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Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina
Address all correspondence and requests for reprints to: Enrique H. Luque, Ph.D., Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Casilla de Correo 242, 3000 Santa Fe, Argentina. E-mail: eluque{at}fbcb.unl.edu.ar.
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Abstract |
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(ER) and ERß mRNA expression in both the preoptic area (POA) and medial basal hypothalamus, and determination of PRL, LH, and testosterone serum levels. On d 30 (peripubertal period), the prostatic periductal stroma of BPA-exposed rats demonstrated a significantly larger layer of fibroblasts than that of controls, whereas on d 120 (adulthood) no significant differences were observed. Moreover, BPA-exposed rats on d 15 exhibited an increase in stromal cellular proliferation compared with controls. Decreased expression of both androgen receptor in prostatic stromal cells and prostatic acid phosphatase in epithelial cells was observed only on d 30 in BPA-exposed males. BPA did not alter POA ER mRNA expression, whereas a 4-fold increase in POA ERß mRNA expression was observed on both d 30 and 120. No alterations were observed in either ER or ERß expression in the medial basal hypothalamus. BPA-exposed males exhibited increased PRL levels only on d 30, whereas a transient increase in serum testosterone levels was observed on d 15. These results support the hypothesis that prenatal exposure to environmental doses of BPA induces both transient and permanent age-dependent alterations in the male reproductive axis at different levels. |
Introduction |
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In laboratory animals, prenatal exposure to BPA and arochlor was shown to modify anogenital distance (AGD), increase prostate size, decrease epididymal weight, and alter estrous cyclicity and plasma LH levels (9, 10, 11). Using low doses of BPA, Steinmetz et al. (12) induced hyperprolactinemia in ovariectomized Fisher 344 rats. In the same rat strain, Khurana et al. (13) showed that exposure of newborns to either BPA or octylphenol induced both delayed and prolonged hyperprolactinemia. Moreover, they reported alterations in estrogen receptor- (ER) mRNA expression in the medial basal hypothalamus (MBH) of female neonates, whereas no differences in hypothalamic ER mRNA expression were observed in male pups. BPA-induced hyperprolactinemia has been postulated to be an indirect mechanism that could increase the incidence of prostate inflammation in the adult rat (14).
In a previous report (15) we demonstrated that in utero exposure to environmentally relevant levels of BPA decreased androgen receptor (AR) expression and modified prostate periductal stromal cell phenotype at d 30 in Wistar rats. In addition, we observed a decrease in prostatic acid phosphatase (PAP) expression, suggesting alterations in prostatic functional activity. Numerous efforts have focused on elucidating the effects of estrogenization during early postnatal life on prostate development and function (16, 17). These studies demonstrated that several effects of neonatal estrogenization on male puberty and the male reproductive tract are transient and that the reversibility of the process is a function of the dose, the end points examined, and the rat strain employed (18, 19). However, little is known about the time course of the alterations observed in males prenatally exposed to environmentally relevant doses of xenoestrogens. Taking into account the above-mentioned findings, the first goal of the present study was to assess the time course of histofunctional disruption of the rat ventral prostate induced by prenatal BPA exposure. The second goal was to evaluate the action of BPA on the male hypothalamic-pituitary-gonadal axis by evaluating several different end points. ER and ERß mRNA expression in the preoptic area (POA) and MBH were scanned using an RT-PCR technique. PRL, LH, and testosterone (T) serum levels were also determined to ascertain whether these parameters were differentially affected.
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Materials and Methods |
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Proestrous females were caged overnight with males of proven fertility. Day 1 of pregnancy was designated as the day that sperm were found in the vagina. Pregnant rats were placed into three experimental groups: dimethylsulfoxide (DMSO) vehicle-treated (control), 25 µg/kg body weight/d BPA (25-BPA), and 250 µg/kg body weight/d BPA (250-BPA).
