摘要:

We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole-cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10nM), identifying it as a Na+ current. Slowly inactivating inward current, activated at potentials more positive than –30 mV, remained in Na+-free medium or in the presence of 1 µM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedi-pine about 70% of this high voltage-activated Ca2+ current was abolished, leaving a slowly inactivating component. No transient Ca2+ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 µM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K+ currents sensitive to membrane voltage and intracellular Ca2+ concentration ([Ca2+]i) were present. The resting membrane potential lay between –20 and –75 mV (mean = –43 ± 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at –20 to –35 mV) for up to 1 h. Apamin, the SK-type Ca2+-dependent K+ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca2+ channel blockade did not. Microfluorimetric detection of [Ca2+]i showed that 10nM GnRH induced [Ca2+]i oscillations. We conclude that Ca2+ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca2+]i fluctuations may activate the apamin-sensitive, Ca2+-dependent SK-type K+ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na+ current spikes may be crucial for activation of the nifedipine- and CGTX-sensitive high-voltage-activated Ca2+<

ISSN:0028-3835    

DOI:10.1159/000126887

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

α-T7 mice are a transgenic line which carries a hybrid transgene composed of the 5’ flanking region of the human glycoprotein hormone α-subunit gene (1.8 kb) linked to the coding region of the oncogene SV40 T-antigen. Large, hemorrhagic, pituitary tumors form in these mice and contain giant, transformed gonadotropes (immunopositive for T-antigen), in addition to normal-appearing gonadotropes (also immunopositive for T-antigen). An additional feature of these tumors is an abundance of neural tissue proliferating throughout the anterior pituitary, concentrated around the giant gonadotropes, and forming synaptoid contacts upon them. Continued study of these mice has demonstrated that the giant gonadotropes contain immunostainable basic fibroblast growth factor (FGF-2), and apparently release the FGF by focal cellular disruption and/or cytoplasmic blebbing. Normal gonadotropes, in control and transgenic mice, were strongly immunopositive for FGF, and appeared intact. In 8- to 13-month-old transgenic mice most of the giant cells were intact, and were surrounded by well-differentiated neural tissue. These giant cells were lightly immunopositive for FGF. Disrupted, giant gonadotropes were more frequent in 2- to 7-month-old transgenic mice, and also were surrounded by well-differentiated neural tissue with many synaptoid contacts. These cells generally were moderately immunopositive for FGF. In neonatal mice, 1-8 days old, precursors of the giant, transformed gonadotropes were identified, primarily, but not exclusively, near the periphery of the anterior pituitary. These cells stained strongly immunopositive for FGF, frequently were focally disrupted, and were associated with slender processes of neural tissue, but no synaptoid contacts. Mouse pituitaries, control and transgenic, were consistently immunonegative for nerve growth factor. We conclude that FGF is present within normal and transformed gonadotropes in α-T7 mice, and that FGF released from giant gonadotropes early in development appears likely to be the neurotrophic factor which induces neural tissue ingrowth into the anterior pituitaries of these

ISSN:0028-3835    

DOI:10.1159/000126888

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

Previous studies from our laboratory demonstrated that chronic testosterone administration to castrated transgenic mice suppressed human follicle-stimulating hormone-β (FSHβ) mRNA levels transcribed from a human transgene to approximately 20% of control values. In the present study we used primary pituitary cultures prepared from the transgenic mice and in vivo experiments in hypogonadal (hpg) mice carrying the human transgene to assess the role of hypothalamic gonadotropin-releasing hormone (GnRH) in this inhibitory action. The levels of human FSHβ mRNA in monolayer cultures of pituitary cells were decreased by 24-hour treatments with 10 nM testosterone propionate or 5α-dihydrotestosterone to 13 and 26% of control values, respectively, in the absence of GnRH. For the in vivo experiments we introduced the 10-kb human FSHβ transgene into the hpg genetic background by selective crossbreeding. Daily injections of 1 µg GnRH for 14 days induced expression of the human FSHβ gene in male and female mice. Maximal effects were obtained by GnRH treatment of gonadectomized, hpg transgenic mice. Human FSHβ mRNA levels rose to approximately 4- or 10-fold that of control males and females, respectively. The stimulation was blocked completely by simultaneous administration of testosterone propionate in males and partially by estradiol in females. Pituitary content of immunoreactive FSH paralleled the mRNA changes. These data suggest that testosterone feedback inhibits the human FSHβ subunit gene directly at the pituitary gland in addition to the indirect mechanism of GnRH suppression. Furthermore, the in vitro data indicate that the suppression of human FSHβ gene expression is at least partly a direct androgen effect that does not require aromatization of testosterone to

