Jorge G. Ramos

Prenatal Exposure to Low Doses of Bisphenol A Alters the Periductal Stroma and Glandular Cell Function in the Rat Ventral Prostate

Abstract 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 α-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.

Sex reversal effects on Caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol A

Exposure to environmental contaminants known as endocrine disruptors (EDs) alters the development and function of reproductive organs in several species. Bisphenol A (BPA) is an estrogenic chemical that leaches from dental materials and plastic food and beverage containers. BPA has been found in sewage, surface and drinking water, and therefore poses a potentially significant risk for human and wildlife. Prenatal exposure of rodents to environmentally relevant doses of BPA alters the development of the reproductive organs of male and female offspring. Species with temperature dependent sex determination (TSD) could act as sentinels of ecosystem health by providing sensitive biomarkers of endocrine disruptors effects. We selected Caiman latirostris as an animal model to study endocrine disruption caused by BPA. The aim of this study was to determine whether exposure in ovum to BPA could cause estrogen-like effects on the reproductive system of C. latirostris. Sex determination and gonadal histoarchitecture were the endpoints evaluated after in ovum exposure to different doses of BPA and 17beta-estradiol (E(2)). We confirmed that C. latirostris is a species with TSD and additionally demonstrated that BPA causes estrogen-like developmental effects by reversing gonadal sex and altering gonadal histoarchitecture. Differences in responses to BPA and E(2) in our in vivo system were on the order of 100-fold. In contrast published in vitro studies have reported differences on the order of 10,000x or more. These results support the utility of C. latirostris, a species in which sex determination is temperature dependent, as a tool in assessing estrogenic activity in vivo and as a sentinel to monitor EDs in aquatic environment.

Neonatal Exposure to Bisphenol A Alters Rat Uterine Implantation-Associated Gene Expression and Reduces the Number of Implantation Sites

Endocrine disrupters have been associated with reproductive pathologies such as infertility and gynecological tumors. Using a rat model of early postnatal exposure to bisphenol A (BPA), we evaluated the long-term effects on 1) female reproductive performance, 2) uterine homeobox A10 (Hoxa10) and Hoxa10-target gene expression, and 3) ovarian steroid levels and uterine estrogen receptor α and progesterone (P) receptor expression. Newborn female rats received vehicle, BPA.05 (0.05 mg/kg · d), BPA20 (20 mg/kg · d), diethylstilbestrol.2 (0.2 μg/kg · d), or diethylstilbestrol 20 (20 μg/kg · d) on postnatal d 1, 3, 5, and 7. A significant decrease in the number of implantation sites was assessed in the xenoestrogen-exposed females. To address the molecular effects of postnatal xenoestrogen exposure on the pregnant uterus, we evaluated the expression of implantation-associated genes on d 5 of pregnancy (preimplantation uterus). All xenoestrogen-treated rats showed a lower expression of Hoxa10. In the same animals, two Hoxa10-downstream genes were misregulated in the uterus. β(3) Integrin, which is up-regulated by Hoxa10 in controls, was decreased, whereas empty spiracles homolog 2, which is down-regulated by Hoxa10, was increased. Furthermore a clear down-regulation of estrogen receptor α and P receptor expression was detected without changes in estradiol and P serum levels. The early exposure to BPA produced a lower number of implantation sites in association with a defective uterine environment during the preimplantation period. Alterations in the endocrine-regulated Hoxa10 gene pathways (steroid receptors--Hoxa10--β(3) integrin/empty spiracles homolog 2) could explain, at least in part, the BPA effects on the implantation process.

Developmental Exposure to Bisphenol A Impairs the Uterine Response to Ovarian Steroids in the Adult

