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PubMed2006

A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

Amino Acid Sequence↗
PubMed1996

Effect of testosterone on maturational gonadotropin subunit messenger ribonucleic acid levels in the goldfish pituitary.

In this study, we investigated the effects of testosterone and a nonaromatizable androgen, 11 beta-hydroxyandrosterone, on maturational gonadotropin (GtH-II) subunit gene expression in the goldfish pituitary. While testosterone treatment at physiological doses resulted in stimulation of GtH-II-alpha and -beta subunit mRNA production, time-course and dose-response studies performed on sexually immature goldfish of mixed sex, using a wider dose range exceeding physiological levels, demonstrated a biphasic response to in vivo androgen treatment. Time-related treatment with testosterone and 11 beta-hydroxyandrosterone (20 micrograms/fish) resulted in an initial inhibition of GtH-II subunit mRNA production (12-24 h) followed by stimulation at 72-96 h. In dose-response studies, treatment for 24 h with testosterone resulted in a significant stimulation at the low physiological doses of 0.2 and 2 micrograms/fish. At the supraphysiological level of 20 micrograms/fish, testosterone treatment resulted in no stimulation or in decreased GtH-II subunit mRNA levels compared to the control values. Similarly, treatment with 11 beta-hydroxyandrosterone resulted in a significant stimulation of GtH-II subunit mRNA levels at low physiological concentrations (0.2 microgram/ fish) and an inhibition, or no stimulation, at higher concentrations (2-20 micrograms/fish). In sexually mature goldfish of mixed sex, the biphasic effect of testosterone was not observed in vivo, and treatment with this steroid resulted in stimulation of GtH-II subunit mRNA production in a dose-related manner. To investigate the direct action of testosterone, studies were carried out using isolated goldfish pituitary fragments from goldfish of mixed sex in vitro. Treatment with testosterone at various concentrations was found to stimulate GtH-II subunit mRNA production in pituitary glands obtained from both sexually immature and sexually mature goldfish. Overall, the present study demonstrates a stimulatory effect of testosterone on GtH-II subunit mRNA levels in goldfish. The observed stimulation of basal GtH-II subunit mRNA production by testosterone occurs, in part, through a direct action at the level of the pituitary in both sexually immature and mature goldfish.

Androsterone↗
PubMed1989

Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish.

The present study examined the influence of GnRH on the in vivo and in vitro secretion of GH in the goldfish (Carassius auratus). Intraperitoneal injection of several GnRH peptides, including a form native to goldfish, salmon GnRH (sGnRH), elevated circulating GH levels in female goldfish. An analog of mammalian GnRH (mGnRH), [D-Ala6,Pro9-NEt] mGnRH (mGnRH-A), at a dosage of 0.1 microgram/g BW increased serum GH levels for up to 48 h after a single ip injection. Goldfish receiving a series of injections of this dose of mGnRH-A also displayed an increased rate of body growth, indicating that the mGnRH-A-induced increase in the circulating GH level was sufficient to accelerate body growth. In vitro experiments using perifused pituitary fragments found that sGnRH stimulated the secretion of GH from the goldfish pituitary in a potent, dose-dependent, and reversible manner. The time course of response and half-maximally effective dose of sGnRH were very similar for both GH and gonadotropin (GTH) secretion in vitro, suggesting that the mechanism(s) mediating the stimulatory actions of GnRH in the goldfish may be similar for both GH and GTH secretion. However, GnRH-induced GH and GTH secretion from the goldfish pituitary can occur independently of each other, as demonstrated by the finding that somatostatin inhibited the GnRH stimulation of GH secretion in vitro, without influencing the GTH response, whereas the dopamine agonist apomorphine inhibited GnRH-induced GTH secretion in vitro, without influencing the GH response. Furthermore, the dopamine antagonist pimozide did not influence serum GH levels, although pimozide potentiated the stimulatory effect of GnRH on GTH secretion in vivo by blocking the endogenous GTH release inhibitory action of dopamine. Results of the present study suggest that the secretion of GH and GTH in the goldfish are regulated, at least in part, through a common releasing factor, GnRH, whereas somatostatin and dopamine appear to act independently as GH and GTH release inhibitory factors, respectively.

