• Development and Reproduction
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• v.26 no.1
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• pp.13-21
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• 2022

Neurokinin B (NKB) is a neuropeptide involved in the regulation of reproductive endocrine system of vertebrate animals, including fish. However, the pathway of NKB action in fish has not been clearly elucidated. In order to clarify the effect of NKB and NKF (neurokinin F) on gonadotropic hormone (GTH) gene expression in the pituitary, we studied the changes of LHβ and FSHβ gene expressions by using two different pituitary culture methods (whole pituitary culture or dispersed pituitary cell culture). Pituitaries were removed from mature female and male Nile tilapia. Changes of LHβ and FSHβ gene expressions were measured and compared after the treatment with NKB or NKF peptides at concentrations 0 to 1,000 nM. Expression of GTH genes in the whole pituitary cultures treated with NKB or NKF peptides did not show significant difference except in female at one concentration when treated with NKF. On the contrary, there were significant changes of GTH gene expressions in the dispersed pituitary cell cultures when treated with NKB and NKF peptides. These results suggest that dispersed pituitary cell culture is more relevant than whole pituitary culture in studying the function of pituitary, and that NKB and NKF could act directly on the pituitary to regulate the expression of GTH genes.

• Archives of Pharmacal Research
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• v.26 no.1
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• pp.34-39
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• 2003

The traditional Asian medicinal herb, roots of Astragalus (A.) membranaceus (Leguminosae), is used for many purposes, some of which are purported to stimulate the release of growth hormone in vivo. Extracts of A. membranaceus were tested to determine whether they stimulate the release of growth hormone in rat pituitary cell culture. A. membranaceus was extracted sequentially with 80% ethanol (fraction A), n-hexane (fraction B); the test compound from the herbal extraction was isolated using silica gel column chromatography and was identified with spectral data. Test compound was also extracted by traditional boiling water methods. Induction of growth hormone in pituitary cell culture was conducted with isolated compounds and extracted fractions of A. Radix (dried roots of A. membranaceus). The fraction A was not active in the rat pituitary cell culture, but the fraction B derived from the ethanol fraction stimulated the release of growth hormone in culture. Six compounds from fraction B (1-6) were isolated and identified previously. The compounds 1,2-benzendicarboxylic acid diisononylester (1), $\beta$-sitosterol (2), and 3-Ο-$\beta$-D-galactopyranosyl-$\beta$-sitosterol (5) did not induce growth hormone release in the culture. Formononetin (3), 9Z, 12Z-octadecadienoic acid (4), stigmast-4-en-6$\beta$-o1-3-one (6) and 98-E, a mixture of 1'-9, 12-octadecadienoic acid (Z,Z)-2',3'-dihydroxy-propylester (7) and 1'-hexadecanoic acid-2',3'-dihydroxy-propylester (8) stimulated the release of growth hormone in the rat pituitary cell culture significantly compared to the control. In conclusions, four compounds isolated from extracts of A. Radix induced growth hormone release in the rat pituitary cell culture. The 98-E isolate was the most active inducer of growth hormone release.

• Korean Journal of Pharmacognosy
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• v.9 no.1
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• pp.33-39
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• 1978

The present study is involved with the prolactin secretion from anterior pituitary gland by ginseng saponin since it was handled down by tradition that ginseng might influence the milk secretion when it was given to nursing mother. To investigate the effect of saponin on the prolactin production or release from the anterior pituitary gland, cell culture study and whole animal studies were carried out. For the cell culture study, enzymatically dispersed anterior pituitary cells of rat anterior pituitary gland in HEPES buffers containing trypsin were used. Ginseng saponin was added to the culture media and the amount of prolactin produced in the cell culture media was determined by radloimmunoassay(RIA) technique. Dose-dependent increases of prolactin with ginseng saponin were observed, whereas, no change was observed without ginseng treatment. For the whole animal study, normal and castrated rats which previously cannulated into the heart via the right juglar vein were used. The prolactin concentration in plasma were determined by using the technique of RIA. In normal rats, prolactin concentration in plasma were elevated dramatically after 1 hour of ginseng saponin administration, whereas, instantaneous increases were observed in castrated rats. For prolactin assay by RIA, NIAMDD Rat Prolactin Kit and NIAMDD Rat Prolactin RP-1 were used as standard. The results indicate that ginseng saponins increase the release of prolactin from the anterior pituitary gland and production of prolactin from the cell in rats.