Timed pregnant rats were assigned to each group (n = 7–9 mothers/treatment group) and then individually housed in stainless steel cages. In our colony, delivery occurs on d 23 between 1230–1400 h. On gestation d 8, a miniature osmotic pump (model 1002, Alza Corp., Palo Alto, CA) was inserted sc over the spine caudal into the scapula. Osmotic pumps were filled with either 25-BPA or 250-BPA (Sigma-Aldrich Corp., St. Louis, MO) dissolved in DMSO (99.9%, molecular biology grade, Sigma-Aldrich Corp.) or only with DMSO in control rats. BPA or its vehicle was administered continuously from d 8 of gestation to the day of parturition (d 23). The BPA solutions were released at a rate of 0.25 µl/h. No signs of acute or chronic toxicity were observed, and no significant differences in weight gain between BPA-exposed and control mothers were recorded during gestation. No differences in litter size or pup body weight were observed at birth or at weaning. Moreover, sex ratios of the litters were comparable in the three groups, and AGD measured at birth and on postnatal d 4 did not differ among females or males of different groups (data not shown).
After parturition, pups were weighed and sexed according to AGD, and litters of eight pups (preferably four males and four females) were left with lactating mothers until sacrifice or weaning on postnatal d 22. Males from a single mother were killed on selected postnatal days representative of prepuberty (d 15), peripuberty (d 30), and adulthood (d 120). There were six to eight animals per group at each time point evaluated. Siblings were excluded from the same experimental group. Pups were injected with bromodeoxyuridine (BrdU; Sigma-Aldrich Corp.; 6 mg/100 g body weight/1.5 ml PBS, ip) 2 h before sacrifice. Animals were killed by decapitation, trunk blood was collected, and serum stored at -20 C until used for hormone assays.
Ventral prostates were microdissected, weighed, and fixed by immersion in 10% formalin buffer for 6 h at room temperature. Fixed tissue was dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Serial sections (5 µm thick) of ventral prostate were mounted on 3-aminopropyl triethoxysilane (Sigma-Aldrich Corp.)-coated slides and dried for 24 h at 37 C. For each prostate specimen, three sections separated at 20-µm intervals were evaluated. To secure uniformity between sections of each animal, a nonparametric ANOVA between sections of the same specimen was performed.
From d 30 and 120 rats, brain tissue blocks containing mainly the POA or MBH were quickly microdissected under a GZ6 series dissecting microscope (Leica Corp., Buffalo, NY). The POA fragment was encompassed by the anterior portion of the anterior commissure, the beginning of the ascending optical tracts, and laterally by a virtual line that is projected from the internal capsule to the external edge of the optical tracts. The MBH fragment was delimited by the beginning of the ascending optical tracts and the mammillary bodies. All microdissections were performed using thick coronal sections so that the ventral portion of the third ventricle was always visible (20). After removal, tissue samples were immediately frozen in liquid nitrogen and stored at -80 C until used for RNA analysis.
Immunohistochemistry and morphometric analysis
The expression of several markers was evaluated by immunohistochemistry to characterize the cellular phenotype and biological behavior of the prostatic tissue (15, 21). Incubation with primary antibodies was performed at 4 C for 14–16 h (Table 1
). Antigens were stained using 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp.), and sections were counterstained with Mayer’s hematoxylin (Biopur, Rosario, Argentina). Each immunohistochemical run included both positive and negative controls. Negative controls were incubated with nonimmune mouse or rabbit serum (Sigma-Aldrich Corp.).
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Image analysis of immunostained tissue sections was performed by color segmentation analysis as previously described (15). Briefly, cytoskeletal protein expression was quantified in the periductal stroma using an automated standard sequence programmed by Auto-Pro macro language. Using consecutive histological sections, the automatic script was performed to measure the percentage of the reference periductal area (relative area) occupied by vimentin (Vim) or -smooth muscle actin (-SMA) cells. The microscope was prepared for Koehler illumination. This was achieved by recording a reference image of an empty field for the correction of unequal illumination (shading correction) and by calibrating the measurement system with a reference slide to determine background threshold values. The reference slides contain a series of tissue sections stained in the absence of primary antibody. The image resolution was set at 640 x 480 pixels, and the final screen resolution was 0.103 µm/pixel.
