Differential regulation of estrogen receptor alpha and beta mRNAs in the rat uterus during pregnancy and labor: possible involvement of estrogen receptors in oxytocin receptor regulation

Oxytocin receptor (OTR) mRNA levels in the uterus dramatically increase in the near term human and rat. Estrogen is believed to be a potent stimulator of OTR mRNA expression. However, estrogen does not stimulate rat OTR mRNA expression on day 18 of pregnancy or in progesterone-treated rats. Thus, the regulation of uterine responsiveness to estrogen in the near term rat appears to be an important mediator of estrogen action. To determine the effect of altering uterine responsiveness to estrogen on OTR induction, uterine ERalpha and ER beta mRNA levels were examined by competitive RT-PCR in pregnant and parturient rats, progesterone-treated ovariectomized (OVX) virgin rats and OVX pregnant rats. In pregnant and parturient rats, OTR mRNA levels were highest at 2200-2230 h on day 21 of pregnancy (P21pm) and during labor when compared with other groups. ERalpha mRNA levels significantly increased during labor compared with days 15-21 of pregnancy. Compared with control animals, ERalpha mRNA levels decreased significantly in OVX virgin rats implanted with tubes containing progesterone for one week; 24 h following the removal of the progesterone tubes, ERalpha mRNA levels were found to be similar to control levels. Estrogen treatment following OVX on day 18 of pregnancy caused increased OTR mRNA levels, whereas ovariectomy alone increased ERbeta mRNA but not ERalpha mRNA. Results from the present study suggest that ERalpha and ERbeta mRNA expressions are differentially regulated in the rat uterus. Moreover, during spontaneous labor our findings appear to suggest that ERalpha plays a more prominent role than ERbeta in mediating estrogen action in the induction of uterine OTR mRNA before labor.     

雌激素对去卵巢大鼠内脏脂肪细胞脂肪因子表达水平的影响

目的 观察雌激素对去卵巢大鼠内脏脂肪细胞瘦素、脂联素、抵抗素和肿瘤坏死因子-α(TNF-α)表达水平的影响,探讨雌激素对体脂分布的影响机制.方法 6周龄Sprauge-Dawley雌性大鼠30只,采用随机数字表法分成3组:假手术组、去卵巢组和去卵巢+戊酸雌二醇组(OVX+E2组),每组10只.术后1周,OVX+E2组大鼠每天按1 mg/kg体重灌胃戊酸雌二醇水溶液,其他组大鼠灌胃等体积蒸馏水.连续给药12周后,腹主动脉取血,迅速剥离内脏脂肪组织.采用全自动生化分析仪检测血脂、血糖.采用免疫组化染色、实时荧光定量RT-PCR和Western印迹检测脂肪细胞瘦素、脂联素、抵抗素和TNF-α的表达.结果 3组血清瘦素、脂联素和抵抗素水平差异无统计学意义(P均＞0.05),但去卵巢组TNF-α水平显著高于假手术组(F=4.785,P＜0.05).免疫组化显示,与假手术组相比,去卵巢组内脏脂肪组织中瘦素表达明显减弱,而脂联素、抵抗素和TNF-α表达明显增强;与去卵巢组相比,OVX+E2组内脏脂肪组织中瘦素表达明显增强,脂联素、抵抗素和TNF-α表达明显减弱(F =3.712 ～5.198,P均＜0.05).3组内脏脂肪细胞瘦素mRNA和蛋白表达水平差异无统计学意义(P均＞0.05);去卵巢组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著高于假手术组,而OVX+E2组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著低于去卵巢组(F=3.175～5.342,P均＜0.05).结论 雌激素可通过下调去卵巢大鼠内脏脂肪细胞脂联素、抵抗素和TNF-α的表达,进而影响去卵巢大鼠体脂再分布.     

Acute temporal regulation of vascular endothelial growth/permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration. Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 +/- 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 +/- 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 +/- 0.30, glands and 1.20 +/- 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (+/-SE) of 71.6 +/- 4.6 nm at 0 h to 101.1 +/- 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.     

Regional induction of c-fos-like protein in rat brain after estradiol administration.

The protooncogene c-fos is induced in rat brain by various forms of physiological stimulation. In this study immunocytochemical staining for a peptide fragment of the c-fos protein was used to assess estradiol's effects on c-fos in rat brain. After estradiol benzoate (100 micrograms/kg) administration to ovariectomized rats, the number of cells staining for c-fos-like protein increased in the anterior medial preoptic area, the medial preoptic area, the medial amygdala nucleus, and the ventromedial nucleus of hypothalamus, all regions rich in estradiol-concentrating cells. This increase peaked between 12-48 h (depending on the region) after estradiol administration and was not observed in several areas with a lower density of estradiol-concentrating cells. In the most affected region, the anterior medial preoptic area, estradiol effects were dose dependent and not altered by progesterone administration. It is not yet clear whether estradiol induces c-fos expression in brain directly or whether c-fos is part of a cascade of mechanisms by which estradiol regulates gene expression.     

