Goldfish Pituitary Gland Research Articles

The inhibitory effects of (gamma)-aminobutyric acid (GABA) on growth hormone secretion in the goldfish are modulated by sex steroids.

Double-labelling studies at the electron microscopic level demonstrated that gamma-aminobutyric acid (GABA)-immunoreactive nerve endings are associated with growth-hormone-secreting cells in the proximal pars distalis of the goldfish pituitary gland, suggesting that GABA may be important for the control of growth hormone release in this species. An in vitro assay for GABA-transaminase activity demonstrated that the pituitary is a site for the metabolism of GABA to succinic acid. In vitro, GABA or the GABA antagonists bicuculline and saclofen did not affect the rate of growth hormone release from dispersed pituitary cells in static incubation. In contrast, intracerebroventricular injection of GABA reduced serum growth hormone levels within 30 min. During the seasonal gonadal cycle, intraperitoneal injection of GABA was without effect in sexually regressed goldfish, but caused a significant decrease in serum growth hormone levels in sexually recrudescent animals. Intraperitoneal implantation of solid silastic pellets containing oestradiol increased serum GH levels fivefold in sexually regressed and recrudescent goldfish; in both groups, GABA suppressed the oestradiol-stimulated increase in circulating growth hormone levels. The effect of oestradiol on basal serum growth hormone levels was specific since progesterone and testosterone were without effect. However, in recrudescent animals treated with progesterone and testosterone, the inhibitory effects of GABA on serum growth hormone levels were absent, indicating a differential role for these steroids in growth hormone release. Taken together, these results demonstrate that GABA has an inhibitory effect on growth hormone release in goldfish.

Molecular cloning of the cDNA encoding the β subunit of thyrotropin and regulation of its gene expression by thyroid hormones in the goldfish, Carassius auratus

A cDNA encoding the β subunit of thyrotropin (TSH) was isolated from a goldfish (Carassius auratus) pituitary gland cDNA library. By comparing the sequence with other teleost TSHβs, a signal peptide of 19 amino acids and a mature hormone of 131 amino acids were predicted for goldfish TSHβ subunits. The resulting putative mature hormone of 131 amino acids had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 51–67% with TSHβs from other teleosts, 38–43% with tetrapod TSHβs, but only 27 and 29% with goldfish GTH-Iβ and -IIβ, respectively. We also examined the effects of thyroid hormones (TH) and thiourea (TU, an inhibitor of TH production) treatments on TSH and GTH subunit gene expressions in the goldfish pituitary gland. After thyroxine (T4) treatment, circulating T4 concentration increased and TSHβ mRNA level decreased. Supressing the amount of circulating T4 and triiodothyronine (T3) by TU treatment increased the TSHβ mRNA level. Moreover, T4 replacement therapy (simultaneous treatment of both TU and T4) caused a high level of circulating T4 and a low level of circulating T3, and a decrease in the TSHβ mRNA level. Thus, changing levels of circulating TH exert a negative feedback on the level of TSH β subunit mRNA in goldfish in vivo. On the other hand, GTH subunit mRNA levels were not affected by changes in the levels of circulating TH.

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus I. Initial morphological, static, and cell column perifusion studies

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pitiitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 n M to 1 μ M [Trp 7, Leu 8]-GnRH (sGnRH) and [ d-Arg 6, Pro 9- N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED 50s of 1.92 ± 0.48 and 0.19 ± 0.08 n M, respectively. [His 5, Trp 7, Tyr 8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED 50s of 0.29 ± 0.13, 0.18 ± 0.11, and 0.19 ± 0.17 n M, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 n M sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.

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.

Gonadotropin production and release in female goldfish (Carassius auratus) after administration of pimozide and an LHRH analogue as studied by electron microscopy and radioimmunoassay

The effect of pimozide and an LHRH-analogue (LHRH-A) on gonadotropic cells of the goldfish pituitary gland were described qualitatively and quantitatively. A scale of four categories was devised to reflect various ultrastructural appearances of the cells. Experimental animals were divided into a control group, a group injected with LHRH-A alone, pimozide alone, and groups receiving these two substances in combination. Fish injected with the single substance were killed 12 h after injection while the groups receiving the combined treatments were killed at 4, 12 and 48 h. Serum levels of gonadotropin measured by radioimmunoassay were used to indicate whether an increase in hormone release had occurred. An immunocytochemical technique, the protein A-gold procedure, assured that the cells studied were gonadotropes. The control group showed variation in the profiles of gonadotropic cells. The single treatment groups showed some increase in secretory inclusions. At 4 h after injection the combined treatment caused a significant increase in hormone granules; at 12 and 48 h there was a gradual decrease in content of secretory products, and an increase in vacuolization. The results indicate that the combined pimozide and LHRH-A treatment stimulated gonadotropin production as well as release.

The dopaminergic innervation of the goldfish pituitary. An immunocytochemical study at the electron-microscope level using antibodies against dopamine.

The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocytochemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.

