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The intermediate filament protein composition in glial cells of goldfish optic nerve differs from that found in glial cells of the goldfish spinal cord and brain. Brain and spinal cord glial cells contain glial fibrillary acidic protein (GFAP), whereas glial cells in the optic nerve contain ON3. The ON3 protein of the goldfish optic nerve was recently identified as the goldfish equivalent to the mammalian type II keratin 8 protein. In addition to the ON3 protein, the goldfish optic nerve also contains a 48-kDa protein. Immunoblotting experiments suggest that this protein is equivalent to the mammalian type I keratin 18 protein, which typically pairs with keratin 8 to form filaments. We show that these proteins are not specific to the optic nerve. The ON3 and 48-kDa proteins of the goldfish optic nerve share common antigenic properties with the predominant keratin pair expressed in the goldfish liver. These proteins are also expressed at low levels in the goldfish brain and spinal cord. In addition RNase protection assays and Northern blots indicate that the mRNA for the ON3 protein in optic nerve is identical to the message found in other goldfish tissues. The expression of ON3 was also examined in cultured glial cells from goldfish spinal cord and optic nerve and cultured fibroblast cells. Analysis of intermediate filament protein expression in cultured glial cells taken from goldfish spinal cord demonstrated the absence of GFAP in these cells and the expression of ON3. This protein was also the predominant intermediate filament protein of cultured optic nerve glial cells and fibroblasts. The differences in the expression of intermediate filament proteins in mammals and lower vertebrates are discussed. In addition, we discuss how the expression of a simple epithelial keratin pair in glial cells of the goldfish optic nerve may be associated with this system's capacity for continuous growth and regeneration.
The fluidity of synaptosomal membrane preparations isolated from goldfish acclimated to 5, 15 and 25 degrees C and from rat has been estimated using the fluorescence polarisation technique with 1,6-diphenyl-1,3,5-hexatriene as probe. Membranes of cold-acclimated goldfish were more fluid than those of warm-acclimated goldfish when measured at an intermediate temperature, indicating a temperature-dependent regulation of this parameter. Similarly, membranes of warm-acclimated goldfish were more fluid than those prepared from rat brain. Liposomes prepared from the purified phospholipids of goldfish and rat synaptosomal preparations showed differences similar to those of the native membranes. Increased membrane fluidity of cold-acclimated goldfish was correlated with a decrease in the proportion of saturated fatty acids of the major phospholipid classes and an increased unsaturation index in choline phosphoglycerides. Rat membranes showed a substantial reduction in unsaturation index and an increase in the proportion of saturated fatty acids compared to the membranes of 25 degrees C-acclimated goldfish. The cholesterol content of synaptosomal membranes of goldfish was unaffected by acclimation treatment. The role of homeoviscous adaptation in the compensation of the rates of membrane processes during thermal acclimation, and upon the resistance adaptation of poikilotherms to extreme temperatures is discussed.
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.
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.
The localization of hyaluronate was studied in the CNS of rat, goldfish and lamprey. Cryostat sections were incubated with glial hyaluronate-binding protein of human origin and stained by indirect immunofluorescence with glial hyaluronate binding protein antibodies not reaching with rat and fish. As previously reported for glial hyaluronate-binding protein and glial fibrillary acidic protein, hyaluronate and glial fibrillary acidic protein had a similar distribution in rat spinal cord and optic nerve, both substances forming ring-like structures around individual myelinated axons. A similar periaxonal distribution was observed in goldfish spinal cord and medulla, except that the rings were much wider, to accommodate the large goldfish axons. The glial fibrillary acidic protein-positive neuroglial tissue forming distinctive structures in goldfish vagal lobes also stained for hyaluronate. In both rat and goldfish spinal cord, motoneurons were surrounded by a hyaluronate coat. Goldfish optic nerve and lamprey spinal cord were hyaluronate-negative and, as previously reported, they stained for keratin but not for glial fibrillary acidic protein. The findings suggest that hyaluronate in CNS fibre tracts in a product of glial fibrillary acidic protein-positive neuroglia. They also suggest that the appearance of glial fibrillary acidic protein-positive neuroglia and the formation of a hyaluronate-bound extracellular matrix are related phenomena in phylogeny.