For ER- and AR-positive cell characterization, 2 sections were evaluated for each prostate specimen, and 30 representative fields in each section were scored using a Dplan x40 objective. Positive cells were expressed as the percent ratio of the total number of epithelial or stromal cells measured in the examined area of the ventral prostate.
To obtain quantitative data regarding PAP expression in ductal epithelial cells, 2 sections for each prostate specimen were evaluated, and 30 representative fields in each section were digitalized and recorded using a Dplan x40 objective. Using Auto-Pro macro language, a second script was created to measure the OD as previously described by Ramos et al. (15). All epithelial cells with positive OD values were considered PAP-positive cells. Results were expressed as the percent ratio of the total number of epithelial cells measured in the examined area.
RNA analysis by RT-PCR
An optimized RT-PCR protocol was employed to analyze the relative expression levels of ER and ERß mRNAs in the POA and MBH on d 30 and 120 in rats in all experimental groups. Total RNA was isolated using the single step, acid guanidinium thiocyanate-phenol-chloroform extraction method (23). Equal quantities (4 µg) of total RNA were reverse transcribed into cDNA with avian myeloblastosis virus reverse transcriptase (12.5 U; Promega Corp., Madison, WI) using 200 pmol random primers (Promega Corp.). Twenty units of ribonuclease inhibitor (RNAout, Invitrogen, Buenos Aires, Argentina) and 100 nmol of a deoxy NTP mixture were added to each reaction tube in a final volume of 30 µl of 1x avian myeloblastosis virus reverse transcriptase buffer. RT was performed at 42 C for 90 min. Reactions were terminated by heating at 97 C for 5 min and cooling on ice, followed by dilution of the reverse transcribed cDNA with ribonuclease-free water to a final volume of 60 µl. RNA incubated under identical conditions, but without reverse transcriptase, served as a negative control. Primer pairs used for amplification of the target genes are shown in Table 2. The primers were selected based upon a previous reference (24) according to the published cDNA sequences of the rat ER (25), ERß (26), and L19 ribosomal protein (27). All amplifications were performed in duplicate. To perform comparative PCR, aliquots of cDNA samples equivalent to 800 ng total RNA input were used in each PCR amplification. Each reaction mixture contained 2.5 U Taq DNA polymerase (Promega Corp.), 2.5 mM MgCl2 (Promega Corp.), 0.2 mM of each of the four deoxy-NTPs (Promega Corp.), and 20 pmol of each primer (Invitrogen) in a final volume of 50 µl of 1x PCR Taq buffer. After initial denaturation at 97 C for 5 min, the reaction mixture was subjected to successive cycles of denaturation at 96 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 2 min. A final extension cycle at 72 C for 15 min was included. The optimal number of cycles for each reaction was determined experimentally to yield linear relationships between signal intensity and cycle number (Fig. 1). The optimal number of cycles was 36 for ER and ERß and 20 for L19 ribosomal protein, using separate reactions for each target gene. In all assays negative controls using RNA without RT and Taq polymerase-negative tubes were performed to minimize the introduction of potential artifacts. All PCR products were cloned using the TA cloning kit (Invitrogen), and specificity was confirmed by DNA sequencing (data not shown).
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and ERß PCR-amplified sequences were normalized with respect to that of the L19 ribosomal protein, allowing relative levels of the specific mRNAs to be expressed in arbitrary units.
Hormone assays
Serum levels of PRL and LH were determined by RIA using the kit provided by the NIDDK as previously described (28, 29). Results were expressed in terms of rat PRL (RP3) and rat LH (RP3) reference preparations. Intra- and interassay coefficients of variation for PRL were 8.1% and 11.4%, respectively, whereas coefficients for LH were 5.4% and 9.8%, respectively. The lowest detectable levels were 0.039 ng/ml for PRL and 0.016 ng/ml for LH. T concentrations were determined by RIA using [1,2-N-3H]T (60 Ci/mmol; NEN Life Science Products, Boston, MA) and a specific antibody (Immunotech Diagnostic, Montréal, Canada) as previously described and validated by Suescun et al. (30). The sensitivity of the assay was 12 pg/ml, and the intraassay coefficients of variation were 7.1%.