Progesterone and medroxyprogesterone acetate effects on central and peripheral allopregnanolone and beta-endorphin levels

The increased use of hormonal therapies has led to the study of the properties of different progestin molecules and their effects on the central nervous system. The central and peripheral levels of neurosteroid allopregnanolone and the opioid peptide beta-endorphin (beta-END) are regulated by estrogens. The aim of the present study was to investigate the effects of a 2-week oral treatment with micronized progesterone or medroxyprogesterone acetate (MPA) alone or in addition to estradiol valerate (E2V) on central and peripheral allopregnanolone and beta-END levels in ovariectomized (OVX) female rats. Thirteen groups of Wistar OVX rats received one of the following treatments: oral progesterone (2, 4 or 8 mg/kg/day); oral MPA (0.05, 0.1 or 0.2 mg/kg/day); E2V (0.05 mg/kg/day); E2V + progesterone (0.05 mg/kg/day + 2, 4 or 8 mg/kg/day), or E2V + MPA (0.05 mg/kg/day + 0.05, 0.1 or 0.2 mg/kg/day) for 14 days. One group of fertile and one group of OVX rats were used as controls. The concentration of allopregnanolone was assessed in the frontal and parietal lobes, hypothalamus, hippocampus, anterior pituitary, adrenals and serum, while the beta-END content was assessed in the frontal and parietal lobes, hypothalamus, hippocampus, anterior and neurointermediate pituitary, and plasma. E2V administration reverted the ovariectomy-induced reduction in allopregnanolone and beta-END. Progesterone and MPA increased allopregnanolone levels in all tissues except in the adrenal gland. The combined administration of progesterone or MPA and E2V determined a further increase in allopregnanolone levels with respect to E2V alone except in the adrenal gland and hippocampus only after MPA treatment. Progesterone did not affect beta-END levels in the frontal and parietal lobes, hippocampus and anterior pituitary, while it caused an increase plasma, hypothalamic and neurointermediate pituitary beta-END levels. MPA only affected beta-END levels in the hippocampus and in the neurointermediate lobe. The combined administration of progesterone or MPA and E2V did not alter the effect of estradiol or it determined a further dose-dependent increase in beta-END levels. In conclusion, this study demonstrates that progesterone and MPA have a similar but not identical effect on central and peripheral allopregnanolone and beta-END levels. Their association with an estrogenic compound does not interfere with the positive effects produced by estrogen on allopregnanolone and beta-END brain content.     

Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes

BACKGROUND & AIMS: We evaluated the effects of ovariectomy (OVX) and estrogen replacement treatment on cholangiocyte proliferation induced by bile duct ligation (BDL). METHODS: BDL (2 weeks) was performed in ovariectomized rats and the proliferative and apoptotic activity compared with normal, with BDL control rats, and with BDL +/- OVX rats treated with 17-beta estradiol. RESULTS: OVX induced a significant (P < 0.01) reduction of bile duct mass in BDL rats. The reduction of bile duct mass induced by OVX was associated with a decreased expression of estrogen receptor (ER)-alpha (2.5-fold) and, mainly, ER-beta (35-fold). Proliferating cellular nuclear antigen (PCNA) expression in cholangiocytes was impaired by OVX, indicating depression of proliferation, whereas terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and Fas positivity were markedly enhanced, indicating activation of Fas-mediated apoptosis. Administration of 17-beta estradiol during BDL in OVX rats induced a normalization of bile duct mass, ER expression, cholangiocyte proliferation, and apoptosis (Fas and TUNEL) in comparison with untreated BDL rats. CONCLUSIONS: Our findings support the role of endogenous estrogens in sustaining the enhanced proliferative and secretory activities of cholangiocytes in cholestasis. On the basis of these data, the hypothesis of an estrogenic functional deficiency in chronic cholestatic liver diseases should merit careful attention.     