Analysis of Variance↗
PubMed1992

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Inhibin and activin are important reproductive regulators in mammalian species and have been demonstrated to be highly conserved in structure. The present study examines the effects of porcine follicular fluid (pFF; a crude inhibin and activin preparation) and purified porcine inhibin-A and activin-A on goldfish gonadotropin-II (GTH-II) release. In studies using primary cultures of dispersed goldfish pituitary cells in static incubation, treatments with pFF, inhibin-A, and activin-A for 10 h caused dose-dependent increase in GTH-II release. In perifusion studies using goldfish pituitary fragments, basal GTH-II release was significantly elevated after 12-h exposure to 500 micrograms/ml pFF. Furthermore, GnRH-induced GTH-II secretion was potentiated by pretreatment with pFF. When pFF was applied in the form of 5-min pulses, a rapid dose-related stimulation of GTH-II was observed. Similarly, challenges with 2-min pulses of 15, 150, and 1500 pM inhibin-A and activin-A stimulated GTH-II release by goldfish pituitary fragments in a rapid and dose-dependent manner. This acute stimulatory action of inhibin on goldfish GTH-II release was completely abolished after pretreatment with specific inhibin antibodies. The acute actions of inhibin and activin on GTH-II release are probably not due to the release of endogenous GnRH from nerve terminals in the pituitary fragments or binding to the GnRH receptors. First, a specific GnRH antagonist did not block the actions of inhibin and activin. Second, dopamine, a potent inhibitor of GnRH-stimulated GTH-II secretion in goldfish, was only partially effective in decreasing inhibin- and activin-induced GTH-II release. Third, the stimulatory effects of inhibin and GnRH on GTH-II release were additive. These lines of evidence also indicate that the mechanisms mediating inhibin and activin stimulation of goldfish GTH-II release may be somewhat different from those of GnRH. These results demonstrate that in contrast with the usual inhibitory effects of inhibin on GTH release in mammals, both inhibin and activin exert long term and acute stimulatory actions on GTH-II release in the goldfish.

Activins↗
PubMed1999

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.

Amino Acid Sequence↗
PubMed1999

Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins.

The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (Mr) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and low-capacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (Ka) of 20.1x10(9) M-1 and a maximum binding capacity (Bmax) of 161 fmol ml-1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with Mr ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with Mr of 66 kDa and a minor band with Mr of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with Mr of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.

Animals↗
PubMed1994

Complex expression of keratins in goldfish optic nerve.

Keratins are the predominant intermediate filament proteins in the nonneuronal cells of the goldfish optic nerve. At least three different keratin pairs are expressed in this tissue, indicating an unexpected complexity. Expression of the type II keratin ON3 in goldfish optic nerve astrocytes predicts the expression of a type I keratin partner. Here we report the cDNA sequence and predicted amino acid sequence of two type I keratins from the goldfish optic nerve, designated GK48 and GK49. The GK48 protein is the goldfish equivalent of mammalian keratin 18 (K18) and is the most likely type I keratin partner to the ON3 protein. The GK49 protein is similar to the GK50 protein, a type I keratin characterized previously from the goldfish optic nerve. The GK48 and ON3 mRNAs are expressed in a variety of goldfish tissues, whereas the expression of GK49 mRNA has a more limited expression. In addition, in situ hybridization experiments show that the expression of the GK48 and ON3 mRNAs are evenly distributed throughout the optic nerve, while the GK49 mRNA is expressed along longitudinal lines. These results show that there is a diversity of keratin expression within different cell types in the goldfish optic nerve.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of the goldfish thyrotropin beta subunit gene including the 5'-flanking region.

The complete gene encoding the beta subunit of thyrotropin (thyroid-stimulating hormone, TSH) was isolated from a goldfish genomic library. The goldfish TSHbeta subunit gene, which is approximately 2.0 kilobase pairs (kb) in length, consisted of three exons and two introns. The first intron was much longer (0.89 kb) than the second intron (0.3 kb) as are TSHbeta genes in mammalian species. On the basis of the location of the first intron, the goldfish TSHbeta gene belongs to the mammalian TSHbeta/FSHbeta gene group, which is distinct from the LHbeta group. Inspection of the 5'-flanking and exon 1 regions of the goldfish TSHbeta gene (1.2 kb) revealed the presence of several putative cis-acting elements, including the negative triiodothyronine (T(3))-responsive element (nTRE), Pit-1 element, and GATA-2 element. Comparison of the goldfish sequence with mammalian TSHbeta promoter sequences showed an identical region, nTRE, in the first exon-intron junction region. An in vitro study using dispersed goldfish pituitary cells showed that T(3) treatment (20 ng/ml) suppressed the TSHbeta mRNA level in the cells. These data indicate that (1) the basic structure of TSHbeta genes is highly conserved in vertebrates and that (2) T(3) acts directly on the pituitary and inhibits TSHbeta gene expression in goldfish, probably via the nTRE in the TSHbeta gene.

