Trout coelomic fluid suitability as Goldfish oocyte extender can be determined by a simple turbidity test.

Regeneration technologies such as androgenesis, intracytoplasmic sperm injection, and nuclear transfer require that handling conditions do not alter oocyte ability to sustain embryo development. One important parameter in the maintenance of oocyte quality in fish is the possibility to prevent oocytes activation during manipulation. In Cyprinid, such activation is known to be delayed when Salmonid coelomic fluid is used as incubation medium. Coelomic fluid however is a biological fluid whose ability to sustain oocyte quality during in vitro incubation may be variable. The purpose of the present work was to explore this variability using Rainbow Trout (Oncorhynchus mykiss) coelomic fluid (TCF) and Goldfish (Carassius auratus) oocytes, and to set up a test which would reflect TCF suitability for Goldfish oocyte incubation. We showed that different TCF induced very different development rates after oocyte incubation for 30 min at 20 °C: at 24h post fertilization (pf) and at hatching, rates ranged between 35% and 110% of the non-incubated controls. When TCF (1 volume) was mixed with tap water (9 volumes), a precipitate developed whose extent was measured by spectrophotometry. This turbidity test proved to be highly correlated to development rates after Goldfish oocyte incubation in TCF (r(2) = 0.83 at hatching, n = 150): TCF with the highest turbidity (> 1.5 absorbance unit at 400 nm) were the ones which altered the most the development rates after incubation (less than 50 % at hatching). This easy and rapid turbidity test can therefore be used as a reliable estimator of TCF suitability for Goldfish oocyte incubation and manipulation.

Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

Cryobanking of fish somatic cells: optimizations of fin explant culture and fin cell cryopreservation.

When gametes or embryos are not available, somatic cells should be considered for fish genome cryobanking of valuable or endangered fish. The objective of this work was to develop a method for fin explant culture with an assessed reliability, and to assess fin cells ability to cryopreservation. Anal fins from goldfish (Carassius auratus) were minced and gently loosened with collagenase before explants were plated at 20 degrees C in L-15 medium supplemented with fetal bovine serum and pH buffering additives. Quantification of cell-donor explants per fin rated the culture success. Cells were successfully obtained from every cultured anal fin (mean = 65% cell-donor explant per fin). All other fin types were suitable except the dorsal fin. Explant plating could be deferred 3 days from fin collecting. Fins from seven other fish species were successfully cultured with the method. After 2-3 weeks, sub-confluent fin cells from goldfish were cryopreserved. Cryopreservation with dimethyl sulfoxide and sucrose at a slow freezing rate allowed the recovery of half the goldfish fin cells. Cells displayed the same viability as fresh ones. 1,2-propanediol was unsuitable when a fast freezing rate was used. The procedure could now be considered for cryobanking with only minimal adaptation to each new species.

In vitro action of leptin on FSH and LH production in rainbow trout (Onchorynchus mykiss) at different stages of the sexual cycle.

The short-term effect of recombinant human leptin (rhleptin) on FSH and LH production (release+intracellular content) was studied in vitro using pituitary cells from male and female rainbow trout during the first gametogenesis cycle. In our rearing conditions, we found a direct action of rhleptin at the pituitary level, which depends on the sexual stage of the fish. No effect of rhleptin on FSH or LH release and cellular content could be detected in immature fish and post-ovulatory females. However, throughout the process of spermatogenesis and ovogenesis, high concentrations (0.5 and 1 x 10(-6)M) of rhleptin stimulated FSH and LH release, without observable action on intracellular content of gonadotropins. A relatively constant response to rhleptin for FSH was observed throughout gonad maturation, while LH response tended to be higher at the first stages of gametogenesis (beginning of spermatogenesis and endogenous vitellogenesis). Preliminary results on the potential interaction of rhleptin and salmon GnRH (sGnRH) suggest a possible synergistic effect of high concentration of rhleptin (10(-6)M) and sGnRH only at restricted phases of gonadal development when the gametogenetic process was already fully started (full spermatogenesis and early vitellogenesis). The direct action of leptin on FSH and LH release, evident only when gametogenesis had already started suggests that leptin is not the unique signal for the activation of the gonadotropic axis but requires a combined action with other promoting factors.

