Chhanda Gupta

Reproductive Malformation of the Male Offspring Following Maternal Exposure to Estrogenic Chemicals

Recently, significant concerns have been placed on the widespread use of chemicals with persistent estrogenic activity for their long-term effects on human health. In this communication, we investigated whether fetal exposure to some of these chemicals at doses consumed by people, has any long-term effect on the reproductive functions of the male offspring. Thus, time-pregnant CD-1 mice were fed diethylstilbestrol (DES), bisphenol A (BPA), and aroclor (aroclor 1016) at an average concentration of 100 ng/kg/day, 50 microg/kg/day, and 50 microg/kg/day, respectively, during Days 16-18 of gestation. A high dose of DES (200 microg/kg/day) was also tested to compare the results of the current study with those of others using the high dose only. The offspring were examined at Day 3, Day 21, and Day 60 following birth. We demonstrated that BPA, aroclor, and the lower dose of DES enhanced anogenital distance, increased prostate size, and decreased epididymal weight. No effect was found on the testicular weight or size. The chemicals also permanently increased androgen receptor (AR) binding activity of the prostate at this dosage. This is the first demonstration that environmental chemicals program AR function permanently at the dosage consumed by the general population. The higher dosage of DES, on the other hand, produced an opposite effect, decreasing prostate weight, prostate AR binding, and anogenital distance, thus confirming the previous reports. To investigate whether the above mentioned effects of the chemicals represent direct or indirect effects, we also tested the effect of the chemicals on prostate development in vitro. Thus fetal urogenital sinus (UGS), isolated at the 17th day of gestation was cultured with the chemicals in the presence and absence of testosterone (10 ng/ml) for 6 days, and prostate growth was monitored by determining the size and branching of the specimen following histology. Results showed that these chemicals induced prostate growth in the presence and absence of testosterone. They also increased androgen-binding activity. Thus, the results of the in vivo studies were reproduced in the in vitro experiments, suggesting a direct effect of these chemicals on the development of fetal reproductive organs. This is the first demonstration that estrogenic chemicals induce reproductive malformation by direct interference with the fetal reproductive organs and not by interfering with the maternal or fetal endocrine system. The chemicals are able to induce malformation even in the absence of fetal testosterone; however, they are more effective in the presence of testosterone.

The role of EGF in testosterone-induced reproductive tract differentiation

EGF is known to modulate a variety of cellular functions including differentiation. The aim of this investigation was to determine the role of EGF in androgen-induced masculine differentiation. Accordingly, a series of experiments were designed and the results are summarized as described below. (1) We found that the specific deprivation of EGF using anti-EGF serum during the period of masculine differentiation in an organ culture bioassay system resulted in the disintegration of the Wolffian system in a dose-dependent manner. (2) Exogenous EGF supplemented in the above experiment corrected the anti-EGF effect, suggesting a specific role of EGF. (3) Anti-EGF serum was also found to disrupt the differentiation even in the presence of exogenous testosterone, suggesting an effect independent of testosterone synthesis. (4) EGF was found to have a direct masculinizing effect both in vivo and in vitro; however, it was not able to mimic all masculinizing effects of testosterone. The mesonephric segment of the Wolffian duct was retained by EGF in the female fetal tract under in vitro conditions, and under in vivo conditions EGF was able to increase anogenital distance and to induce epididymis in some female fetal mice. (5) We were able to detect an EGF-like material in the fetal genital tract during differentiation and found that the level of this material increased with advancement of differentiation. Thus, it appears from the above results that EGF plays a role in testosterone-induced reproductive tract differentiation.

Modulation of androgen receptor (AR)-mediated transcriptional activity by EGF in the developing mouse reproductive tract primary cells.

