Receptor and Biological Response to Estriol in the Fetal Uterus of Guinea Pig

Abstract

The binding of ³H-estriol was examined in the fetal uterus of guinea pig. The physico-chemical characteristics of the binding of ³H-estriol to macromolecules are similar to the typical receptor protein for estrogens. Different estrogens (estriol, estradiol, estrone and diethylstilbestrol) compete with this binding but progesterone and testosterone have no effect. The binding affinity has a Kd of 5.5 ± 1.6 ± 10^?10M. By ultra-centrifugation in sucrose gradient, two specific components with sedimentation coefficients of 8 and 45 are found. Competition studies suggest that the same specific binding sites may be present for estriol (E₃) and for estradiol. The s.c. administration of E₃ to the pregnant guinea pig (1 mg/day per kg body weight for 3 days) provokes two biological responses in the fetal uterus: a uterotrophic effect and a significant increase in the progesterone receptor. The increase in the fetal uterine weight is 50–70% in relation to the non-treated animals and the progesterone receptor concentration is 10–14 times higher than in the control animals. These effects are similar (or slightly higher) than in animals primed with equimolecular quantities of estradiol. In contrast, single daily injections of E₃ to newborn guinea pig, results only in weak uterotrophic activity.

It is concluded that estriol is capable of causing a biological response in the uterus during intra-uterine life.     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.     

Estrogen and Progesterone Receptors in the Uterus of the Vervet Monkey

Abstract

Experimental conditions for the optimal measurement of estrogen (ER) and progesterone (PR) receptors in normal vervet monkey (Cercopithecus aethiops pygerythrus) uteri are described. The uteri of this primate were found to contain relatively high concentrations of both ER and PR. Levels of ER ranged from 151 to 822 femtomoles per mg protein (mean for group assayed is 327±165 femtomoles per mg protein). PR assays were performed on the same cytosols and the levels ranged from 444 to 2267 femtomoles per mg protein (mean of 1285±511 femtomoles per mg protein). Mean K_d values for the ER- and PR-ligand complexes were found to be 3.15±1.4x10^-10 M and 2.38±0.2x10^-9 M respectively, within the group analysed (n=21). The ratio of PR to ER varied between 1.1 and 13.1 with a mean of 4.5±2.4. Ligand specificity studies revealed that [³H]-17β-estradiol binding to the ER could only be inhibited by estrogens or estrogen analogues. The PR however exhibited an affinity for a wider range of ligand types. In low ionic strength buffers both ER and PR sedimented as ±8S type molecules in the presence or absence of 10mM sodium molybdate. Both receptors dissociated into smaller components, following a short exposure to 0.4 M KCl and subsequent centrifugation in a gradient containing 0.4 M KCl.     

雌激素、孕激素对雌激素受体、孕激素受体阴性子宫内膜腺癌细胞系JEC裸鼠皮下移植瘤生长的影响

目的探讨一定浓度雌激素(E2)及孕激素(P)对雌激素受体(ER)和孕激素受体(PR)阴性的子宫内膜腺癌细胞系JEC裸鼠移植瘤的影响。方法选用人子宫内膜腺癌细胞系JEC为研究对象,Balb/c.nu裸鼠皮下注射8×105个/0.2mLJEC细胞,分别注射E2、P及NS连续一周,观察肿瘤的生长及病理学变化情况。结果各组肿瘤体积、瘤体重量及瘤细胞核的积分光密度值依次为E2组>NS组>P组,差异有统计学意义(P<0.01)。结论 ER和PR阴性的JEC细胞生长可受到雌激素和孕激素的调控,一定浓度的雌激素能促进瘤体的生长,而孕激素则对瘤体的生长有抑制效应。     

PMSG或FSH同期发情处理对小鼠卵巢、输卵管和子宫中孕激素受体分布的影响

目的探讨孕马血清促性腺激素（pregnant mare serum gonadotropin,PMSG）和促卵泡激素（folliclestimulating hormone,FSH）同期发情处理的小鼠卵巢、输卵管和子宫中孕激素受体（progesterone receptor,PR）分布的差异。方法 10只8周龄KM系雌鼠,随机分为PMSG和FSH两个组,第一次处理后48 h取其卵巢、输卵管、子宫进行固定,免疫组织化学法观察各组织中PR的分布。结果两组小鼠卵巢、输卵管和子宫内膜中均有PR表达;其中PMSG组初级卵泡、次级卵泡和输卵管的阳性率均显著高于FSH处理组（P〈0.05）;PMSG组各级卵泡的平均吸光度值均显著高于FSH处理组（P〈0.05）;PMSG处理组子宫内膜上皮与腺上皮中的阳性率显著高于FSH组（P〈0.05）,而基质和子宫内膜上皮中的平均吸光度值显著低于FSH组（P〈0.05）。结论 PMSG和FSH同期发情处理不同程度影响小鼠卵巢、输卵管和子宫中PR的表达分布;其中PMSG处理组小鼠卵巢、输卵管和子宫上皮中PR表达普遍显著高于FSH处理组。     