The effects of topiramate and sex hormones on energy balance of male and female rats

OBJECTIVE: The effects of topiramate (TPM) on components of energy balance were tested in male and female rats that were (i) left intact, (ii) castrated or (iii) castrated with replacement therapies consisting of testosterone administration in orchidectomized (OCX) rats and of estradiol or progesterone treatments in ovariectomized (OVX) rats. METHODS: TPM was mixed into the diet and administered at a dose of 60 mg per kg of body weight. Male and female rats were treated for 28 and 35 days, respectively. At the end of the treatment period, variables of energy balance and determinants of lipid and glucose metabolism were assessed. RESULTS: TPM reduced energy and fat gains in both male and female rats either in the absence or in the presence of hormone replacement therapies. In male rats, it also decreased food intake, protein gain and energetic efficiency. In female animals, TPM reduced energetic efficiency while it stimulated lipoprotein lipase activity in brown adipose tissue. TPM also reduced plasma glucose and plasma leptin levels in female rats as well as plasma insulin and liver triglycerides in male animals. As expected, castration and sex hormones also strongly influenced energy balance. In male rats, OCX led to a decrease in energy and protein gains that was blocked by treatment with testosterone. In female rats, OVX caused increases in energy, fat and protein gains that were prevented by treatment with estradiol. CONCLUSION: In female rats, the effects of TPM on fat and energy gains were clearly not influenced by the sex hormone status of the rats. In male animals, there was also no interaction of TPM and the status of sex hormones on energy balance, suggesting that OCX and testosterone minimally interfere with the action of TPM on energy balance. The effects of TPM on energy balance were accounted for by a decrease in energetic efficiency, resulting from an effect exerted by the drug on both energy intake and thermogenesis. The present results also suggest that TPM can enhance insulin sensitivity.     

Regulation of angiotensinogen gene expression by estrogen.

OBJECTIVE: Clarification of the role of estrogen in the regulation of angiotensinogen gene expression in multiple tissues. DESIGN: The effect of 17 beta-estradiol (E2; 10 micrograms/100 mg body weight) administration in ovariectomized (OVX) rats upon angiotensinogen messenger RNA (mRNA) levels in multiple tissues was assessed. Confounding ovarian factors were thus removed by studying the animals in the castrate state. Controls consisted of OVX and intact female rats. METHODS: Adult female Sprague-Dawley rats were ovariectomized and experiments begun 21 days postsurgery. Animals were injected with E2 and studied after 0, 1, 4, and 24 h of treatment. Levels of angiotensinogen mRNA were determined by Northern blot analysis using beta-actin mRNA as an internal standard. RESULTS: A single angiotensinogen mRNA species with molecular size of approximately 1800 bp was observed in rat liver, aorta, kidney, cardiac atria, hypothalamus and whole brain. Little or no angiotensinogen mRNA was identified in the pituitary gland. Angiotensinogen mRNA was most abundant in rat liver, hypothalamus, aorta and progressively less abundant in whole brain, cardiac atria and kidney. A twofold induction of hepatic angiotensinogen mRNA levels in E2-OVX rats was observed by 4h. The angiotensinogen mRNA levels in kidney were threefold higher by 4 h compared with OVX control animals. In aorta, the angiotensinogen mRNA level was also threefold higher by 1 h after E2 treatment. No significant effect of estradiol treatment was observed in cardiac atria although the level of angiotensinogen mRNA was higher in intact female rats compared with OVX controls. CONCLUSION: These results suggest that estrogen modulates angiotensinogen gene expression in a tissue-specific manner.     

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.     

Effects of long-term treatment with resveratrol and subcutaneous and oral estradiol administration on pituitary function in rats

Hormone replacement therapy (HRT) has been used for several decades to treat menopausal discomforts. However, in the light of recent studies that draw attention to the potential hazards of conventional HRT, various attempts have been undertaken to search for alternatives to classical HRT. Phytoestrogens are claimed to be capable of positively influencing menopausal symptoms, including hot flushes. We designed a long-term study of 3 months to assess the effects of subcutaneous and orally fed 17beta-estradiol (E2), as well as the actions of resveratrol (RES) on pituitary function in female rats. Our results have demonstrated that RES binds with a 10-fold lower affinity to estrogen receptor (ER)-alpha than to ERbeta. The data from the in vivo study revealed that a dosage of 5 microg and 50 microg RES/kg bodyweight per day given to ovariectomized (OVX) rats achieved serum levels of 1.0 and 8.1 microM respectively. Long-term treatment of OVX rats with RES revealed no estrogenic potential on pituitary function in vivo as assessed by LH and prolactin secretion and by regulation of mRNAs for LHalpha, LHbeta, and GnRH receptor. Subcutaneous treatment with E2 in silastic capsules exerted stronger effects on LH and prolactin secretion, as well as on LHbeta, LHalpha, GnRH receptor, and ERbeta mRNA regulation compared with orally applied estradiol benzoate despite comparable serum levels. Levels of aryl hydrocarbon receptor (AhR) mRNA in the pituitary were increased following OVX and attenuated by long-term E2 treatment, whereas RES did not modulate AhR mRNA expression.