ISSN:0028-3835    

DOI:10.1159/000126929

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Pituitary transcription factor-1 (Pit-1 or GHF-1) is a transcription factor specific to the anterior pituitary and is involved in the expression and regulation of the growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone (TSH) β-subunit genes. The expression of these three genes can be modulated by changes in the hormone environment and it is thought that some of these effects are mediated through Pit-1, but little is known about the physiological regulation of this transcription factor. Therefore, we first asked whether Pit-1 gene expression is modified as a result of changes in the in vivo gonadal steroid environment and if this could be correlated with changes in GH and/or PRL mRNA levels. Secondly, we sought to determine if sex steroids affect the mRNA levels of these three peptides by acting at the level of the pituitary and whether these effects are androgen or estrogen mediated. Finally, how sex steroids modulate the response of these three genes to the hypothalamic neuropeptides growth hormone-releasing hormone (GHRH) and somatostatin (SS) was analyzed. To this end, we compared Pit-1 GH and PRL mRNA levels in the anterior pituitary of intact, castrated, and castrated testosterone-replaced adult male rats. In addition, primary cultures of adult male pituitaries were used to study the direct effects of both androgens and estrogens on Pit-1 GH, and PRL mRNA levels. In situ hybridization histochemistry was used to compare relative levels of Pit-1 GH and PRL mRNA. Densitometric analysis of the in vivo studies showed that castration resulted in a 57, 40 and 55% decline in Pit-1 GH and PRL mRNA signal levels, respectively. Furthermore, replacement with testosterone (T) at the time of castration completely prevented the decline in all three mRNA species (ANOVA: Pit-1 mRNA, p < 0.0001; GH mRNA, p < 0.0001; PRL mRNA, p < 0.0001). In vivo, both T (10–7M) and estradiol (10–9 M) were capable of stimulating Pit-1 mRNA and PRL mRNA levels, while dihydrotestosterone (DHT; 10–7M) had no effect. There was no effect of any of these steroid treatments on GH mRNA levels in vitro. Addition of GHRH to the cultures increased GH mRNA levels, as well as those of Pit-1 and PRL, and SS had the opposite effect on GH mRNA levels. Whereas the GH response to GHRH was not significantly modified by exposure to sex steroids, the effect of SS was. The presence of sex steroids was capable of modifying the Pit-1 and PRL responses to both GHRH and SS. These results clearly indicate that changes in circulating levels of sex steroids modulate the expression of Pit-1 in the anterior pituitary and that these changes can be correlated with commensurate modifications in GH and PRL mRNA levels. Furthermore, the effect on both Pit-1 and PRL mRNA levels occurs, at least in part, at the level of the anterior pituitary and is an estrogen-receptor-mediated event. In contrast, the effects of gonadal steroids on GH mRNA levels are less direct and are most likely mediated at the level of the hypothalamus, as well as through modulation of the response of the somatotroph to hypothalamic factors. We conclude that the transcription factor Pit-1 is actively regulated physiologically and may be involved in mediating some of the effects of sex steroids and hypothalamic factors on the synthesis of certain anterior pituitary ho