ISSN:0028-3835    

DOI:10.1159/000126889

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

The present studies investigated the role of glucocorticoid receptors (GR) in the inhibitory effects of acute and chronic immobilization stress on pituitary luteinizing hormone (LH) release. Systemic administration of the GR antagonist, RU486, significantly attenuated the acute decline in circulating LH observed in intact male rats immobilized by encasement within paper cocoons. Whereas vehicle-injected controls exhibited a significant reduction in plasma LH between +1 and +5 h of stress, animals given subcutaneous (sc) injections of 2.5 mg RU846/kg did not exhibit a reduction in circulating LH until 4 h after initiation of stress. Plasma LH levels in the GR-treated group were significantly elevated compared to the vehicle controls between +1 and +3 h of stress. Repetitive exposure to the same stress stimulus 24 and 48 h later resulted in decreased plasma LH levels in the vehicle-treated rats, but not in the animals injected sc with RU486. Other studies showed that intracere-broventricular (icv) administration of RU486 (10.0 µg/rat) also blunted the inhibitory effects of acute and chronic immobilization stress on pituitary LH release. In experiments designed to evaluate whether activation of central GR can influence the magnitude and/or temporal characteristics of the LH secretory response to acute inhibitory stress, it was observed that animals pre-treated by icv injection of the GR agonist, RU362, exhibited a greater reduction in plasma LH levels during the first hour of stress, as compared to rats pretreated with vehicle alone

ISSN:0028-3835    

DOI:10.1159/000126890

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

The neuropeptide galanin (GAL) has been implicated in a variety of neuroendocrine functions and has been shown to be regulated by gonadal hormones in several brain regions. We have used slice binding and quantitative autoradiography techniques to determine whether the activation of GAL pathways across puberty in female rats is associated with changes in the density of GAL binding in telencephalic and diencephalic regions as we previously observed in male rats. We have also asked whether sex differences in GAL immunoreactivity and GAL gene expresion detected in some brain regions would be paralleled by sex differences in 125I-GAL-binding density in adult male and female rat brains. To control for intrinsic differences in the level of endogenous GAL synthesis and release, brain slices from prepubertal female and adult male and female rats were treated with guanosine 5’-triphosphate (GTP) to induce dissociation of endogenous GAL from its binding sites prior to incubation with radiolabeled ligand. 125I-GAL binding was significantly reduced in seven brain regions of adult compared with prepubertal female rats. These regions included the islands of Calleja (p < 0.03), the medial amygdaloid nucleus, posterodorsal division (p < 0.05), median eminence (p ≤ 0.02), medial habenular nucleus (p < 0.05), rhomboid thalamic nucleus (p < 0.05), and paraventricular (p < 0.05) and intermediodorsal (p ≤ 0.02) thalamic nuclei. Only one region, the lateral preoptic area, exhibited significantly enhanced 125I-GAL binding in adult female (p ≤ 0.04) compared with prepubertal animals. The distribution and density of 125I-GAL-binding sites were similar in adult male and female rat brains and no statistically significant effect of sex or interaction of sex with brain region were detected by ANOVA. These results suggest that gonadal hormones and/or some other factors associated with puberty may regulate central GAL receptor number and/or affinity in discrete regions of the female rat brain. The regional diversity of this regulation suggests that multiple factors are i

ISSN:0028-3835    

DOI:10.1159/000126891

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

γ-Aminobutyric acid (GABA) exerts an inhibitory action on gonadotropin-releasing hormone (GnRH) release from the hypothalamus. In vivo, this inhibitory action appears to be mediated via the GABAA receptor since in ovariectomized (ovx) rats and sheep direct application of muscimol (MUS), a GABAA agonist, into the preoptic area (POA), the site were the GnRH cell bodies are located, caused an immediate reduction of LH release. This effect may be the result of an inhibition of GnRH release but also GnRH biosynthesis may be affected. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) we now addressed the question, whether an acute inhibition of the GnRH pulse generator in ovx rats by GABA involves reduction of GnRH biosynthesis as determined by GnRH mRNA levels in micropunches of the POA. To activate either the GABAA or GABAB receptor, we injected intraventricularly (icv) MUS or baclofen (BAC). Intracerebroventricular injection of 10 nmol MUS caused a rapid and lasting inhibition of LH release from about 7.5 ng/ml (pretreatment value) to approximately 1.5 ng/ml. Neither application of BAC or saline (control injections) affected LH secretion. Two hours after icv injections, rats were decapitated and GnRH mRNA levels were determined. MUS induced a pronounced decrease of GnRH levels in the POA (control rats: 2.26 pg GnRH mRNA; MUS-treated rats: 0.85 pg, n = 10/ group). BAC was without any effect on GnRH mRNA levels. Thus, we confirm the inhibitory action of GABA on LH release in vivo which is exerted via the A-subtype of the receptor. We suggest that this effect, in addition to the possible actions of MUS on other neuronal systems relevant to central regulation of LH secretion, is at least in part due to a similarly rapid suppression of GnRH gene expression in the POA and/or accelerated GnRH mRNA turnover

