ERα mRNA Research Articles

PSF Promotes ER-Positive Breast Cancer Progression via Posttranscriptional Regulation of ESR1 and SCFD2.

Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer, yet long-term treatment often causes acquired resistance, which results in recurrence and metastasis. Recent studies have revealed that RNA-binding proteins (RBP) are involved in tumorigenesis. Here, we demonstrate that PSF/SFPQ is an RBP that potentially predicts poor prognosis of patients with ER-positive breast cancer by posttranscriptionally regulating ERα (ESR1) mRNA expression. Strong PSF immunoreactivity correlated with shorter overall survival in patients with ER-positive breast cancer. PSF was predominantly expressed in a model of tamoxifen-resistant breast cancer cells, and depletion of PSF attenuated proliferation of cultured cells and xenografted tumors. PSF expression was significantly associated with estrogen signaling. PSF siRNA downregulated ESR1 mRNA by inhibiting nuclear export of the RNA. Integrative analyses of microarray and RNA immunoprecipitation sequencing also identified SCFD2, TRA2B, and ASPM as targets of PSF. Among the PSF targets, SCFD2 was a poor prognostic indicator of breast cancer and SCFD2 knockdown significantly suppressed breast cancer cell proliferation. Collectively, this study shows that PSF plays a pathophysiologic role in ER-positive breast cancer by posttranscriptionally regulating expression of its target genes such as ESR1 and SCFD2. Overall, PSF and SCFD2 could be potential diagnostic and therapeutic targets for primary and hormone-refractory breast cancers. SIGNIFICANCE: This study defines oncogenic roles of RNA-binding protein PSF, which exhibits posttranscriptional regulation in ER-positive breast cancer.

Sex Differences in Cognitive Impairment Induced by Cerebral Microhemorrhage.

It has been suggested that cerebral microhemorrhages (CMHs) could be involved in cognitive decline. However, little is known about the sex-dependency of this effect. Using a multimodal approach combining behavioral tests, in vivo imaging, biochemistry, and molecular biology, we studied the cortical and hippocampal impact of a CMH in male and female mice (C57BL/6J) 6weeks post-induction using a collagenase-induced model. Our work shows for the first time that a single cortical CMH exerts sex-specific effects on cognition. It notably induced visuospatial memory impairment in males only. This sex difference might be explained by cortical changes secondary to the lesion. In fact, the CMH induced an upregulation of ERα mRNA only in the female cortex. Besides, in male mice, we observed an impairment of pathways associated to neuronal, glial, or vascular functions: decrease in the P-GSK3β/GSK3β ratio, in BDNF and VEGF levels, and in microvascular water mobility. The CMH also exerted spatial remote effects in the hippocampus by increasing the number of astrocytes in both sexes, increasing the mean area occupied by each astrocyte in males, and decreasing hippocampal BDNF in females suggesting a cortical-hippocampal network impairment. This work demonstrates that a CMH could directly affect cognition in a sex-specific manner and highlights the need to study both sexes in preclinical models.

FSH Levels Are Related to E-cadherin Expression and Subcellular Location in Nonfunctioning Pituitary Tumors.

