Rupesh K. Gupta

Bisphenol A Impairs Follicle Growth, Inhibits Steroidogenesis, and Downregulates Rate-Limiting Enzymes in the Estradiol Biosynthesis Pathway

Bisphenol A (BPA) is used as the backbone for plastics and epoxy resins, including various food and beverage containers. BPA has also been detected in 95% of random urine samples and ovarian follicular fluid of adult women. Few studies have investigated the effects of BPA on antral follicles, the main producers of sex steroid hormones and the only follicles capable of ovulation. Thus, this study tested the hypothesis that postnatal BPA exposure inhibits antral follicle growth and steroidogenesis. To test this hypothesis, antral follicles isolated from 32-day-old FVB mice were cultured with vehicle control (dimethyl sulfoxide [DMSO]), BPA (4.4-440 μM), pregnenolone (10 μg/ml), pregnenolone + BPA 44 μM, and pregnenolone + BPA 440 μM. During the culture, follicles were measured for growth daily. After the culture, media was subjected to ELISA for hormones in the estradiol biosynthesis pathway, and follicles were processed for quantitative real-time PCR of steroidogenic enzymes. The results indicate that BPA (440 μM) inhibits follicle growth and that pregnenolone cotreatment was unable to restore/maintain growth. Furthermore, BPA 44 and 440 μM inhibit progesterone, dehydroepiandrosterone, androstenedione, estrone, testosterone, and estradiol production. Pregnenolone cotreatment was able to increase production of pregnenolone, progesterone, and dehydroepiandrosterone and maintain androstenedione and estrone levels in BPA-treated follicles compared with DMSO controls but was unable to protect testosterone or estradiol levels. Furthermore, pregnenolone was unable to protect follicles from BPA-(44-440 μM) induced inhibition of steroidogenic enzymes compared with the DMSO control. Collectively, these data show that BPA targets the estradiol biosynthesis pathway in the ovary.

Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro

Any insult that affects survival of ovarian antral follicles can cause abnormal estradiol production and fertility problems. Phthalate esters (PEs) are plasticizers used in a wide range of consumer and industrial products. Exposure to these chemicals has been linked to reduced fertility in humans and animal models. Di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) decrease serum estradiol levels and aromatase (Arom) expression, prolong estrous cycles, and cause anovulation in animal and culture models. These observations suggest PEs directly target antral follicles. We therefore tested the hypothesis that DEHP (1-100 microg/ml) and MEHP (0.1-10 microg/ml) directly inhibit antral follicular growth and estradiol production. Antral follicles from adult mice were cultured with DEHP or MEHP, and/or estradiol for 96 h. During culture, follicle size was measured every 24 h as a measurement of follicle growth. After culture, media were collected for measurement of estradiol levels and follicles were subjected to measurement of cylin-D-2 (Ccnd2), cyclin-dependent-kinase-4 (Cdk4), and Arom. We found that DEHP and MEHP inhibited growth of follicles and decreased estradiol production compared to controls at the highest doses. DEHP and MEHP also decreased mRNA expression of Ccnd2, Cdk4, and Arom at the highest dose. Addition of estradiol to the culture medium prevented the follicles from DEHP- and MEHP-induced inhibition of growth, reduction in estradiol levels, and decreased Ccnd2 and Cdk4 expression. Collectively, our results indicate that DEHP and MEHP may directly inhibit antral follicle growth via a mechanism that partially includes reduction in levels of estradiol production and decreased expression of cell cycle regulators.

Di (2-ethylhexyl) phthalate inhibits growth of mouse ovarian antral follicles through an oxidative stress pathway.