Morphoregulator genes like members of the Hox gene family regulate uterine development and are associated with endocrine-related processes such as endometrial proliferation and differentiation in the adult uterus. Exposure to neonatal endocrine disruptors could affect signaling events governed by Hox genes, altering the developmental trajectory of the uterus with lasting consequences. We investigated whether neonatal exposure to bisphenol A (BPA) alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment as well as in the adult. Moreover, we studied whether xenoestrogen exposure may affect the adult uterine response to hormonal stimuli. Newborn females received vehicle, 0.05 mg/kg.d BPA, 20 mg/kg*d BPA, or diethylstilbestrol (0.2 microg/kg*d) on postnatal d 1, 3, 5, and 7). At postnatal d 8, real time RT-PCR assays showed a decrease in Hoxa10 and Hoxa11 expression in all xenoestrogen-treated groups. To evaluate the long-term effects, we used adult ovariectomized rats with hormonal replacement. The subepithelial stroma in BPA- and diethylstilbestrol-treated animals showed an impaired proliferative response to steroid treatment associated with a silencing of Hoxa10 but not associated with changes in the methylation pattern of the Hoxa10 promoter. BPA animals showed that the Hoxa10 reduction was accompanied by an increased stromal expression of the silencing mediator for retinoic acid and thyroid hormone receptor. The spatial coexpression of steroid receptors Hoxa10 and silencing mediator for retinoic acid and thyroid hormone receptor was established using immunofluorescence. Our data indicate that postnatal BPA exposure affects the steroid hormone-responsiveness of uterine stroma in adulthood. Whether this impaired hormonal response is associated with effects on uterine receptivity and decidualization is currently under investigation.

Cellular Turnover in the Rat Uterine Cervix and Its Relationship to Estrogen and Progesterone Receptor Dynamics

Abstract Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor α (ERα) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERα and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERα and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERα were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERα was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERα and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.

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.

Endosulfan modulates estrogen-dependent genes like a non-uterotrophic dose of 17β-estradiol

The estrogenic activity of environmentally relevant doses of endosulfan was investigated using an animal model. Ovariectomized adult rats were injected once a day for 3 days with sesame oil (control), 0.02mg/kg/day 17beta-estradiol (an uterotrophic dose; UE(2)), 0.0002mg/kg/day 17beta-estradiol (a non-uterotrophic dose; NUE(2)), or 0.006, 0.06, 0.6 or 6mg/kg/day endosulfan. After 24h of treatment, the uteri were weighed (uterotrophic assay) and the luminal epithelial cell height (LECH) and progesterone receptor (PR), and estrogen receptor alpha (ERalpha) protein levels were measured. PR, ERalpha, and complement factor-3 (C3) mRNAs were evaluated using real-time PCR. Uterine weight and LECH were only increased in UE(2)-treated rats. PR, ERalpha and C3 expression levels were modified in most of the endosulfan-treated groups, showing an identical pattern of expression to the NUE(2)-group. Our results show that the pesticide endosulfan mimics non-uterotrophic E(2) actions, strengthening the hypothesis that endosulfan is a widespread xenoestrogen.

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.

Effects of Neonatal Exposure to Bisphenol A on Steroid Regulation of Vascular Endothelial Growth Factor Expression and Endothelial Cell Proliferation in the Adult Rat Uterus

Abstract Hormonally controlled vascular changes play a key role in endometrial development and in the differentiation process necessary for implantation. Vascular endothelial growth factor (VEGF) has emerged as one of the central regulators of the uterine vasculature. Hormonal perturbations during neonatal development may alter sex steroid-dependent regulation of VEGF and may ultimately affect fertility later in life. The aim of this study was to determine whether neonatal exposure to the environmental estrogenic chemical bisphenol A (BPA) affects the adult rat uterine response to hormonal stimuli. Newborn female rats were given s.c. injections of vehicle, BPA (0.05 mg/kg per day or 20 mg/kg per day) or diethylstilbestrol (0.2 μg/kg per day) on Postnatal Days 1, 3, 5, and 7. To evaluate the long-term effects, rats were ovariectomized at Postnatal Day 80 and submitted to hormonal replacement. Rats neonatally exposed to xenoestrogens showed a decreased induction of uterine endothelial proliferation and a decreased Vegf mRNA expression in response to ovarian steroid treatment. Also, although the estrogen receptor alpha (ESR1) expression was lower in subepithelial cells than in controls, a higher expression of silencing mediator of retinoic acid and thyroid hormone receptor (NCOR1, also known as SMRT) corepressor was evidenced in the same compartment. The results indicate that disturbed Vegf expression in BPA rats could be the result of changes in endocrine pathways, such as an altered induction of ESR1 and/or NCOR1 expression. Because of the importance of VEGF in the implantation process, our data suggest that neonatal BPA exposure might have negative consequences on female fertility.

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.