Progesterone Receptor mRNA Expression Research Articles

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10−9 M or 1 × 10−6 M BPA; or c) 1 × 10−12 M, 1 × 10−9 M or 1 × 10−6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. Total PR expression was also increased with 1 × 10−6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10−12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10−9 M BPA or 1 × 10−12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10−9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.

Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17β-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

Local sex steroid hormone milieu in the bovine oviduct ipsilateral and contralateral to preovulatory follicle or corpus luteum during the periovulatory phase

Estradiol-17β (E2) and progesterone (P4) regulate oviductal functions, providing a suitable environment for the transport and maturation of gametes, fertilization, and embryonic development. In addition to the E2 and P4 nuclear receptors, estrogen receptor (ESR) α and β, nuclear progesterone receptor (PGR), nongenomic mechanisms through G protein-coupled estrogen receptor (GPER1), and progesterone receptor membrane component (PGRMC) 1 and 2 mediate E2 and P4 actions. This study aimed to characterize the local endocrine environment of the oviduct by examining the oviductal E2 and P4 concentrations and their receptors' mRNA expression during the periovulatory phase. The bovine oviducts were collected in a slaughterhouse and the days postovulation were estimated according to state of the ovaries and the uterus. Samples of the ampulla and isthmus ipsilateral and contralateral to the preovulatory follicle or corpus luteum were collected on Days 19 to 21, Days 0 to 1, Days 2 to 4, and Days 5 to 7 of the estrous cycle. The effects of the estrous cycle phase and oviductal region (ampulla and isthmus) and side (ipsilateral and contralateral) were analyzed by 3-way ANOVA. Moreover, to clarify the regulatory mechanisms of the mRNA expression of hormone receptors, the effects of E2 and P4 on mRNA expression in the oviduct were examined by multiple linear regression. The oviductal endocrine milieu on Days 19 to 21 was characterized by an E2-dominant environment with high E2 and low P4, high ESR1 and PGR mRNA expression, and low ESR2, GPER1, and PGRMC2 mRNA expression, whereas the corresponding on Days 0 to 1 was characterized by the endocrine milieu without hormone dominance. The environment on Days 2 to 4 and Day 5 to 7 was characterized by opposite tendency of oviductal hormone concentrations and their receptors' mRNA expression to Days 19 to 21. Additionally, the ipsilateral oviduct had the more P4-dominant endocrine milieu, with lower E2 and higher P4 concentrations, and different expression of ESR1/2, GPER1, PGR, and PGRMC2 mRNA when compared with the contralateral oviduct on Days 2 to 4 and Days 5 to 7, except for PGRMC1. Although oviductal E2 and P4 influenced the mRNA expression of ESR1/2, GPER1, PGR, and PGRMC1/2, their effects were different between regions and sides. In summary, the oviductal endocrine milieu varies according to the estrous cycle phase and the oviductal region and side, which may be involved in the estrous cycle phase-specific and oviductal region-specific and side-specific functions.

Progesterone's role in deep infiltrating endometriosis: Progesterone receptor and estrogen metabolism enzymes expression and physiological changes in primary endometrial stromal cell culture

To study progesterone signaling activation, we measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) using a cytosensor microphysiometer and assessed progesterone receptor (PR) and estrogen metabolism enzymes mRNA expression in cultured endometrial cells from women with deep infiltrating endometriosis and healthy controls using real-time quantitative PCR. This study was conducted at a University hospital and included patients with and without deep infiltrating endometriosis (DIE). Primary endometrial stromal cells (ECs) from women with DIE and controls were treated with 17β-estradiol and progesterone prior to microphysiometer measurements and qPCR evaluations. Decreased progesterone responsiveness and decreased total nuclear PR and HSD17B1 mRNA expression were observed in cultured ECs from women with deep infiltrating endometriosis relative to those from control samples before and after hormone treatment. These cells also showed increased 17β-hydroxysteroid dehydrogenases types 2 (HSD17B2) relative to control group and increased expression of aromatase (CYP19) after exposure to progesterone. These physiological and expression patterns observed in ECs cultures from women with DIE reinforces previous findings in the literature supporting the progesterone resistance hypothesis in the pathogenesis of endometriosis.

