Tessie Paulose

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.

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.

Impact of environmental factors and poverty on pregnancy outcomes.

Studies have indicated that various societal factors such as toxicant exposure, maternal habits, occupational hazards, psychosocial factors, socioeconomic status, racial disparity, chronic stress, and infection may impact pregnancy outcomes. These outcomes include spontaneous abortion, preterm birth, alterations in the development of the fetus, and long-term health of offspring. Although much is known about individual pregnancy outcomes, little is known about the associations between societal factors and pregnancy outcomes. This manuscript reviews some of the literature available on the effects of the above-mentioned societal factors on pregnancy outcomes and examines some potential remedies for preventing adverse pregnancy outcomes in the future.

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.

Methoxychlor-Induced Ovarian Follicle Toxicity in Mice: Dose and Exposure Duration-Dependent Effects

BACKGROUND Methoxychlor (MXC) is specifically known to target ovarian antral follicles, increasing atresia (death via apoptosis) in them. This is of concern because females are born with a finite pool of ovarian follicles. Only limited studies have explored the phenomenon of a reduced fertility threshold for effect based on the percentage of antral follicle atresia. METHODS In this article, we report on adult female CD-1 mice exposed intraperitoneally to various doses of MXC for 5, 10, 20, and 30 days. In the 20-day treatment, mice were dosed with either the vehicle or MXC at 64 or 96 mg/kg/day, whereas in the 30-day treatment, mice were dosed with vehicle or MXC at 48, 64, or 96 mg/kg/day. The mice that were dosed with MXC for 30 days were also mated with untreated males for a determination of overall fertility. RESULTS A significantly increased percentage (50%) of atretic antral follicles was observed only after 20 and 30 days of treatment. Specifically, mice treated with MXC64 for 20 and 30 days had an increased percentage of atretic antral follicles compared with vehicle-treated mice. Interestingly, mice dosed with MXC96 had an increased percentage of atretic antral follicles after 30 days, but not after 20 days of treatment compared with vehicle-treated mice. Overall fertility of the mice was not different compared with controls. CONCLUSIONS The results indicate that as much as a 50% increase in atretic antral follicles does not affect the immediate fertility of the mice.

Estrogen receptor alpha overexpressing mouse antral follicles are sensitive to atresia induced by methoxychlor and its metabolites.

Methoxychlor (MXC) and its metabolites bind to estrogen receptors (ESRs) and increase ovarian atresia. To test whether ESR alpha (ESR1) overexpressing (ESR1 OE) antral follicles are more sensitive to atresia compared to controls, we cultured antral follicles with vehicle, MXC (1-100 μg/ml) or metabolites (0.1-10 μg/ml). Results indicate that MXC and its metabolites significantly increase atresia in ESR1 OE antral follicles at lower doses compared to controls. Activity of pro-apoptotic factor caspase-3/7 was significantly higher in ESR1 OE treated antral follicles compared to controls. ESR1 OE mice dosed with MXC 64 mg/kg/day had an increased percentage of atretic antral follicles compared to controls. Furthermore, pro-caspase-3 levels were found to be significantly lower in ESR1 OE ovaries than controls dosed with MXC 64 mg/kg/day. These data suggest that ESR1 OE ovaries are more sensitive to atresia induced by MXC and its metabolites in vitro and in vivo compared to controls.

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.

11.19 – Ovarian Toxicology

In the mammalian ovary, the development of follicles is a complex process that begins with the formation of primordial follicles and culminates either in a normal atretic degradation process or in the release of an oocyte for fertilization. Female reproduction depends, in part, on successful development of follicles. However, large numbers of toxicants target the follicles compromising female reproduction. This chapter describes the processes occurring and factors controlling primordial follicle formation, activation, growth, and development to the ovulatory stage. Further, this chapter provides information about deleterious effects and mechanisms that some environmental chemicals use to damage the follicle and thereby impair ovarian function. Although many mechanisms of ovotoxicity have been elucidated, knowledge of factors that control follicle development may allow the design of studies that evaluate new potential endpoints of toxicity. This may help to develop new strategies of prevention or treatment of toxicant-induced ovotoxicity.