ContextGonadotroph pituitary neuroendocrine tumors (PitNETs) can express follicle-stimulating hormone (FSH) and luteinizing hormone (LH) or be hormone negative, but they rarely secrete hormones. During tumor development, epithelial cells develop a mesenchymal phenotype. This process is characterized by decreased membranous E-cadherin and translocation of E-cadherin to the nucleus. Estrogen receptors (ERs) regulate both E-cadherin and FSH expression and secretion. Whether the hormone status of patients with gonadotroph PitNETs is regulated by epithelial-to-mesenchymal transition (EMT) and ERs is unknown.ObjectivesTo study the effect of EMT on hormone expression in gonadotroph nonfunctioning (NF)-PitNETs.DesignMolecular and clinical analyses of 105 gonadotroph PitNETs. Immunohistochemical studies and real-time quantitative polymerase chain reaction were performed for FSH, LH, E-cadherin, and ERα. Further analyses included blood samples, clinical data, and radiological images.SettingAll patients were operated on in the same tertiary referral center.ResultsNF-PitNET with high FSH expression had decreased immunohistochemical staining for membranous E-cadherin (P < .0001) and increased staining for nuclear E-cadherin (P < .0001). Furthermore, high FSH expression was associated with increased ERα staining (P = .0002) and ERα mRNA (P = .0039). Circulating levels of plasma-FSH (P-FSH) correlated with FSH staining in gonadotroph NF-PitNET (P = .0025). Tumor size and invasiveness was not related to FSH staining, E-cadherin, or ERα. LH expression was not associated with E-cadherin or ERα.ConclusionIn gonadotroph PitNETs, FSH staining is related to E-cadherin, ERα expression, and circulating levels of P-FSH. There was no association between FSH staining and invasiveness. The clinical significance of these findings will be investigated in ongoing prospective studies.

Heregulin controls ERα and HER2 signaling in mammospheres of ERα-positive breast cancer cells and interferes with the efficacy of molecular targeted therapy

Estrogen receptor (ER)α and the human epidermal growth factor receptor (HER) family are inversely expressed in ERα-positive cancer in association with resistance to hormonal therapy, but the mechanism underlying their relationship remains unknown. We analyzed the effect of HER family ligands on the expression of ER and the HER family in ERα-positive MCF-7 and T47D breast cancer cell lines in 3D spheroid culture. Here, we demonstrated for the first time that heregulin-1β (HRG), a HER3 and HER4 ligand, most effectively regulated ER/HER family expression by decreasing ERα mRNA expression and increasing HER family mRNA expression. HRG treatment attenuated fulvestrant-mediated growth inhibition, and promoted the migration of MCF-7 cells. Moreover, HRG increased the CD44+/CD24− cell fraction and side population cells, both of which are recognized as prospective breast cancer stem cell markers. HRG activated both phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways. Inhibitors of these pathways reduced the growth of MCF-7 cells, but the addition of HRG has different effects on these pathways. HRG blocked the inhibitory effect of mTOR inhibitors, such as rapamycin and everolimus, on cell growth but not that of a PI3K inhibitor. Furthermore, HRG slightly decreased the inhibitory effect of an AKT inhibitor on cell growth. In contrast, HRG enhanced the MEK inhibitor-induced inhibition of cell growth. These findings suggest that HRG-stimulated signaling pathways allow ERα-positive breast cancer cells to escape from growth inhibition caused by everolimus, via MAPK signaling and/or other signaling pathways. Everolimus improves progression-free survival in combination with exemestane as second-line therapy for metastatic hormone receptor-positive breast cancer. Our study suggests that HRG is a novel target for ERα-positive breast cancer therapy.

MON-008 Estrogen-Responsive and -Unresponsive Gene Expressions Promoted by Enhancers Specifically Hypomethylated in Endometriotic Cells May Become a Molecular Marker in Endometriosis Lesions

BACKGROUND: Endometriosis is an estrogen-dependent, inflammatory disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. We sought to define an aberrant gene expression derived from an epigenetic background in endometriosis.Objective: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined estrogen receptor (ER)-dependent and -independent gene expressions promoted by active enhancers specifically hypomethylated in endometriotic cells. Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma.Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was examined using RT-PCR. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies and eRNA expression analysis. Using ChIP-seq and ChIA-PET analysis in silico, ER-specific loci within gene bodies and the up- and downstream regions were extracted. ER-dependent gene expression was examined using estradiol or SERM.Results: ER expression in endometriotic cells.1) Relative expression of ERα mRNA was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is at a comparable level of the ERα. 3) ERβ2 mRNA expression was at a comparable level of the ERβ1. From our DNA methylation and gene expression analysis, 6 genes were selected and classified into 3 categories: estrogen-responsive genes with specific methylation (ESR1 and ESR2) or without any methylation (TGFα and GREB1), and estrogen-unresponsive but upregulated genes depending on specific hypomethylation (GATA6 and CYP19). 4) ChIP-seq and ChIA-PET analysis in silico suggested the presence of ER-specific loci within gene bodies and the up- and downstream in estrogen-responsive genes. 5) ChIP and eRNA expression analysis predicted active enhancer regions both in estrogen-responsive and -unresponsive genes. 6) In response to estrogen, TGFα and GREB1 expressions were upregulated, but ESR1 and ESR2 showed marginal responses.Conclusion: We focused on estrogen-responsive and -unresponsive genes linked to the epigenetic environment of endometriotic lesions, and revealed a facet of gene expression in endometriotic cells.