We have produced and characterized a monoclonal antibody, MH1, which selectively labels rod horizontal cells and Müller cells in the goldfish retina. Biochemical and tissue distribution studies indicate that MH1 may recognize four out of five classes of intermediate filament proteins in goldfish: vimentin, desmin, glial fibrillary acidic protein (GFAP), and keratin, but not neurofilament. The intermediate filament which is labeled strongest in the retina is vimentin. In the goldfish retina, the only type of horizontal cells recognized by MH1 appear to be rod horizontal cells. This result suggests that the rod horizontal cell, an interneuron, and Müller (glial) cells share a common antigen: vimentin, which is usually only expressed in mesenchymal origin cells. The development of rod horizontal cells in the goldfish retina was also studied using MH1. The cells were not labeled by MH1 until 4-6 weeks posthatching, a stage in which the animals are already visually active. MH1 also did not label any horizontal cell in the region close to the ora terminalis in the goldfish retina. These results suggest that either the emergence and maturation of rod horizontal cells occur late during goldfish retinal development or the expression of vimentin itself occurs late in the development of rod horizontal cells.
Both crucian and carp egg-mRNAs were found to mediate the transformation of goldfish tail from the double (veil-shaped) to the single (fork-shaped). Livers of these single tailed goldfish were tested for genetic change using lactate dehydrogenase isozymes (LDH) as marker. Electrophoresis of liver extracts on starch gel showed that the liver specific LDH-C4 isozymes of both the egg-mRNA and liver-mRNA injected goldfish resembled the control goldfish except an additional intermediate band. The migration distance of this new band matched the third band of the hybrids (carp X goldfish). These findings lead to the conclusion that carp egg-mRNA contains a fraction partly responsible for liver development. Rat liver was found possessing the cathodally migrating C4 isozymes. Injection of rat liver-mRNA into goldfish eggs resulted in the intensification of the liver specific C4 isozymes. It appears therefore that the input informational molecules (liver-mRNA) and the output molecular products (liver C4 isozymes) are causally related. This causal relationship would pave the way for genetic manipulation in higher organisms.
The metabolic clearance rate (MCR) and initial half-disappearance time (T(1/2)i) of salmon gonadotropin-releasing hormone (sGnRH) and its agonist analog [D-Arg6,Pro9-NEt]-sGnRH (sGnRH-A) were investigated in goldfish, following a single intraarterial injection of radioiodinated sGnRH and sGnRH-A. The tissue uptake of radioiodinated sGnRH-A was also investigated. 125I-sGnRH had a MCR of 0.01173 ml min-1 35 g-1 and a (T(1/2)i) of 32.38 min; 125I-sGnRH-A had a MCR of 0.0192 ml min-1 35 g-1 and a (T(1/2)i) of 72.95 min. In the tissue uptake experiments, high levels of accumulated labeled sGnRH-A were generally found in the gills, kidney, liver, and pituitary compared to air bladder, brain, gonad, eyes, and muscle. Coinjection of excess amount of cold sGnRH-A caused decreased uptake of labeled sGnRH-A only in pituitary, but increased accumulation of labeled sGnRH-A in some other tissues. Our results show a correlation between the T(1/2)i and the affinity of the peptides for a serum GnRH binding protein present in the goldfish, suggesting that the formation of a hormone-binding protein complex may decrease the MCR of GnRH in the circulation in goldfish. Our data also indicate specific uptake of 125I-sGnRH-A by the pituitaries of both male and female goldfish, confirming the pituitary as a major target organ of GnRH in goldfish.
