Gene structure of chum salmon somatolactin, a presumed pituitary hormone of the growth hormone/prolactin family.

From a chum salmon genomic DNA library, clones coding for somatolactin (SL), a presumed pituitary hormone belonging to the GH/PRL family, were isolated and analyzed. The salmon SL gene was 16 kilobases (kb) in length, this being the largest among mammalian GH (approximately 2 kb) and PRL (approximately 10 kb) and fish GH (approximately 4 kb) genes. As in the case of mammalian GH/PRL genes, the salmon SL gene was comprised of five exons in contrast to rainbow trout and Atlantic salmon GH genes, which each have six exons. Upstream from the transcriptional initiation site were found one TATA box identical to those of mammalian PRL genes and four consensus sequences corresponding to the Pit-1/GHF-1 binding element which is essential to the expression of mammalian GH and/or PRL genes. The similarity in amino acid sequences and organization of the SL gene with mammalian GH/PRL genes indicates that the SL gene is quite likely produced from an ancestral gene common to GH/PRL genes by gene duplication. Based on these considerations, an evolutionary model for the GH/PRL/SL gene family was made and shows the common ancestral gene to originate not from duplication of a primordial gene but from shuffling of independent exons and a regulatory sequence.

cDNA cloning of eel (Anguilla anguilla) somatolactin.

The nucleotide sequence of the cDNA coding for eel somatolactin (SL), a pituitary hormone belonging to the growth hormone (GH)/prolactin (PRL) family, has been determined. The full-length eel SL cDNA (1213 bp) encodes a protein of 229 amino acids (aa), with a putative signal peptide of 24 aa and a mature protein of 204 aa. Eel SL contains seven Cys residues found to be characteristic of SLs, and two potential N-glycosylation sites. Significant sequence homology between eel and seven other fish SLs (42% aa identity, 63% aa similarity) reveal SL to be highly conserved. A higher sequence identity of SL to GH than PRL is suggested by the comparison of these hormones in eel and chum salmon.

Fluorescent and radiolabelling of pepsin-digested human glomerular basement membrane with a newly developed hydroxy-coumarin derivative (CASE).

The labelling of pepsin-digested human glomerular basement membrane (pHGBM) with a newly developed fluorescent iodine acceptor 7-hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl ester (CASE) is described. The binding of a monoclonal antibody to pHGBM was assessed by radiobinding assays, and when directly iodinated pHGBM was used there was no apparent binding. When CASE was conjugated to pHGBM prior to iodination 11% binding was achieved. CASE acting as an iodine acceptor may be useful for proteins containing few or inaccessible tyrosine residues or which are destroyed by introduction of 125I. Since CASE is fluorescent, small amounts of material can be detected during isolation prior to iodination.

Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae).

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.

Pronounced seasonal rhythms in plasma somatolactin levels in rainbow trout.

The aim of this study was to establish whether there are seasonal or age-related changes in circulating levels of somatolactin (SL) in rainbow trout (Onchorhynchus mykiss). SL levels were determined in blood sampled at monthly intervals over a 2-year period from a population of rainbow trout maintained under a natural daylength and temperature regime (North-West England, latitude 54 degrees 20' N). SL levels displayed a distinct circannual cycle, with peak levels in summer (17-20 micrograms/l) and lowest levels occurring in winter (0.2-2 micrograms/l). This variation in SL levels was closely correlated with water temperature (P < 0.001) but was out of phase with changes in daylength. Plasma SL levels were significantly higher in both mature male and female fish compared with immature fish. Plasma prolactin levels were determined to provide information on a hormone structurally related to SL, and also because mammalian prolactin is known to show distinct seasonal cycles. However, trout prolactin, in contrast to SL, did not show a pronounced seasonal pattern, although prolactin levels were significantly, but inversely, correlated with water temperature (P < 0.001).

Somatolactin, a novel pituitary protein: purification and plasma levels during reproductive maturation of coho salmon.

Somatolactin (SL), a newly discovered fish pituitary protein belonging to the GH/prolactin family, was isolated from coho salmon (Oncorhynchus kisutch). Antibodies were raised to purified coho SL, and a homologous radioimmunoassay was developed and validated. The assay was specific for SL as indicated by the absence of cross-reactivity with coho salmon GH, gonadotrophins I and II and less than 0.2% cross-reaction to prolactin. Serial dilutions of plasma and pituitary extracts from Oncorhynchus species including coho salmon, chinook salmon and rainbow trout were parallel to the coho salmon SL standard curve. Displacement curves for dilutions of Atlantic salmon (Salmo salar) plasma, but not pituitary extract were parallel to the standards. Plasma levels of SL were measured in coho salmon throughout the final year of reproductive maturation. During the period of gonadal growth, plasma SL levels increased and were highly correlated to oestradiol levels in females and 11-ketotestosterone levels in males. Peak levels of SL were observed at the time of final maturation and spawning in both sexes. It is hypothesized that SL may regulate some physiological aspect of reproduction.