Amino Acid Sequence↗
PubMed1977

Trysinase positive oculocutaneous albinism in the goldfish, Carassius auratus l., and ultrastructural and biochemical study of the eye.

Ultrastructural studies, and cytochemical and biochemical determinations of tyrosinase activity were conducted on the pigment epithelium of albino and xanthic goldfish eyes. In eyes of xanthic goldfish, two types of melanosomes are present, spherical and elongated. Melanized melanosomes are absent in the eyes of the albino goldfish, but elongated lamellar premelanosomes are observed. Internal vesicles are present in both melanosome types in the pigment epithelium of the xanthic goldfish but are absent in premelanosomes of the albino. There are also differences in the distribution of lipid droplets, smooth endoplasmic reticulum and Golgi complexes with the latter two being more abundant in the albino. Tyrosinase was not identified cytochemically; however, the enzyme was demonstrated biochemically in the pigment epithelia of both albino and xanthic goldfish. The enzyme is associated with the particulate and soluble fractions fo both types of eyes. Particulate albino tyrosinase may be solubilized by triton X-100 treatment. Tyrosinase inhibitors are present in the particulate fractions of both albino and xanthic goldfish eyes. Thus, in the goldfish, ocular albinism appears to be a multiple defect at the molecular and ultrastructural levels.

Albinism↗
PubMed1987

Activity of position-8-substituted analogs of mammalian gonadotropin-releasing hormone (mGnRH) and chicken and lamprey gonadotropin-releasing hormones in goldfish.

Several vertebrate gonadotropin-releasing hormones (GnRH) and analogs were tested for activity in vivo in goldfish. Each peptide was administered intraperitoneally to goldfish, pretreated with pimozide or vehicle for pimozide, and changes in serum levels of gonadotropin were determined. Pimozide potentiates the activity of GnRH in vivo in goldfish by blocking the endogenous gonadotropin release-inhibitory activity of dopamine; relative potencies of GnRH peptides become evident in vivo in goldfish pretreated with pimozide (R. Peter et al. (1985), Gen. Comp. Endocrinol. 58, 231-242). Mammalian GnRH (mGnRH) was used as reference standard. [Try3, Leu5, Glu6, Trp7, Lys8]-GnRH (lamprey GnRH), [Gln8]-GnRH (chicken GnRH-I), and [His5, Trp7, Try8]-GnRH (chicken GnRH-II) caused increases in serum gonadotropin level similar in magnitude to mGnRH. [His5, D-Arg6, Trp7, Tyr8]-GnRH is superactive in the goldfish. [Asn8]-, [Met8]-, [Phe8]-, and [Ser8]-GnRH had activity similar to mGnRH in goldfish; [His8]-, [Ile8]-, and [Leu8]-GnRH had a lower level of activity; [Glu8]-GnRH had no apparent activity. The results indicate that there is no particular requirement for a hydrophobic or hydrophilic amino acid, or for a positively charged amino acid in position 8 of mGnRH for activity in vivo in the goldfish; a negatively charged amino acid in position 8 is detrimental for activity.

Animals↗
PubMed1989

Relationship between serum growth hormone levels and the brain and pituitary content of immunoreactive somatostatin in the goldfish, Carassius auratus L.