Effects of UHRF1 on Estrogen Receptor and Proliferation, Invasion and Migration of BCPAP Cells in Thyroid Papillary Carcinoma

To investigate the effects of ubiquitin-like PDH and ring finger domain 1 (UHRF1) on the expression ratio of estrogen receptor (ER) α/ERβ, and to explore the experimental mechanism of UHRF1 affecting the proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma. The protein and mRNA expressions of UHRF1, ERα and ERβ in normal thyroid Nthy-ori3-1 cells and thyroid papillary carcinoma BCPAP cells were detected by Western blot and qRT-PCR. BCPAP cells were treated with Scrambled siRNA and UHRF1 siRNA, respectively. The expressions of ER α and ER β mRNAs were detected by qRT-PCR. MTT and Transwell were used to determine the proliferation, invasion and migration in each group of BCPAP cells. Compared with Nthy-ori3-1 cells, the expressions of UHRF1 and ERα proteins and mRNAs in BCPAP cells were significantly up-regulated ( P<0.05), while the expressions of ERβ protein and mRNA were significantly down-regulated ( P<0.05). Compared with the control group and Scrambled siRNA group, the expression of ER α mRNA in BCPAP cells transfected with UHRF1 siRNA was significantly decreased ( P<0.05), while the expression of ER β mRNA was significantly increased ( P<0.05). The proliferation, invasion and migration of BCPAP cells transfected with UHRF1 siRNA were significantly decreased ( P<0.05). UHRF1 upregulates ERα/ERβ expression ratio and promotes proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma.

Good prognosis for follicular lymphoma with estrogen receptor α-positive follicular dendritic cells.

Follicular lymphoma (FL) has a meshwork of follicular dendritic cells (FDCs). We previously demonstrated the presence of estrogen receptor alpha (ERα)+CD23+ FDCs in grades 1‐2 FL. The significance of FDCs as a prognostic factor in FL remains unknown. The current study aimed to compare clinicopathological features, including prognosis, between FL with and without ERα+ FDCs. This study evaluated the clinicopathological significance of ERα expression in 70 FL patients by immunostaining. The presence of ERα mRNA on FDCs from 5 FL patients was confirmed by CD21/ERα double staining (immunohistochemistry and in situ hybridization). We defined patients with frequent ERα expression as the ERαhigh group and those with infrequent ERα expression as the ERαlow group. Thirty‐two patients were assigned to the ERαhigh group (45.7%), and 38 patients were assigned to the ERαlow group (54.3%). Both overall survival (OS) and progression‐free survival (PFS) were significantly better in the ERαhigh group than in the ERαlow group (OS, log‐rank, P = .0465; PFS, log‐rank, P = .0336). Moreover, high ERα expression on FDCs was an independent prognostic factor for OS in both the univariate ([hazard ratio] HR, 0.163; P = .0260) and multivariate (HR, 0.050; P = .0188) analyses and for PFS in both the univariate (HR, 0.232; P = .0213) and multivariate (HR, 0.084; P = .0243) analyses. ERα mRNA expression was detected in CD21+ FDCs within the neoplastic follicles of FL patients. In conclusion, a neoplastic follicular microenvironment with ERα‐positive FDCs might affect the grade and presence of the follicular pattern of FL and improve patient prognosis.