Change of Ras and its guanosine triphosphatases (GTPases) during development and regression in bovine corpus luteum

The aim of this study was to determine the change of Ras and its guanosine triphosphatases (GTPases) proteins in the bovine corpus luteum (CL) during estrous cycle and investigate protein-protein interaction between hormone receptors and Ras proteins via angiogenetic and apoptotic factors using bioinformatics database. The bovine CLs at proliferation phase (PP), secretion phase (SP), and regression phase (RP) were dissected from abattoir ovaries (n = 4/stage), whole of the tissue samples was used to analyze two-dimensional electrophoresis (2-DE), mRNA, and protein analysis. The protein-protein interaction between the Ras GTPases proteins and hormone receptors were analyzed using Search Tool for the Retrieval of Interacting Genes (STRING) database. The Ras protein activator like 3 (RASAL3), Ras GTPase activating protein 3 (RASA3), Ras guanine nucleotide exchange factors 1 beta (RasGEF1B) were discovered by the 2-DE and mass spectrometry in bovine CLs, and the protein spots of RASA3 and RASAL3 were significantly increased in the SPCL compared to the PPCL, whereas the RasGEF1B was reduced in the PPCL (P < 0.05). The mRNA and proteins expression of progesterone receptor, estrogen receptor alpha (ERα), vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang1), VEGF receptor2 (VEGFR2), and Tie2 were significantly increased, but intrinsic and extrinsic apoptotic factors were decreased in PPCL and SPCL compared to RPCL (P < 0.05). Based on STRING database, we determined that RasGEF1B is activated by ERα via VEGFA and VEGFR2, then RasGEF1B activates H-Ras and R-Ras. In addition, the RasGAP protein was significantly increased, however, the RasGEF, H-Ras and R-Ras proteins were reduced in SPCL compared to PPCL and RPCL (P < 0.05). In summary, the RasGEF and Ras proteins were raised during the development, whereas the RasGAP was increased when development was completed, then the Ras and its GTPases dramatically decreased at the regression in bovine CL. In conclusion, these results suggest that Ras and Ras GTPases could be changed during development and regression, activated by the ERα via angiogenetic signaling during proliferation, and may be important to understanding of the Ras and its GTPases system for estrous cycle in bovine CL.

Causal associations of autoimmune thyroiditis and papillary thyroid carcinoma: mRNA expression of selected nuclear receptors and other molecular targets.

Potential causal associations of autoimmune thyroiditis (AIT) and papillary thyroid carcinoma (PTC) have been studied previously. The mRNA expression patterns of thyroid hormone receptors (TR), retinoid receptors (RAR), rexinoid receptors (RXR), dihydroxyvitamin D3 receptors (VDR), and progesterone receptors (PR) in PTC tissue of patients without autoimmune thyroiditis (PTC/AIT-) and in PTC tissue of patients with coexisting AIT (PTC/AIT+) have been investigated in order to judge whether the observed changes may take part in the promotion and progression of thyroid cancer. Tumours with or without AIT were classified histologically and the semiquantitative PCR was performed. The results revealed that there was decreased expression of TRα, TRβα, RARα and PR mRNA in PTC/AIT+ tumours when compared with PTC/AIT- tumours. Decreased expression of RARα in PTC/AIT+ was detected when compared with PTC/AIT- patients. A similar effect of AIT was observed with a decrease in RARγ expression in PTC/AIT+ patients. On the other hand, there was an increased expression of VDR in thyroid tumours (PTC/AIT+) when compared with PTC/AIT-. PR mRNA was decreased in the thyroid tumours of PTC/AIT+ patients when compared with PTC/AIT- patients. In addition, there was an increased expression of MKi67 and complement C3 in PTC of PTC/AIT+ when compared with PTC/AIT-. In the PTC/AIT+ group, a decreased level of IGF-1 mRNA was found when compared with the PTC/AIT- group. According to the significant differences of the studied markers in PTC/AIT+ compared with PTC/AIT-, it was indicated that AIT may be a predisposing factor for the development of PTC.

