Classical Estrogen Receptors and ERα Splice Variants in the Mouse

Estrogens exert a variety of effects in both reproductive and non-reproductive tissues. With the discovery of ERα splice variants, prior assumptions concerning tissue-specific estrogen signaling need to be re-evaluated. Accordingly, we sought to determine the expression of the classical estrogen receptors and ERα splice variants across reproductive and non-reproductive tissues of male and female mice. Western blotting revealed that the full-length ERα66 was mainly present in female reproductive tissues but was also found in non-reproductive tissues at lower levels. ERα46 was most highly expressed in the heart of both sexes. ERα36 was highly expressed in the kidneys and liver of female mice but not in the kidneys of males. ERβ was most abundant in non-reproductive tissues and in the ovaries. Because the kidney has been reported to be the most estrogenic non-reproductive organ, we sought to elucidate ER renal expression and localization. Immunofluorescence studies revealed ERα66 in the vasculature and the glomerulus. It was also found in the brush border of the proximal tubule and in the cortical collecting duct of female mice. ERα36 was evident in mesangial cells and tubular epithelial cells of both sexes, as well as podocytes of females but not males. ERβ was found primarily in the podocytes in female mice but was also present in the mesangial cells in both sexes. Within the renal cortex, ERα46 and ERα36 were mainly located in the membrane fraction although they were also present in the cytosolic fraction. Given the variability of expression patterns demonstrated herein, identification of the specific estrogen receptors expressed in a tissue is necessary for interpreting estrogenic effects. As this study revealed expression of the ERα splice variants at multiple sites within the kidney, further studies are warranted in order to elucidate the contribution of these receptors to renal estrogen responsiveness.     

Differential Effects of Bisphenol A and Diethylstilbestrol on Human,Rat and Mouse Fetal Leydig Cell Function

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10⁻¹² to 10⁻⁵ M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5–10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1–3 days. BPA concentrations as low as 10⁻⁸ M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10⁻⁵ M BPA were required. Similarly, 10⁻⁸ M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10⁻⁵ and 10⁻⁶ M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10⁻⁸ M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.     

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.     

Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β

Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions.     

Bisphenol a exposure causes meiotic aneuploidy in the female mouse

BACKGROUND: There is increasing concern that exposure to man-made substances that mimic endogenous hormones may adversely affect mammalian reproduction. Although a variety of reproductive complications have been ascribed to compounds with androgenic or estrogenic properties, little attention has been directed at the potential consequences of such exposures to the genetic quality of the gamete. RESULTS: A sudden, spontaneous increase in meiotic disturbances, including aneuploidy, in studies of oocytes from control female mice in our laboratory coincided with the accidental exposure of our animals to an environmental source of bisphenol A (BPA). BPA is an estrogenic compound widely used in the production of polycarbonate plastics and epoxy resins. We identified damaged caging material as the source of the exposure, as we were able to recapitulate the meiotic abnormalities by intentionally damaging cages and water bottles. In subsequent studies of female mice, we administered daily oral doses of BPA to directly test the hypothesis that low levels of BPA disrupt female meiosis. Our results demonstrated that the meiotic effects were dose dependent and could be induced by environmentally relevant doses of BPA. CONCLUSIONS: Both the initial inadvertent exposure and subsequent experimental studies suggest that BPA is a potent meiotic aneugen. Specifically, in the female mouse, short-term, low-dose exposure during the final stages of oocyte growth is sufficient to elicit detectable meiotic effects. These results provide the first unequivocal link between mammalian meiotic aneuploidy and an accidental environmental exposure and suggest that the oocyte and its meiotic spindle will provide a sensitive assay system for the study of reproductive toxins.     

Exposure to bisphenol A disrupts meiotic progression during spermatogenesis in adult rats through estrogen-like activity

The effect of bisphenol A (BPA) on the reproductive system is highly debated but has been associated with meiotic abnormalities. However, evidence is lacking with regard to the mechanisms involved. In order to explore the underlying mechanisms of BPA-induced meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 0, 2, 20 or 200 μg/kg body weight (bw)/day for 60 consecutive days. 17β-Estradiol (E₂) was administered at 10 μg/kg bw/day as the estrogenic positive control. Treatments with 200 μg/kg bw/day of BPA and E₂ significantly decreased sperm counts and inhibited spermiation, characterized by an increase in stage VII and decrease in stage VIII in the seminiferous epithelium. This was concomitant with a disruption in the progression of meiosis I and the persistence of meiotic DNA strand breaks in pachytene spermatocytes,and the ataxia–telangiectasia-mutated and checkpoint kinase 2 signal pathway was also activated; Eventually, germ cell apoptosis was triggered as evaluated by terminal dUTP nick-end labeling assay and western blot for caspase 3. Using the estrogen receptor (ER) antagonist ICI 182780, we determined that ER signaling mediated BPA-induced meiotic disruption and reproductive impairment. Our results suggest that ER signaling-mediated meiotic disruption may be a major contributor to the molecular events leading to BPA-related male reproductive disorders. These rodent data support the growing association between BPA exposure and the rapid increase in the incidence of male reproductive disorders.     

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.     

