Yuchen Li

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.

Protective effect of theaflavins on cadmium-induced testicular toxicity in male rats

Male Sprague-Dawley rats were treated with cadmium (Cd) (0.4 mg/kg body weight, s.c., once a day) and three concentrations of theaflavins (50, 100 or 200mg/kg body weight, orally, once a day) for five weeks to evaluate the protective role of theaflavins on Cd-induced testicular toxicity. After five weeks, serum sex hormone levels, sperm characteristics, DNA damage, oxidant-antioxidant status, Cd content in several organs were measured. The results showed that a low dose of Cd caused testicular toxicity, which was represented by decreased serum testosterone levels, induction of DNA damage, increased malondialdehyde (MDA) content, Cd accumulation in several organs. Administration of theaflavins led to a dose-dependent alleviation Cd-induced damage in testis, including enhanced serum testosterone levels, improved sperm characteristics and abrogation of DNA damage. Theaflavins may also reduce the production of Cd-induced MDA content, decrease Cd concentration in liver, testis and blood, increase Cd content in urine and feces. These findings suggest the use of theaflavins as a potential therapeutic agent for Cd-induced testicular toxicity.

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.

Protective effect of theaflavins on homocysteine-induced injury in HUVEC cells in vitro.

Abstract: A model of homocysteine-induced injury in vascular endothelial cells was established to evaluate the protective role of theaflavins on homocysteine-injured human vascular endothelial cells (HUVECs). The cells were co-incubated with 3 concentrations of theaflavins (5, 10, or 20 mg/L) and 0.5 mM homocysteine for 24 hours. The morphology and viability of the cells were determined, and the DNA damage was detected by a comet assay. Superoxide dismutase, malondialdehyde, glutathione peroxidase, nitric oxide, nitric oxide synthase, and endothelin-1 were measured. The results showed that theaflavins can reduce the changes in and damage of homocysteine-injured HUVECs, increase the viability of homocysteine-injured HUVECs, and alleviate DNA damage induced by homocysteine. These results indicate that theaflavins can inhibit homocysteine-induced injury of HUVECs. Further studies showed that theaflavins may reduce the production of homocysteine-induced reactive oxygen species and partly modulate the secretory dysfunction of vascular endothelial cells caused by homocysteine. This finding indicates that the mechanism by which theaflavins inhibit homocysteine-induced injury may relate to their antioxidant activity and the regulation of the secretion of endothelium-derived factors. These findings suggest that theaflavins may be beneficial in the prevention of atherosclerosis and cardiovascular disease.

Di(2-ethylhexyl) phthalate (DEHP) influences follicular development in mice between the weaning period and maturity by interfering with ovarian development factors and microRNAs

Although studies have shown that di(2‐ethylhexyl) phthalate (DEHP) can disrupt ovarian function, few reports have focused on follicular development in mice between the weaning period and maturity, especially with respect to microRNA (miRNA) expression. In this study, 21‐day‐old ICR mice were administered DEHP at doses of 0, 100, 400, and 1600 mg/(kg d) for 6 weeks by gavage. After DEHP administration, a significant decrease in the expression of follicle development‐related factors (including c‐kit, kitl, gdf9, and atm) was observed by quantitative real‐time PCR (RT‐PCR), but no significant difference in the proteins encoded by these genes was observed by Western blot. Bisulfite sequencing suggested that the total methylation percentages of promoter regions of these genes were not notably altered after DEHP exposure. However, RT‐PCR revealed a significantly increased expression of ovarian miRNAs (let‐7b, miR‐17‐5p miR‐181a, and miR‐151), which inhibit follicular granulosa cell proliferation. Overall, this study showed that DEHP administration from weaning to maturity could suppress the mRNA expression of follicular development factors, and this effect was not achieved through changes in the methylation of DNA in CpG islands of development factors. In addition, DEHP was shown to induce miRNAs to inhibit the proliferation of follicular granulosa cells and the anti‐apoptosis function of KITL and GDF9 while increasing bax/bcl2 expression to further promote the apoptosis of granulosa cells.

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.