Nuclear Receptors CAR, ERα and RORα Communicate via Phosphorylation to Differentially Regulate the SULT1E1 Gene Expression in Diabetic and Nondiabetic Human Livers

Studies showed that SULT1E1 is differentially expressed in nondiabetic and diabetic human as well as mouse livers. Here, we intended to investigate the molecular mechanism of the differential expression of SULT1E1 in diabetic and nondiabetic human livers. Utilizing chromatin immunoprecipitation (ChIP) in male human livers and coimmunoprecipitation (CoIP) assays, we report that nuclear receptors CAR, ERα and RORα communicated via phosphorylation of their respective DNA binding domains to differentially regulate the expression of SULT1E1 gene in nondiabetic and diabetic (type 2) male human livers. We studied the functional role of ERα and RORα with respect to SULT1E1 expression in human primary hepatocytes by treating the cells with siRNAs against ERα or RORα and SULT1E1 mRNAs were suppressed along with ERα or RORα mRNAs. Moreover, promoter analysis, electrophoretic mobility shift assays and cell‐based reporter assays in COS‐1 cells identified three DNA elements: ERα response element (h1E1‐ERE) and RORα response element (h1E1‐RORE) within the enhancer region and a TATA box within the proximal promoter that were critical for the regulation of SULT1E1 gene in human liver cells. ChIP assays using antibodies against ERα, p‐Ser212 ERα, RORα or p‐Ser100 RORα and primers targeting the proximal SULT1E1 promoter revealed that both receptors were phosphorylated and enriched on the proximal region of SULT1E1 gene promoter in nondiabetic but not in diabetic male human livers. Similar observations were also made in ChIP assays performed with antibody against CAR. Moreover, CoIP assay in HepG2 cells overexpressing ERα WT or its ERα S212D mutant and RORα WT, RORα S100A or RORα S100D displayed a stronger interaction between ERα S212D and RORα S100D and this interaction was further enhanced with CAR cotransfection. Altogether, phosphorylation driven interaction between nuclear receptors CAR, p‐Ser212 ERα and p‐Ser100 RORα was critical for a positive expression SULT1E1 gene in human livers and lack thereof might be responsible for the observed downregulation of SULT1E1 in diabetic livers.Support or Funding InformationNIEHS ‐ NIH Intramural Research Program

Perfluorooctanoic Acid (in the Presence of Fetal Bovine Serum) Induces Proliferation in ERα Positive and ERα Negative Breast Cancer Cell Lines

Perfluorooctanoic acid (PFOA) is a synthetic chemical belonging to a larger group of fluorotelomers. These compounds have been used in the production of both industrial and consumer products as surfactants and due to their chemical nature are environmentally persistent pollutants. Individuals can be exposed to PFOA through ingesting PFOA‐contaminated water or food. PFOA has been shown to have a half‐life of approximately 4 years in humans. While the long‐term effects of PFOA are largely unknown, there is increasing evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates peroxisome‐proliferator‐activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα).Previous experiments in our lab demonstrated that PFOA treatment of MCF‐7 breast cancer cells (an ERα positive cell line) decreased expression of ERα mRNA and protein, and decreased cell viability by ~20% within 48h of treatment compared to DMSO controls. However, these cells were treated in the absence of fetal bovine serum (FBS). When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF‐7 cells treated with 50μM and 100μM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in this PFOA‐induced proliferation, we carried out additional experiments in MCF‐7 cells along with experiments in another ERα‐positive cell line, T47D, as well as an ERα‐negative cell line, MDA‐MB‐231. There was a tendency for increased viability in all three cell lines when treated with PFOA, although only the MDA‐MB‐231 cells and the T47D cells showed a statistically significant (~20%) increase at 48h with 50μM PFOA and 100μM PFOA, respectively. These data suggest that the PFOA‐induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF‐7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.Support or Funding InformationAugusta University Department of Biological Sciences, Augusta University Center for Undergraduate Research and Scholarship

Spatial and temporal changes in the expression of steroid hormone receptors in mouse model of endometriosis.