Data analysis
Statistical analyses were performed using the Kruskal-Wallis one-way ANOVA, and significance between groups was determined using the Dunn’s posttest. P < 0.05 was accepted as indicating a significant difference between groups. Nonlinear correlations were determined by sigmoidal approximation (31).
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Results |
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. There were no significant differences in the weights of ventral prostates between BPA-treated and control animals at any of the ages studied. At a histological level, the stroma surrounding the epithelial ducts of the adult control prostate is primarily comprised of -SMA+ cells that encompass the basal membrane of the epithelial ducts. In the distal tips, the smooth muscle cell layer is separated from the basal membrane in a discontinuous fashion by a sheath of fibroblasts along the prostatic ducts. This cellular arrangement allows the smooth muscle cells to come into direct contact with the epithelium. In the proximal tips, the covering fibroblasts became multilayered such that the smooth muscle cells directly contacted the ductal basal membrane sporadically. The fibroblastic sheath was recognized in serial histological sections by the staining pattern of the Vim+/-SMA- cells. On postnatal d 15, no differences were observed between BPA-exposed animals and controls in the relative area occupied by Vim+ (Fig. 2, A vs. D) and/or -SMA+ (Fig. 2, G vs. J) cells. In contrast, on d 30 in BPA-exposed groups, the Vim+/-SMA- cells (fibroblasts) exhibited a uniform, continuous halo surrounding the proximal and distal ducts; the halo was thicker around the proximal ducts than around the distal ducts (Fig. 2E). In addition, the -SMA+ cells (smooth muscle cells) formed a discontinuous layer in both distal and proximal ducts; these zones were thinner on d 30 in BPA-exposed animals (Fig. 2K) than in controls (Fig. 2H). In proximal and distal regions on d 30, the relative area occupied by Vim+/-SMA- cells in the periductal ring was significantly larger in BPA-exposed groups than in controls (Fig. 3A). On the same day postnatally, the relative area occupied by -SMA+ cells was smaller in BPA-exposed groups than in controls (Fig. 3B). No differences were observed in the relative areas occupied by Vim+ or -SMA+ cells between 25-BPA- and 250-BPA-exposed groups. During adulthood (d 120) the periductal stroma was mainly comprised of smooth muscle cells that maintained close contact with the epithelial basal membrane in both distal and proximal tips (Fig. 2, C and I). The enlarged sheath of fibroblasts observed in BPA-exposed males on d 30 was absent on d 120 (Fig. 2F). Furthermore, on d 120 the relative area occupied by Vim+/-SMA- or -SMA+ cells did not differ in either the distal or proximal region between BPA-exposed and control groups (Fig. 3, A and B, and Table 4).
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). However, a significant increase in cellular proliferation was detected in the periductal stroma of d 15 BPA-exposed rats (Fig. 3D). Proliferative indexes in periductal stroma during the peripubertal period (d 30) and adulthood (d 120) were not affected by BPA exposure (Table 4).
Prenatal BPA exposure induces transient changes in AR and PAP expression in the ventral prostate, whereas ER was not altered
Epithelial and stromal compartments were negative for ER in all groups studied (Table 4). Epithelial cells (basal and glandular) were strongly stained for AR in both proximal and distal prostate regions in all groups studied. No significant differences were observed between groups (data not shown). On postnatal d 15, more than 50% of the periductal stroma cells were AR+ in all groups studied (Fig. 3E). The AR staining intensity was higher in smooth muscle cells than in fibroblasts. On d 30 in controls, more than 60% of the periductal stromal cells remained AR+; however, a significant decrease in AR+ stromal cells was observed in BPA-exposed males (Fig. 3E). At adulthood (d 120), a sharp decrease in AR expression was observed in the ventral prostate periductal stroma of controls, with no significant differences observed between groups (Fig. 3E and Table 4).