ISSN:0028-3835    

DOI:10.1159/000126892

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

Correlations between the specific binding of radiolabelled adrenoligands in frontal cortex and hypothalamus, and plasma testosterone levels were investigated in inbred mouse strains. Binding values were structure and genotype dependent. There were significant negative intrastrain and interstrain correlations between hypothalamic binding of 3H-clonidine, an imidazoline with α2-adrenoceptor agonist specificity, and resting testosterone levels. Neither binding of the α1 -adrenoceptor antagonist 3H prazosin nor of the β-adrenoceptor antagonist 3H-dihydroalprenolol (DHA) correlated significantly with hormone concentrations. The inverse correlation between hypothalamic 3H-clohidine-binding and plasma testosterone levels suggests a possible role of α2-adrenoceptors in the negative regulation of testosterone plasma lev

ISSN:0028-3835    

DOI:10.1159/000126893

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

The metabolite of dihydrotestosterone, 5α-androstane-3α,17β-diol (3α-Diol), is a potent inhibitor of estrogen-induced gonadotropin and prolactin secretion and lordosis behavior in the female rat. This study examined whether 3α-Diol can counteract the ultrastructural changes which are known to occur in the ventromedial nucleus (VMN) of the hypothalamus following estrogen treatment. Ovariectomized rats were treated with estradiol (E2; n = 7), 3α-Diol (n = 5), E2 and 3α-Diol (n = 6), or received control (n = 6) treatments. E2 was administered in subcutaneous capsules for two discontinuous 2-hour periods separated by 5 h, a ‘pulsed’ treatment regimen known to mimic the timing of endogenous E2 action and to influence neuronal ultrastructure in the VMN. Animals given 3α-Diol received subcutaneous injections (6 mg/kg) 3 h prior to each implantation of E2 or empty capsules. Control animals received vehicle 3 h prior to implantation of blank capsules. Animals were perfused 24 h after initial hormone treatment and neurons from the ventrolateral portion of the VMN were examined using electron microscopy. Separately, both E2 and 3α-Diol treatment increased somal and nuclear size, altered somal and nuclear shape, and increased the numbers of lysosomes present in the cytoplasm above control levels. E2 treatment resulted in increased stacking of the rough endoplasmic reticulum while 3α-Diol treatment resulted in an unusual plexiform rough endoplasmic reticulum distribution. In contrast, combined treatment with E2 and 3α-Diol resulted in cells which were similar to ovariectomized control cells on these measures. All steroid treatments decreased the amount of heterochromatin present within the nucleus compared to that seen in controls. Thus, 3α-Diol influences the ultrastructural characteristics of neurons within the VMN in a manner somewhat though not altogether similar to E2. However, 3α-Diol given in combination with E2 counteracts or prevents the actions of E2 within th

ISSN:0028-3835    

DOI:10.1159/000126894

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

Cnemidophorus uniparens is a unisexual species of whiptail lizard of hybrid origin whereas C. inornatus is a sexual species and the maternal ancestor of C. uniparens. Together they represent an excellent model system for investigating the evolution of hormone-brain-behavior relationships. Normal circulating estradiol (E) concentrations in C. uniparens are approximately 5-fold lower than those of female C. inornatus in a similar reproductive state. Experiments were performed to determine whether (i) C. uniparens is more sensitive to E, and (ii) whether the difference in sensitivity is correlated with differences in estrogen receptor (ER)-mRNA expression in the brain. Dose-response curves reveal that ovariectomized C. uniparens are more responsive than ovariectomized C. inornatus to exogenous estradiol 17β-benzoate (EB). EB is more effective in C. uniparens at inducing receptive behavior and progesterone receptor (PR) gene expression in the ventromedial nucleus of the hypo-thalamus (VMH). In situ hybridization analysis of ER-mRNA expression revealed no species differences in ER-mRNA content in the VMH of ovariectomized animals. Treatment of ovariectomized animals with EB resulted in a greater induction of ER-mRNA expression in the VMH of C. uniparens compared to C. inornatus. These results indicate that the differences in behavioral sensitivity to E lie in the estrogen target neurons in the brain region controlling receptive behavior, the VMH, and that the difference in sensitivity cannot be explained by species differences in the basal rate of ER gene expression

ISSN:0028-3835    

DOI:10.1159/000126895

出版商:S. Karger AG    

年代:1995

数据来源: Karger

摘要:

In vivo and in vitro prolactin (PRL)-synthesizing and PRL·releasing activity of fetal (days 12–22) and early postnatal (days 1–10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18 12-15 % of monodispersed pituitary cells displayed PRL immuno-positivity, but only 3-5% of PRL·positive cells were plaque-forming, i.e. releasedPRL. By the end of gestation 19-25% and on postnatal day 1042-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL·positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke’s pouch cells have a substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions. This study is the first demonstrating PRL release of individual fetal lactotrophs from day 18 of intra-uterine life onwards and the ontogenetic change in the percentage of functionally active and inactive subpopulations of PRL cells. Further, the observations are in line with the assumption that the dopaminergic inhibition of PRL release may start in early postna

ISSN:0028-3835    

DOI:10.1159/000126896

出版商:S. Karger AG    

年代:1995

数据来源: Karger