Cloning, characterization, and comparative activity of turbot IGF-I and IGF-II.

IGF peptides belong to a complex system that is known to play a major role in the control of growth and development in mammals. Even if studies performed in nonmammalian species tend to demonstrate an important function of these molecules, use of heterologous ligands, especially in fish, partly limit our knowledge of the physiological role(s) of IGFs. We report in this study the cloning, production, and characterization in an evolved fish, the turbot Psetta maxima, of mature IGF-I and IGF-II. The deduced 68-amino-acid IGF-I and 70-amino-acid IGF-II show 75% and 74% sequence identity with their mammalian counterparts, respectively, confirming the high sequence homology observed in other species. The development of a simple and efficient system for the production and purification of both IGF-I and IGF-II in Escherichia coli was used to investigate the in vitro regulation of GH release in the turbot. Our results demonstrated for the first time in a Euteleost species that both peptides specifically inhibited GH release. Both hormones were equally potent in inhibiting GH release from dispersed pituitary cells, with maximal inhibitory effects of 92% and 91% at 1 nM doses after 12 days of culture, respectively. The biologically active recombinant turbot IGFs that we obtained will allow us to further investigate potential and perhaps the specific role(s) of these hormones in turbot as, in contrast with mammals, growth in fish is potentially continued during "adult" life.

Effect of egg deprivation on sex steroids, gonadotropin, prolactin, and growth hormone profiles during the reproductive cycle of the mouthbrooding cichlid fish Oreochromis niloticus.

Various hormones were analyzed during the course of a reproductive cycle in the cichlid fish Oreochromis niloticus: plasma levels of the gonadal steroids 17beta-estradiol (E2), testosterone (T), 17, 20beta-OH progesterone (17,20beta-P), gonadotropin (taGtH), and plasma and pituitary concentrations of prolactin (tiPRL(I) and tiPRL(II)) and growth hormone (tiGH). Two categories of fish were sampled and sacrificed on days 1 and 3 postspawning and at 3-day intervals thereafter: typical incubating females (INC), and nonincubating females (NI), deprived of their eggs just after spawning. Such deprivation is known to suppress maternal behavior and to accelerate ovarian development and especially vitellogenesis, thus shortening the mean interspawning interval. In both groups, variations of the plasma concentrations of E2 and T appeared to depend on ovarian stages, and differences between groups appeared to reflect underlying differences in the kinetics of ovarian development. The observation of noticeable levels of 17,20beta-P in plasma before spawning, when high values of taGtH could also be detected in NI females, suggests the implication of this progestin in the control of final maturation events, as in some other teleosts. Moreover, 17,20beta-P, which was still detected a few days after spawning, but at low concentrations and only in the plasma of INC females, might play a role at the beginning of the reproductive cycle in incubating females (maternal behavior and/or slowing down of ovarian growth). The pituitary and plasma profiles of both tiPRLs isoforms appeared to depend mainly on the kinetics of ovarian development in each group of fish, suggesting a role during the beginning of vitellogenesis. However, the variance of plasma tiPRL(II), which was significantly enhanced during maternal behavior in INC females, also suggests an implication of this hormone in the control of that behavior. Concerning tiGH, comparison of the plasma profiles in INC and NI fish also suggest an influence on the control of maternal behavior, but a main effect of starvation of INC during mouthbrooding cannot be excluded.

Differential effect of insulin-like growth factor I on in vitro gonadotropin (I and II) and growth hormone secretions in rainbow trout (Oncorhynchus mykiss) at different stages of the reproductive cycle.