Plasma somatolactin concentrations in salmonid fish are elevated by stress.

The preliminary finding that plasma levels of somatolactin (SL) were markedly elevated following stress caused by confinement in chinook salmon (Oncorhynchus tshawytscha) prompted a more detailed study of SL dynamics during stress. SL levels have been determined in the plasma of rainbow trout (Oncorhynchus mykiss) during exposure to acute (0-30 min) and short (0-24 h) periods of stress resulting from handling and confinement. The results show that SL levels increase rapidly within minutes following the onset of stress, reach a peak between 1 and 2 h, decline over the next 3 h, and then show an additional increase again by 24 h. During acute stress caused by confinement, the increase in plasma SL levels occurred within 2 min, thus showing a more rapid response than cortisol. This suggests that the response is mediated directly by the hypothalamus and is not a result of a feedback mechanism. The results also demonstrate that SL secretion in response to stress is at least partially under genetic control. In the short-term stress experiment, progeny of fish selected as high responders or low responders to stress, based on the magnitude of the plasma cortisol levels induced by stress, were used, and these fish showed similarly accentuated or attenuated release of SL following stress. These results clearly demonstrate that non-specific environmental stress causes rapid activation of SL-secreting cells in the pars intermedia, suggesting that this hormone has an important role in the adaptive response of fish to stress.

Detection of a novel sequence change in the major form of alpha-MSH isolated from the intermediate pituitary of the reptile, Anolis carolinensis.

Intermediate pituitaries of the reptile, Anolis carolinensis, were separately pulse labeled with [3H]Trp and [3H]Tyr. The major form of alpha-MSH was purified by immunoprecipitation and isolated by reverse phase HPLC. Tryptic peptide analysis indicated that the [3H]Trp-labeled C-terminal fragment of Anolis alpha-MSH had the same retention time as mammalian ACTH(9-13) amide; however, the [3H]Tyr-labeled N-terminal fragment did not coelute with either mammalian ACTH(1-8) or N-acetyl-ACTH(1-8). Purification of alpha-MSH from 76 Anolis intermediate pituitaries confirmed that a sequence change had occurred in the N-terminal region of Anolis alpha-MSH. The tissues were acid extracted and purified by Sephadex G-25 chromatography and reverse phase HPLC to yield 4.5 micrograms of purified Anolis alpha-MSH for amino acid composition analysis and automated Edman degradation sequence analysis. The major form of Anolis alpha-MSH is nonacetylated and has the following novel primary sequence: Ser-Tyr-Ala-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro(Val-amide). The presence of Val-amide was verified by immunological analysis, tryptic peptide analysis and amino acid composition analysis.

Fish p53 as a possible biomarker for genotoxins in the aquatic environment.

The p53 gene is a tumour suppressor gene which has a fundamental role in cell cycle control and division, and in mammals certain genotoxic agents induce specific mutations in p53, leading to tumourigenesis. Fish have been investigated as models for studying carcinogens, but as yet very little data exists that links exposure to specific chemicals with the aetiology of tumours found in wild populations. In this study, p53 was sequenced from five species of fish with a view to the possible use of mutations in the highly conserved domains of p53 to identify genotoxins in the aquatic environment. A 0.8 kb fragment of the cDNA encompassing the conserved DNA-binding domain of p53 was sequenced in three Oncorhynchus salmonid fish: coho (O. kisutch), chum (O. keta), and chinook (O. tshawytscha) and full-length p53 cDNAs were sequenced in the puffer fish (Tetraodon miurus) and the barbel (Barbus barbus). The full-length puffer fish and barbel p53 cDNAs were 1834 bp and 1790 bp in length, encoding a 367 aa protein and a 369 aa protein, respectively. The deduced aa sequences of the p53 cDNA in the Oncorhynchus salmon shared a 100% identity in the five conserved regions (I-V). Comparisons of the deduced aa sequences for puffer fish and barbel p53 with other fish p53s revealed a high homology within the conserved DNA binding domain (68-86% for puffer fish and between 66-88% for barbel). "Conserved" domain I was not highly conserved in fish, as it is in mammals, and, therefore, conserved domains II-V are most likely to provide the valuable sequences in fish p53 for use in mutational studies to fingerprint genotoxins in the aquatic environment.

Molecular characterisation of a putative Atlantic salmon (Salmo salar) odorant receptor.