• Korean Journal of Poultry Science
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• v.23 no.3
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• pp.113-119
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• 1996

A series of experiments were conducted to investigate the role of protein kinase C (PKC) as a second messenger in vasoactive intestinal peptide (VIP) mediated prolactin secretion. Primary pituitary cells (106 cells/treatment) were separated from laying hens and incubated in M-199 with 5% chicken serum and 5% fetal calf serum. The VIP(0.1 $\pi$M) treatment enhanced prolactin Secretion into media upto 9-fold during 48-h incubation. The phorbol 12-myristate 13-acetate (PMA), a PKG agonist, increased prolactin secretion upto 2-fold at 0.1 nM PMA (P<0.01), and the prolactin secretion was not significantly higher than this concentration. Staurosporine (ST; 1.0$\pi$M) a PKC antagonist, decreased by 70% of 0.1 $\pi$M VIP-stimulated prolactin secretion and by 48% of 10 ${\mu}$M PMA-stimulated prolactin secretion (P<0.01). However, pituitary cell prolactin content did not differ in any treatment (P>0.05). In conclusion, these results indicate that the PKC second messenger system is involved in VIP-stimulated prolactin release in chicken primary pituitary cell culture.

• Archives of Pharmacal Research
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• v.3 no.2
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• pp.85-86
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• 1980

Dopaminergic influences on prolactin release from lactotrophs have been studied using the rat anterior pituitary cell culture. The prolactin inhibiting activity of hypothalamic extracts was examined in relation to dopamine. Dopamine inhibited prolactin secretion from the rat anterior pituitary cell culture in a dose dependent fashion. The median effective dose was $2{\times}10^{-7}$ / M and the maximal inhibition (70-90 % of the control value) was shown by 10$^{-5}$ / M dopamine. Further increase in dopamine concentration did not result in any further inhibition of prolactin secretion.nhibition of prolactin secretion.

• Animal cells and systems
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• v.13 no.4
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• pp.429-437
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• 2009

The control mechanism of gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH) release was studied using cultured pituitary cell or cultured whole pituitary obtained from Testosterone (T) treated and control immature rainbow trout. The release of FSH was not changed by salmon type GnRH (sGnRH), chiken-II type (cGnRH-II), GnRH analogue ([des-$Gly^{10}D-Ala^6$] GnRH ethylamide) and GnRH antagonist ([Ac-3, 4-dehydro-$Pro^1$, D-p-F-$Phe^2$, D-$Trp^{3,6}$] GnRH) in cultured pituitary cells of T-treated and control fish. Indeed, FSH release was not also altered by sGnRH in cultured whole pituitary. All tested drugs had no effect on the release of LH in both culture systems of control fish. The levels of LH, in contrast, such as the pituitary content, basal release and responsiveness to GnRH were increased by T administration in both culture systems. In addition, the release of LH in response to sGnRH or cGnRH-II induced in a dose-dependent manner from cultured pituitary cells of T-treated fish, but which is not significantly different between in both GnRH at the concentration examined. Indeed, LH release was also increased by sGnRH in cultured whole pituitary of T-treated fish. GnRH antagonist suppressed the release of LH by sGnRH ($10^{-8}\;M$) and GnRH analogue ($10^{-8}\;M$) stimulation in a dose-dependent manner from cultured pituitary cells of T-treated fish, and which were totally inhibited by $10^{-7}\;M$ GnRH antagonist. These results indicate that the sensitivity of pituitary cells to GnRH is elevated probably through the T treatment, and that GnRH is involved in the regulation of LH release. GnRH-stimulated LH release is inhibited by GnRH antagonist in a dose-dependent manner. The effects of gonadal steroids on FSH levels are less clear.

• Development and Reproduction
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• v.4 no.2
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• pp.237-242
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• 2000

Growth hormone releasing hormone (GHRH), the major hypothalamic stimulus of GH secretion from the anterior pituitary gland, has been found to be present in several extrahypothalamic sites including placenta testis, ovary and anterior pituitary gland. The present study was performed to elucidate the role of pituitary GHRH on proliferation of cells derived from rat anterior pituitary gland. The GHRH content of pituitary tissue, cultured pituitary cells, and the conditioned media was evaluated by radioimmunoassay (RIA). Primary cultures of pituitary cells derived from adult rats were prepared by enzymatic dispersion. Significant amounts of GHRH-like molecules were detected in both pituitary tissue and cell cultures by GHRH RIA. Competition curves with increasing amounts of tissue extracts and conditioned media were parallel with those of standard peptide, indicating that the pituitary GHRH-like material is similar to authentic GHRH. To analyze specific cell types responsible for producing GHRH in anteroior pituitary, cell fractionation technique combined with GHRH RIA was performed. In cell fractionation experiment, the highest level of GHRH content was found in gonadotrope enriched-fraction and followed by somatotrope-, lactotrope- and thyrotrope-fraction. Treatment of pituitary cells with GHRH resulted in a dose-dependent increase in [$^3$H] thymidine incorporation. The mitogenic effect of GHRH could be mediated by typical oncogenic activation since the GHRH induced transient increase in c-fos mRNA levels with peak response at 30 minutes. The present study demonstrated that i) the pituitary GHRH expressed in the rat anterior pituitary gland can be secreted, ii) among the various cell types, gonadotropes and somatotorpes are the major GHRH source, and iii) the GHRH treatment increased the [$^3$H] thymidine incorporation and c-fos transcriptional activity in the pituitary cell culture. These findings suggested that GHRH could participated in the paracrine and/or autocrine regulation of cell proliferation, as well as promoting growth hormone secretion.