Estrogenic Exposure Alters the Spermatogonial Stem Cells in the Developing Testis,Permanently Reducing Crossover Levels in the Adult

Bisphenol A (BPA) and other endocrine disrupting chemicals have been reported to induce negative effects on a wide range of physiological processes, including reproduction. In the female, BPA exposure increases meiotic errors, resulting in the production of chromosomally abnormal eggs. Although numerous studies have reported that estrogenic exposures negatively impact spermatogenesis, a direct link between exposures and meiotic errors in males has not been evaluated. To test the effect of estrogenic chemicals on meiotic chromosome dynamics, we exposed male mice to either BPA or to the strong synthetic estrogen, ethinyl estradiol during neonatal development when the first cells initiate meiosis. Although chromosome pairing and synapsis were unperturbed, exposed outbred CD-1 and inbred C3H/HeJ males had significantly reduced levels of crossovers, or meiotic recombination (as defined by the number of MLH1 foci in pachytene cells) by comparison with placebo. Unexpectedly, the effect was not limited to cells exposed at the time of meiotic entry but was evident in all subsequent waves of meiosis. To determine if the meiotic effects induced by estrogen result from changes to the soma or germline of the testis, we transplanted spermatogonial stem cells from exposed males into the testes of unexposed males. Reduced recombination was evident in meiocytes derived from colonies of transplanted cells. Taken together, our results suggest that brief exogenous estrogenic exposure causes subtle changes to the stem cell pool that result in permanent alterations in spermatogenesis (i.e., reduced recombination in descendent meiocytes) in the adult male.     

Bisphenol A exposure modifies methylation of imprinted genes in mouse oocytes via the estrogen receptor signaling pathway

Bisphenol A (BPA), a synthetic additive used to harden polycarbonate plastics and epoxy resin, is ubiquitous in our everyday environment. Many studies have indicated detrimental effects of BPA on the mammalian reproductive abilities. This study is aimed to test the potential effects of BPA on methylation of imprinted genes during oocyte growth and meiotic maturation in CD-1 mice. Our results demonstrated that BPA exposure resulted in hypomethylation of imprinted gene Igf2r and Peg3 during oocyte growth, and enhanced estrogen receptor (ER) expression at the levels of mRNA and protein. The relationship between ER expression and imprinted gene hypomethylation was substantiated using an ER inhibitor, ICI182780. In addition, BPA promoted the primordial to primary follicle transition, thereby speeding up the depletion of the primordial follicle pool, and suppressed the meiotic maturation of oocytes because of abnormal spindle assembling in meiosis I. In conclusion, neonatal exposure to BPA inhibits methylation of imprinted genes during oogenesis via the ER signaling pathway in CD-1 mice.     

Pancreatic Insulin Content Regulation by the Estrogen Receptor ERα

The function of pancreatic β-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ERα and ERβ, are important molecules involved in glucose metabolism, yet their role in pancreatic β-cell physiology is still greatly unknown. In this report we show that both ERα and ERβ are present in pancreatic β-cells. Long term exposure to physiological concentrations of 17β-estradiol (E2) increased β-cell insulin content, insulin gene expression and insulin release, yet pancreatic β-cell mass was unaltered. The up-regulation of pancreatic β-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ERα and ERβ agonists as well as ERαKO and ERβKO mice suggests that the estrogen receptor involved is ERα. The up-regulation of pancreatic insulin content by ERα activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis

Introduction

Estrogen (E2) delays onset and decreases severity of experimental arthritis. The aim of this study was to investigate the importance of total estrogen receptor alpha (ERα) expression and cartilage-specific ERα expression in genetically modified mice for the ameliorating effect of estrogen treatment in experimental arthritis.

Methods

Mice with total (total ERα^-/-) or cartilage-specific (Col2α1-ERα^-/-) inactivation of ERα and wild-type (WT) littermates were ovariectomized, treated with E2 or placebo, and induced with antigen-induced arthritis (AIA). At termination, knees were collected for histology, synovial and splenic cells were investigated by using flow cytometry, and splenic cells were subjected to a T-cell proliferation assay.

Results

E2 decreased synovitis and joint destruction in WT mice. Amelioration of arthritis was associated with decreased frequencies of inflammatory cells in synovial tissue and decreased splenic T-cell proliferation. E2 did not affect synovitis or joint destruction in total ERα^-/- mice. In Col2α1-ERα^-/- mice, E2 protected against joint destruction to a similar extent as in WT mice. In contrast, E2 did not significantly ameliorate synovitis in Col2α1-ERα^-/- mice.

Conclusions

Treatment with E2 ameliorates both synovitis and joint destruction in ovariectomized mice with AIA via ERα. This decreased severity in arthritis is associated with decreased synovial inflammatory cell frequencies and reduced splenic T-cell proliferation. ERα expression in cartilage is not required for estrogenic amelioration of joint destruction. However, our data indicate that ERα expression in cartilage is involved in estrogenic effects on synovitis, suggesting different mechanisms for the amelioration of joint destruction and synovitis by E2.     

