Isabel Hernández-Ochoa

Increased Sensitivity of Estrogen Receptor Alpha Overexpressing Antral Follicles to Methoxychlor and Its Metabolites

Methoxychlor (MXC), an organochlorine pesticide, and its metabolites, mono-hydroxy MXC (MOH) and bis-hydroxy MXC (HPTE) are known ovarian toxicants and can cause inhibition of antral follicle growth. Since these chemicals bind to estrogen receptor alpha (ESR1), we hypothesized that ovaries overexpressing ESR1 (ESR1 OE) would be more susceptible to toxicity induced by MXC and its metabolites because the chemicals can bind to more ESR1 in the antral follicles. We cultured antral follicles from controls and ESR1 OE mouse ovaries with either the vehicle dimethylsulfoxide (DMSO), MXC, MOH, or HPTE. The data show that at 96 h, the cultured antral follicles from ESR1 OE antral follicles are more susceptible to toxicity induced by MXC, MOH, and HPTE because low doses of these chemicals cause follicle growth inhibition in ESR1 OE mice but not in control mice. On comparing gene expression levels of nuclear receptors in the cultured antral follicles of ESR1 OE and control follicles, we found differential messenger RNA (mRNA) expression of Esr1, estrogen receptor beta (Esr2), androgen receptor (Ar), progesterone receptor (Pr), and aryl hydrocarbon receptor (Ahr) between the genotypes. We also analyzed mRNA levels of Cyp3a41a, the enzyme metabolizing MOH and HPTE, in the cultured follicles and found that Cyp3a41a was significantly lower in DMSO-treated ESR1 OE follicles compared with controls. In ESR1 OE livers, we found that Cyp3a41a levels were significantly lower compared with control livers. Collectively, these data suggest that MXC and its metabolites cause differential gene expression in ESR1 OE mice compared with controls. The results also suggest that the increased sensitivity of ESR1 OE mouse ovaries to toxicity induced by MXC and its metabolites is due to low clearance of the metabolites by the liver and ovary.

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.

The role of the aryl hydrocarbon receptor in the female reproductive system.

In recent years, many studies have emphasized how changes in aryl hydrocarbon receptor (AHR)-mediated gene expression result in biological effects, raising interest in this receptor as a regulator of normal biological function. This review focuses on what is known about the role of the AHR in the female reproductive system, which includes the ovaries, Fallopian tubes or oviduct, uterus and vagina. This review also focuses on the role of the AHR in reproductive outcomes such as cyclicity, senescence, and fertility. Specifically, studies using potent AHR ligands, as well as transgenic mice lacking the AHR-signaling pathway are discussed from a viewpoint of understanding the endogenous role of this ligand-activated transcription factor in the female reproductive lifespan. Based on findings highlighted in this paper, it is proposed that the AHR has a role in physiological functions including ovarian function, establishment of an optimum environment for fertilization, nourishing the embryo and maintaining pregnancy, as well as in regulating reproductive lifespan and fertility. The mechanisms by which the AHR regulates female reproduction are poorly understood, but it is anticipated that new models and the ability to generate specific gene deletions will provide powerful experimental tools for better understanding how alterations in AHR pathways result in functional changes in the female reproductive system.

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E₂) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E₂ metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E₂, testosterone, androstenedione, and progesterone (P₄) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3β hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.

Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa

Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.

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.

Methoxychlor inhibits growth and induces atresia through the aryl hydrocarbon receptor pathway in mouse ovarian antral follicles

Methoxychlor (MXC) is an organochlorine pesticide used against pests that attack crops, vegetables, and livestock. MXC inhibits growth and induces atresia (death) of mouse ovarian antral follicles in vitro. Since several studies indicate that many chemicals act through the aryl hydrocarbon receptor (AHR) pathway, the current study tested the hypothesis that MXC binds to the AHR to inhibit growth and induce atresia of antral follicles. The data indicate that MXC binds to AHR. Further, a relatively high dose of MXC (100μg/ml) inhibits growth and induces atresia in both wild-type (WT) and AHR null (AHRKO) follicles, whereas a lower dose of MXC (10μg/ml) inhibits growth and induces atresia in WT, but not in AHRKO follicles. These data indicate that AHR deletion partially protects antral follicles from MXC induced slow growth and atresia. Collectively, these data show that MXC may act through the AHR pathway to inhibit follicle growth and induce atresia in antral follicles of the ovary.

Follicle-stimulating hormone responsiveness in antral follicles from aryl hydrocarbon receptor knockout mice

BackgroundPrevious studies have demonstrated that pre-pubertal aryl hydrocarbon receptor knockout (AHRKO) mice have slow antral follicle growth and reduced capacity to produce estradiol compared to wild-type (WT) mice. Although previous studies have suggested that this is likely due to a reduced ability of the AHRKO follicles to respond to follicle-stimulating hormone (FSH), this possibility was not directly tested. Thus, the goal of these studies was to test the hypothesis that low FSH responsiveness is responsible for the slow growth and reduced estradiol production observed in pre-pubertal AHRKO versus WT antral follicles.MethodsAntral follicles from WT and AHRKO mice were cultured with varying amounts of FSH (0–15 IU/mL) for up to 7 days, and subjected to measurements of growth, FSH receptor and steroidogenic regulator expression, sex steroid hormone levels, and inhibin beta-A expression. General linear models (GLM) for repeated measures were used to compare follicle diameters over time among treatments. If the global tests from GLM were significant, Tukey’s tests were used for pairwise comparisons. Remaining comparisons among groups were performed using one-way analysis of variance followed by Tukey’s post hoc test.ResultsThe results indicate that FSH stimulated growth in both WT and AHRKO follicles, but that high levels of FSH (10–15 IU/mL) were required for AHRKO follicles to reach maximal growth, whereas lower levels of FSH (5 IU/mL) were required for WT follicles to reach maximal growth. Further, FSH stimulated expression of FSH receptor, steroidogenic factors, and inhibin beta-A as well as production of steroid hormones in both WT and AHRKO follicles, but the degree of stimulation differed betw een WT and AHRKO follicles. Interestingly, FSH treatment increased expression of FSH receptor, some steroidogenic regulators, inhibin beta-A, and steroid hormone production more in AHRKO follicles compared to WT follicles.ConclusionsCollectively, these data suggest that the slow growth, but not reduced steroidogenesis in AHRKO follicles, is due to their reduced ability to respond to FSH compared to WT follicles. These data also suggest that the AHR may contribute to the ability of FSH to stimulate proper follicle growth, but it may not contribute to FSH-induced steroidogenesis.

Genetically induced estrogen receptor α mRNA (Esr1) overexpression does not adversely affect fertility or penile development in male mice.

Previously, we reported that estrogen receptor α mRNA (Esr1) or protein (ESR1) overexpression resulting from neonatal exposure to estrogens in rats was associated with infertility and maldeveloped penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing Esr1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls as a result of Esr1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P < .05) lower as compared with controls; however, the weight of the testis, the morphology of the testis and epididymis, and the plasma and testicular testosterone concentration were not different from controls. Hence, genetically induced Esr1 overexpression alone, without an exogenous estrogen exposure during the neonatal period, is unable to adversely affect the development of the penis as well as other male reproductive organs, except for limited, but significant, reductions in weights of the seminal vesicles and BS/LA muscles.