The olfactory system of fish is extremely important as it is able to recognise and distinguish a vast array of odorous molecules that are involved in behaviours paramount to survival. This is achieved by the activation of a diverse multigene family of G-protein coupled receptors through odorous ligand binding. Using molecular techniques, the nucleotide sequence of the cDNA coding for an Atlantic salmon (Salmo salar) odorant receptor (ASOR1) has been determined. The full-length cDNA (1260 nt) encodes a protein of 320 amino acid residues, including one potential N-linked glycosylation site, within the short extracellular amino terminal of the receptor. Hydrophobicity analysis revealed seven hydrophobic regions within the amino acid sequence, corresponding to possible positions of the transmembrane domains characteristic of the G-protein coupled receptor superfamily. Several conserved motifs unique to odorant receptors were also present. Through characterisation of this receptor, we hope to increase the understanding of the mechanisms underlying olfaction in salmonid species.

Molecular characterization of the first non-mammalian p73 cDNA.

The p53 gene is believed to be mutated or deficient in over 50% of human tumours, and is therefore considered to be instrumental in the process of carcinogenesis. Recently in humans, homologues of p53 (such as p73 and p63) have been isolated. In our studies in fish, we have been isolating tumour suppressor genes with a view to their potential use to study genotoxins in the aquatic environment. In this paper, we report the characterisation of the first non-mammalian p73 cDNA, isolated from barbel (Barbus barbus), a freshwater cyprinid fish indigenous to UK rivers. The deduced barbel p73 amino acid sequence has a high homology with human p73 alpha: the proteins are 641 and 636 aa in length, respectively, and there is a 72% identity over the entire sequence length of the protein (over 90% in the putative DNA binding domain). The level of conservancy for p73 is considerably higher across class (from man to fish), than for p53 and it may therefore have particular value in studies on environmental mutagenesis. Northern analysis showed expression of three p73 mRNA transcripts/homologues. The patterns of p73 tissue expression in the barbel differed from the expression of p53 mRNA, suggesting specific functional roles for the two genes.

Overwinter fasting and re-feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilisation.

This study investigated the roles of cortisol and growth hormone (GH) during a period of fasting in overwintering salmonid fish. Indices of carbohydrate (plasma glucose, liver glycogen), lipid (plasma free fatty acids (FFAs)) and protein metabolism (plasma protein, total plasma amino acids) were determined, together with plasma GH, cortisol and somatolactin (SL) levels at intervals in three groups of rainbow trout (continuously fed; fasted for 9 weeks then fed; fasted for 17 weeks). In fasted fish, a decline in body weight and condition factor was accompanied by reduced plasma glucose and hepatic glycogen and increased plasma FFA. No consistent elevation of plasma GH occurred until after 8 weeks of fasting when plasma GH levels increased ninefold. No changes were observed in plasma total protein and AA until between weeks 13 and 17 when both were reduced significantly. When previously fasted fish resumed feeding, plasma glucose and FFA, and hepatic glycogen levels rapidly returned to control values and weight gain resumed. No significant changes in plasma cortisol levels, related to feeding regime, were evident at any point during the study and there was no evidence that SL played an active role in the response to fasting. The results suggest that overwinter fasting may not represent a significant nutritional stressor to rainbow trout and that energy mobilisation during fasting may be achieved without the involvement of GH, cortisol or SL.

Exposure to exogenous 17beta-oestradiol disrupts p450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas).

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (p450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17beta-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of p450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on p450aromB mRNA expression in the gonads; E2 up-regulated p450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, p450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on p450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.

Changes in placental progesterone receptors in term and preterm labour.

Currently, preterm labour is associated with increased fetal mortality and morbidity and is often associated with elevated levels of inflammatory cytokines. However, the exact mechanisms that lead to this pathology are not fully elucidated. In the present study evidence was obtained using a specific membrane progesterone receptor (mPR) agonist, Org OD 02-0, that the progestin antagonism of apoptotic effects of a cytokine, IL-1ß, on human placental BeWo cells is mediated through mPRs. Therefore the aim of this study was to determine whether the gene expression of mPRs and all other known progesterone receptors changes in human placentas at term and during labour. Quantitative PCR (qPCR) in clinical samples revealed a 2.8 fold decrease of mPRß in labouring comparing to non-labouring tissues and 4.6 fold higher levels of mPRγ in preterm mPRγ compared to term placentas. The ratio of mPRα to PR-B was increased in term compared to preterm samples, whereas it was decreased in labour compared to non-labour placentas. There was also a high correlation between mPRα and PGRMC1 expression irrespective of pathologies. Collectively, our data indicates that changes in the ratios of progesterone receptors rather than individual fluctuations might affect progesterone signalling at the placental level.

Immunocytochemical study of the somatolactin cells in the pituitary of Pacific salmon, Oncorhynchus nerka, and O keta at some stages of the reproductive cycle.