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer that has been shown to inhibit growth of mouse antral follicles, however, little is known about the mechanisms by which DEHP does so. Oxidative stress has been linked to follicle growth inhibition as well as phthalate-induced toxicity in non-ovarian tissues. Thus, we hypothesized that DEHP causes oxidative stress and that this leads to inhibition of the growth of antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice (age 31-35days) were cultured with vehicle control (dimethylsulfoxide [DMSO]) or DEHP (1-100μg/ml)±N-acetyl cysteine (NAC, an antioxidant at 0.25-1mM). During culture, follicles were measured daily. At the end of culture, follicles were collected and processed for in vitro reactive oxygen species (ROS) assays to measure the presence of free radicals or for measurement of the expression and activity of various key antioxidant enzymes: Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX) and catalase (CAT). The results indicate that DEHP inhibits the growth of follicles compared to DMSO control and that NAC (0.25-1mM) blocks the ability of DEHP to inhibit follicle growth. Furthermore, DEHP (10μg/ml) significantly increases ROS levels and reduces the expression and activity of SOD1 compared to DMSO controls, whereas NAC (0.5mM) rescues the effects of DEHP on ROS levels and SOD1. However, the expression and activity of GPX and CAT were not affected by DEHP treatment. Collectively, these data suggest that DEHP inhibits follicle growth by inducing production of ROS and by decreasing the expression and activity of SOD1.

The aryl hydrocarbon receptor affects mouse ovarian follicle growth via mechanisms involving estradiol regulation and responsiveness.

Abstract The aryl hydrocarbon receptor (AHR) is a known transcription factor. Although studies indicate that Ahr-deficient (AhRKO) mice have defects in female reproduction, only a few studies have examined the role of AHR in the ovary. Previous studies have suggested, without directly testing, that AhRKO mice have slower follicular growth than wild-type (WT) mice. Therefore, the first objective of the present study was to examine whether AhRKO follicles grow slower than WT follicles and if so, to determine whether the mechanism by which Ahr affects follicular growth is through effects on antrum size, granulosa cell proliferation, and regulators of cell cycle progression. Since estradiol (E2) is critical for the normal growth of ovarian follicles, the second objective of the present study was to determine the role of Ahr in regulating E2 production and responsiveness. The third objective of the present study was to determine whether E2 replacement restores follicular growth of AhRKO follicles to WT levels in vitro. We found that AhRKO follicles grew slower than WT follicles in vitro. While AhRKO and WT follicles had similar antrum sizes, AhRKO follicles showed decreased granulosa cell proliferation and reduced mRNA and protein levels of cell cycle regulators, as compared to WT follicles. Furthermore, the AhRKO mice had lower serum and follicle-produced E2 levels and showed decreased Esr1 and Esr2 mRNA levels compared to WT mice. Finally, E2 treatment of AhRKO follicles restored follicular growth to WT levels in vitro. Collectively, these findings suggest that the AHR affects follicular growth via mechanisms that involve E2 regulation and responsiveness.

Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone production in cultured mouse antral follicles

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant found in consumer products that causes ovarian toxicity. Antral follicles are the functional ovarian units and must undergo growth, survival from atresia, and proper regulation of steroidogenesis to ovulate and produce hormones. Previous studies have determined that DEHP inhibits antral follicle growth and decreases estradiol levels in vitro; however, the mechanism by which DEHP elicits these effects is unknown. The present study tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and steroidogenesis to inhibit antral follicle functionality. Antral follicles from adult CD-1 mice were cultured with vehicle control or DEHP (1-100 μg/ml) for 24-96 h to establish the temporal effects of DEHP on the follicle. Following 24-96 h of culture, antral follicles were subjected to gene expression analysis, and media were subjected to measurements of hormone levels. DEHP increased the mRNA levels of cyclin D2, cyclin dependent kinase 4, cyclin E1, cyclin A2, and cyclin B1 and decreased the levels of cyclin-dependent kinase inhibitor 1A prior to growth inhibition. Additionally, DEHP increased the mRNA levels of BCL2-associated agonist of cell death, BCL2-associated X protein, BCL2-related ovarian killer protein, B-cell leukemia/lymphoma 2, and Bcl2-like 10, leading to an increase in atresia. Further, DEHP decreased the levels of progesterone, androstenedione, and testosterone prior to the decrease in estradiol levels, with decreased mRNA levels of side-chain cleavage, 17α-hydroxylase-17,20-desmolase, 17β-hydroxysteroid dehydrogenase, and aromatase. Collectively, DEHP directly alters antral follicle functionality by inhibiting growth, inducing atresia, and inhibiting steroidogenesis.

