Liying Gao

Exposure to an Environmentally Relevant Phthalate Mixture Causes Transgenerational Effects on Female Reproduction in Mice

Phthalates are used in consumer products and are known endocrine-disrupting chemicals. However, limited information is available on the effects of phthalate mixtures on female reproduction. Previously, we developed a phthalate mixture made of 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% di-isononyl phthalate, 8% di-isobutyl phthalate, and 5% benzylbutyl phthalate that mimics human exposure. We tested the effects of prenatal exposure to this mixture on reproductive outcomes in first-filial-generation (F1) female mice and found that it impaired reproductive outcomes. However, the impact of this exposure on second-filial-generation (F2) and third-filial-generation (F3) females was unknown. Thus, we hypothesized that prenatal exposure to the phthalate mixture induces multigenerational and transgenerational effects on female reproduction. Pregnant CD-1 dams were orally dosed with vehicle (tocopherol-stripped corn oil) or a phthalate mixture (20 and 200 µg/kg/d, 200 and 500 mg/kg/d) daily from gestational day 10 to birth. Adult F1 females born to these dams were used to generate the F2 generation and adult F2 females born to F1 females were used to generate the F3 generation. F2 and F3 females were subjected to tissue collections and fertility tests. Prenatal phthalate mixture exposure increased uterine weight, anogenital distance, and body weight; induced cystic ovaries; and caused fertility complications in the F2 generation. It also increased uterine weight, decreased anogenital distance, and caused fertility complications in the F3 generation. These data suggest that prenatal exposure to the phthalate mixture induces multigenerational and transgenerational effects on female reproduction.

Prenatal exposure to an environmentally relevant phthalate mixture disrupts reproduction in F1 female mice.

&NA; Phthalates are used in a large variety of products, such as building materials, medical devices, and personal care products. Most previous studies on the toxicity of phthalates have focused on single phthalates, but it is also important to study the effects of phthalate mixtures because humans are exposed to phthalate mixtures. Thus, we tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects female reproduction in mice. To test this hypothesis, pregnant CD‐1 dams were orally dosed with vehicle (tocopherol‐stripped corn oil) or a phthalate mixture (20 and 200 &mgr;g/kg/day, 200 and 500 mg/kg/day) daily from gestational day 10 to birth. The mixture was based on the composition of phthalates detected in urine samples from pregnant women in Illinois. The mixture included 35% diethyl phthalate, 21% di(2‐ethylhexyl) phthalate, 15% dibutyl phthalate, 15% diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. Female mice born to the exposed dams were subjected to tissue collections and fertility tests at different ages. Our results indicate that prenatal exposure to the phthalate mixture significantly increased uterine weight and decreased anogenital distance on postnatal days 8 and 60, induced cystic ovaries at 13 months, disrupted estrous cyclicity, reduced fertility‐related indices, and caused some breeding complications at 3, 6, and 9 months of age. Collectively, our data suggest that prenatal exposure to an environmentally relevant phthalate mixture disrupts aspects of female reproduction in mice. HighlightsPrenatal exposure to a phthalate mixture disrupts F1 estrous cyclicity.Prenatal exposure to a phthalate mixture induces F1 ovarian cysts.Prenatal exposure to a phthalate mixture decreases F1 female fertility‐related indices.Prenatal exposure to a phthalate mixture induces F1 breeding complications.

Bisphenol A Exposure, Ovarian Follicle Numbers, and Female Sex Steroid Hormone Levels: Results From a CLARITY-BPA Study

Bisphenol A (BPA) is an industrial chemical found in thermal receipts and food and beverage containers. Previous studies have shown that BPA can affect the numbers and health of ovarian follicles and the production of sex steroid hormones, but they often did not include a wide range of doses of BPA, used a small sample size, focused on relatively short-term exposures to BPA, and/or did not examine the consequences of chronic BPA exposure on the ovaries or steroid levels. Thus, this study was designed to examine the effects of a wide range of doses of BPA on ovarian morphology and sex steroid hormone production. Specifically, this study tested the hypothesis that prenatal and continuous BPA exposure reduces ovarian follicle numbers and sex steroid hormone levels. To test this hypothesis, rats were dosed with vehicle, ethinyl estradiol (0.05 and 0.5 μg/kg body weight/d), or BPA (2.5, 25, 250, 2500, and 25,000 μg/kg body weight/d) from gestation day 6 until 1 year as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). Ovaries and sera were collected on postnatal days 1, 21, and 90, and at 6 months and 1 year. The ovaries were subjected to histological evaluation of follicle numbers and the sera were subjected to measurements of estradiol and progesterone. Collectively, these data indicate that BPA exposure at some doses and time points affects ovarian follicle numbers and sex steroid levels, but these effects are different than those observed with ethinyl estradiol exposure and some previous studies on BPA.

