Glyphosate induces epithelial mesenchymal transition-related changes in human endometrial Ishikawa cells via estrogen receptor pathway.

Glyphosate based herbicides are the most commonly used herbicide in the world. We aimed to determine whether glyphosate (Gly) induces epithelial mesenchymal transition (EMT) - related changes in a human endometrial carcinoma cell line (Ishikawa cells), and whether the estrogen receptor (ER) pathway is involved in these changes. Ishikawa cells were exposed to Gly (0.2 µM and 2 µM) or 17ß-estradiol (E2: 10-9 M). We detected that Gly increased cell migration and invasion ability compared to vehicle, as did E2. Moreover, a down regulation of E-cadherin mRNA expression was determined in response to Gly, similar to E2-effects. These results show that Gly promotes EMT-related changes in Ishikawa cells. When an ER antagonist (Fulvestrant: 10-7 M) was co-administrated with Gly, all changes were reversed, suggesting that Gly might promote EMT-related changes via ER-dependent pathway. Our results are interesting evidences of Gly effects on endometrial cancer progression via the ER-dependent pathway.

Postnatal exposure to endosulfan affects uterine development and fertility.

Endosulfan is an organochlorine pesticide (OCP) used in large-scale agriculture for controlling a variety of insects and mites that attack food and non-food crops. Although endosulfan has been listed in the Stockholm Convention as a persistent organic pollutant to be worldwide banned, it is still in use in some countries. Like other OCPs, endosulfan is bioaccumulative, toxic and persistent in the environment. Human unintentional exposure may occur through air inhalation, dietary, skin contact, as well as, via transplacental route and breast feeding. Due to its lipophilic nature, endosulfan is rapidly absorbed into the gastrointestinal tract and bioaccumulates in the fatty tissues. Similar to other OCPs, endosulfan has been classified as an endocrine disrupting chemical (EDC). Endocrine action of endosulfan on development and reproductive function of males has been extensively discussed; however, endosulfan effects on the female reproductive tract have received less attention. This review provides an overview of: i) the fate and levels of endosulfan in the environment and human population, ii) the potential estrogenic properties of endosulfan in vitro and in vivo, iii) its effects on uterine development, and iv) the long-term effects on female fertility and uterine functional differentiation during early gestation.

Epigenetic Dysregulation of Dopaminergic System by Maternal Cafeteria Diet During Early Postnatal Development.

Dopamine is a neurotransmitter crucial for motor, motivational, and reward-related functions. Our aim was to determine the effect of a palatable maternal diet on the transcriptional regulation of dopaminergic-related genes during perinatal development of rat offspring. For that, female offspring from dams fed with a control (CON) or a cafeteria (CAF) diet were sacrificed on embryonic day 21 (E21) and postnatal day 10 (PND10). Using micropunch techniques, ventral tegmental area (VTA) and nucleus accumbens (NAc) were isolated from brain's offspring. Bioinformatic analysis of the promoter regions, mRNA quantification and methylation studies were done. The increase in tyroxine hidroxylase (TH), dopamine receptor (DRD) 1 and ghrelin receptor (GHSR) expression in VTA and NAc from E21 to PND10 was correlated with changes in DNA methylation of their promoter regions. Maternal diet did not affect the expressionpatternsin E21. At PND10, maternal CAF diet decreased the transcription of TH, GHSR, DRD2 and dopamine transporter (DAT) in VTA. Interestingly, the changes in TH, DRD2 and DAT expression were related to the methylation status of their promoters. In NAc, maternal CAF diet reduced DRD1, DRD2 and DAT expression in the offspring at PND10, although alternations in the methylation patterns were only detected in DAT promoter. These results show the importance of maternal nutrition and provide novel insights into the mechanisms through which maternal junk-food feeding can affect reward system during development and early postnatal life. Particularly important is the expression decline of DRD2 given its physiological implication in obesity and addiction.

Exposure of neonatal female rats to bisphenol A disrupts hypothalamic LHRH pre-mRNA processing and estrogen receptor alpha expression in nuclei controlling estrous cyclicity.