The short-term effect of insulin-like growth factor I (IGF-I) on GTH I (FSH-like), GTH II (LH-like), and GH production by cultured rainbow trout pituitary cells was studied in immature fish of both sexes, at early gametogenesis and in spermiating and periovulatory animals. IGF-I had no effect on basal GTH I and GTH II release, whereas it always inhibited basal GH, showing decreasing intensity with the gonad maturation. In absence of IGF-I, GTH I and GTH II cells were always responsive to GnRH, whereas no response was observed for GH cells whatever the sexual stage. The action of IGF-I on the sensitivity to GnRH differs between GTH and GH cells. The former requires a coincubation with IGF-I (10(-6) M)/GnRH to show an increase in sensitivity, independent of the sexual stage. To be responsive to GnRH, the GH cells require longer exposure to IGF-I, the efficiency of which decreases with gonad maturation. The action of IGF-I (10(-6) M) on GTH cell sensitivity to GnRH does not seem to be related to a mitogenic effect or to an improvement in cell survival. It seems to be IGF-I specific, not passing via the insulin receptor. Certain hypotheses on the putative role of IGF-I and GnRH as a link between growth and puberty are suggested.

Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss).

In order to evaluate potential interactions between somatotropic and gonadotropic axes in rainbow trout (Oncorhynchus mykiss), changes in pituitary content of the specific messenger RNA of growth hormone (GH) and gonadotropin (GTH) alpha- and beta-subunits were studied during gametogenesis with respect to pituitary and plasma hormone concentrations. Quantitative analyses of mRNA and hormones were performed by dot blot hybridization and homologous RIA on individual fish according to stage of spermatogenesis and oogenesis. All transcripts were detectable in 9-month-old immature fish. GH, GTH IIbeta, and GTH alpha increased moderately throughout most of gametogenesis and then more dramatically at spermiation and during the periovulatory period. GTH Ibeta mRNA increased first from stage I to V in males and more abruptly at spermiation, while in females GTH Ibeta transcripts increased first during early vitellogenesis and again around ovulation. Pituitary GH absolute content (microgram/pituitary, not normalized with body weight) increased slowly during gametogenesis and more abruptly in males during spermiation. In the pituitary of previtellogenic females and immature males, GTH I beta peptide contents were 80- to 500-fold higher than GTH II beta peptide contents. GTH I contents rose regularly during the initial phases of vitellogenesis and spermatogenesis and then more abruptly in the final stages of gonadal maturation, while GTH II contents show a dramatic elevation during final oocyte growth and maturation, in postovulated females, and during spermiogenesis and spermiation in males. Blood plasma GTH II concentrations were undetectable in most gonadal stages, but were elevated during spermiogenesis and spermiation and during oocyte maturation and postovulation. In contrast, plasma GTH I was already high ( approximately 2 ng/ml) in fish with immature gonads, significantly increased at the beginning of spermatogonial proliferation, and then increased again between stages III and VI to reach maximal levels ( approximately 9 ng/ml) toward the end of sperm cell differentiation, but decreased at spermiation. In females, plasma GTH I rose strongly for the first time up to early exogenous vitellogenesis, decreased during most exogenous vitellogenesis, and increased again around ovulation. Our data revealed that patterns of relative abundance of GTH Ibeta mRNA and pituitary and plasma GTH I were similar, but not the GTH II patterns, suggesting differential regulation between these two hormones at the transcriptional and posttranscriptional levels. Pituitary and plasma GH changes could not be related to sexual maturation, and only a weak relationship was observed between GH and gonadotropin patterns, demonstrating that no simple connection exists between somatotropic and gonadotropic axes at the pituitary level during gametogenesis.

Individual diurnal plasma profiles of thyroid hormones in rainbow trout (Oncorhynchus mykiss) in relation to cortisol, growth hormone, and growth rate.

In order to characterize the individual diurnal plasma profiles of triiodothyronine (T3) and thyroxine (T4) in rainbow trout (Oncorhynchus mykiss), blood samples from 41 fish were taken every hour during a 24-hr period, through a catheter inserted into the dorsal aorta. The possible influences of day-night alternation, sex, and diet (feed intake, time of meals) on thyroid hormone (TH) profiles were analyzed. The existence of relations between diurnal plasma profiles of T3, T4, T3/T4 ratio, and those of the growth hormone (GH), cortisol (previously described in Gomez et al., J. Exp. Zool. 274, 171-180, 1996), and the growth rate was monitored. Average daily T3 and T4 concentrations were, respectively, 2.6 +/- 0.2 and 5.5 +/- 0.3 ng/ml (n = 41). Our study showed little or no variation in plasma T3 concentrations during one 24-hr period, while those of T4 fluctuated markedly. T4 peaks occurred from a baseline of 4.0 +/- 0.2 ng/ml at a frequency of 2.5 +/- 0.2 peaks/24 hr, with an amplitude of 3.0 +/- 0.4 ng/ml, and a duration of 4.3 +/- 0.4 hr. There was a significant difference between the average circulating T3 level during the day and that at night (2.4 +/- 0.2 vs 2.7 +/- 0.2 ng/ml). No influence of sex or food factors was observed on daily TH concentrations. TH peaks occurred irregularly and asynchronously without apparent influence of day-night alternation, sex, and diet. The growth rate was significantly correlated with the daily T3 concentration (r = 0.77), but not with T4. No significant relationships were found between daily concentrations of T3, T4, GH, and cortisol. The absence of a relationship between TH and GH concentrations suggests that, in salmonids, GH may have no observable short-term action on the conversion of T4 to T3.