Effects of isoliquiritigenin on ovarian antral follicle growth and steroidogenesis

Isoliquiritigenin is a botanical estrogen used as a dietary supplement. Previous studies show that other botanical estrogens affect ovarian estradiol synthesis, but isoliquiritigenins effects on the ovary are unknown. Thus, this study tested the hypothesis that isoliquiritigenin inhibits ovarian antral follicle growth and steroidogenesis. Antral follicles from CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or isoliquiritigenin (0.6μM, 6 μM, 36 μM, and 100 μM) for 48-96h. During culture, follicle diameters were measured daily to assess follicle growth. After culture, media were collected for hormone assays and follicles were collected for gene expression analysis of steroidogenic enzymes. Isoliquiritigenin inhibited antral follicle growth and altered estradiol, testosterone, and progesterone levels. Additionally, isoliquiritigenin altered the mRNA levels of cytochrome P450 steroid 17-α-hydroxylase 1, aromatase, 17β-hydroxysteroid dehydrogenase 1, and steroidogenic acute regulatory protein. These data indicate that exposure to isoliquiritigenin inhibits growth and disrupts steroid production in antral follicles.

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1-F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice.

Prenatal exposure to di(2-ethylhexyl) phthalate disrupts ovarian function in a transgenerational manner in female mice

Abstract Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer found in polyvinyl chloride products such as vinyl flooring, plastic food containers, medical devices, and childrens toys. DEHP is a ubiquitous environmental contaminant and is a known endocrine disrupting chemical. Little is known about the effects of prenatal DEHP exposure on the ovary and whether effects occur in subsequent generations. Thus, we tested the hypothesis that prenatal exposure to DEHP disrupts ovarian functions in the F1, F2, and F3 generations of female mice. To test this hypothesis, pregnant CD-1 mice were orally dosed with corn oil (vehicle control) or DEHP (20 and 200 µg/kg/day and 200, 500, and 750 mg/kg/day) daily from gestation day 10.5 until birth (7–28 dams/treatment group). F1 females were mated with untreated males to obtain the F2 generation, and F2 females were mated with untreated males to produce the F3 generation. On postnatal days 1, 8, 21, and 60, ovaries were collected and used for histological evaluation of follicle numbers and sera were used to measure progesterone, testosterone, 17β-estradiol, luteinizing hormone, and follicle stimulating hormone levels. In the F1 generation, prenatal exposure to DEHP disrupted body and organ weights, decreased folliculogenesis, and increased serum 17β-estradiol levels. In the F2 generation, exposure to DEHP decreased body and organ weights, dysregulated folliculogenesis, and disrupted serum progesterone levels. In the F3 generation, DEHP exposure accelerated folliculogenesis. These data suggest that prenatal exposure to DEHP leads to adverse multigenerational and transgenerational effects on ovarian function. Summary Sentence Developmental exposure to di(2-ethylhexyl) phthalate adversely affects ovarian functions in multiple generations of mice.

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.

Prenatal Exposure to Di(2-Ethylhexyl) Phthalate Causes Long-Term Transgenerational Effects on Female Reproduction in Mice

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer in many consumer products. Although DEHP is a known endocrine disruptor, little is known about the effects of DEHP exposure on female reproduction. Thus, this study tested the hypothesis that prenatal DEHP exposure affects follicle numbers, estrous cyclicity, and hormone levels in multiple generations of mice. Pregnant CD-1 mice were orally dosed with corn oil (vehicle control) or DEHP (20 and 200 µg/kg/d and 500 and 750 mg/kg/d) from gestational day 11 until birth. The F1 females were mated with untreated males to create the F2 generation, and the F2 females were mated with untreated males to create the F3 generation. At 1 year, ovaries, hormones, and estrous cycles were analyzed in each generation. Prenatal DEHP exposure altered estrous cyclicity (750 mg/kg/d), increased the presence of ovarian cysts (750 mg/kg/d), and decreased total follicle numbers (750 mg/kg/d) in the F1 generation. It also decreased anogenital distance (200 µg/kg/d) and altered follicle numbers (200 µg/kg/d and 500 mg/kg/d) in the F2 generation, and it altered estrous cyclicity (20 and 200 µg/kg/d and 500 and 750 mg/kg/d) and decreased folliculogenesis (200 µg/kg/d and 500 mg/kg/d) in the F3 generation. Further, prenatal DEHP increased estradiol levels (F1 and F3), decreased testosterone levels (F1, F2, and F3), decreased progesterone levels (F2), altered gonadotropin hormone levels (F1 and F3), and decreased inhibin B levels (F1 and F3). Collectively, these data show that prenatal exposure to DEHP has multigenerational and transgenerational effects on female reproduction and it may accelerate reproductive aging.

Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro.

Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles.

Monohaloacetic acid drinking water disinfection by-products inhibit follicle growth and steroidogenesis in mouse ovarian antral follicles in vitro.

Water disinfection greatly reduced the incidence of waterborne diseases, but the reaction between disinfectants and natural organic matter in water leads to the formation of drinking water disinfection by-products (DBPs). DBPs have been shown to be toxic, but their effects on the ovary are not well defined. This study tested the hypothesis that monohalogenated DBPs (chloroacetic acid, CAA; bromoacetic acid, BAA; iodoacetic acid, IAA) inhibit antral follicle growth and steroidogenesis in mouse ovarian follicles. Antral follicles were isolated and cultured with either vehicle or DBPs (0.25-1.00mM of CAA; 2-15μM of BAA or IAA) for 48 and 96h. Follicle growth was measured every 24h and the media were analyzed for estradiol levels at 96h. Exposure to DBPs significantly inhibited antral follicle growth and reduced estradiol levels compared to controls. These data demonstrate that DBP exposure caused ovarian toxicity in vitro.