Aromatase CYP19A1, progesterone receptor PGR and estrogen receptor ESR1 gene expression in biopsy specimens of endometrioid heterotopia and endometrial tissue by reverse transcription PCR

Hypothesis/aims of study. Endometriosis is one of the most pressing problems of gynecology. Clarifying the expression of the estrogen receptor (ESR1) and the progesterone receptor (PGR) genes and polymorphisms in the aromatase (CYP19A1) gene in endometriosis will expand the understanding of the pathogenesis of the disease and the causes of resistance to its therapy. The objective of this study was to conduct a comparative analysis of mRNA expression of PGR, ESR1 and CYP19A1 genes in paired samples of the eutopic endometrium and peritoneal endometrioid lesions in order to search for predictive markers of response to hormonal therapy. In the future, this may allow personalizing the selection of hormonal preparations for the treatment of endometriosis.&#x0D; Study design, materials and methods. Reverse transcription real-time PCR made it possible to evaluate CYP19A1, PGR and ESR1 gene expression levels in studied tissue samples from 22 patients with endometriosis and 9 women in the comparison group.&#x0D; Results. Quantitative analysis revealed a high heterogeneity in the expression level of the studied genes, in both the endometrium and endometrioid lesions from patients with endometriosis. In the endometrium of patients in the comparison group, the heterogeneity of the expression level was observed only for the ESR1 gene.&#x0D; Conclusion. Our findings suggest a high variability in CYP19A1, ESR1 and PGR gene expression levels in the endometrium and peritoneal foci in patients with endometriosis. This information indicates the need for an individual approach to prescribing targeted therapy, since it is obvious that the effect of treatment will depend primarily on the availability of a therapeutic target in a particular patient. The absence of a typical expression pattern for each of the genes in patients with endometriosis indicates the heterogeneity of the disease and the need to develop a molecular classification of this common pathology.

Relationships Between Neurofibromatosis-2, Progesterone Receptor Expression, the Use of Exogenous Progesterone, and Risk of Orbitocranial Meningioma in Females.

Background: The pathogenesis of meningioma in females and its association with exogenous progesterone is remained unclear. This study was aimed to examine expression of Progesterone receptor (PR) and Neurofibromatosis-2 (NF2) and assess their relationships to history of exogenous progesterone use and risk of meningioma.Methods: Our study was a case-control study that involves 115 females, 40 cases who diagnosed with orbito-cranial meningioma and 75 controls of healthy, that has been presented in previous study. The demographic characteristics, reproductive factors, and history of progesterone use were obtained in–depth face-to-face interviews. PR and NF2 mRNA were assessed by real-time quantitative polymerase chain reaction (RT-qPCR) on serum specimens.Results: The mean age of participants in cases vs. controls were 46.6 ± 6.2 vs. 46.5 ± 7.45 (P = 0.969). The expression of PR and NF2 in cases was significantly lower than in controls. The longer duration of progesterone exposure was significantly associated with lower expression of PR and NF2. Significant association between lower expression of PR (OR 11.7; 95% CI 4.17–32.9; P < 0.001 comparing the lowest quartile vs. 3 highest quartile of PR) and NF2 (OR 4.23; 95% CI 1.85–9.67; P = 0.001 comparing the 2 lowest quartiles vs. 2 highest quartiles) with increased risk of meningioma were also reported.Conclusion: In this study we showed that the longer the exposure to exogenous progesterone, the lower the expression of PR and NF2 mRNA in the serum. Low expression of PR and NF2 were associated with higher risk of meningioma, suggesting that low PR expression and inactivation of NF2 might play a key role in progesterone-associated meningioma tumorigenesis and may be potential clinical marker for females at higher risk of meningioma.