Production of recombinant insulin-like growth factor-II in the development of a radioimmunoassay in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) insulin-like growth factor-II (IGF-II) cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into an expression vector (parHS3) for production by Escherichia coli. Recombinant rainbow trout IGF-II (rtIGF-II) was recovered from bacterial inclusion bodies, solubilized, and purified by gel filtration chromatography under reducing conditions. After refolding of the peptide by dialysis at basic pH, we obtained a final yield of 820 micrograms/liter culture medium. The rtIGF-II appeared as a single band estimated at 80% and with molecular mass of 7.5 kDa. A homologous radioimmunoassay (RIA) for the measurement of IGF-II in trout plasma has been developed using rtIGF-II as antigen, radiolabeled tracer, and standard. RIA sensitivity was 0.1 ng/ml and the ED50 was 0.75 +/- 0.06 ng/ml. Intra- and interassay coefficients of variation were 4.04% (n = 6) and 4.56% (n = 9), respectively, at ED50 levels. Cross-reactivities of 1 and 0.1% were found for recombinant coho salmon (Oncorhynchus kisutch) IGF-I and coho salmon insulin, respectively. No cross-reactivities were found for recombinant human IGF-I and IGF-II, porcine insulin, or a variety of salmonid pituitary hormones. The interference of IGF binding proteins (IGFBP) in the RIA has been tested with and without extraction (acid/ethanol extraction or C-25 acid chromatography) of fed and fasted trout plasma samples. Parallel displacement curves were obtained for unextracted and C-25-extracted plasma but not for acid/ethanol-extracted plasma. Comparison of IGF-II levels from these samples showed a possible interference of IGFBP in the RIA in fed (19.6 +/- 1.56 ng/ml unextracted and 25.25 +/- 1.21 ng/ml extracted) and fasted (21.58 +/- 1.02 ng/ml unextracted and 9.86 +/- 1.68 ng/ml extracted) trout plasma. Finally, the displacement curves for unextracted plasma from rainbow trout, Couch's sea bream (Pagrus pagrus), and carp (Cyprinus carpio) were parallel to that of the rtIGF-II standard and not strictly parallel for tilapia (Oreochromis niloticus) and catfish (Silurus glanis) plasma. No significant cross-reaction occurred with eel (Anguilla anguilla) plasma. The rainbow trout IGF-II RIA reported in this study has low variability and is highly sensitive. Therefore it is a reliable method for measuring IGF-II levels in salmonids and some nonsalmonid fish species.

Consequences of food restriction on short-term growth variation and on plasma circulating hormones in Oreochromis niloticus in relation to sex.