Prostatic acid phosphatase immunostaining was restricted to the glandular epithelial cells of the proximal and distal ducts (Fig. 2, M–R, and Table 4). PAP was expressed throughout the cytoplasm; however, staining was concentrated in the apical region. In controls there was a sharp increase in the percentage of PAP+ cells and in the staining intensity from d 15–30 (Fig. 2, M vs. N), which was sustained on d 120 (Fig. 2O). In contrast, BPA-exposed animals did not exhibit this sharp increase on d 30 (Fig. 2Q), and levels of PAP expression returned to control values on d 120 (Figs. 2R and 3F).
Prenatal BPA exposure permanently up-regulates ERß mRNA expression in the male POA
The relative expression of ER and ERß mRNAs in the POA and MBH of d 30 and 120 animals was evaluated by RT-PCR. Validation of the RT-PCR assays is summarized in Fig. 1. For amplification in the exponential phase of PCR, different numbers of cycles were tested for each mRNA. All linear correlation coefficients were greater than 0.97 between 32 and 38 cycles for ER and ERß, and between 16 and 22 cycles for L19 ribosomal protein. Optimization of RT-PCR conditions was achieved using total RNA from a pool of control and BPA-exposed rat hypothalami. Quantitative analysis revealed a strong linear relationship between the amount of starting RNA and the intensity of the generated PCR signals (Fig. 1).
ER and ERß expression were differentially modulated by BPA in the POA (Fig. 4, A and B). Both doses of BPA employed induced a significant increase in POA ERß mRNA expression in peripubertal d 30 animals (P < 0.01; Fig. 4A). Overexpression of ERß mRNA in the POA was maintained on d 120 of BPA-exposed males (P < 0.01; Fig. 4B), suggesting that this was a permanent effect. On the other hand, BPA treatment did not alter POA ER mRNA expression levels, and neither ER nor ERß mRNA quantities were modified in the MBH of BPA-exposed groups in comparison with controls (Fig. 4, A and B).
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, A and C). No differences in PRL serum levels were observed between prepubertal (d 15) males prenatally exposed to BPA and control animals. On d 30, BPA-exposed males exhibited significantly higher PRL levels compared with controls; however, no differences were observed between 25-BPA- and 250-BPA-exposed groups (Fig. 5A). Upon adulthood (d 120), PRL serum levels in BPA-exposed males returned to normal (Fig. 5A). LH serum levels did not differ significantly between BPA-exposed animals and controls on any of the postnatal days examined (Fig. 5B), although BPA-exposed groups exhibited a high degree of variability.
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mRNA expression in both brain regions analyzed. Our results are in agreement with previous findings (13) showing no alterations in ER and ERß mRNA expression in the medial hypothalamus of neonatally BPA- and octylphenol-exposed male rats. However, this is the first report showing a permanent increase in ERß mRNA expression in the POA of prenatally BPA-exposed rats. Previous reports have demonstrated that ERß mRNA expression is sexually dimorphic in several nuclei of the rat POA and that orchidectomy of male neonates completely reverses the sexual phenotype of ERß expression in the anteroventral periventricular nucleus (37). In utero BPA exposure could disrupt ERß expression in sexual dimorphic structures, either modifying the total number of ERß-positive cells or affecting neuronal migration patterns during critical periods of brain maturation (38, 39). It has been shown that ER expression in the male rat brain is necessary to maintain normal sexual behavior patterns such as mounting or ejaculation (40). Because ERß can repress transcriptional activity of ER through the formation of ER/ERß heterodimers (41), and in utero BPA exposure reduces both the motivation to explore and anxiety in male pups (42), a possible relationship between the overexpression of ERß in the POA and abnormal sexual behavior should not be ruled out. It has been established that LH-releasing hormone (LHRH) neurons of the POA in male rats also coexpress ERß mRNA/protein (43). Moreover, repression of the LHRH promoter by estradiol has been shown in LHRH-producing GT1 cells (44). In our experimental setting, prenatal exposure to BPA resulted in a marked increase in POA ERß mRNA expression levels; therefore, it is reasonable to hypothesize that prenatal exposure to xenoestrogens may disrupt the sensitive negative feedback mechanisms normally regulating LHRH secretion. In the mouse, ERß expression in the anterior hypothalamus from the late gestational to the prepubertal period is higher in males, which exhibit high levels of locally produced estrogen after aromatization of