Analysis of the prognostic relevance of sex-steroid hormonal receptor mRNA expression in muscle-invasive urothelial carcinoma of the urinary bladder.

Muscle-invasive urothelial carcinoma of the urinary bladder (UCB) often recurs following radical cystectomy (RC). An altered expression of sex-steroid hormone receptors has been associated with oncological outcomes of UCB and may represent therapeutic targets. Here the expression of different hormone receptors was measured on mRNA levels in patients treated by RC and associated with outcomes. Androgen receptor (AR), estrogen receptor 1 (ESR1), and progesterone receptor (PGR) mRNA expression was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in RC samples of 87 patients with a median age of 66 (39-88) years. Univariate and multivariate analyses were performed to test associations with pathological and clinical characteristics as well as recurrence-free (RFS) and disease-specific survival (DSS). AR mRNA expression was lower in comparison with ESR1 and PGR expression (p < 0.0001). In univariate analysis, high expression levels of AR were associated with reduced RFS (HR 2.8, p = 0.015) and DSS (HR 2.8, p = 0.010). High AR mRNA expression and a positive lymph node status were independent predictors for reduced RFS (HR 2.5, p = 0.0049) and DSS (HR 3.4, p = 0.009). In patients with low AR mRNA expression, an increased ESR1 and PGR mRNA expression were associated with reduced RFS and DSS. High expression levels of AR are significantly associated with adverse outcome in patients with muscle-invasive UCB following RC. ESR1 and PGR expression status can further stratify patients with low AR expression into subgroups with significantly reduced RFS and DSS. Therapeutic targeting of AR may influence outcomes in patients with UCB.

Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells.

Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.

Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

Prenatal undernutrition suppresses sexual behavior in female rats

Infectious, psychological and metabolic stresses in the prenatal and early neonatal period induce long-lasting effects in physiological function and increase the risk of metabolic disorders later in life. We examined the sexual behavior of female rats that were subjected to undernutrition in the prenatal period. Eight pregnant rats were divided into two groups: a maternal normal nutrition group (mNN; n = 4) and a maternal undernutrition group (mUN; n = 4), which received 50% of the daily food intake amount of the mNN group from gestation day 13 to delivery. Nine and seven female offspring were randomly selected from the mNN and mUN groups, respectively. Vaginal opening (VO), estrous cycle length, sexual behavior and mRNA expression levels of the factors that regulate sexual behavior were observed. In the mUN group, VO day was later, the estrous cycle was longer, and the lordosis quotient and lordosis rating were lower than in the mNN group; such differences were not seen in other sexual performances, such as ear wiggles, darts, kick bouts and box. The hypothalamic mRNA expression level of progesterone receptor (PR) A + B and oxytocin (OT) were significantly lower in the mUN group than in the mNN group. These findings indicated that prenatal undernutrition disrupted puberty onset, the estrous cycle, sexual behavior and hypothalamic mRNA expression of PR and OT in female rat pups.

MiR-194-3p Represses the Progesterone Receptor and Decidualization in Eutopic Endometrium From Women With Endometriosis.

Progesterone resistance in the eutopic endometrium (EuE) is suggested to be a critical factor for decreased endometrial receptivity and implantation failure in reproductive-aged women with endometriosis. Altered expression of miRNAs has been reported to play an important role in the pathophysiology of endometriosis-associated infertility. However, the underlying mechanisms of aberrant progesterone receptor (PR) and deficient decidualization regulated by miRNAs in endometriosis have not been thoroughly elucidated. The goal of this study was to explore the regulation and roles of miR-194-3p in aberrant PR expression and impaired decidualization in endometrial stromal cells (ESCs) from the EuE of women with mild or minimal endometriosis. Using a series of studies, we observed decreased PR mRNA expression and an increasing PR-A/PR-B mRNA ratio trend in the midsecretory phase of the EuE of women with minimal or mild endometriosis (n = 19) compared with controls (n = 14); the increased expression of miR-194-3p in the endometriosis group was consistent with previous microarray analysis. We also found that PR protein levels were inhibited by the transfection of ESCs with an miR-194-3p mimic and upregulated by miR-194-3p inhibition. As predicted by the bioinformatic analysis, the 3'-untranslated region luciferase assay indicated the direct regulation of PR expression by miR-194-3p. Furthermore, miR-194-3p overexpression inhibited the in vitro decidualization of ESCs via both cellular morphological changes and prolactin levels. Therefore, our study demonstrated that miR-194-3p contributes to progesterone resistance in endometriosis, which hinders fertility by repressing the levels of PR and decidualization in the EuE. Thus, miR-194-3p regulation is a future therapeutic strategy for endometriosis.