In tilapia, there is a sex-related growth difference between males and females. This study tried to detect any correlation between the somatic growth and the plasma endocrine status. For this, individually marked (Floytags) male and female tilapia (BW 82 +/- 10 g) were either starved or fed on different daily food rations (1, 2, or 3% of the biomass) during 15 days. We have found that specific growth rates (SGR) were positively and significantly related to feeding levels. Growth hormone (GH) plasma levels tended to increase with the decrease in food levels, and thus with the decrease in growth rate. No significant correlation was found between GH levels and SGR. Triiodothyronine (T3) levels in well-fed fish were higher than those in restricted fish (0 and 1%), but no differences in thyroxine (T4) levels were observed. No significant relationship was found between plasma levels of steroid hormones and feeding ration, even though 11-ketotestosterone (11-KT) levels tended to increase with the ration in fed males. SGR were not significantly different between males and females at the same feeding level, but taken as a whole, they were significantly different in favor of males (P < 0.05). There was no important difference in GH levels between the two sexes. Steroid hormones were, in general, higher in males for 11-KT and in females for 17 beta-estradiol (17 beta-E2). Males and females exhibited significant differences in T3 levels (respectively 4.25 +/- 0.18 and 2.71 +/- 0.09 pmol/ml), whatever the food ration, but no significant differences in T4 levels were observed except in the high-ration group. The correlation between T3 levels and SGR was low but stronger in males (r2 = 0.21; n = 90) than in females (r2 = 0.10; n = 105). The slope of the log-log regression of T3 levels with body weight was much lower in females (b = 0.87) than in males (b = 1.31). This relationship suggests the involvement of T3 in tilapia growth and probably in the differential growth between males and females. In both males and females, a significant but low correlation was observed between T3 and 11-KT levels (respectively r2 = 0.12; n = 82 and r2 = 0.08; n = 89), while no correlation was found between the levels of T3 and 17 beta-E2. T3 plasma levels were found to be the most different parameter between males and females. This hormone seemed to be involved in the control of somatic growth, and could explain the differential growth rate between males and females.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II >>> insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish.

Characterization of growth hormone nycthemeral plasma profiles in catheterized rainbow trout (Oncorhynchus mykiss).

This study was conducted in order to characterize the nycthemeral plasma profiles of growth hormone (GH) in 41 prepubertal (2+) rainbow trout (Oncorhynchus mykiss). The possible influences of day-night alternation and of the food factor (quantity ingested, timing of the feed) on these plasma profiles were also analysed. Blood samples were taken every hour during a 24-hour period through a catheter inserted in the dorsal aorta. An assay of cortisol levels showed that the fish were in satisfactory physiological condition. The plasma concentrations of GH fluctuated significantly during the course of a nycthemere. The basal level was very low (0.32 +/- 0.01 ng/ml), and was interrupted by peaks that occurred at a rate of 2.1 +/- 0.1 peaks/24 hr, an amplitude of 2.0 +/- 0.3 ng/ml, and a duration of 3.5 +/- 0.2 hr. As a result, mean GH levels over a 24-hr period were low (0.7 +/- 0.1 ng/ml). Peaks occurred irregularly and asynchronously in individual fish and displayed no rhythmicity. Our study shows that there is a sexual difference in GH profiles in rainbow trout. No significant difference was observed between daily and nightly levels of GH (0.6 +/- 0.07 vs. 0.8 +/- 0.1 ng/ml). No influence of the diet on the plasma profiles of GH was observed. Average levels of GH over a 24 hr period are not significantly correlated with the growth rate of the fish. This study shows that circulating levels of GH in rainbow trout are markedly lower than in other vertebrates.

Enzyme-linked immunosorbent assay (ELISA) for rainbow trout (Oncorhynchus mykiss) vitellogenin.

1. A specific and simple enzyme-linked immunoassay for rainbow trout (Onchorynchus mykiss) vitellogenin (Vtg) is described. This assay is performed using a rabbit antiserum for Vtg purified from trout plasma. 2. This assay is based upon the competition between soluble Vtg and Vtg adsorbed on microtiter plates, for the rabbit anti-Vtg antibody binding sites. 3. The adsorbed Vtg-antibody complexes are revealed through the peroxidase-antiperoxidase antibody, which is colored by o-phenylendiamin. This assay can be performed in a day and a night. 4. Under our conditions, 90-20% of binding gave a sensibility range of 33-1473 ng/ml. With almost a 50% binding yield (335 ng/ml) the intra-assay coefficient of variation (CV) was 5.2% (n = 26) and the inter-assay CV was 12.5% (n = 5). 5. There was low immunological cross-reactivity with sera from other salmonids and with ovary extracts. Extracts of liver from oestrogenized male rainbow trout yielded displacements parallel to the vitellogenin standard and to mature female serum or oestrogenized male serum. 6. This enzyme immunoassay is simple and easy to use. Its great specificity allows its use only for the rainbow trout species.