Endometriosis is recognized as a steroid hormone-dependent disorder. However, controversies exist regarding the status of the steroid hormone receptor expression in endometriotic tissues. The purpose of this study was to determine the ontogeny of cellular changes in the expression of estrogen receptors (ERα, ERβ), G protein-coupled estrogen receptor 1 (GPER1), and progesterone receptors (PRs) in endometriosis using a mouse model. We used the autologous uterine tissue transfer mouse model and studied the mRNA and protein expression of ERα, ERβ, GPER1, and PR in ectopic lesions at 2, 4, and 8weeks of induction of endometriosis. As compared to endometrium of controls, in the ectopic endometrium, ERα is reduced while ERβ was elevated in stromal cells; however, Gper1 and PR levels are reduced in both stromal and epithelial cells in a time-specific manner. There is a high inter-animal variation in thelevels of these receptors in ectopic endometrium as compared to controls; the levels also varied by almost 100-fold within the same lesion resulting in "micro-heterogeneity." The expression of all these receptors also deferred between two lesions from the same animal. In the endometriotic tissue, there is extensive inter-animal and intra-lesion heterogeneity in the expression of ERα, ERβ, GPER1, and PR. These changes are not due to the influence of the peritoneal environment but appear to be tissue intrinsic. We propose that the variable outcomes in hormonal therapy for endometriosis could be possibly due to heterogeneity in the expression of steroid hormone receptors in the ectopic endometrium.

The effects of estradiol are modulated in a tissue-specific manner in mice with inducible inactivation of ERα after sexual maturation.

Mouse models with lifelong inactivation of estrogen receptor-α (ERα) show that ERα is the main mediator of estrogenic effects in bone, thymus, uterus, and fat. However, ERα inactivation early in life may cause developmental effects that confound the adult phenotypes. To address the specific role of adult ERα expression for estrogenic effects in bone and other nonskeletal tissues, we established a tamoxifen-inducible ERα-inactivated model by crossing CAGG-Cre-ER and ERαflox/flox mice. Tamoxifen-induced ERα inactivation after sexual maturation substantially reduced ERα mRNA levels in cortical bone, trabecular bone, thymus, uterus, gonadal fat, and hypothalamus, in CAGG-Cre-ERαflox/flox (inducible ERαKO) compared with ERαflox/flox (control) mice. 17β-estradiol (E2) treatment increased trabecular bone volume fraction (BV/TV), cortical bone area, and uterine weight, while it reduced thymus weight and fat mass in ovariectomized control mice. The estrogenic responses were substantially reduced in inducible ERαKO mice compared with control mice on BV/TV (-67%), uterine weight (-94%), thymus weight (-70%), and gonadal fat mass (-94%). In contrast, the estrogenic response on cortical bone area was unaffected in inducible ERαKO compared with control mice. In conclusion, using an inducible ERαKO model, not confounded by lack of ERα during development, we demonstrate that ERα expression in sexually mature female mice is required for normal E2 responses in most, but not all, tissues. The finding that cortical, but not trabecular bone, responds normally to E2 treatment in inducible ERαKO mice strengthens the idea of cortical and trabecular bone being regulated by estrogen via different mechanisms.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

Effect of cassic acid on immunity and immune-reproductive genes transcription in Clarias gariepinus against Edwardsiella tarda