Structure-activity relationships of 2, 4-disubstituted pyrimidines as dual ERα/VEGFR-2 ligands with anti-breast cancer activity

Both ERα and VEGFR-2 are important targets for cancer therapies. Here a series of 2, 4-disubstituted pyrimidine derivatives were designed, synthesized and evaluated as dual ERα/VEGFR-2 ligands. Most of the derivatives exhibited potent activities in both enzymatic and cellular assays. Structure-activity relationship studies showed that a hydrogen-bonding interaction in the head section is important factors for the enhancement of ERα-binding affinity. The most potent compound II-9OH, an analog of 2-(4-hydroxylphenyl)pyrimidine, was 19-fold more efficacious than tamoxifen in MCF-7 cancer cells and exhibited the best ERα binding affinity (IC50 = 1.64 μM) as well as excellent VEGFR-2 inhibition (IC50 = 0.085 μM). Furthermore, this dual targeted compound II-9OH exerted significantly antiestrogenic property via suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7 cells and also showed obvious in vivo angiogenesis inhibitory effects in CAM assay. An induction of apoptosis and a decrease in cell migration, accompanied by transduction inhibition of Raf-1/MAPK/ERK pathway, were observed in MCF-7 cells after treatment with II-9OH, suggesting that II-9OH is a promising candidate for the development of multifunctional agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.

Effects of Estrogen Therapy on the Serotonergic System in an Animal Model of Perimenopause Induced by 4-Vinylcyclohexen Diepoxide (VCD).

Chronic exposure to 4-vinylcycloxene diepoxide (VCD) in rodents accelerates the natural process of ovarian follicular atresia modelling perimenopause in women. We investigated why estrogen therapy is beneficial for symptomatic women despite normal or high estrogen levels during perimenopause. Female rats (28 d) were injected daily with VCD or oil for 15 d; 55-65 d after the first injection, pellets of 17β-estradiol or oil were inserted subcutaneously. Around 20 d after, the rats were euthanized (control rats on diestrus and estradiol-treated 21 d after pellets implants). Blood was collected for hormone measurement, the brains were removed and dorsal raphe nucleus (DRN), hippocampus (HPC), and amygdala (AMY) punched out for serotonin (5-HT), estrogen receptor β (ERβ), and progesterone receptor (PR) mRNA level measurements. Another set of rats was perfused for tryptophan hydroxylase (TPH) immunohistochemistry in the DRN. Periestropausal rats exhibited estradiol levels similar to controls and a lower progesterone level, which was restored by estradiol. The DRN of periestropausal rats exhibited lower expression of PR and ERβ mRNA and a lower number of TPH cells. Estradiol restored the ERβ mRNA levels and number of serotonergic cells in the DRN caudal subregion. The 5-HT levels were lower in the AMY and HPC in peristropausal rats, and estradiol treatment increased the 5-HT levels in the HPC and also increased ERβ expression in this area. In conclusion, estradiol may improve perimenopause symptoms by increasing progesterone and boosting serotonin pathway from the caudal DRN to the dorsal HPC potentially through an increment in ERβ expression in the DRN.