Recently, a role for epidermal growth factor in male sexual differentiation was reported from different laboratories. We demonstrated that androgen receptor (AR) mediates the EGF-induced effects. The mechanism, by which EGF modulates AR mediated activity, is not known and the current studies were designed to investigate the role of AR. Using mesenchymal cell preparation from the 18-day fetal reproductive tract, first, we determined whether EGF induced sexual differentiation by enhancing AR gene expression. Thus, AR mRNA and AR protein levels were measured in response to EGF-treatment using RT-PCR based analysis of AR mRNA and Western blot analysis of AR protein level respectively. Both of these analysis detected presence of AR gene expression in this cell preparation, however no effect of EGF was observed in AR protein or AR mRNA expression. Next, we examined whether EGF enhanced AR mediated transcriptional activity in the developing tract. Using the mesenchymal cells, co-transfected with AR expression vector (pSV-AR) and androgen response element linked to luciferase reporter vector (pMAMneoLUC) we assessed AR-mediated transcriptional activity in response to EGF treatment in the presence and absence of testosterone. The results showed that androgen stimulated the luciferase activity in a dose dependent manner, as expected. EGF also enhanced such activity; however, the response was significantly lower than that by androgen. EGF, however, produced striking enhancement of the androgen-induced transcriptional activity when used with androgen. EGF and testosterone produced no stimulation of the luciferase activity either alone or in combination in the cells lacking AR expression vector, suggesting a role for AR in the effect of EGF and testosterone. Flutamide, an AR antagonist, also blocked the enhancement of luciferase activity induced by EGF and testosterone, further confirming the role of AR in the effect of EGF and testosterone. Thus, it appears that EGF-modulation of sexual differentiation involves enhancement of AR-mediated transcriptional activity and not enhancement of AR gene expression. Additionally, it appears that EGF modulates sexual differentiation in the presence of testosterone possibly by potentiating the testosterone-effect.

ACTIVATION OF ANDROGEN RECEPTOR IN EPIDERMAL GROWTH FACTOR MODULATION OF FETAL MOUSE SEXUAL DIFFERENTIATION

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male sexual differentiation. The mechanism by which EGF modulates male sexual differentiation has not been determined and investigation has been made to assess the role for androgen receptor (AR) in mediating the EGF-induced effect. We report that EGF, like androgen, stabilized the Wolffian duct in the 13-day female specimen, grown in organ culture. Anti-AR, flutamide and cyproterone acetate blocked the Wolffian duct-stabilizing effect of EGF. EGF also induced cell proliferation of the fetal reproductive tract in a dose-dependent manner and a combination of physiological dosages of EGF and androgen-induced cell proliferation synergistically, suggesting an interactive effect of these two drugs. Cyproterone acetate blocked both EGF-induced normal cell proliferation and the synergistic cell proliferation induced by combination of EGF and androgen suggesting a role of AR in the effects of EGF. The role of AR was further assessed by determining the effect of EGF on AR binding directly. It was shown that EGF stimulated androgen binding activity of the male fetal reproductive tract cells significantly by increasing the number of binding sites by 3-fold with slight decrease in binding affinity. Thus, it appears that AR plays a role in mediating EGF-modulation of sexual differentiation.

Activation of Phospholipases during Masculine Differentiation of Embryonic Genitalia

Abstract We have investigated whether the androgen-induced masculine differentiation of the sex organs involves an induction of phospholipases. We have measured phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase A2 in the reproductive tract of male and female mouse (CD-I) fetuses at the 18th day of gestation. We report here that (1) the activity of these two enzymes is higher in the male genitalia than in the female genitalia; (2) exogenous testosterone at the 13th to 17th day of pregnancy induces both phospholipase A2 and phospholipase C in the female fetal genitalia; and (3) prenatal administration of cyproterone acetate, an antiandrogen, known to produce feminized males, completely prevents the stimulation of phospholipase A2 and C by testosterone in the female fetuses. In the male fetuses, however, cyproterone acetate inhibits the PLC activity but is unable to alter phospholipase A2 activity. These findings provide evidence that the mechanism by which testosterone organizes the genitalia may involve a modification of phospholipases A2 and C.

Further characterization of the testosterone inducible protein fraction from the mouse fetal male reproductive tract inducing masculine differentiation in vitro