In this study, the relationships between endogenous brain and pituitary immunoreactive somatostatin (irSRIF) and circulating growth hormone (GH) levels in the goldfish were examined using two approaches. First, the amount of irSRIF in extracts of the pituitary gland and various brain regions was measured by radioimmunoassay several times throughout the year and was compared to serum GH levels at each time. The amounts of irSRIF in extracts of the pituitary gland, hypothalamus, and telencephalon were found to be inversely related to seasonal changes in serum GH levels, such that irSRIF was highest in these regions when serum GH levels were lowest (November and February). Conversely, irSRIF in these regions was lower in May, June, and July when serum GH levels were highest. These results suggest that endogenous irSRIF in the pituitary and forebrain may participate in the regulation of seasonal changes in serum GH levels in the goldfish. In extracts from other brain regions (thalamus + midbrain and cerebellum + medulla), some changes in the amount of irSRIF were observed among the various sample times, but these variations were not related to changes in serum GH levels. In a second set of experiments, the origin of irSRIF fibers innervating the goldfish pituitary gland was examined by using brain lesioning techniques to destroy regions of the forebrain known to contain irSRIF perikarya and fibers, and subsequently measuring the amount of irSRIF in the pituitary gland. Lesions in the preoptic area of the forebrain resulted in increased serum GH levels concomitant with a decrease in pituitary irSRIF content. This provides direct evidence that the preoptic area is the origin of a somatostatinergic projection inhibiting GH secretion from the goldfish pituitary. Lesions centered in the nucleus lateral tuberis (NLT) pars anterioris did not influence serum GH levels or the pituitary content of irSRIF. In contrast, more posterior lesions centered in the NLT pars posterioris (NLTp) resulted in a dramatic reduction in the amount of irSRIF in the pituitary. This suggests that the majority of irSRIF projections to the goldfish pituitary pass through the area destroyed by the lesion centered in the NLTp; it is also possible that perikarya within this area may be the origin of at least some of the irSRIF-containing fibers in the goldfish pituitary. Together, results from the present study provide evidence of a functional relationship between circulating levels of GH and endogenous brain and pituitary irSRIF in the goldfish.

Animals↗
PubMed1999

Predominant expression of group-II metabotropic glutamate receptors in the goldfish brain.

Group-II metabotropic glutamate (mGlu) receptors (mGlu2/3 receptors) were highly expressed in various regions (telencephalon, optic tectum, and cerebellum, but not vagal lobe) of the goldfish brain. In the goldfish telencephalon, expression of mGlu2/3 receptors was even higher than in the rat cerebral cortex. In contrast, mGlu5 receptors showed low levels of expression in all goldfish brain regions, whereas mGlu1a receptors were only expressed in the goldfish cerebellum. Pharmacological activation of group-II mGlu receptors with the selective agonists, 2R,4R-4-aminopyrrolidine-2, 4-dicarboxylic acid and (2S,2'R,3'R)-2-(2,3-dicarboxycyclopropyl) glycine, reduced the evoked release of glutamate from goldfish brain synaptosomes, whereas agonists of group-I and -III mGlu receptors (3, 5-dihydroxyphenylglycine and L-2-amino-4-phosphonobutanoate) were inactive. The predominance of group-II over group-I mGlu receptors in the goldfish brain may provide a natural defense against excitotoxic neuronal death and contribute to the unusually high resistance of goldfish against hypoxic brain damage.

Animals↗
PubMed2001

Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages.

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leukocytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The proteins were identified as truncated forms of transferrin, having approximate molecular weights (MW) of 33, 35, and 37kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did not enhance NO production by LPS-stimulated goldfish macrophages, but enzymatic cleavage of this precursor form correlated with enhanced production of NO by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in response to both mixed lymphocyte reactions (MLR) and the mitogenic stimulation of goldfish kidney leukocytes. Time course analysis revealed that 24h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin products appeared in the supernatants of cultured cells, which was related to the on-set of NO-inducing activity of these preparations. To confirm these findings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish transferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine transferrin. Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity, indicating that this response was not an artifact of bovine serum components (i.e. bovine transferrin). Anti-bovine and anti-carp transferrin polyclonal antibodies identified the presence of truncated forms of transferrin in the active fractions of FPLC-separated mitogen-stimulated leukocyte supernatants prepared in the presence of bovine or carp serum, respectively. Thus, our results suggest a novel role for fish transferrin as one of the factors that mediates teleost macrophage antimicrobial functions.

Animals↗
PubMed1999

Postprandial, seasonal and sexual variations in cholecystokinin gene expression in goldfish brain.