• Korean Journal of Pharmacognosy
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• v.34 no.3 s.134
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• pp.256-262
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• 2003

Lonicerae Flos (LF) has been used as an anticancer, anti-viral, and anti-inflammatory agent in traditional herbal medicine. In this study, induction of rat growth hormone (rGH) by addition of methanol (MeOH) extract of LF of Lonicerae (L.) Folium or several constituents of L. Folium were carried out in the pituitary cell culture system. Induced rGH level by addition of 70% MeOH extract of LF was increased to $732.65{\pm}105.64%$ of control (n=18, p<0.01), however, the other sequential fractions were not significantly different from the control. Ochnaflavone, a constituent of L. Folium, induced rGH level in the cell culture to $329.73{\pm}160.00%$ of control (n=6, p<0.01). An I..v. injection of the MeOH extract of LF did not increase plasma rGH level in anesthetized rats. Unfortunately, the MeOH extract of LF induced prolactin and LH release about 7 and 5 fold of the control, respectively (p<0.05, each). In conclusions, 70% MeOH extract of LF exerted induction of rGH release in rat pituitary cell culture. Further studies to investigate mechanisms of the inducded rGH by LF are in progress.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.33 no.1
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• pp.55-59
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• 2000

A specific and sensitive sandwich enzyme-immunoassay (EIA) using Avidin-Biotin complex was developed for the measurement of GTH II levels in pituitary content and pituitary cell culture medium of the rainbow trout-(Oncorhpchus mykiss). Biotin-salmon GTH II rabbit IgG (sefondary antibody) wai purified by a protein A sepharose affinity chromatography column and that was biotinylated by using Biotin-N-hydroxysuccinimide ofter (BNHS). Non-biotin salmon GTH II rabbit IgG (first antibody) was obtained only through a protein A sepharose affinity chromatography column. The assay was performed by the so-called 'sandwich' method using a microtiter plate, A dose-response curve was obtained between $0.12 to 125 ng/ml$ of salmon GTH II. The displacement curves for pituitary extraction and pituitary cell culture medium of testosterone-treated rainbow trout were Parallel to the standard curie. The intra-assay and inter-assay coefficients of variation (CV) were $8.2{\%} (N=5) and 12.5{\%} (N=6)$, respectively, This assay system was used to measure the amount of GTH II that accumulated in the culture medium of dispersed pituitary cells in testosterone-treated immature rainbow trout, The accumulation was increased with the amount or salmon gonadotropin-releasing hormone. GTH II values determined by the present method were well correlated with those determined by radioimmunoassay. As a result, this assay system was found to be suitable for the measurement of GTH II for pituitary extraction and pituitary culture medium in many salmonid fishes.

• Fisheries and Aquatic Sciences
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• v.14 no.4
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• pp.379-388
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• 2011

The mechanism by which gonadotropin-releasing hormone (GnRH) and dopamine (DA) control gonadotropin (GTH) release was studied in male and female rainbow trout using cultured pituitary cells obtained at different reproductive stages. The mechanisms of follicle-stimulating hormone (FSH) release by GnRH and DA could not be determined yet. However, basal and salmon-type GnRH (sGnRH)- or chicken-II-type GnRH (cGnRH-II)- induced luteinizing hormone (LH) release increased with gonadal maturation in both sexes. LH release activity was higher after sGnRH stimulation than cGnRH-II stimulation at maturing stages in both sexes. The GnRH antagonist ([Ac-3, 4-dehydro-$Pro^1$, D-p-F-$Phe^2$, D-$Trp^{3,6}$] GnRH) suppressed LH release by sGnRH stimulation in a dose-dependent manner, although the effect was weak in maturing fish. The role of DA as a GTH-release inhibitory factor differs during the reproductive cycle: the inhibition of sGnRH-stimulated LH release by DA was stronger in immature fish than in maturing, ovulating, or spermiated fish. DA did not completely inhibit sGnRH-stimulated LH release, and DA alone did not alter basal LH release. Relatively high doses ($10^{-6}$ or $10^{-5}M$) of domperidone (DOM, a DA D2 antagonist) increased LH release, which did not change with reproductive stage in either sex. The potency of DOM to enhance sGnRH-stimulated LH release was higher in maturing and ovulated fish than in immature fish. These data suggest that LH release from the pituitary gland is controlled by dual neuroendocrine mechanisms by GnRH and DA in rainbow trout, as has been reported in other teleosts. The mechanism of control of FSH release, however, remains unknown.