This study examines the effects of neonatal exposure to the endocrine disruptor bisphenol A (BPA) on the neural network that controls estrous cyclicity. From postnatal day 1 (PND1) to PND7, female pups were injected with vehicle (control) or BPA (BPA.05: 0.05mg/kg-d, BPA20: 20mg/kg-d). At PND100 BPA.05-females showed alterations in estrous cyclicity and BPA20-females were incapable of producing an estradiol-induced LH surge. By real-time PCR we determined that hypothalamic expression of mature LH-releasing hormone (LHRH) mRNA was increased in BPA.05 and decreased in BPA20-females. Furthermore, unprocessed intron A-containing LHRH RNA was decreased in the cytoplasm of hypothalamic cells of both groups. Immunohistochemistry revealed that estrogen receptor alpha protein was up-regulated in anteroventral periventricular and down-regulated in arcuate nucleus of both groups. Our results show that BPA permanently disrupts hypothalamic LHRH pre-mRNA processing and steroid receptors expression in nuclei that control estrous cyclicity in adult rats.

Neonatal exposure to bisphenol A alters estrogen-dependent mechanisms governing sexual behavior in the adult female rat.

This study examines the effects of neonatal exposure to the endocrine disruptor bisphenol A (BPA) on the hypothalamic circuitry controlling the female sexual behaviors of adult rats. From postnatal day 1 (PND1) to PND7, pups were injected with corn oil (control) or BPA (BPA20: 20mg/kg-d; BPA.05: 0.05 mg/kg-d) and at PND85 the rats were bilaterally ovariectomized (OVX). At PND100, OVX-rats received estradiol alone or estradiol and progesterone to evaluate estrogen-dependent gene expression in the hypothalamus and sexual behavior. In BPA-exposed females, estrogen receptor alpha (ER alpha) expression was down-regulated in both the medial preoptic (MPN) and ventromedial nucleus (VMHvl), while repressor of estrogen receptor activity (REA) expression was up-regulated in the VMHvl. Interestingly, BPA-exposed females displayed significantly lower levels of proceptive behavior. Our results show that BPA permanently alters the hypothalamic estrogen-dependent mechanisms that govern sexual behavior in the adult female rat.

Mast cell degranulation in rat uterine cervix during pregnancy correlates with expression of vascular endothelial growth factor mRNA and angiogenesis.

Vascular growth of the uterine cervix during pregnancy is associated with mast cell (MC) degranulation. To better understand the mechanism underlying this process, uterine cervices of intact pregnant rats were dissected and endothelial cell proliferation was measured by a bromodeoxyuridine incorporation technique. Total vascular endothelial growth factor (VEGF) mRNA expression and the relative abundance of VEGF splice variants (120, 164, and 188) were determined by RT-PCR. VEGF protein expression was evaluated by immunohistochemistry. To investigate the role of MCs on cervical angiogenesis, a second set of pregnant animals were treated with an MC stabilizer (disodium cromoglycate) to inhibit MC degranulation. Furthermore, 17beta-estradiol (E(2)) serum levels were established by RIA. In intact pregnant rats, VEGF mRNA expression was positively correlated with endothelial cell proliferation and circulating E(2) levels. All selected splice variants of VEGF gene were detected and their relative abundance did not show any change throughout pregnancy. Animals treated with disodium cromoglycate showed a decrease in endothelial cell proliferation and in VEGF mRNA expression compared with controls. Relative abundance of VEGF mRNA splice variants and E(2) serum levels showed no differences between these experimental groups. These results show a time-dependent correlation between VEGF mRNA expression and E(2) serum levels in the uterine cervix of intact pregnant rats, while MC stabilizer-treated animals reduced the VEGF expression without modifying E(2) serum levels. We suggest that cervical angiogenesis during pregnancy could be regulated by a mechanism which involves endogenous E(2) and chemical mediators stored in MC granules via a VEGF-dependent pathway.

The estrogen receptor alpha sigma3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus.

The gene for estrogen receptor alpha (ER alpha) has been shown to be under complex hormonal control and its activity can be regulated by mRNA alternative splicing. Here we examined the regulation of ER alpha transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER alpha mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-beta estradiol (E2) (0.05 microg/rat or 5 microg/rat), progesterone (P4) (1 mg/rat) or a combination of both hormones for 4 days. Animals were killed 24 h after the last injection and uterine horns were removed. In order to determine whether ER alpha mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER alpha protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER alpha mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER alpha mRNA and protein levels were high, demonstrating a constitutive expression of the ER alpha gene in the uterus. When animals received P4 or the high dose of E2, a significant decrease in both ER alpha mRNA and protein was observed in the uterus. However, when rats were protein was treated with the low dose of E2, only the ER alpha down-regulated; no changes were observed in ER alpha mRNA expression. In addition to the full-length ER alpha mRNA, OVX control rat uteri expressed three shorter transcripts: sigma3, sigma4 and sigma3,4 (lacking exon 3, exon 4, or both 3 and 4 respectively). Surprisingly, when OVX animals were treated with P4, the low dose of E2 or a combination of both steroids, expression of the sigma3 isoform was completely abolished. During the estrous cycle, all ER alpha mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the sigma3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E2 concentrations and the level of control in the ER alpha transcription-translation cascade. Moreover, the alternative splicing of the ER alpha primary transcript is influenced by the hormonal milieu, suggesting that these events could affect the estrogen responsiveness of the rat uterus during the estrous cycle.