Recently, we identified a protein fraction with phospholipase A2 (PLA2) stimulatory activity (named as PLSP in previous publications) from the fetal male reproductive tract which induced masculine differentiation of the Wolffian duct in vitro. The role of the PLA2-stimulatory activity of this protein was not clear from the past investigations and the present study was designed to elucidate its role. PLSP as expected stimulated snake venom PLA2 and it induced masculinization not only in male explant but also in female explants in a dose-dependent manner in the absence of gonads. However, neither its PLA2-stimulatory activity nor its masculinizing activity was affected after passing it through a PLA2-Sepharose column (a compound expected to eliminate any PLA2 binding component) suggesting that the PLA2-stimulatory activity of PLSP was not at the level of PLA2 enzyme. To investigate whether modulation of the PLA2 substrate by PLSP played a role in producing PLA2-stimulatory activity, we determined the effect of increasing amounts of unlabeled substrate on the PLA2-stimulatory activity induced by PLSP. However, no change in the PLA2-stimulatory activity of PLSP by unlabeled substrate was noticed suggesting against this possibility. The PLA2-stimulatory activity of PLSP, however, was found completely abolished when it was subjected to extensive dialysis, while its protein composition and masculinizing activity remained unaffected. Thus, it appears that PLA2-stimulatory activity of PLSP is associated with a small compound co-purified with masculinizing protein of PLSP and this activity plays no role in inducing masculinization by PLSP.

Anti-masculinizing action of estradiol and cyproterone acetate: Regulation of a protein fraction with phospholipase-A2 stimulatory and masculinizing activities

Recently we have identified a protein fraction (55-63 K) from male and testosterone-exposed female mouse genital tract, which stimulates phospholipase A2 (PLA2) and induces masculine differentiation in an undifferentiated mouse genital explant, suggesting a role of this protein in the action of testosterone. In the current study we have further investigated the role of this protein by determining whether anti-masculinizing agents, namely, estradiol and cyproterone acetate, have any effect on the production of this protein. The results described here indicate that a protein fraction containing PLA2 stimulatory activity was present in both control male and estradiol- or cyproterone acetate-exposed male fetal genital tract. However the specific activity of the PLA2-stimulatory protein was significantly higher in the control males than in the experimental males. We did not find any major difference in the behavior of this protein fraction in various chromatographic steps except that in CM-sepharose column; the PLA2-stimulatory activity from the male preparation was eluted in two overlapping peaks with 0.3 and 0.25 M NaCl and that from the treated males was eluted only with 0.25 M NaCl. The SDS-gel analysis of this protein fraction revealed a doublet band (55 and 63 K) in control samples and primarily a 63 K band in experimental samples. The protein fraction from all these sources showed a significant difference in their biological activity. The control male preparation induced Wolffian duct whereas the estradiol sample was completely ineffective and the cyproterone acetate sample was partially effective in inducing Wolffian duct. Thus, it appears that the protein fraction has a role in the masculinizing action of testosterone.

Choline Deficiency, Lipid Peroxidation, Liver Cell Surface Receptor Alterations and Liver Tumor Promotion

The two-stage model of carcinogenesis, originally demonstrated in the skin of mice and rabbits, has been extended to several other organ systems including the liver1,2. The importance of tumor promotion in the genesis of human cancers has been recognized3, and the better understanding of the mechanism of tumor promotion would be critical in the formulation of the overall strategies for the prevention of human cancers. Considerable insights have been gained for the mechanisms of action of phorbol esters, the classical and perhaps most intensively studied skin tumor promoter4 Studies of liver carcinogenesis during the past 15 years have identified a number of promoters with diverse properties1, but the mechanisms of their action remain elusive.

Male Accessory Glands: Molecular Mechanism of Development

Over the past decade, knowledge of human sexual differentiation has greatly expanded to understand the processes involved in the development of male accessory glands. Sexual differentiation occurs in three consecutive levels. The chromosomal sex is determined by distribution of sex chromosomes during meiosis. This is followed by gonadal sex determination by the Sry locus on the Y chromosome, leading to development of the testis. The testis induces two hormones, namely, anti-Mullerian hormone (AMH) and testosterone, determining the somatic sex. AMH causes regression of Mullerian ducts whereas testosterone maintains the Wolffian ducts and induces male genital tract and male phenotype formation. In the absence of the sex-determining region of the Y chromosome (SRY), the ovary develops from the indifferent gonad. In the absence of AMH and testosterone, the Mullerian ducts constitutively differentiate and the Wolffian duct regresses resulting in the formation of a female external phenotype. Present knowledge on the role of different factors mediating development of male accessory glands is based on key experiments performed by several investigators (1–5). In 1940, Jost demonstrated a role for AMH using a rabbit embryo, castrated in utero (1). In 1942, Raynaud demonstrated a role of testosterone during embryonic development (2) and in 1967, Ohno identified the Sry locus determining the testis (3).