The present study was investigated the dietary administration of cassic acid (CA) on growth, innate immunity, transcription profiles of estrogen and follicle-stimulating hormones as well as lysozyme enzyme determined in Clarias gariepinus against Edwardsiella tarda. The weight gain (WG), protein efficacy ratio (PER), and feed conversion ratio (FCR) were significantly improved in infected fish fed dietary administration with CA at 5 and 10 mg kg−1 diets. The survival is higher (96.7% and 98.3%) in the infected groups fed at 5 and 10 mg kg−1 CA diets. The red (RBC) and white (WBC) blood cells, hemoglobin (Hb), and packed cell volume (PCV) was found significantly high in the infected fish feeding at 5 and 10 mg kg−1 CA diets. Total protein and albumin were significantly increased with 5 and 10 mg kg−1 CA diets among weeks 1–4 while the globulin and albumin: globulin ratio increased of these diet only after week 2. The phagocytic and respiratory burst activities were enhanced statistically the infected fish fed at 10 mg kg−1 CA diet group whereas the production of superoxide anion (SOA) and nitric oxide (NO) were significantly increased at 5 and 10 mg kg−1 CA diets. The lymphocyte proliferation and myeloperoxidase (MPO) activity were significantly high the infected fish when fed at 5 and 10 mg kg−1 CA diets after 2nd week whereas the alternate complement activity (ACP), generation of reactive oxygen species (ROS), and lysozyme activity (Lyz) were observed at 5 and 10 mg kg−1 CA diets among weeks 1–4. The accumulative mortality was 10% in infected fish fed at 1 and 5 mg kg−1 CA diets whereas 15% mortality found with 10 mg kg−1 CA diet. The highest levels of estrogen receptor alpha (ERα) mRNA expression found in gonad while the highest levels of follicle stimulating hormone-beta subunit (FSH-β) mRNA expression found in testis of the infected fish given at 5 and 10 mg kg−1 CA diets. The up-regulation of chick-type lysozyme (c-Lyz) mRNA was observed at 5 and 10 mg kg−1 CA diets after 2nd week while goose-type lysozyme (g-Lyz) mRNA was up-regulation amongst weeks 1–4 of these diets. The present study suggested that E. tarda challenged fish after feeding with 5 and 10 mg kg−1 CA diets did not affect growth and hemato-biochemical parameter, but it enhanced nonspecific immune system and improving ERα, FSH-β, c-Lyz, and g-Lyz mRNA expression in C. gariepinus against E. tarda.

English

Recognized for its traditional roles, estrogen is ever-present in all vertebrates, regulates reproduction by binding and activating estrogen receptors (ERs), and also controls several functions of vertebrates, including reproductive immune, and central nervous systems. In order to access any other possible functions of the estrogen receptors in the development of the juvenile Hybrid grouper (Epinephelus fuscoguttatus â™€× Epinephelu polyphekadion ♂), full-length of ERs cDNA sequences were isolated and analyses were found to be 2391 bp for hgERα, 2626 bp for gERb1 and 2339 bp for hgERb2, respectively. The results of amino acid and phylogenetic analysis revealed that each hgER was grouped in consistent taxonomic groups of perciformes and demonstrated great evolutional conservation in functional domains. Real-time PCR examination discovered that the receptors expressed in all tissues examined, though, at a different level, the ERα mRNA level expressed higher than ERβ1, and ERβ2 in tissues examined. The ERα mRNA level of expression was found to be highest in the tissue of the heart, followed by muscle, and liver. The ERβ1 mRNA level was greatest in heart tissue, trailed by liver and muscle and ERβ2 was highest in the heart trailed by stomach and liver. The minimal expression was recorded in the kidney, the gill, and the brain for ERα, ERβ1, and ERβ2 respectively. These results put forward that steroid hormone estrogen receptors might be playing a significant part in the controlling of social behavior complexity, plasticity behavior, and the assessment of a gratifying inducement in Hybrid grouper. Key words: Estrogen receptors, Real-time POR, tissue expression, hybrid grouper.