Estrogen regulates histone deacetylases to prevent cardiac hypertrophy

The development and progression of cardiac hypertrophy often leads to heart failure and death, and important modulators of hypertrophy include the histone deacetylase proteins (HDACs). Estrogen inhibits cardiac hypertrophy and progression in animal models and humans. We therefore investigated the influence of 17-β-estradiol on the production, localization, and functions of prohypertrophic (class I) and antihypertrophic (class II) HDACs in cultured neonatal rat cardiomyocytes. 17-β-Estradiol or estrogen receptor β agonists dipropylnitrile and β-LGND2 comparably suppressed angiotensin II–induced HDAC2 (class I) production, HDAC-activating phosphorylation, and the resulting prohypertrophic mRNA expression. In contrast, estrogenic compounds derepressed the opposite effects of angiotensin II on the same parameters for HDAC4 and 5 (class II), resulting in retention of these deacetylases in the nucleus to inhibit hypertrophic gene expression. Key aspects were confirmed in vivo from the hearts of wild-type but not estrogen receptor β (ERβ) gene–deleted mice administered angiotensin II and estrogenic compounds. Our results identify a novel dual regulation of cardiomyocyte HDACs, shown here for the antihypertrophic sex steroid acting at ERβ. This mechanism potentially supports using ERβ agonists as HDAC modulators to treat cardiac disease.     

aP2-Cre-Mediated Inactivation of Estrogen Receptor Alpha Causes Hydrometra

In this study we describe the reproductive phenotypes of a novel mouse model in which Cre-mediated deletion of ERα is regulated by the aP2 (fatty acid binding protein 4) promoter. ERα-floxed mice were crossed with transgenic mice expressing Cre-recombinase under the control of the aP2 promoter to generate aP2-Cre/ERα^flox/flox mice. As expected, ERα mRNA levels were reduced in adipose tissue, but in addition we also detected an 80% reduction of ERα levels in the hypothalamus of aP2-Cre/ERα^flox/flox mice. Phenotypic analysis revealed that aP2-Cre/ERα^flox/flox female mice were infertile. In line with this, aP2-Cre/ERα^flox/flox female mice did not cycle and presented 3.8-fold elevated estrogen levels. That elevated estrogen levels were associated with increased estrogen signaling was evidenced by increased mRNA levels of the estrogen-regulated genes lactoferrin and aquaporin 5 in the uterus. Furthermore, aP2-Cre/ERα^flox/flox female mice showed an accumulation of intra-uterine fluid, hydrometra, without overt indications for causative anatomical anomalies. However, the vagina and cervix displayed advanced keratosis with abnormal quantities of accumulating squamous epithelial cells suggesting functional obstruction by keratin plugs. Importantly, treatment of aP2-Cre/ERα^flox/flox mice with the aromatase inhibitor Letrozole caused regression of the hydrometra phenotype linking increased estrogen levels to the observed phenotype. We propose that in aP2-Cre/ERα^flox/flox mice, increased serum estrogen levels cause over-stimulation in the uterus and genital tracts resulting in hydrometra and vaginal obstruction.     

Regional Difference in Sex Steroid Action on Formation of Morphological Sex Differences in the Anteroventral Periventricular Nucleus and Principal Nucleus of the Bed Nucleus of the Stria Terminalis

Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-β (ERβ). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERβ genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERβ gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.     

Naringenin modulates skeletal muscle differentiation via estrogen receptor α and β signal pathway regulation

Several experiments sustain healthful benefits of the flavanone naringenin (Nar) against chronic diseases including its protective effects against estrogen-related cancers. These experiments encourage Nar use in replacing estrogen treatment in post-menopausal women avoiding the serious side effects ascribed to this hormone. However, at the present, scarce data are available on the impact of Nar on E2-regulated cell functions. This study was aimed at determining the impact of Nar on the estrogen receptor (ERα and β)-dependent signals important for 17β-estradiol (E2) effect in muscle cells (rat L6 myoblasts, mouse C2C12 myoblasts, and mouse skeletal muscle satellite cells). Dietary relevant concentration of Nar delays the appearance of skeletal muscle differentiation markers (i.e., GLUT4 translocation, myogenin, and both fetal and slow MHC isoforms) and impairs E2 effects specifically hampering ERα ability to activate AKT. Intriguingly, Nar effects are specific for E2-initiating signals because IGF-I-induced AKT activation, and myoblast differentiation markers were not affected by Nar treatment. Only 7 days after Nar stimulation, early myoblast differentiation markers (i.e., myogenin, and fetal MHC) start to be accumulated in myoblasts. On the other hand, Nar stimulation activates, via ERβ, the phosphorylation of p38/MAPK involved in reducing the reactive oxygen species formation in skeletal muscle cells. As a whole, data reported here strongly sustain that although Nar action mechanisms include the impairment of ERα signals which drive muscle cells to differentiation, the effects triggered by Nar in the presence of ERβ could balance this negative effect avoiding the toxic effects produced by oxidative stress .

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0425-3) contains supplementary material, which is available to authorized users.     

Mouse Leydig Cells with Different Androgen Production Potential Are Resistant to Estrogenic Stimuli but Responsive to Bisphenol A Which Attenuates Testosterone Metabolism

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E₂) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E₂ ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.