Effect of cadmium on kitl pre‐mRNA alternative splicing in murine ovarian granulosa cells and its associated regulation by miRNAs

In this study, we established an in vitro exposure model of murine ovarian granulosa cells to observe the effect of Cd on alternative splicing of the kitl pre‐mRNA and subsequently to explore the role of kitl gene expression regulation‐related miRNAs through miRNA prediction, miRNA chip, bioinformatics and real‐time quantitative polymerase chain reaction analyses. Our results showed that the kitl1/kitl2 mRNA ratio was significantly different (P < 0.05) at different dosages and times. The miRNA chip analysis showed that the miRNA expression profiles for the Cd treatment were significantly changed, and the expression of 29 miRNAs involved in alternative splicing of the kitl pre‐mRNA was changed. The gene ontology analysis showed that the target gene functions of these 29 miRNAs were mainly enriched in the biological processes of cell metabolism regulation, post‐transcriptional regulation of mRNA, interleukin‐6‐mediated signal transduction, cell cycle, cell proliferation, differentiation and migration. The pathway enrichment analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in the Ras signaling pathway, the Rap1 signaling pathway, the Foxo signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the carcinogenic pathway. Polymerase chain reaction verification results showed that compared to the control group, the variation trends in the expression of mmu‐miR‐27a‐3p, mmu‐miR‐34b‐5p, mmu‐miR‐297a‐3p, mmu‐miR‐129‐5p and mmu‐miR‐107‐3p in the 4 hour 10 μm Cd treatment group were basically the same as that of the chip result. Our results indicate that Cd exposure can affect alternative splicing of the kitl pre‐mRNA in ovarian granulosa cells, and miRNAs play regulatory roles in the alternative splicing of kitl.

Activity of MPF and expression of its related genes in mouse MI oocytes exposed to cadmium

Research has revealed that cadmium can disrupt ovarian function; however, few reports have focused on MI oocytes meiotic progression, especially the activity of maturation promoting factor (MPF) and its related genes (Cdk1, Ccnb1, and Cdc25b) expression. In this study, GV oocytes cultured in vitro for 0, 6, and 9 hours with five groups (control and doses of 0.05, 0.5, 2.5, and 5 μM Cd). At the same dose of cadmium but different exposure time: compared with 0h, Periodic changes in MPF activity were changed and continuously increased over time. The mRNA and protein expression of each MPF-related gene in different cadmium dose groups were changed compared with that of 0h. At the same exposure time but different dose of cadmium: compared with control group, MPF activity, mRNA and protein expressions of each MPF-related gene in all the cadmium exposure groups were increased at 9h after exposure. Cadmium maintains the high MPF activity in mouse MI oocytes during its meiotic process and disturbs the periodic change of MPF activity; meanwhile, cadmium exposure promotes the syntheses of MPF-related gene, which may be one of the molecular mechanisms for the maintenance of high MPF activity, and ultimately prevents the meiotic progression in oocytes.

Cadmium induces ovarian granulosa cell damage by activating PERK-eIF2α-ATF4 through endoplasmic reticulum stress

Abstract This study aimed to investigate whether cadmium induces ovarian granulosa cell damage by activating protein kinase R‐like endoplasmic reticulum kinase (PERK)‐eIF2&agr;‐ATF4 through endoplasmic reticulum (ER) stress and to elucidate the underlying regulation mechanism. Two models of cadmium exposure were established. In one model, ovarian granulosa cells isolated from 21‐day‐old female Sprague Dawley rats were cultured in vitro for 36 h and exposed to CdCl2 (0, 5, 10, and 20 &mgr;M), and in another model, a human ovarian granulosa tumor cell line (COV434) was used to construct the binding immunoglobulin protein (BIP)‐knockdown cell line sh‐BIP and exposed to 0 and 20 &mgr;M CdCl2. After exposure to cadmium for 12 h, the expression mRNA and protein levels of BIP, p‐PERK, and p‐eIF2&agr; were determined in the two models. miRNAs related to BIP were also detected in granulosa cells after cadmium exposure. We found that mRNA and protein levels of all factors were upregulated in each cadmium‐dose group, except for BIP mRNA expression in the 5 &mgr;M Cd group. The BIP gene was knocked down in COV434 cells before exposure to cadmium. All factors were upregulated in COV434 cells exposed to Cd, and the expression of the p‐eIF2&agr; protein was downregulated in sh‐BIP cells exposed to Cd. In addition, no differences in BIP‐related miRNAs were detected in cadmium‐exposed rat ovarian granulosa cells versus the control group. Cadmium induces ovarian granulosa cell damage by inducing ER stress.