ISSN:0028-3835    

DOI:10.1159/000126930

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Specific cells of the hypophyseal pars tuberalis (PT) have been associated with the transmission of photoperiodic stimuli to the endocrine system. However, their principal secretory products have not been identified yet. Therefore we studied the expression of several adenohypophyseal hormones and their subunits (TSH, FSH, LH, common α-chain, GH, ACTH, PRL, α- and γ-MSH, β-lipotropin) by immunocytochemistry, Northern blot analysis and in situ hybridization in the sheep pituitary. Only the common α-chain of glycoprotein hormones could be detected in ovine PT-specific cells by immunocytochemistry while antibodies directed against the β-chains of LH, FSH, TSH and β-lipotropin labeled single cells in the PT but failed to detect these antigens in PT-specific cells. In situ hybridization and Northern blot analysis with antisense oligonucleotides against the common α-chain, β-LH, β-FSH, β-TSH, PRL and POMC revealed the expression of these subunits in the ovine PT. The mRNA of the common α-chain, β-TSH and, to a far lower extent, PRL and POMC were found throughout the entire pars tuberalis while β-FSH and β-LH could only be detected in cells of the caudal PT. Hence, GH-mRNA and GH immunoreactivity were exclusively found in the pituitary pars distalis. Compared to these results – obtained under the short photoperiod (winter) – we found clear ultrastructural signs of altered secretory activity in PT-specific cells of animals exposed to the long photoperiod (summer); the common α-chain immunoreactivity was nearly absent in PT-specific cells of summer animals. However, no seasonal influence on gene transcription or translation for other adenohypophyseal hormone was observed. These findings suggest that ovine PT-specific cells, which are only immunopositive for the common α-chain, are capable to express different mRNAs of adenohypophyseal hormones. Although it remains elusive how gene transcription and translation are related in this cell type, the presence of an mRNA pool for hormone subunits leads to the speculation that – at least in the sheep – hormone synthesis is mainly regulated at the translational level and that secretion of hormones may b

ISSN:0028-3835    

DOI:10.1159/000126931

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

A novel cDNA clone, CR16, was isolated from a rat hippocampal cDNA library and characterized for responses to corticosteroids and regional expression. The 4-kb RNA was increased 3-fold by treatment of adrenalectomized (ADX) rats with corticosterone (CORT). Overlapping cDNA totaling 4,374 nt were used to define an open reading frame of 1,356 nt beginning 191 nt from the 5’-end and encoding a 45-kD protein containing 32% proline. CR16 has no obvious homologies to GenBank or protein databases. CR16 RNA was detected by in situ hybridization in neuron-rich layers of the hippocampal formation, layers II, III and VI of the cerebral cortex, thalamus, ventromedial nucleus of the hypothalamus, bed nucleus of the stria terminalis, lateral septal nucleus, nucleus accumbens, olfactory bulb, inferior colliculus, pons and inferior olive. The CR16 RNA has low prevalence in the hippocampus and cortex (<10 pg/µg total RNA) and is elevated 3-fold in both structures in a dose-dependent manner by CORT in ADX rats. Treatment of ADX rats with aldosterone (ALDO), CORT, or RU28362 increased CR16 RNA to similar levels in the hippocampus while ALDO had minimal effects on the level of CR16 RNA relative to CORT or RU28362 in the cortex. Neither shaking stress (2 h) nor 2 h CORT significantly elevated CR16 RNA in the hippocampus, suggesting that its response to elevated CORT is not rapid. ADX lowered CR16 RNA levels by 50% relative to intact rats while low-level CORT replacement (≧4 ng/ml serum CORT) significantly elevated CR16 RNA 2-fold in ADX rats. These results are consistent with both the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) regulating the CR16 gene. This gene will be useful in dissecting the role of MR and GR in CNS neu

ISSN:0028-3835    

DOI:10.1159/000126932

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Toward the end of pregnancy, mammalian mothers undergo a cascade of hormonal, neural and somatic events that culminate in birth and lactation. Milk ejection, the final step in lactation, is regulated by high-frequency, synchronized firing of oxytocinergic neurons in the magnocellular nuclei of the hypothalamus. This synchronization of neural activity may be regulated by an increase in the cell-to-cell coupling of magnocellular neurons by connexins. Direct intercellular channels between neurons are formed by the connexin 32 protein. The present study examined the pattern of connexin 32 mRNA levels using semiquantitative in situ hybridization histochemistry with isotopically labeled cRNA probes complementary to connexin 32 and connexin 43, found in neurons/oligodendrocytes and astrocytes, respectively. Connexin 32 mRNA levels were relatively low in the supraoptic nucleus of virgin females and were dramatically up-regulated during late pregnancy. Immediately after birth, connexin 32 mRNA levels dropped and were similar to levels in virgins. On the 13th day of lactation when dye-coupling between magnocellular neurons is high, connexin 32 message levels were again very high. Levels of the astrocytic connexin 43 were no different in lactating and virgin animals. These results demonstrate that there are two peaks of connexin 32 expression, one immediately preceding parturition and one during lactation. So far, only the second peak of connexin 32 is known to be related to an increase in dye-coupling of magnocellular neurons in the supraoptic nucleus, suggesting that in this case, the elevation of connexin 32 message levels leads to the subsequent increase in intercellular coupling.

