N-hexane inhalation during pregnancy alters DNA promoter methylation in the ovarian granulosa cells of rat offspring.

The N-hexane-induced impact on the reproductive system of the offspring of animals exposed to n-hexane has caused great concern. Pregnant Wistar rats inhaled 500, 2 500 or 12 500 ppm n-hexane during gestational days 1-20. Clinical characteristics and developmental indices were observed. Ovarian granulosa cells were extracted from F1 rats, the number of follicles was determined in ovarian slices and promoter methylation was assessed using MeDIP-Chip. Several methods were used to analyze the scanned genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering and KEGG pathway analysis. The results indicated that the live pups/litter ratio was significantly lowest in the 12 500 ppm group. A significant decrease in secondary follicles and an increase in atresic follicles were observed in the 12 500 ppm group. The number of shared demethylated genes was higher than that of the methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the 500 ppm and control groups were different from those of the 2500 and 12 500 ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1 and Srd5a1 promoters were hypermethylated in the n-hexane-exposed groups. These results indicate that the developmental toxicity of n-hexane in F1 ovaries is accompanied by the altered methylation of promoters of genes associated with apoptotic processes and steroid hormone biosynthesis.

Metabolomic changes in follicular fluid induced by soy isoflavones administered to rats from weaning until sexual maturity.

Female Wistar rats at 21 days of age were treated with one of three concentrations of soy isoflavones (SIF) (50, 100 or 200mg/kg body weight, orally, once per day) from weaning until sexual maturity (3 months) in order to evaluate the influence of SIF on ovarian follicle development. After treatment, the serum sex hormone levels and enumeration of ovarian follicles of the ovary were measured. The metabolic profile of follicular fluid was determined using HPLC-MS. Principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA) was used to identify differences in metabolites and reveal useful toxic biomarkers. The results indicated that modest doses of SIF affect ovarian follicle development, as demonstrated by decreased serum estradiol levels and increases in both ovarian follicle atresia and corpora lutea number in the ovary. SIF treatment-related metabolic alterations in follicular fluid were also found in the PCA and PLS-DA models. The 24 most significantly altered metabolites were identified, including primary sex hormones, amino acids, fatty acids and metabolites involved in energy metabolism. These findings may indicate that soy isoflavones affect ovarian follicle development by inducing metabolomic variations in the follicular fluid.

Soy isoflavones administered to rats from weaning until sexual maturity affect ovarian follicle development by inducing apoptosis.

Twenty-one-day-old female Wistar rats were treated daily with orally administered soy isoflavones (SIFs) at concentrations of 50, 100, or 200 mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian follicle development was evaluated. At the end of the treatment period, the ultrastructure of the ovarian granulosa cells was examined by transmission electron microscopy. The apoptotic cell death of ovarian granulosa cells was detected using TUNEL staining. The mRNA expression levels of caspase-3, caspase-8, caspase-9, Bcl2, Bax, and Fas were determined by real-time quantitative PCR. The protein expression levels of caspase-3, Bcl2, Bax, and Fas were determined by western blotting. Our data showed that exposure to SIFs resulted in morphological changes consistent with ovarian granulosa cell apoptosis. The percentage of TUNEL-positive granulosa cells was increased. The mRNA expression levels of the apoptosis-related genes caspase-3, caspase-8, caspase-9, Bax, and Fas increased significantly. The protein levels of Bax, Fas, and cleaved caspase-3 were also increased. These results indicate that the exposure of rats to modest doses of SIFs from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis. The mechanism of SIF-induced alterations in ovarian follicle development may involve the activation of Fas-mediated and Bcl2/Bax-mediated apoptotic signaling pathways.

Prepubertal bisphenol A exposure interferes with ovarian follicle development and its relevant gene expression.

Bisphenol A (BPA) is recognized as one of several environmental estrogens. Pre-puberty is an important part of reproductive system development, and even a short-term exposure to BPA during this period may cause serious damage to the reproductive system. In this study, Pre-puberty female Wistar rats were exposed to BPA for one week. The effects of BPA on ovarian structure and function were assessed. The expression levels of follicle development-related genes were analyzed. Our study showed that BPA reduced rat ovarian weights and follicle numbers, and interferes with the constituent ratio of follicles. With increasing doses of BPA, the expression of factor in the germline alpha (FIGLA) and oocyte-specific histone H1 variant (H1FOO) genes decreased, and anti-mullerian hormone (AMH) genes expression increased, suggesting that BPA exposure during the pre-pubertal period may inhibit the development of ovaries, and follicle development-related genes may play certain roles in this process.

Continuous cadmium exposure from weaning to maturity induces downregulation of ovarian follicle development-related SCF/c-kit gene expression and the corresponding changes of DNA methylation/microRNA pattern.

Cadmium (Cd) impairs ovary structure and function in mature animals. However, the influence of Cd on follicle development from weaning to maturity is obscure. In the current study, 21-day-old Wistar rats were administered Cd chloride at doses of 0, 0.5, 2.0 and 8.0 mg/kg body weight once a day for eight weeks by gavage. After administration, a significant decrease in ovarian wet weight, ovarian/body weight ratios, and primordial follicles, in addition to an increase in atresic follicles, were observed. Transmission electron microscopy and TUNEL assay confirmed the increase of follicle apoptosis as Cd concentration increased. Real-time quantitative PCR and Western blotting showed a significantly decreased expression of follicle development-related factors, stem cell factor (SCF) and c-kit. Bisulfite sequencing suggested that the total methylation percentages of SCF/c-kit promoter region were not obvious change after Cd exposure. Real-time quantitative PCR revealed a significantly increased expression of miR-193, miR-221 and miR-222, which regulate c-kit, in the 2.0 mg/kg and 8.0 mg/kg treatment groups. Overall, this study proved that Cd administration from weaning to maturity could damage follicle development, suggesting that SCF/c-kit might play an important role in this effect. In addition, microRNAs might play a role in c-kit protein downregulation.