The relationship between drugs elevating intracellular cAMP levels and gonadotropin (GTH)-releasing hormone (GnRH) in the stimulation of GTH secretion in the goldfish was investigated using dispersed goldfish pituitary cells in primary culture. In static incubation experiments, activation of adenylyl cyclase by forskolin and the inhibition of cAMP phosphodiesterase by 3 isobutyl-1-methylxanthine (IBMX) increased cAMP release and stimulated GTH secretion. The addition of membrane permeant cAMP analogs, 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), and dibutyryl cAMP also increased GTH release, suggesting that elevation of cAMP levels can induce GTH secretion. In the goldfish, dopamine is a physiological inhibitor of GTH release. Application of the dopamine agonist apomorphine decreased the GTH responses to forskolin, 8Br-cAMP, and salmon GTH-releasing hormone (sGnRH). The ability of agents that elevate cAMP levels to mimic GnRH action on GTH release suggests that cAMP may mediate GnRH-stimulated GTH secretion in the goldfish; however, this possibility was not substantiated by results from further experiments. In 2-hr static incubation studies, the GTH responses to sGnRH and chicken GnRH-II (cGnRH-II) were enhanced by coincubations with forskolin, IBMX, and 8Br-cAMP. The magnitudes of these enhancements were at least additive, if not synergistic. The levels of cAMP released into the media were unaffected by treatment with sGnRH and cGnRH-II, either in the absence or in the presence of IBMX. Replacement of normal testing media with Ca(2+)-deficient media (without Ca2+ salts and in the presence of 0.1 mM EGTA) decreased sGnRH and cGnRH-II stimulation of GTH release but did not affect forskolin and 8Br-cAMP actions. These results indicate that sGnRH and cGnRH-II stimulation of short term (less than or equal to 2-h) GTH release in the goldfish is not mediated by cAMP. The kinetics of the interactions between sGnRH, forskolin, and IBMX were also investigated in cell column perifusion studies. Applications of 5-min pulses of forskolin and IBMX stimulated rapid increases in GTH release; the latencies of these responses were similar to that observed with sGnRH. The simultaneous applications of sGnRH with either forskolin or IBMX resulted in GTH responses that were of greater magnitude and longer duration than those in response to sGnRH alone. These results together indicate that elevation of cAMP levels can potentiate the GTH response to the native GnRHs by increasing the magnitude of the acute GTH release and by prolonging the duration of GnRH action; however, cAMP does not appear to be involved directly in mediating GnRH stimulation of GTH release.(ABSTRACT TRUNCATED AT 400 WORDS)
In goldfish, the gonadal steroid, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), functions as a potent preovulatory female sex pheromone which stimulates rapid elevations in serum gonadotropin (GtH) levels and subsequent increases in milt production in males. GtH secretion in goldfish is known to be regulated by the stimulatory actions of gonadotropin-releasing hormone (GnRH) and the inhibitory actions of dopamine (DA). This study specifically examined whether the 17,20 beta-P-induced elevation in male GtH is caused by pheromone-mediated changes in DA inhibition at the level of the pituitary. First, we have demonstrated that dihydroxyphenylacetic acid (DOPAC) is the primary metabolite of DA catabolism in the brain and pituitary gland of goldfish. Second, we measured changes in circulating levels of GtH and changes in pituitary content of DA and its metabolite, DOPAC, as well as possible alterations in DA turnover rate (DOPAC/DA ratio) following short-term exposure of male goldfish to water-borne 17,20 beta-P. Water-borne 17,20 beta-P consistently increased serum GtH levels in males within 20 min of exposure and maintained elevated levels for up to 120 min. Although changes in pituitary DA content were not observed during periods of high GtH release, coincident reductions in pituitary levels of DOPAC were measured within 45 min of exposure to the pheromone. More importantly, there was a significant decrease in the rate of DA turnover in the pituitary, as assessed by comparing the ratio of DOPAC to DA present, at 20, 45, and 120 min of exposure. Since the reduction of DA turnover in the pituitary is inversely correlated with periods of increased GtH release, the present results suggest that water-borne 17,20 beta-P causes an abatement of DA release to the pituitary. Based on the latency of the GtH response to water-borne 17,20 beta-P, a rapid reduction of DA turnover in the pituitary appears to be at least part of the neuroendocrine trigger for 17,20 beta-P-induced GtH release in male goldfish.