Copper and lead exposures disturb reproductive features of primary endometrial stromal and epithelial cells

This study investigates if Cu and Pb act as endocrine disruptors affecting endometrial cells. Primary EnSCs and EnECs were exposed to Cu (0, 50, 100 and 200 μM) or Pb (0, 30, 100 and 500 μM) and assessed for viability, decidualization, apoptosis and proliferation on EnSCs, and wound healing and adhesion capabilities on EnECs. Cu exposure decreased significantly cell viability in a dose-dependent manner. Cu and Pb negatively affected in vitro decidualization, showing a significant decrease in PRL secretion. HOXA10 and ERα mRNA levels significantly decreased in decidualized cells (dEnSCs) exposed to Cu. Cu and Pb decreased adhesion and regeneration capability in EnEC. This study reveals that Cu and Pb could negatively affect endometrial functionality, compromising the decidualization process and disrupting endometrial regeneration and embryo adhesion. Therefore, special care should be taken considering heavy metals exposure if pregnancy is being pursued.

Epigenetic changes associated with increased estrogen receptor alpha mRNA transcript abundance during reproductive maturation in chicken ovaries

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that regulates cellular responses to estrogens and transcription processes of target genes. In this study, changes in DNA methylation and histone modifications in the promoter region and Exon 1 of the ERα gene were analyzed to ascertain epigenetic changes associated with increased ERα mRNA abundance during reproductive maturation from 90 (egg production not yet initiated) to 160 (after egg production was initiated) d of age (d post-hatching) in chicken ovaries. The results indicate there was no difference in CpG methylation at the promoter and Exon 1 except at the region analyzed with primer pairs F2 and R2, where percentage of methylated CpG of Sites 2 and 8 after reproductive maturation was greater compared with before reproductive maturation. By using the chromatin immunuoprecipitation (ChIP) assay combined with SYBR green quantitative PCR, effects of histone modifications were evaluated, including histone H3K4 di + tri methylation, H3K9 phosphorylation and trimethylation, H3K36 methylation and H3K27 acetylation on chicken ERα mRNA transcript abundance. The results indicated that there was a greater histone H3K27 acetylation and lesser H3K36 trimethylation associated with increased abundance of ERα mRNA transcript in chicken ovaries after reproductive maturation (90 compared with 160 d of age). In consistent with this finding, the relative abundance of transcriptional coactivator p300 mRNA transcript and protein in the ovaries was markedly greater in reproductively mature than immature chickens. Findings provide insights into the epigenetic regulations of the chicken ERα gene expression that is required for chicken ovarian development.

The estrogenic proliferative effects of two alkylphenols and a preliminary mechanism exploration in MCF-7 breast cancer cells.

Bisphenol A (BPA) and 4-cumylphenol (4-CP), as estrogen-like chemicals, are ubiquitous in the environment media and associated with the occurrence and development of hormone-dependent tumors. However, the combinatorial effects of these two structurally similar alkylphenols are not well informed. In the present study, the classic breast cancer cell line MCF-7 was used as in vitro model to estimate the estrogenic proliferative effects of BPA and 4-CP. MTT assay, reactive oxygen species, cell apoptosis, cell cycle, and real-time fluorescent quantitative Step One Plus Real-time PCR System (Applied Biosystems, CA, USA) were applied to explore their proliferative mechanisms. MTT results showed that both BPA and 4-CP ranging from 10-9 to 10-5 M stimulated cell proliferation in a nonmonotonic dose-response manner. Along with the proliferative effects, cell cycle was progressed from G0/G1 to S and G2/M phase. Meanwhile, the expression levels of ERα, pS2, and Bcl-2 mRNA were also upregulated. In contrast, 4-CP and BPA at high dose (10-4 M) obviously displayed antiproliferative effects in MCF-7 cells via inducing cell apoptosis and blocking cell cycle in G0/G1 phase. As expected, the relative expression levels of ERα, pS2, and Bcl-2 mRNA were decreased, whereas Bax mRNA was increased. Interestingly, the proliferative or antiproliferative effects of 4-CP were higher than that of BPA. Moreover, coexposure of lower concentrations BPA and 4-CP significantly induced cell proliferation in a synergistic manner. These findings indicated that the potential environmental risks of coexposure of BPA and 4-CP were greater than either of them.