猪雌激素受体（ESR）基因对产仔数性状的影响

猪产仔数是重要的经济性状,产仔数的提高将会大大地增加商品肉猪的产量,给现代养猪生产带来巨大的经济效益。ＥＳＲ基因是影响猪产仔数的主效基因,而且与猪的生长发育性状及酮体性状之间不存在负的基因多效性影响。采用ＰＣＲ－ＲＦＬＰｓ的方法,通过对５个不同品种的、特别是我国地方品种的２６２头母猪进行了基因多态性分析,研究表明ＥＳＲ基因在这种种群对产仔数均有极显著影响,ＥＳＲ位点ＢＢ基因型母猪平均比ＡＡ基因型母     

Reversibility of the Stabilization Effect of Sodium Molybdate on Uterine Estrogen and Progesterone Receptors of the Vervet Monkey

Abstract

Sodium molybdate affected the stability of vervet monkey (Cercopithecus aethiops pygerythrus) uterine estrogen (ER) and progesterone (PR) receptors. Yields of receptors were invariably higher (20 - 40 %) when cytosols were prepared in the presence of 10mM sodium molybdate. No changes were observed in the binding affinities for the natural ligands as reflected in dissociation at 0°C and 20°C was not affected in the presence or absence of molybdate. Stability studies at 37°C indicated both receptors to be more resistant to inactivation in the presence of molybdate. Dissociation of ER and PR was biphasic, indicating the existence of slow (SDC), as well as fast dissociating (FDC) complexes. Rate constants of dissociation were significantly affected by the presence of sodium molybdate Although no significant changes in the sedimentation coefficeints were observed, marked differences in the actual gradient profiles could be illustrated in the presence or absence of sodium molybdate. Observed effects could only be partially reversed in sedimentation dialysis experiments. Proteolytic inhibitors phenlymethylsulfonylfluoride (PMSF) and leupeptin had no inhibitive effect on the molybdate stabilization of ER and PR.     

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.     

雌二醇和孕酮对小鼠卵母细胞体外成熟的影响

目的研究雌二醇（E2）、孕酮（P4）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）后48 h,摘取卵巢获得未成熟卵母细胞,分别在单独含不同浓度的E2或P4的成熟液中,或在同时含有不同浓度E2和P4的成熟液中,进行体外成熟,并对经过E2和P4处理成熟的卵母细胞进行体外受精。结果经15-16 h的成熟培养,各E2处理组的卵母细胞第一极体排出率虽然均略高于对照组,但它们之间无显著差异;各组卵母细胞体外受精48 h后,1000 ng/mL E2处理组的卵裂率显著低于其他各处理组和对照组,但各组的囊胚率之间无显著差异。各P4处理组的极体率和卵裂率,与对照组相比无显著差异,但是各处理组的囊胚率均极显著低于与对照组;两种激素协同处理组中[1000 ng/mL E2＋1000 ng/mL P4]组的极体率显著高于其他各处理组;而与对照组的极体率差异不显著。结论P4对小鼠卵母细胞的后期发育能力有一定的抑制作用,而E2却对小鼠卵母细胞的成熟及其发育潜力无明显作用。     

Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

雌激素和孕激素对不同发情方式小鼠子宫内膜中孕激素受体分布的影响

目的探讨雌（Estrogen,E2）、孕激素（Progesterone,P4）对同期发情与自然发情小鼠子宫内膜中孕激素受体（Progesterone receptor,PR）分布的影响。方法45只同日龄雌鼠,根据处理方式的不同随机分为5组：自然发情组（对照组）、同期发情组、卵巢摘除组、P4处理组和E2处理组,5组小鼠在见栓后第4、6、8天分别取样后,采用免疫组织化学法观察小鼠子宫内膜中PR的分布变化情况。结果免疫组织化学染色结果显示,5个处理组小鼠子宫内膜的三种细胞中都有PR存在;同期发情组小鼠子宫内膜中三种类型细胞PR的表达与自然发情组差异有显著性（P〈0.05）;P4处理组小鼠子宫内膜中三种类型细胞PR的表达在见栓第4、6天显著低于卵巢摘除组（P〈0.05）;E2处理组小鼠子宫内膜腺上皮和间质中PR在第4、6、8天时都显著高于卵巢摘除组（P〈0.05）,而在腔上皮中则显著低于卵巢摘除组（P〈0.05）。结论同期发情处理与自然发情小鼠的子宫内膜上PR的分布,都受E2和P4的特异诱导而变化。     