Mast cells degranulation affects angiogenesis in the rat uterine cervix during pregnancy.

During pregnancy, it is essential that sufficient nutrients are supplied by the vascular system to support the dramatic modifications of the rat uterine cervix. Angiogenesis refers to the growth of new blood vessels from pre-existing microcirculation and mast cells have been associated with this process. This study examined the modifications of the vascular compartment and the distribution of mast cells on cervical tissue during pregnancy. Using disodium cromoglycate as a mast cell stabilizer, we determined the effects of the mast cell degranulation on cervical angiogenesis. Mast cell distribution and their degranulation status were evaluated by immunohistochemistry. Endothelial cell proliferation was measured by bromodeoxyuridine incorporation. Vascular areas (absolute and relative) and maturation indices were assessed by quantitative immunohistochemistry of von Willebrand factor and alpha-smooth muscle actin respectively. Mast cells were predominantly observed during the first half of pregnancy in the perivascular zones. The values of bromodeoxyuridine incorporation, absolute vascular area and vascular maturation index exhibited a significant increase throughout pregnancy. All animals that received mast cell stabilizer showed more than 40% of non-degranulated mast cells. Treated rats exhibited a decrease in endothelial proliferation and in relative vascular area; in addition, a large proportion of mature blood vessels was observed, suggesting a diminished level of new vessel formation. The effects of the mast cell stabilizer were sustained beyond the end of treatment. This is the first report that brings evidence that mast cell degranulation could be a necessary process to contribute to the normal angiogenesis of the rat cervix during pregnancy. Further investigations are needed to elucidate the possible implications of abnormal vascular development of the uterine cervix on the physiological process of ripening and parturition.

Collagen remodelling in the guinea-pig uterine cervix at term is associated with a decrease in progesterone receptor expression.

In human and guinea-pig parturition, progesterone withdrawal and estrogen action are not mediated by changes in their circulating levels. Instead, these events might be promoted by changes in the responsiveness of the uterus and cervix to progesterone and estrogen via changes in their receptors. In this study, the guinea-pig model was used to investigate whether high levels of progesterone and estrogen at term are associated with regional changes in PR and ERalpha levels in uterus and cervix. PR and ERalpha profiles were established in both subepithelium and the muscular layer of the cervix and the lower uterine horns during pregnancy, parturition and postpartum; while collagen remodelling was measured in the subepithelium. Our data showed that collagen remodelling involved in cervical ripening is temporally and spatially associated with a decrease in PR, whereas high expression of ERalpha is observed. This association was found in the subepithelium of the cervical tissue but not in the same region of the uterus. The muscular region of the cervix and uterus also present a transiently decreased expression of PR while ERalpha levels remain high. Thus, the present results indicate that, before parturition, diminished responsiveness of the cervix to progesterone might be caused by a decrease in PR levels and that this may be the mechanism of functional progesterone withdrawal. The guinea-pig was further validated as an animal model for human parturition studies.

Phenotypic modulation of fibroblastic cells in the mucous layer of the human uterine cervix at term.

The uterine cervix is a dynamic structure with a high capacity to adapt to different, even opposing, roles during the sequence of physiological events of gestation (for example, acting as a barrier to retain the fetus during pregnancy and dilating to allow delivery at term). Histoarchitectural changes of the uterine cervix allow its successful adaptation. The aim of this study was to investigate whether fibroblastic cell plasticity, described in the lamina propria of the rat uterine cervix at term, could be observed in women too. Biopsy specimens of non-pregnant and intrapartum human cervices were studied under the transmission electron microscope, and cytoskeletal differentiation markers were identified by immunohistochemistry under the light microscope. Desmin-positive cells were present in the mucous layer of the cervix during labour. These cells displayed cytoplasmic processes (typical of myofibroblasts) that also stained positively for vimentin. The main ultrastructural features for defining the myofibroblast under the electron microscope were also observed in these cells. However, cervices of non-pregnant women contained resident fibroblasts at the same location. Examination of the differentiation repertoire of fibroblastic cells in the mucous layer of the uterine cervix resulted in the characterization of myofibroblasts at term. The implications of the plasticity of fibroblastic-myofibroblastic cells in the physiological changes displayed in the uterine cervix during pregnancy, labour and postpartum involution require further investigation.