testosterone (45). Thus, increased expression of the ERß subtype in the POA may contribute to a defective maturation of LHRH control circuits. These events could explain the higher T levels observed in BPA-exposed males during peripubertal periods. On the other hand, constitutive trans-activation of the vasopressin gene promoter by the unliganded ERß has been demonstrated using in vitro experiments (46). A similar interaction between high levels of ERß detected in BPA-treated males with the LHRH gene also cannot be ruled out, arising as an LHRH mRNA transcription control mechanism in a ligand-independent fashion (43, 46). Confirmation of this hypothesis awaits further investigation. Hyperprolactinemia is a well-established consequence of neonatal xenoestrogen exposure in estrogen-sensitive rats such as the Fisher 344 strain (12, 13, 14). However, little is known about the persistence of this phenomenon beyond the peripubertal age. Our results, using a Wistar-derived strain, indicate that the hyperprolactinemia is transient and is evident only in d 30 rats. It is not clear whether the xenoestrogen-induced hyperprolactinemia affects the onset of puberty and/or fertility in male rats. These issues will need to be addressed in future experiments.
In the present study, we did not observe significant differences in ventral prostate weights, AGD, or pups’ body weights between BPA-exposed and control animals. These results are in accordance with previous studies that used a similar range of BPA doses (47). However, we demonstrated in a previous study (15) that prenatal exposure to BPA alters the differentiation pattern of the periductal stromal cells in the rat ventral prostate on postnatal d 30. These results taken together suggest that differences in organ weight-related end points would not have enough sensitivity to significantly evaluate the effects of low doses of BPA. In BPA-exposed groups, the presence of a thick layer of Vim+/-SMA- cells in the periductal zone contrasts with the multicellular smooth muscle Vim-/-SMA+ layer observed in controls. Herein, we demonstrated that the presence of a thicker layer of fibroblasts in the periductal stroma on d 30 is the consequence of a modified proliferative status during early development (d 15). Contemporary with this disruption of cellular dynamics in BPA-exposed rats, increased T serum levels were observed. A cause-effect relationship between higher T serum levels and the modified proliferative indexes in the periductal stroma cannot be ruled out. Previous work has shown that ER expression is confined to mesenchymal cells only during early development of ventral prostate (48). In agreement with this study and confirming our previous report (15), control groups were negative for ER in both epithelial and stromal compartments, and BPA did not modify this pattern of expression. Decreased PAP expression observed in BPA-exposed groups could be mediated by either a direct effect of BPA on the columnar epithelial cells or through an indirect consequence of primary events occurring in the stroma (16, 17, 49). Stromal signals are believed to be critical in determining the decision of epithelial cells to undergo proliferation, apoptosis, or differentiation. The decreased AR expression observed in the periductal stromal cells on d 30 may affect the androgen-signaling pathway resulting in decreased PAP expression, although a direct effect of BPA on the epithelial cells also cannot be ruled out. During adulthood, no differences were observed in the periductal stromal architecture. Prostatic acid phosphatase expression in columnar epithelial cells during adulthood was increased in all groups analyzed, and the differences observed on d 30 were not present on d 120. In addition, functional relationships have been established between hyperprolactinemia and prostatic tissue disorders in rats (14). As the histoarchitectural and functional changes that were observed in the ventral prostates of BPA-exposed rats are temporally associated with elevated serum PRL levels, further studies will need to be undertaken to elucidate the possible relationships between these phenomena. The transient characteristic of the changes observed in the ventral prostates and in T and PRL serum levels could be the result of early organizational effects that are associated with the actions of exogenous or endogenous estrogens during organogenesis and development (1). Our hypothesis is that organizational effects induced by low doses of xenoestrogens during early development stages could be expressed during critical periods such as puberty, when the hormonal milieu exhibits a high degree of variation, and compensatory mechanisms (such as negative feedback systems) are not fully developed.