Rat scotophobin A increased dark avoidance in goldfish in dark and light avoidance shuttlebox experiments, controlled for general and light cycling-induced swimming activity. A possible site of action for scotophobin was suggested by the reports that dark avoidance was also increased in goldfish by pinealectomy, a treatment which increased shock sensitivity as well. It was found that scotophobin alone decreased the voltage required to induce tail-flip contractures in goldfish. The pineal gland was further implicated in the mode of action of scotophobin when it was found that this peptide suppressed the norepinephrine-induced aggregation of goldfish chromatophores whose state is in part controlled by pineal melatonin. Pinealectomized goldfish became insensitive to the effects of scotophobin upon both light-dark preference and chromatophore aggregation state. There observations strongly suggest that the pineal gland is required for the action of scotophobin.
Monoclonal antibodies against the inhibitory glycine receptor of rat spinal cord were used to identify corresponding receptor polypeptides in goldfish CNS. Both Western blot analysis and quantitative receptor immunoassays revealed crossreacting antigens in goldfish brain membranes. A polypeptide of 46 kDa molecular weight is immunologically related to the 48 kDa alpha subunit of the mammalian receptor. Similarly, a large receptor-associated protein of 93 kDa is present both in goldfish and mammals. Throughout the goldfish CNS, glycine-displaceable [3H]strychnine binding codistributes with the alpha subunit protein as determined immunologically. Glycine receptor contents were highest in goldfish medulla oblongata, medium in optic tectum and mesencephalon, whereas little or no receptor was detected in cerebellum, olfactory bulb, and spinal cord. Immunohistochemistry confirmed that the alpha subunit antigen and the 93 kDa protein were located in the plasma membrane of neurons and concentrated in small clusters found on the soma and dendrites. These data indicate that immunological properties and cellular distribution of glycine receptors are conserved from fish to mammals.
The development of substance P-like immunoreactivity (SPLI) in the goldfish brain was studied by means of the indirect peroxidase-antiperoxidase technique and an antibody to substance P. By 80 h after fertilization, the first SPLI-cell bodies appear in the ventricular zone of the future diencephalon and the first SPLI-fibers appear in the olfactory bulbs. Two days after hatching (which occurs at 100 h after fertilization), SPLI fibers connecting the olfactory bulbs and hypothalamus are seen. In the optic tectum SPLI-fibers appear for the first time 5 days after hatching. In the brain stem, SPLI-cell bodies appear in juvenile animals 40 days after hatching. The highest number and intensity of SPLI-cell bodies and fibers are found in the area postrema. SPLI-cell bodies are also seen in the gustatory nucleus, nucleus ambiguous, reticular formation of the medulla, dorsal motor nucleus of the vagus and commissural nucleus of Cajal. The significant information gained from the present study is: 1. The rostro-caudal sequence in which the SPLI appears in the developing nuclei of the goldfish brain 2. The reduction of SPLI-cell bodies in some nuclei with age Thus, in the brain stem, SPLI-cell bodies that were labeled in juvenile goldfish were not seen in adults. This might be due to changes in the rate of axonal transport, changes of the SP phenotype during development or cell death. The developmental sequence and relative timing in which SPLI-cell bodies appear in the goldfish, rat and mice are similar.