Effect of side of the corpus luteum and pregnancy on estrogen and progesterone receptor expression and localization in the endometrium of mares

The effect of side of corpus luteum on uterine gene expression and protein localization of estrogen receptor α (ERα) and progesterone receptor (PR) in healthy cyclic and pregnant mares 13 days after ovulation (day 0) was investigated. Transcervical biopsies were performed to collect endometrium ipsilateral and contralateral regarding the side of corpus luteum on day 13 post-ovulation in cyclic (n = 6) and pregnant (n = 6) mares. Blood samples were collected daily from day 0 until the day of biopsy for 17β-estradiol (E2) and progesterone (P4) determinations. Receptor expression was determined by immunohistochemistry and transcript expression by real time RT-PCR. Serum E2 and P4 concentrations were not affected by reproductive status. The contralateral horn presented higher percentage of positive cells for ERα than the ipsilateral horn (P < .05), but side did not affect PR. ERα showed low staining and no main effect of pregnancy was found, but pregnant mares had lower protein expression of PR (19.8 vs. 40.4 ± 5.3%, P < .01). The contralateral horn tended to present higher expression of ERα mRNA (1.33 vs. 0.97 ± 0.17, P < .10) and PR mRNA (1.96 vs. 1.57 ± 0.52, P < .09). ERα mRNA relative expression was lower in the pregnant group (0.88 vs. 1.44 ± 0.19, P < .05). The interaction of reproductive status and side of corpus luteum tended to affect PR mRNA expression as pregnant mares had a lower PR mRNA content in the ipsilateral horn than cyclic mares. To our knowledge, this is the first study that describes the behavior of steroid receptors in the endometrium of mares regarding side of corpus luteum.

Sex-steroid receptors, prostaglandin E2 receptors, and cyclooxygenase in the equine cervix during estrus, diestrus and pregnancy: Gene expression and cellular localization

The cervix is a dynamic structure that undergoes dramatic changes during the estrous cycle, pregnancy and parturition. It is well established that hormonal changes, including estrogens, progestogens and prostaglandins, regulate the expression of key proteins involved in cervical function. The arachidonic acid cascade is important in the remodeling and relaxation of the cervix in the days preceding parturition. Despite the complexity of this mechanism, regulation of cervical function has received little study in the mare. Therefore, the objective of this study was to compare the expression of estrogen receptor α (ESR1) and β (ESR2), progesterone receptor (PGR), prostaglandin E2 type 2 (PTGER2) and type 4 (PTGER4) receptors as well as cyclooxygenase-1 (PTGS1) and -2 (PTGS2) in the equine cervical mucosa and stroma during estrus, diestrus and late pregnancy using qPCR. Immunohistochemistry was used to localize ESR1, ESR2, PGR, PTGER2 and PTGER4 receptors in these regions of the cervix. Relative mRNA expression of ESR1 and PGR was greater during estrus and diestrus than in late pregnancy in both the mucosa and stroma of the cervix. Expression of PTGER2 was highest in the cervical stroma during late pregnancy compared to either estrus or diestrus. Moreover, PTGS1 expression in mucosa and PTGS2 in stroma was greater during late pregnancy compared with estrus, but not diestrus. Immunostaining for ESR1, ESR2, PGR, PTGER2 and PTGER4 was consistently detected in the nucleus and cytoplasm of epithelium of the endocervix as well as the smooth muscle cytoplasm of the cervix in all stages evaluated. Immunolabeling in smooth muscle nuclei was detected for ESR1 and PGR in estrus, diestrus and late pregnancy, and for ESR2 in estrus and late pregnancy stages. The changes noted in late gestation likely reflect preparation of the equine cervix for subsequent parturition.

