia; sGnRH and DA both increased GH secretion, although sGnRH elicited considerably greater responses at lower doses. Two forms of GHRH increased GH levels, although the unavailability of the homologous peptide prevented an accurate evaluation of its importance in regulating GH secretion. The thyroid axis (TRH, TSH, and T3) stimulates both synthesis and release of GH, although TRH did not appear to have a direct effect on the level of the pituitary.

The effects of gonadal development and sex steroids on growth hormone secretion in the male tilapia hybrid (Oreochromis niloticus × O. aureus).

Profiles of plasma growth hormone (GH) in male tilapia hybrid (Oreochromis niloticus x O. aureus) were measured and compared at different times of the year. The profiles did not appear to be repetitive, however, differences in their nature were observed at the different seasons; the most erratic profiles were seen in the height of the reproductive season (July), while the peaks were more subdued in the spring and disappeared in the autumn. Peaks in male fish were more prominent than in the females when measured in July. Perifused pituitary fragments from fish with a high GSI responded to salmon gonadotropin-releasing hormone (sGnRH) analog (10 nM-1 µM), while those from fish with a low GSI barely responded to even the highest dose. Exposure of perifused pituitary fragments from sexually-regressed fish to carp growth hormone-releasing hormone (cGHRH; 0.1 µM) or sGnRH (I µM) stimulated GH release only after injection of the fish with methyl testosterone (MT; 3 injections of 0.4 mg kg (1)). The same MT pretreatment did not alter the response to dopamine (DA; 1 or 10 µM). GH pituitary content in MT-treated fish was lower than in control fish, which may be explained by the higher circulating GH levels in these fish, but does not account for the increased response to the releasing hormones. Castration abolished the response of cultured pituitary cells to sGnRH (I fM-100 nM) without altering either their basal rate of secretion or circulating GH levels. Addition of steroids to the culture medium (MT or estradiol at 10 nM for 2 days) enabled a GH response to sGnRH stimulation in cells from sexually regressed fish. Pituitary cells which had not been exposed to steroids failed to respond to sGnRH, although their response to forskolin or TPA was similar to that of steroid-exposed cells. It would appear, therefore, that at least one of the effects of the sex steroids on the response to GnRH is exerted proximally to the formation of cAMP, or PKC, presumably at the level of the receptor. An increase in the number of receptors to the GH-releasing hormones, following steroid exposure, would explain also the changing nature of the GH secretory profile in different stages of the reproductive season.

Isolation of growth hormone and in vitro translation of mRNA isolated from pituitaries of the gilthead sea bream Sparus aurata.

Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of beta-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from 35S-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.

Intracellular mediation of GnRH action on GTH release in tilapia.

The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala(6), Pro(9)-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca(2+) ionophore (A23187; 1-100 µM) or ionomycin (0.02-10 µM). The response of perifused pituitary to GnRH was reduced by nimodipine (1-10 µM) indicating that Ca(2+) influx via voltage-sensitive Ca(2+) channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 µM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25-125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33-330 µM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 µM) in the presence of GnRH-antagonist ([Pro(2,6), Trp(3)]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca(2+) influx through voltage-sensitive Ca(2+) channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)