ISSN:0028-3835    

DOI:10.1159/000126933

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

This study was designed to (1) examine the ability of fetal diencephalic neurons cultured in vitro to express aromatase mRNA and (2) evaluate the involvement of several environmental factors which may regulate the development and differentiation of the neurons in the central nervous system. Brain cells from fetal mice at various developmental stages were cultured as tissue slices and as primary monolayer cells, and the expression levels of aromatase mRNA in the cultured cells were measured by a quantitative reverse transcription-polymerase chain reaction method using an internal standard. On cultured slices of the diencephalic region from fetal mice on embryonic day 12 (E12), E13, or E15 on collagen-coated membranes, the expression level of the mRNA continued to increase for the initial 2-3 days as that in vivo. Time-dependent increase of aromatase mRNA was also observed for 3 days in E13 neuronal cells dissociated with papain and cultured on poly L-Lys-coated dishes in serum-free medium. However, no significant time-dependent increase of the aromatase mRNA level was observed in E10 or El 1 brain cells cultured by either method. These findings suggest that the developmental increase of aromatase mRNA in diencephalic neurons is an endogenous characteristic and probably genetically determined after E12.

ISSN:0028-3835    

DOI:10.1159/000126934

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

To determine whether estrogen and androgens either alone or in combination downregulate estrogen receptors in the brain, ovariectomized/adrenalectomized female rats received one of the following four treatments: (1) one subcutaneously placed Silastic capsule containing 10% estradiol in cholesterol, (2) one capsule containing 10% estradiol and two capsules containing 100% 5α-dihydrotestosterone (DHT), (3) two capsules containing DHT, or (4) empty Silastic capsules (control animals). Animals were killed 4 or 8 days after capsule insertion and the occupied, unoccupied and total estrogen receptor content in specific brain nuclei was determined by quantitative in vitro autoradiography. To determine if the effects of the androgen were reversible, DHT capsules were removed after 4 days from half of the estradiol + DHT-treated rats, and the animals were killed 4 days later. Estradiol downregulated estrogen receptor expression in the periventricular preoptic area, medial preoptic area, bed nucleus of the stria terminalis (BNST), arcuate nucleus (ARC), ventromedial nucleus (VMN), and medial and cortical amygdala, decreasing receptor content by 30–41 % in animals treated for 4 days, and by 44-60% in animals treated for 8 days with estradiol alone. DHT treatment in combination with estradiol further decreased estrogen receptor content in the BNST, ARC and VMN, relative to the estradiol-only animals. DHT in the absence of estrogen was without effect. In animals in which the DHT capsules were removed after 4 days of exposure, allowing the estradiol to remain for a further 4 days, estrogen receptor levels were indistinguishable from those measured in control animals treated for 8 days with estradiol alone. These results demonstrate that sustained estrogen exposure downregulates levels of estrogen receptor in the brain and confirm that DHT synergizes with estrogen in inducing this response in some, but not all, target neuronal grou