Novel SERMs based on 3-aryl-4-aryloxy-2H-chromen-2-one skeleton - A possible way to dual ERα/VEGFR-2 ligands for treatment of breast cancer

There is considerable interest in developing new SERMs as multifunctional agents in women's health. Development of dual selective estrogen receptor modulators/VEGFR-2 inhibitors (SERMs/V-2I) has been an attractive strategy for the discovery of new breast cancer therapeutic agents. Our previous efforts led to the preparation of a series of 3-aryl-4-anilino-2H-chromen-2-ones endowed with potent estrogen receptor binding affinity and anti-proliferative efficacy. In this study, various structurally related 3-aryl-4-anilino/aryloxy-2H-chromen-2-one analogues were rationally designed, synthesized and evaluated as a new chemo-type of dual ERα and VEGFR-2 inhibitors. Most of the derivatives exhibited potent activities in both enzymatic and cellular assays. SAR investigation revealed that introducing of bioisosteric O atom at the C-4 position of coumarin scaffold is beneficial to improve the inhibitory potency, especially in ERα binding affinity assay. Furthermore, most of the piperidyl substituted compounds showed better inhibitory activity against MCF-7 and Ishikawa cells than lead compounds BL-18d, tamoxifen and Vandetanib. Optimization of the hit compound, identified in an ERα binding affinity assay, led to compound 42d, exhibiting an IC50 for ERα binding affinity of 2.19 μM while retaining an excellent inhibition on VGFR-2 as well as a potent suppression on the growth of angiogenesis-related cells. In RT-PCR assay, 42d exerted significantly antiestrogenic property via suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7 cells, which was consistent with the ERα antagonistic property of a selective estrogen receptor modulator. Further mechanism investigation demonstrated that compound 42d could inhibit the activation of VEGFR-2 and subsequent signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells. All these results together with molecular modeling studies open a new avenue for the development of multifunctional agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.

Effect of growth hormone on steroid concentrations and mRNA expression of their receptor, and selected egg-specific protein genes in the chicken oviduct during pause in laying induced by fasting

This study was undertaken to examine the effect of growth hormone (GH) treatment during pause in laying on (1) the concentration of steroids in blood plasma and oviduct tissues, (2) the expression of mRNA of steroid receptors, and (3) the mRNA expression of selected egg-specific proteins in the chicken oviduct. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from Day 10 onward. Birds were divided into three groups: control (n = 18) fed ad libitum, subjected to pause in laying (n = 18), and subjected to pause in laying and injected every day with 200 μg/kg BW of chicken GH (chGH; n = 18). The oviduct was isolated from hens of each group on Days 6 (when the oviduct was regressed), 13 (during oviduct recrudescence), and 17 or 20 (rejuvenated oviduct) of the experiment. Fasting caused a decrease in plasma concentrations of progesterone (P4), testosterone, and estradiol on Day 6 and a reduction in tissue concentrations of these steroids on Days 6 and 13. Fasting also caused an increased relative expression of estrogen receptor α and β (ERα, ERβ) and progesterone receptor (PR) in the magnum and shell gland on Day 6, increased ERα and PR in the magnum on Days 13 and 17 or 20, and increased androgen receptor (AR) mRNA in the magnum on Days 6 and 13 and in the shell gland on Day 13. A fasting-induced elevation in ovocalyxin-36 mRNA expression on Day 6 and a decrease in avidin mRNA on Days 6 and 13 and in ovocleidin-116 on Day 13 were also observed (P < 0.05 to P < 0.001). Administration of chGH abolished the fasting-induced decrease in the concentration of steroids in plasma and tissues. Furthermore, chGH enhanced the effect of fasting on mRNA expression of PR, ERα, and avidin in the magnum on Day 6, and ERα in the shell gland on Day 13. The gene expression of ovalbumin on Days 6 and 13, ovocalyxin-36 and ovocleidin-116 on Day 6 was decreased in chGH-treated chickens. In contrast, the expression of ovalbumin on Day 17 or 20 was increased (P < 0.05 to P < 0.001). The results obtained indicate that, by alterations in the concentration of steroid hormones and their receptor expression in the chicken oviduct, GH determines the rate of regression and rejuvenation of this organ during molting. Moreover, changes in the expression of selected egg proteins indicate that GH might be the regulator of the secretory activity of the hen oviduct.