Light and electron microscopic identification of gonadotrophic cells in the pituitary gland of the goldfish by means of immunocytochemistry

Immunocytochemical techniques were used at the light and electron microscopical levels in order to localize and to characterize the gonadotrophs in the goldfish pituitary gland by means of antibodies to carp gonadotrophin (c-GTH) or its subunit (c-GTHβ). At the light microscopical level antibodies to c-GTH reacted weakly with cells located in the rostral pars distalis (RPD) and strongly with cells of the proximal pars distalis (PPD). The labeling was restricted to the proximal pars distalis when antibodies to c-GTHβ were employed. The PAP and colloidal-gold postembedding procedures demonstrated that two cell types of the PPD react with both immune sera. These cells correspond to the so-called globular and nonglobular basophils of the goldfish pituitary. The labeling was located over the small secretory granules and the large globules. A relationship was noted between the intensity of the labeling and the electron density of the globules.

Ultrastructural immunocytochemistry of gonadotrophs in the goldfish pituitary gland.

The immunocytochemical distribution of gonadotropin (GTH) in the goldfish pituitary gland was studied applying the peroxidase-antiperoxidase (PAP) method and the protein A-gold technique at light- and electron-microscopic levels, respectively, with an antiserum raised against silver carp GTH. In the light-microscopic immunocytochemistry, PAS-positive cells in the proximal pars distalis showed strong reaction with the antiserum. Gold particles wee concentrated both on globules (large) and on granules (small) of the gonadotrophs (PAS-positive cells) in the electron-microscopic immunocytochemistry. Other cells in the pituitary gland, including thyrotrophs, displayed no immunoreactivity with the antiserum at the dilutions tested. These results indicate, not only immunocytochemical distribution of GTH both in globules and in granules in the gonadotrophs, but also the high purity of the antigen (silver carp GTH) and specificity of the antiserum.

Ultrastructural identification of catecholaminergic fibers in the goldfish pituitary. A high-resolution radioautographic study after in vitro 3H-dopamine administration.

The monoaminergic innervation of the goldfish pituitary gland was studied by means of light- and electron-microscopic radioautography after in vitro administration of 3H-dopamine. The tracer was specifically incorporated and retained by part of the type-B fibers innervating the different lobes of the pituitary. In the rostral pars distalis labeled fibers were most frequently observed in contact with the basement membrane separating the neurohypophysis and the adenohypophysis. In the proximal pars distalis and the pars intermedia, labeled profiles were detected in the neural tissue and in direct contact with the different types of secretory cells. According to the previous data concerning the uptake and retention of tritiated catecholamines in the central nervous system, it is assumed that the labeled fibers are mainly catecholaminergic (principally dopaminergic). This study provides morphological evidence for a neuroendocrine function of catecholamines in the goldfish.

Ultrastructural immunocytochemistry of growth hormone cells in the goldfish pituitary gland

The immunocytological activity of an antiserum to carp growth hormone was studied on the goldfish pituitary gland, using a peroxidase-antiperoxidase ultrastructural technique. The reaction was specific for secretory granules in cells in the proximal pars distalis categorized as growth hormone cells on the basis of ultrastructural characteristics. The results support the specificity of the antiserum for growth hormone and provide firm identification of the growth hormone cells in goldfish.

The effects of photoperiod and temperature on the release of prolactin from the pituitary gland of the goldfish, Carassius auratus L.

A number of experiments were conducted to investigate the effects of photoperiod and temperature on prolactin release from the goldfish pituitary gland. Fish were acclimated to different photoperiods and temperatures, and also were subjected to a change in either of these two parameters after different acclimation conditions. Serum and pituitary samples were collected and analyzed by radioimmunoassay for prolactin levels. In other experiments samples for prolactin analysis were taken every 3 h intermittently over a period of 3 days from fish that were acclimated to different photoperiod and temperature conditions. Longer photoperiods and higher temperatures caused pituitary prolactin release. Serum prolactin changed on a circadian rhythm and the rhythm was modified depending on the length of the photoperiod.

FURTHER STUDIES ON THE THYROIDAL CONTROL OF RADIOPHOSPHORUS METABOLISM AS DETERMINED BY THE EFFECTS OF THYROXINE AND PITUITARY EXTRACT ON THIOUREA AND RADIOIODINE TREATED GOLDFISH, CARASSIUS AURATUS L.

Goldfish, Carassius auratus L., were treated with radioiodine and thiourea. In the former the thyroid follicles were completely destroyed, and in the latter they had become hypertrophied and hyperplastic and were devoid of colloid. The follicular cell height had been greatly increased. When they were kept in aquaria with radiophosphorus, the uptake after either type of treatment was similar and it was half of that of the controls.The uptake of P32 in the thiourea treated fish increased by 41.9% after acetone-dried goldfish pituitary gland injections, whereas in radioiodine treated fish there was no significant change.Radioiodine treated fish, when injected with thyroxine, increased their uptake of P32 by 50% but it was even then less than that of the controls. When thiourea treated fish were injected with thyroxine, their P32 uptake was restored to the level of that of the controls.The conclusion drawn from the present investigations is that in fishes, which lack parathyroid glands, the thyroid gland exercises a great influence over the metabolism of phosphorus and that the pituitary gland has also an indirect control through its thyrotrophic hormone.