ISSN:0028-3835    

DOI:10.1159/000126935

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The present study tested the hypotheses that exposure to morphine in utero (10 mg/kg twice a day on gestation days 11–18) and acute estrogen treatment in adulthood alter µ-opioid regulation of hypothalamic norepinephrine (NE) release in sexually mature rats. Both basal and KCl-stimulated NE releases were measured in superfused hypothalamic and preoptic area (POA) slices preloaded with 3H-NE. The µ-opioid receptor agonist D-Ala2, MePhe4-Gly-ol5-enkephalin (DAMGO; 10 or 100 nM) or the opioid antagonist naloxone (1 µM) was applied to some slices 15 min prior to KC1 stimulation. Prenatal morphine had no effect on basal or KCl-stimulated release of preloaded 3H-NE from hypothalamic and POA slices from adult male progeny. Neither the µ-opioid agonist DAMGO nor the nonspecific antagonist naloxone significantly affected KCl-evoked overflow of 3H-NE in slices from either brain region of male rats. Adult female offspring were ovariectomized (OVX), and some were injected with a replacement dose of estrogen 48 h prior to sacrifice. Prenatal morphine had no effect on basal or KCl-stimulated release of 3H-NE from hypothalamic or POA slices or on the response of slices to opioid drugs. Estrogen treatment modestly increased KCl-evoked release of 3H-NE from POA slices from females. Moreover, there was a significant interaction between opioid drugs and estrogen treatment on KCl-evoked overflow of 3H-NE. In hypothalamic and POA slices from OVX females, DAMGO reduced KCl-evoked efflux of 3H-NE. This inhibitory effect of DAMGO was not apparent in slices from estrogen-treated females. In addition, naloxone increased KCl-stimulated NE release in slices from both hypothalamus and POA of estrogen-treated female rats but not control OVX females. Thus, prenatal exposure to morphine does not alter basal, KCl-evoked or µ-opioid modulation of NE efflux from hypothalamic or POA slices in adult progeny of either sex. However, treatment of adult, OVX females with estrogen attenuates µ-opioid inhibition and promotes the appearance of naloxone facilitation of KCl-evoked 3H-NE release from hypothalamic and POA

ISSN:0028-3835    

DOI:10.1159/000126936

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Recently, anatomical evidence was presented that the mammalian circadian clock located in the suprachiasmatic nuclei (SCN) may utilize GABA to transmit diurnal information to the dorsomedial hypothalamus (DMH). The present study provides further physiological evidence for the involvement of this GABAergic projection in the regulation of diurnal rhythms. Infusion of the GABA agonist muscimol in the region of the DMH completely blocked the daily increase of plasma melatonin during darkness and reduced sympathetic output in the pineal gland resulting in lower pineal melatonin production, as measured with transpineal microdialysis. Further experiments in SCN-lesioned animals indicated that the origin of this inhibitory input to the DMH is indeed the SCN. The results of this study imply that the SCN can influence the sympathetic outflow of the hypothalamus through its GABA-containing projection. Furthermore, the present results probably explain the previously reported strong inhibitory effect of benzodiazepines on plasma melatonin in both animals and humans.

ISSN:0028-3835    

DOI:10.1159/000126937

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The cytokine interleukin-1 (IL-1) is present in the brain and is known to cause a variety of neuroendocrine and immune effects in the rodent; the neuropeptide corticotropin-releasing hormone (CRH) plays a critical role in mediating many of these effects. Little is known about these neuropeptide interactions in the primate. We have therefore examined the effects of IL-1α on the release of CRH in the ovariectomized rhesus monkey in vitro and in vivo. In 3 animals, the effect of IL-1α on CRH release from the superfused hypothalamus was studied in vitro. The hypothalamus was divided in half and fragments from each half were superfused separately. Mean CRH release was 262 ± (SE) 46 pg/20 min and increased to 1,340 ± 470 pg/20 min after exposure to IL-1α (p < 0.05). The effect of IL-1α on CRH release into cerebrospinal fluid (CSF) in vivo was studied in 8 animals with chronic cannulas implanted in the lateral ventricle for IL-1 infusion; indwelling catheters were also placed via lumbar puncture and threaded into the cervical area for CSF collection. CSF was collected at a rate of 800 µl/h during a 4-hour baseline period and for 4–8 h after intracerebroventricular infusion of 4.2 µg of IL-1α. CRH increased significantly over time in CSF after IL-1α infusion; the mean concentration of CRH increased from 83 ± 17 pg/ml during the baseline period to 203 ± 40 pg/ml after IL-1α infusion (p < 0.01). We conclude that IL-1 stimulates central CRH release in the primate and that the effects of cytokines on the release of this important neuromodulator can be monitored in chronically cannulated a

ISSN:0028-3835    

DOI:10.1159/000126938

出版商:S. Karger AG    

年代:1996

数据来源: Karger