In summary, in utero exposure to environmentally relevant levels of BPA increased ERß mRNA expression in the anterior hypothalamic structures of the pubertal male rat, and this overexpression persisted into adulthood. In contrast, prenatal exposure to BPA transiently affects the ventral prostate of peripubertal rats by decreasing AR expression, altering proliferative activity in the periductal stromal cells, and decreasing PAP expression. All of these morphological and functional changes that were observed in the ventral prostate on d 30 were transient, as they were not observed during adulthood. In addition, other transient effects observed were increases in T and PRL serum levels in prepubertal BPA-exposed rats, whereas no differences were detected during adulthood. A close time dependence of the biological responses to xenoestrogens during development together with the differential estrogen sensitivity of the various rat strains employed and/or end points examined could explain at least in part the extraordinary variability in the in vivo results obtained to date regarding endocrine disrupters (1, 10, 47, 50, 51).
The findings reported in this study support the hypothesis that environmental xenoestrogen exposure may be associated with low seminal quality and other male reproductive dysfunctions that affect human fertility (2, 3).
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Acknowledgments |
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Footnotes |
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1 J.G.R. and J.V. contributed equally to this work. 
Abbreviations: AGD, Anogenital distance; AR, androgen receptor; BPA, bisphenol A; BrdU, bromodeoxyuridine; DMSO, dimethylsulfoxide; ER, estrogen receptor; LHRH, LH-releasing hormone; MBH, medial basal hypothalamus; PAP, prostatic acid phosphatase; POA, preoptic area; -SMA, -smooth muscle actin; T, testosterone; Vim, vimentin.
Received December 26, 2002.
Accepted for publication March 19, 2003.
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and ß mRNA expression in rat testis during postnatal development. J Endocrinol 165:345–357[Abstract]
-aminobutyric acid to the ventromedial nucleus of the hypothalamus during embryonic development. J Comp Neurol 405:88–98[CrossRef][Medline]
in the uterus. Proc Natl Acad Sci USA 97:5936–5941 (ER)- and ERß-expressing GTI-7 GnRH neurons. Endocrinology 140:5045–5053 and ß. Endocrinology 141:4056–4064This article has been cited by other articles:
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  J. Varayoud, J. G. Ramos, V. L. Bosquiazzo, M. Munoz-de-Toro, and E. H. Luque Developmental Exposure to Bisphenol A Impairs the Uterine Response to Ovarian Steroids in the Adult Endocrinology, November 1, 2008; 149(11): 5848 - 5860. [Abstract] [Full Text] [PDF]
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 L. Monje, J. Varayoud, E. H Luque, and J. G Ramos Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor {alpha} transcripts with alternative 5'-untranslated regions in the female rat preoptic area J. Endocrinol., July 1, 2007; 194(1): 201 - 212. [Abstract] [Full Text] [PDF]
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 R. M. Facciolo, M. Madeo, R. Alo, M. Canonaco, and F. Dessi-Fulgheri Neurobiological Effects of Bisphenol A May Be Mediated by Somatostatin Subtype 3 Receptors in Some Regions of the Developing Rat Brain Toxicol. Sci., December 1, 2005; 88(2): 477 - 484. [Abstract] [Full Text] [PDF]
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J Varayoud, J G Ramos, L Monje, V Bosquiazzo, M Munoz-de-Toro, and E H Luque The estrogen receptor {alpha} {Sigma}3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus J. Endocrinol., July 1, 2005; 186(1): 51 - 60. [Abstract] [Full Text] [PDF]
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