The Ability of the Aryl Hydrocarbon Receptor to Regulate Ovarian Follicle Growth and Estradiol Biosynthesis in Mice Depends on Stage of Sexual Maturity

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of environmental chemicals and regulates many physiological functions, including processes in female reproduction. Previous studies demonstrated that Ahr deletion leads to slow ovarian follicle growth because of impaired estradiol production and reduced gonadotropin responsiveness in prepubertal mice. These studies, however, did not determine how Ahr deletion impairs estradiol production or whether the effects of Ahr deletion on follicle growth and estradiol production persist in adulthood. Thus, the present study evaluated the effect of Ahr deletion on steroid precursors in the estradiol biosynthesis pathway. Furthermore, this study evaluated follicle growth and estradiol biosynthesis in wild-type (WT) and Ahr knockout (AhrKO) antral follicles at different stages of sexual maturity. AhrKO antral follicles from prepubertal mice had slower growth, produced lower estradiol levels, and had reduced cyclin D2 (Ccnd2) expression compared to WT follicles. AhrKO follicles from adult mice, however, produced higher androgen levels and expressed higher levels of Ccnd2 compared to WT follicles. Furthermore, AhrKO follicles from adult mice had growth to that of WT follicles. These findings suggest that the AHR regulates follicle growth by altering factors involved in the estradiol biosynthesis pathway as well as key regulators of follicle growth and that this role of AHR depends on stage of sexual maturity.

Methoxychlor inhibits growth of antral follicles by altering cell cycle regulators.

Methoxychlor (MXC) reduces fertility in female rodents, decreases antral follicle numbers, and increases atresia through oxidative stress pathways. MXC also inhibits antral follicle growth in vitro. The mechanism by which MXC inhibits growth of follicles is unknown. The growth of follicles is controlled, in part, by cell cycle regulators. Thus, we tested the hypothesis that MXC inhibits follicle growth by reducing the levels of selected cell cycle regulators. Further, we tested whether co-treatment with an antioxidant, N-acetyl cysteine (NAC), prevents the MXC-induced reduction in cell cycle regulators. For in vivo studies, adult cycling CD-1 mice were dosed with MXC or vehicle for 20 days. Treated ovaries were subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA) staining. For in vitro studies, antral follicles isolated from adult cycling CD-1 mouse ovaries were cultured with vehicle, MXC, and/or NAC for 48, 72 and 96 h. Levels of cyclin D2 (Ccnd2) and cyclin dependent kinase 4 (Cdk4) were measured using in vivo and in vitro samples. The results indicate that MXC decreased PCNA staining, and Ccnd2 and Cdk4 levels compared to controls. NAC co-treatment restored follicle growth and expression of Ccnd2 and Cdk4. Collectively, these data indicate that MXC exposure reduces the levels of Ccnd2 and Cdk4 in follicles, and that protection from oxidative stress restores Ccnd2 and Cdk4 levels. Therefore, MXC-induced oxidative stress may decrease the levels of cell cycle regulators, which in turn, results in inhibition of the growth of antral follicles.

Mono-hydroxy methoxychlor alters levels of key sex steroids and steroidogenic enzymes in cultured mouse antral follicles