Prenatal exposure to low doses of bisphenol A alters the periductal stroma and glandular cell function in the rat ventral prostate.

Environmental estrogens (xenoestrogens) are chemicals that bind to estrogen receptor, mimic estrogenic actions, and may have adverse effects on both human and wildlife health. Bisphenol A (BPA), a monomer used in the manufacture of epoxy resins and polycarbonate has estrogenic activity. In male rodents prenatal exposure to BPA resulted in modifications at the genital tract level. Our objective was to examine the effects of in utero exposure to low, environmentally relevant levels, of the xenoestrogen BPA on proliferation and differentiation of epithelial and stromal cells on the prepubertal rat ventral prostate. To characterize the periductal stromal cells phenotype the expression of vimentin and smooth muscle alpha-actin was evaluated. Androgen receptor (AR) and prostatic acid phosphatase (PAP) expression were also evaluated in epithelial and stromal compartments. Prenatal exposure to BPA increases the fibroblastic:smooth muscle cells ratio and decreases the number of AR-positive cells of periductal stroma of the ventral prostate. In contrast, no differences in AR expression were observed in epithelial cells between control and BPA-treated groups. No changes in proliferation patterns were observed in epithelial and stromal compartments; however, the expression of PAP was diminished in prostate ductal secretory cells of rats in utero exposed to BPA. Our results suggest that prenatal exposure to BPA altered the differentiation pattern of periductal stromal cells of the ventral prostate. These findings are significant in light of the data on human prostate cancers where alterations in the stroma compartment may enhance the invasive and/or malignant potential of the nascent tumor.

Characterization of fibroblastic cell plasticity in the lamina propria of the rat uterine cervix at term.

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never alpha-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.

Detection of bromodeoxyuridine in formalin-fixed tissue. DNA denaturation following microwave or enzymatic digestion pretreatment is required.

The present study was designed to assess the influence of antigen retrieval and/or DNA denaturation on the quantitative estimation of bromodeoxyuridine (BrdU) in formalin-fixed paraffin-embedded tissue. Specimens of small intestine from rats injected with BrdU were routinely fixed and embedded in paraffin. For antigen retrieval, sections were pretreated with microwave irradiation or enzymatically (pepsin or trypsin). Acid hydrolysis was used as a DNA denaturation method. Immunostaining of BrdU-labeled cells was performed. The best results, regarding tissue morphology and immunostaining, were obtained with microwave pretreatment followed by acid hydrolysis. Enzymatic pretreatment resulted in damage of tissue morphology and/or high background staining. Microwave alone, without DNA denaturation, resulted in a lower percentage of BrdU positive cells. The significance of validation studies is emphasized when the level of positivity for a prognostic marker, such as BrdU, is assessed.

Estrogen and progesterone modulation of eosinophilic infiltration of the rat uterine cervix.

Ripening of the rat cervix involves widespread collagenolysis that follows an eosinophilic leukocyte infiltration. The hormonal control of these events is not well understood. The aims of this study were to investigate the mechanism through which progesterone (P) and 17beta-estradiol (E(2)) modulate eosinophilic invasion and to determine if this event is protein synthesis mediated. Cervical eosinophilic invasion was measured in intact rats during the second half of pregnancy and compared with values from ovariectomized (O) pseudopregnant (PSP) rats treated with P and E(2) in doses that mimicked the levels of pregnancy. Other O-PSP rats were treated with an E(2) antagonist (tamoxifen) and the antiprogestin RU-486. To study the role of protein synthesis in eosinophilic invasion of the cervix, rats were treated with actinomycin-D (an inhibitor of mRNA synthesis), and animals were sacrificed on D21 or D22 to evaluate eosinophilic invasion. Rats treated with E(2) showed high levels of infiltration and tamoxifen blocked this E(2) effect. On the other hand, P antagonized the stimulatory effects of E(2) on eosinophilic invasion, however when the P and E(2) treated rats were injected with RU-486 the inhibitory effect of P was reversed. In intact pregnant rats a sharp rise in eosinophilic infiltration was detected on D23, 20 h after the fall of serum P. Finally, E(2) treated rats injected with actinomycin-D had no invasion of eosinophils. In conclusion, the estrogen-triggered eosinophil invasion is affected by the classic estrogen receptor antagonist tamoxifen and by the mRNA synthesis blocker actinomycin-D suggesting a genomic action of E(2). Furthermore, the estrogen effect is blocked by P and this inhibition is reversed by RU-486.