雌激素和多巴胺对大鼠垂体组织中雌激素受体基因表达的影响

目的研究雌激素和多巴胺激动剂对雌激素受体在大鼠垂体组织表达的作用。方法20只成年雌性Wistar大鼠,切除卵巢后,随机分2组：（1）对照组（n=5）,皮下植入空白硅胶管;（2）雌激素组（n=15）皮下植入含有乙烯雌酚的硅胶管,8周后,两组各处死5只大鼠,雌激素组剩余大鼠（n=10）取出硅胶管,随机再分2组,安慰剂组（n=5）给予自来水灌胃,多巴胺组（n=5）给予溴隐亭（多巴胺激动剂）灌胃,用药4周后处死动物。放免法测定血清PRL水平,用反转录一聚合酶链反应（RT-PCR）方法检测ERs在各组垂体组织中的表达,以β-actin作为内参照,借助于计算机凝胶成像系统分析表达量。结果ERa,ERβ以及TERP在各组大鼠垂体组织均有表达,其中ERα和TERPmRNA水平在雌激素组明显高于对照组（P〈0．001）,在安慰剂组和多巴胺组的表达无明显差别。结论大鼠垂体组织中存在ER的表达,雌激素对ERα和TERP的表达具有升调节作用,多巴胺不影响雌激素受体的表达。     

The Effects of Estradiol on Estrogen Receptor and Glutamate Transporter Expression in Organotypic Hippocampal Cultures Exposed to Oxygen--Glucose Deprivation

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) α and β and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17β-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERα protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERβ was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERβ and, consequently, of the genes influenced by this receptor.Helena Cimarosti and Ross D. O’Shea, equal first authors.     

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Abstract: Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17β-estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17β-estradiol (10 μg/0.2 ml), progesterone (0.72 mg/0.2 ml). 17β-estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [³H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 ± 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16-23%. Chronic treatment of ovariectomized rats with 17β-estradiol progesterone, or their combination increased to the same extent and uniformly [³H]-GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17β-estradiol and/ or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.     

Effect of Puromycin on Guinea Pig Lymphotoxin Release and Lectin-Induced Cellular Cytotoxicity

In order to confirm the role of guinea pig lymphotoxin (GLT) in lectin-induced cellular cytotoxicity (LICC), the effect of puromycin, a potent enhancer of GLT activity, on the LICC to target L · P3 cells induced by phytohemagglutinin (PHA) was investigated under serum-free conditions. LICC was completely inhibited by puromycin, when it was added at the initiation of LICC culture, because of the inhibition of the release of GLT from the effector lymph node cells. However, LICC was markedly enhanced when puromycin was added several hours after the initiation of LICC culture. The interpretation of these facts is that GLT release can be inhibited by puromycin, but that the GLT already released exerts an enhanced cytotoxic effect on the target cells in the presence of puromycin. Enhancement of the cytotoxicity by the addition of puromycin several hours after the initiation of LICC culture was observed even after the removal of the GLT present in the supernatant, suggesting that the morphologically intact target cells were already affected by GLT in the early stages of LICC culture.     

ER、EGFR在分化型甲状腺癌中的表达及其临床意义

目的:探讨雌激素受体(ER)和表皮生长因子受体(EGFR)在分化型甲状腺癌(DTC)中的表达及其意义.方法:用免疫组化SP法检测45例(DTC)组织中ER和EGFR的表达.结果:ER在DTC组中的阳性率为37.3％,明显高于良性腺瘤和正常组(P＜0.05);EGFR在DTC组中的阳性率为57.7％,明显高于两对照组(P＜0.05).ER在DTC中的表达与性别和年龄有关;EGFR的表达与DTC中的淋巴结转移有关.结论:ER和EGFR的表达可以作为鉴别甲状腺肿瘤良恶性的指标EGFR可作为甲状腺癌患者预后的指标.     

Effect of Phenylbutazone on Phagocytosis and Intracellular Killing by Guinea Pig Polymorphonuclear Leukocytes

The anti-inflammatory drug phenylbutazone has been found to inhibit both engulfment and intracellular killing of E. coli by guinea pig peritoneal polymorphonuclear (PMN) leukocytes. The bactericidal activity of leukocytic homogenates was also inhibited by the drug. Addition of the drug at various time intervals to a phagocytic reacting system caused an almost immediate cessation of bactericidal activity. Metabolic studies showed that the drug sharply curtailed glucose-l-(14)C and (14)C-formate oxidation of both resting and phagocytizing PMN leukocytes. These data indicated an effect upon the hexose monophosphate shunt and H(2)O(2) formation. Further investigation showed that the sites of inhibition were on glucose-6-phosphate and 6-phosphogluconate dehydrogenase. These inhibitions resulted in decreased H(2)O(2) production. It is suggested that H(2)O(2) activates lysosomes and subsequently complexes with the lysosomal enzyme, myeloperoxidase. This complex is a potent bactericidal agent in the phagocyte.