Role of IGF-I in the control of GH secretion in rainbow trout (Oncorhynchus mykiss).

To study the role of IGF-I on GH secretion, we looked firstly for IGF-I binding sites in the central nervous system. IGF-I presents a single class of binding sites on brain membranes and pituitary extracts. Their affinity constants (Ka) were 11.44 +/- 4.66 and 4.42 +/- 1.37 x 10(9) M-1, respectively and their capacity (Bmax) were 119.83 +/- 46.21 and 73.65 +/- 20.87 fmoles/mg, respectively. In a second step IGF-I and bGH action on GH release was tested in vitro and in vivo. IGF-I inhibited GH release by pituitary cell cultures while bGH did not, suggesting direct action of IGF at the pituitary level and indirect action of GH, possibly mediated by IGF-I. IGF-I injected into catheterization fish induced a rapid inhibition of GH release, while bGH induced a delayed one. This timing supports a direct effect of IGF-I on GH release and the indirect effect of bGH. So peripheral IGF-I can play a role on GH secretion, perhaps as a mediator of GH action. This could explain the delayed fluctuation of GH and IGF-I plasma levels previously observed. This strong relationship between plasma IGF and GH secretion in trout seems to be different from that in mammals in which systemic IGF does not seem to play a predominant role in regulating GH secretion.

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.

Isolation and culture of somatotrophs from the pituitary of the rainbow trout: immunological and physiological characterization.

The aim of the present paper was to obtain somatotroph- and gonadotroph-enriched populations from collagenase dispersed pituitaries of male rainbow trout. Inasmuch as the percentage of immunoreactive gonadotrophs and somatotrophs present in pituitaries was higher at spermiation than at the beginning of spermatogenesis, we tried such a cell separation with fish at this stage of spermatogenesis. Cells were fractionated using their differences in buoyant density with centrifugation in Percoll solutions. The use of Percoll linear gradients (1.110 to 1.027 g/ml) showed that somatotroph cells have a density of between 1.102 and 1.064 g/ml whereas gonadotrophs are spread over the range of the gradient. It was thus possible, by using linear or discontinuous Percoll gradients, to obtain 95 to 67% (mean 80%) enriched somatotropic cell fractions while no enriched gonadotropic cell fractions were collected. The fractionated cells kept their ability to be cultured and to be responsive to specific secretagogues. Somatostatine induced a 80 to 85% decrease in growth hormone release per somatotroph in the initial cell suspension as well as in the different cell fractions. On the other hand, the basal growth hormone release per cell was lower in the fractions containing cells with a density lower than 1.062 g/ml. Inversely, the gonadotrophs have a basal release per cell independent of their density, and this is also available for their responsiveness to salmon gonadotropin-releasing hormone.

Seasonal changes in circulating growth hormone (GH), hepatic GH-binding and plasma insulin-like growth factor-I immunoreactivity in a marine fish, gilthead sea bream,Sparus aurata.

We have studied the seasonal relationship between growth and circulating growth hormone (GH), hepatic GH-binding and plasma insulin-like growth factor-I immunoreactivity in gilthead sea bream,Sparus aurata. The seasonal increase in plasma GH levels preceded by several weeks the summer increase in growth rates. In contrast, a marked increase in hepatic GH-binding with a high degree of endogenous GH occupancy was found during the period of maximum growth which suggests an enhanced sensitivity of liver to GH action. Thus, circulating levels of immunoreactive IGF-I, probably derived from the liver in response to GH action, were positively correlated with growth throughout the experimental period although a consistent relationship between growth and circulating GH was not found. In spite of this, we consider that, in gilthead sea bream, as in several other teleosts, the availability of endogenous GH can limit growth. Thus, under environmental conditions of suboptimal growth, a single intraperitoneal injection of recombinant rainbow trout GH (rtGH) induced over the dose range tested (0.75, 1.5, 3 µg g BW(-1)) an increase in plasma IGF-I-like immunoreactivity comparable to that seen during the period of maximum growth.

Growth hormone (GH) and reproduction: a review.

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.