In a previous study, the labeling pattern of three proteins (alpha, beta and gamma) in goldfish brain was found to change after the animals successfully acquired a new pattern of behavior. In the present study, these proteins were isolated from the brain cytoplasmic fraction, purified by successive gel electrophoresis and used as antigent to immunize rabbits. Antisera containing antibodies to two of the proteins (beta and gamma) were obtained. These gave single precipitin bands when plated against the antigens and a mixture of the total cytoplasmic proteins. The distribution of beta and gamma in brain subcellular fractions and in a variety of goldfish tissues was determined by immunodiffusion methods. gamma was specific to brain. The beta protein cross-reacted but was not identical to a widely distributed substance in plasma, liver and kidney. Both beta and gamma appear to be species specific in that no cross-reactivity was obtained with mouse, chick or rat brain proteins. Immunological methods, in combination with double labeling experiments were used to establish that the beta and gamma antigens were proteins which were normally present in goldfish brain. Both the beta and gamma antisera were equally capable of specifically precipitating the proteins which were differentially labeled after training as well as purified proteins of the same molecular weight present in the brains of control animals. These results suggest that the acquisition of a new pattern of behavior can increase the demand for the synthesis of specific proteins (beta and gamma) normally present in goldfish brain.
The metabolism of 3 brain cytoplasmic proteins (alpha, beta, and gamma) increases markedly when goldfish acquire a new pattern of behavior. Antisera specific to beta and beta + gamma proteins were prepared and injected into the fourth ventricle of the brains of trained animals at 8 and 24 h after the initiation of training. When tested 3 days later, such goldfish (N = 98) could not recall the training; whereas trained goldfish (N = 97) receiving non-immunized rabbit serum had complete recall of the behavior. Also no amnesia was obtained in control experiments in which trained goldfish were injected with an antiserum to a neural surface membrane protein NS-6. The fact that antisera to beta + gamma had no toxic effects was demonstrated by injecting them prior to training; no effects on the rate of acquisition and recall of the behavior was found. The antisera to beta + gamma were effective in inhibiting recall of the training when they were injected any time between 3 h up to 48 h after training; no effect was obtained at 72 h post training. These results are consistent with the hypothesis that beta and gamma might have some functional role in the plasticity of the CNS.
Homologous desensitization of gonadotropin-releasing hormone (GnRH) was investigated using goldfish pituitary fragments in vitro. The two native GnRH peptides, sGnRH [( Trp7, Leu8]-GnRH) and cGnRH-II [( His5, Trp7, Tyr8]-GnRH) were administered either continuously or in pulsatile fashion at different frequencies and concentrations. Continuous treatment (60 min) with either sGnRH or cGnRH-II at 10(-7), 10(-8), and 10(-9) M resulted in desensitization of goldfish pituitary in a biphasic fashion, characterized by an initial rapid peak of GTH release (phase 1), followed by a lower sustained release of GTH remaining at a stable concentration above the basal level (phase 2). Pititary fragments were then washed for 60 min and further treated continuously (60 min) with the same concentrations of sGnRH or cGnRH-II (second treatment). Total sGnRH- or cGnRH-II-induced GTH release during the second treatment period was significantly lower than that observed during the initial treatment period, depending upon the concentration of the peptides. The second phase of GTH release was more pronounced at lower concentrations compared to that observed following 10(-7) M treatment, especially for sGnRH. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses of 10(-7), 10(-8), and 10(-9) M given every 20 min) resulted in significant desensitization of the pituitary GTH release. Reduction of pulse frequency to 2 min treatment every 60 min resulted in a lower degree of desensitization; little or no desensitization was observed following treatment with 10(-8) and 10(-9) M cGnRH-II or 10(-9) M sGnRH. A further reduction in frequency to 2-min pulses of sGnRH or cGnRH-II (10(-7) or 10(-8) M) given every 90 min did not result in desensitization of the pituitary GTH release. In summary, the present study demonstrates that GnRH-induced desensitization is dependent on both pulse frequency and concentration in the goldfish pituitary. These findings support the hypothesis that pulsatile secretion of the native GnRH peptides may be essential for maintenance of normal pituitary GTH release in goldfish.