Molecular Cloning and Abiotic Stress Expression Analysis of Gtpase Era Gene in Mulberry (Morus Alba L.)

Era (E. coli RAS-like protein) forms a unique family of GTPase, its functions in cell cycle control by coupling cell division with growth rate. In the present paper, a cDNA sequence coding GTPase Era mRNA was cloned from mulberry. Sequence analysis showed that its open reading frame (ORF) is 1275 bp in length encoding a protein of 424 amino acids. The GTPase Era gene had era domain and belonged to the FeoB_N superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the GTPase gene from various species showed that the mulberry was closely related to Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum pennellii, Nicotiana sylvestris and Nicotiana tubacum. The expression patterns of GTPase Era treated under drought, low temperature and salt stresses were examined using real-time quantitative PCR (RT-qPCR). The expression level of the mRNA had a significant change under drought, cold and salt stress treatments compared to the normal growth environment with the maximum expression on third day for drought stress, the sixth day for the low temperature stress and the seventh day for the salt stress. These data provide a better understanding of the molecular basis of signal transduction during stress responses in mulberry trees

Effects of Puerarin on the Ovariectomy-Induced Depressive-Like Behavior in ICR Mice and Its Possible Mechanism of Action.

Daily treatment of ovariectomized (OVX) ICR mice with puerarin, a glycosyl isoflavone isolated from the root bark of Pueraria candollei var. mirifica, and 17β-estradiol attenuated ovariectomy-induced depression-like behavior, as indicated by a decrease in immobility times in the tail suspension test (TST) and the forced swimming test (FST), an increase in the uterine weight and volume, a decrease in serum corticosterone levels, and dose-dependently normalized the downregulated transcription of the brain-derived neurotrophic factor (BDNF) and estrogen receptor (Erβ and Erα) mRNAs. Like 17β-estradiol, puerarin also inhibited ovariectomy-induced suppression of neurogenesis in the dentate gyrus of the hippocampus (increased the number of doublecortin (DCX)-immunosuppressive cells). These results suggest that puerarin exerts antidepressant-like effects in OVX animals, possibly by attenuating the OVX-induced hyperactivation of the HPA axis and/or normalizing the downregulated transcription of BDNF and ER mRNA in the brain.

Blocking of Estradiol Receptors ERα, ERβ and GPER During Development, Differentially Alters Energy Metabolism in Male and Female Rats

Estradiol not only participates in the regulation of energy metabolism in adulthood, but also during the first stages of life as it modulates the alterations induced by under- and over-nutrition. The objectives of the present study were to determine: 1) If estradiol is involved in the normal programming of energy metabolism in rats; 2) If there is a specific window of time for this programming and 3) If males and females are differentially vulnerable to the action of this hormone. Estrogen receptors (ER) α, ERβ and GPER were blocked by their specific antagonists MPP, PHTPP and G15, respectively, from postnatal day (P) 1 (the day of birth) to P5 or from P5 to P13. Physiological parameters such as body weight, fat depots and caloric intake were then analysed at P90. Hypothalamic AgRP, POMC, MC4R, ERα, ERβ and GPER mRNA levels and plasma levels of estradiol, were also studied. We found that blocking ER receptors from P5 to P13 significantly decreases long-term body weight in males and hypothalamic POMC mRNA levels in females. The blocking of ERs from P1 to P5 only affected plasma estradiol levels in females. The present results indicate programming actions of estradiol from P5 to P13 on body weight in male and POMC expression in female rats and emphasize the importance of including both sexes in metabolic studies. It is necessary to unravel the mechanisms that underlie the actions of estradiol on food intake, both during development and in adulthood, and to determine how this programming differentially takes place in males and females.