Recently, we described the complete nucleotide sequence of cholecystokinin (CCK) mRNA and the deduced amino acid sequence of the precursor on the basis of molecular cloning and sequence analysis of cDNA from goldfish brain. In this study, we investigated the hypothesis that CCK has a role in feeding behavior by examining CCK gene expression in the brain of goldfish using Northern blot. We showed that CCK gene is widely but differentially expressed in broad areas of the goldfish brain, including the olfactory bulbs, telencephalon and preoptic region, hypothalamus, optic tectum-thalamus and posterior brain regions, with highest levels in hypothalamus. We found that CCK mRNA levels in goldfish olfactory bulbs, telencephalon-preoptic region, optic tectum-thalamus, and posterior brain were influenced by sex at least sometime of the seasonal gonadal cycle, with female fish having higher levels than males during at least one of the four seasonal sampling times. We also observed a transient and acute increase in the CCK mRNA levels in the olfactory bulbs, telencephalon-preoptic region, hypothalamus, and posterior brain at 120 min after a meal. These widespread postprandial changes in CCK gene expression in goldfish brain indicate that CCK peptides have multiple roles in regulation of feeding behavior in goldfish. This supports the idea that CCK plays a role as a satiety factor in goldfish.

Animals↗
PubMed1999

Cloning and characterization of goldfish activin betaA subunit.

We have cloned a full-length cDNA coding for activin betaA subunit from a goldfish brain and pituitary cDNA library, which represents the first for activin betaA in fish. Sequence analysis of goldfish activin betaA shows that this peptide is highly conserved across vertebrates. The mature region of goldfish activin betaA shares 81% amino acid identity with that of humans. Messenger RNA of goldfish activin betaA is expressed in a variety of tissues including ovary, testis, brain and liver, suggesting a wide range of physiological roles for activin A in the goldfish. The identity of the cloned goldfish activin betaA was confirmed by expressing the protein in the Chinese hamster ovary (CHO) cells followed by detection of the specific activin activity in the culture medium using erythroid differentiation factor (EDF) assay with F5-5 cells. Stable CHO cell lines producing high level of recombinant goldfish activin A were established and characterized.

Activins↗
PubMed2001

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus.

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.

Animals↗
PubMedDate not supplied

Differential distribution of synaptotagmin immunoreactivity among synapses in the goldfish, salamander, and mouse retina.

Synaptotagmin I is the leading candidate for the calcium sensor that triggers exocytosis at conventional synapses. However, physiological characterization of the calcium sensor for phasic release at the ribbon-style synapses of the goldfish Mb1 bipolar cell demonstrates a lower than predicted affinity for calcium, suggesting that a modified or different sensor triggers exocytosis at this synapse. We examined synaptotagmin immunolabeling in goldfish retina using two different antibodies directed against synaptotagmin epitopes that specifically labeled the expected 65-kDa protein on western blots of goldfish and mouse retinal membranes. The first antiserum strongly labeled conventional synapses in the inner plexiform layer (IPL), but did not label the ribbon-style synapse-containing synaptic terminals of goldfish Mb1 bipolar cells or photoreceptors. The second antibody also specifically labeled the expected 65-kDa protein on western blots but did not label any synapses in the goldfish retina. A third synaptotagmin antibody that performed poorly on western blots selectively labeled goldfish photoreceptor terminals. These results suggest that synaptotagmin may exist in at least three distinct "forms" in goldfish retinal synapses. These forms, which are differentially localized to conventional synapses, bipolar cell, and photoreceptor terminals, may represent differences in isoform, posttranslational modifications, epitope availability, and protein-binding partners. Labeling with these antibodies in the salamander and mouse retina revealed species-specific differences, indicating that synaptotagmin epitopes can vary across species as well as among synapses.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of goldfish opsin sequences.

Five opsin cDNA clones were isolated from a goldfish retina cDNA library and sequenced. On the basis of homology to previously characterized visual pigments, one clone was identified as goldfish rod opsin and a second as a goldfish red cone opsin. Two rhodopsin-like clones were found to be similar to the chicken green opsin, a pigment which shares properties with both rod and cone pigments. A fifth clone was equally homologous to human blue cone opsin and human rod opsin. In order to characterize the spectral properties of the encoded pigments, the five clones were expressed in tissue culture cells and the apoproteins reconstituted with 11-cis-retinal. The wavelength of maximal absorption for goldfish rhodopsin is 492 nm and for the fifth pigment, identified as the goldfish blue pigment, 441 nm. Pigments encoded by the two rhodopsin-like clones absorb at 505 and 511 nm and are likely to correspond to the goldfish green pigment previously characterized by microspectrophotometry. The putative red cone opsin cDNA may encode a pigment that is a polymorphic variant of goldfish red since it absorbs maximally at 525 nm.

Amino Acid Sequence↗