Cells of the intermediate lobe of salmonids, homologous to the PAS-positive cells of other teleost species, cannot be differentiated by normal staining techniques, but can be immunostained with an antiserum against cod somatolactin (SL). Immunocytochemical techniques were applied to pituitary sections of two Pacific salmon, Oncorhynchus nerka and O. keta. Samples of immature or early maturing fish were collected in the Pacific Ocean and from mature spawning fish from hatcheries near Seattle and Willard (Washington). SL cells were rather small and moderately immunoreactive in immature fish. They were slightly enlarged in the early stages of gonadal development and more often contacted the basal lamina through processes with terminal swellings, suggesting granule release into perivascular spaces. In spawning fish, cells were enlarged and frequently more granulated, showing a wide contact with the basal lamina and a proximodistal transport of granules. In addition, large and more or less degranulated cells were noted, also indicating an active release of SL granules. Spawning females tended to have more SL cells than equivalent males. The gradual stimulation of SL synthesis and release during sexual maturation suggests that SL may be involved in the control of some steps of reproduction as previously shown by the increase in SL plasma levels in maturing coho salmon.

Immunoreactive somatolactin cells in the pituitary of young, migrating, spawning and spent chinook salmon, Oncorhynchus tshawytscha.

Immunocytochemical techniques using an antiserum to cod somatolactin (SL) demonstrated the presence of SL cells in the intermediate lobe of the pituitary in Oncorhynchus tshawytscha. The cells were small in yearling fish. Two groups of maturing fish were studied. In the spring run salmon collected in April and May during the upstream migration, the SL cells appeared stimulated. In September, during spawning, SL cell stimulation was maximal with indices of hypertrophy and degranulation often more marked in females than in males. In the other group, salmon of the fall run collected in the Pacific Ocean in August had well developed gonads, large gonadotropes and abundant SL cells. In spawning salmon (September) the SL cells were stimulated, mainly in females. However, the final stimulation was less intense than in spring run spawning fish. The SL cells were smaller, without evident granule release, but still abundant in spent salmon of the fall run caught at the end of November. Various factors (time spent in rivers before spawning, starvation, decalcification, stress, hypothalamic influences) were considered which might explain differences between spring and fall run salmon. These observations suggest that SL may play a role in the control of gonadal maturation in chinook salmon as it may also do in sockeye and chum salmon previously studied, and that SL cells may be sensitive to the ambient salinity.

Plasma somatolactin levels in coho salmon (Oncorhynchus kisutch) during smoltification and sexual maturation.

Somatolactin (SL) is a novel pituitary hormone recently characterized in several fish species. Structural analyses have shown that SL belongs to the growth hormone/prolactin family, and that it is a highly conserved protein. SL is synthesized by the periodic acid/Schiff-positive cells in the pars intermedia, but has an as yet unidentified function(s). We have recently developed a homologous radioimmunoassay for coho salmon SL and measured plasma levels of SL during two stages of the coho salmon life cycle, smoltification and sexual maturation. During smoltification, plasma levels of SL changed almost in parallel with plasma levels of thyroxine; levels increased as morphological indices of smoltification appeared and decreased as smoltification was completed. Following this period, SL levels remained low until the spring prior to spawning. In a separate study, plasma levels of SL were measured in sexually maturing coho salmon that remained in fresh water throughout their life cycle. During the year of sexual maturation, plasma levels of SL gradually increased from the spring onward, reaching peak levels at the time of spawning in November and December. These data are similar to those previously reported for sexually maturing coho salmon that were maintained in seawater prior to spawning (Rand-Weaver et al. 1992). Therefore, increases in plasma SL levels occurred in sexually maturing fish irrespective of whether they were maintained in fresh water or seawater. Peak levels at spawning were higher than those observed during smoltification. Possible roles for SL in metabolism and reproduction are discussed.

A rapid procedure for the isolation of bioactive growth hormone.

A two-step isolation method is described for the purification of growth hormone and prolactin from rat pituitaries. Following alkaline extraction, the sample was chromatographed on Sephadex G-75. Growth hormone and prolactin, which eluted in the same gel filtration fraction, were separated on a reversed-phase HPLC column. The isolated proteins migrated to the same position as rat growth hormone and prolactin standards. Further confirmation that the purified proteins were indeed growth hormone and prolactin was provided by immunoblotting using several mammalian antisera and N-terminal amino acid sequencing. The purified rat growth hormone was shown to be biologically active when tested in the hypophysectomized rat body weight gain assay. Although rat prolactin remains to be tested, it seems likely that this hormone would also have biological activity. The present procedure is of general applicability and particularly useful when only small amounts of pituitary glands are available for extraction.