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by decreasing antral follicle numbers and increasing follicular death. MXC is metabolized in the body to mono-hydroxy MXC (mono-OH). Little is known about the effects of mono-OH on the ovary. Thus, this work tested the hypothesis that mono-OH exposure decreases production of 17β-estradiol (E₂) by cultured mouse antral follicles. Antral follicles were isolated from CD-1 mice (age 35-39 days) and exposed to dimethylsulfoxide (DMSO), or mono-OH (0.1-10 μg/mL) for 96 h. Media and follicles were collected for analysis of sex steroid levels and mRNA expression, respectively. Mono-OH treatment (10 μg/mL) decreased E(2) (DMSO: 3009.72±744.99 ng/mL; mono-OH 0.1 μg/mL: 1679.66±461.99 ng/mL; 1 μg/mL: 1752.72±532.41 ng/mL; 10 μg/mL: 45.89±33.83 ng/mL), testosterone (DMSO: 15.43±2.86 ng/mL; mono-OH 0.1μg/mL: 17.17±4.71 ng/mL; 1 μg/mL: 13.64±3.53 ng/mL; 10 μg/mL: 1.29±0.23 ng/mL), androstenedione (DMSO: 1.92±0.34 ng/mL; mono-OH 0.1 μg/mL: 1.49±0.43ng/mL; 1 μg/mL: 0.64±0.31 ng/mL; 10 μg/mL: 0.12±0.06 ng/mL) and progesterone (DMSO: 24.11±4.21 ng/mL; mono-OH 0.1μg/mL: 26.77±4.41 ng/mL; 1 μg/mL: 20.90±3.75 ng/mL; 10 μg/mL: 9.44±2.97 ng/mL) levels. Mono-OH did not alter expression of Star, Hsd3b1, Hsd17b1 and Cyp1b1, but it did reduce levels of Cyp11a1, Cyp17a1 and Cyp19a1 mRNA. Collectively, these data suggest that mono-OH significantly decreases levels of key sex steroid hormones and the expression of enzymes required for steroidogenesis.

Co-treatment of mouse antral follicles with 17β-estradiol interferes with mono-2-ethylhexyl phthalate (MEHP)-induced atresia and altered apoptosis gene expression

Mono-2-ethyhexyl phthalate (MEHP) is a metabolite of a plasticizer found in many consumer products. MEHP inhibits mouse ovarian follicle growth by reducing 17β-estradiol (E2) production. Yet, whether MEHP causes follicle death (atresia) is unclear. We hypothesized that MEHP causes atresia by altering apoptosis gene expression, and that E2 co-treatment blocks these effects. Follicles were exposed to MEHP (0.36-36μM)±E2 for 48-96h to determine the effect of MEHP±E2 on atresia and gene expression. MEHP increased atresia, but this effect was blocked by co-treatment with E2. MEHP increased the expression of the pro-apoptotic gene Aifm1, but decreased that of the pro-apoptotic gene Bok and the anti-apoptotic gene Bcl2l10. E2 interfered with MEHP-induced changes in Aifm1 and Bcl2l10. Our findings suggest that decreased E2 levels are required for MEHP-induced follicle atresia and that Aifm1, Bok, and Bcl2l10 are involved in this process.

Methoxychlor and Its Metabolites Inhibit Growth and Induce Atresia of Baboon Antral Follicles

Methoxychlor (MXC), an organochlorine pesticide, inhibits growth and induces atresia of antral follicles in rodents. MXC metabolites, mono-OH MXC (mono-OH) and bis-OH MXC (HPTE), are thought to be more toxic than the parent compound. Although studies have examined the effects of MXC in rodents, few studies have evaluated the effects of MXC in primates. Therefore, the present study tested the hypothesis that MXC, mono-OH, and HPTE inhibit growth and induce atresia of baboon antral follicles. To test this hypothesis, antral follicles were isolated from adult baboon ovaries and cultured with vehicle (dimethylsulfoxide; DMSO), MXC (1–100 μg/ml), mono-OH (0.1–10 μg/ml), or HPTE (0.1–10 μg/ml) for 96 hr. Growth was monitored at 24 hr intervals. After culture, follicles were processed for histological evaluation of atresia. MXC, mono-OH, and HPTE significantly inhibited follicular growth and increased atresia compared to DMSO. Moreover, the adverse effects of MXC and its metabolites on growth and atresia in baboon antral follicles were observed at lower (100-fold) doses than those causing similar effects in rodents. These data suggest that MXC and its metabolites inhibit growth and induce atresia of baboon antral follicles, and that primate follicles are more sensitive to MXC than rodent follicles.