Wenchang Zhang

Cadmium exerts toxic effects on ovarian steroid hormone release in rats.

This study examined the toxic effects of cadmium on the function of sexual hormone release in the ovaries of rats and the mechanism of this dysregulation. In vivo, adult female rats were randomly assigned to four groups based on weight. Cadmium was given ip 5 days/week for 6 weeks at doses of 1.0, 0.5, 0.25 and 0mg/kg. Following euthanasia, the ovaries were removed and placed into culture medium for 3h. The levels of progesterone (P) and estadiol (E) in the culture medium were then measured by radioimmunoassay. In vitro studies were done using the ovaries of adult rats in different stages of estrous (proestrus, estrus, metestrus and diestrus). Individual ovaries were collected, placed into culture medium and exposed to 0, 0.1, 1, or 2mM of CdCl(2) for 3h, at which time the levels of P and E in the ovary culture serum were determined by radioimmunoassay. The in vivo results showed that the levels of P and E in the ovary culture serum (5.3+/-1.4 ng/ml and 1.4+/-0.4 pg/ml, respectively) decreased significantly in the high dose group compared to the control (9.2+/-0.9 ng/ml and 3.9+/-0.7 pg/ml, respectively). In vitro, there were significant differences in P and E in between the different concentrations of cadmium and also between the different stages of the estrous cycle. These data suggest that cadmium can inhibit P and E release in ovaries. This may represent an important mechanism of endocrine disruption.

Effect of low dose bisphenol A on the early differentiation of human embryonic stem cells into mammary epithelial cells

It has been previously reported that bisphenol A (BPA) can disturb the development of mammary structure and increase the risk of breast cancer in experimental animals. In this study, an in vitro model of human embryonic stem cell (hESC) differentiation into mammary epithelial cells was applied to investigate the effect of low dose BPA on the early stages of mammogenesis. A newly established hESC line was directionally differentiated into mammary epithelial cells by a well-established three-dimensional (3D) culture system. The differentiated mammary epithelial cells were characterized by immunofluorescence and western blotting assay, and were called induced differentiated mammary epithelial cells (iDMECs) based on these data. The hESCs were treated with low doses of BPA range 10(-9)-10(-6)M during the differentiation process, with DMSO as the solvent control and 17-β-estrodiol (E2) as the estrogen-positive control. Our results showed that low dose BPA and E2 could influence the mammosphere area of iDMECs and upregulate the expression level of Oct4 and Nanog proteins, while only BPA could downregulate the expression of E-cadherin protein. Taken together, this study provides some insights into the effects of low dose BPA on the early differentiation stage of mammary epithelial cells and suggests an easier canceration status of iDMECs under the effect of low dose BPA during its early differentiation stage.

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.

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.

Cadmium exposure in newborn rats ovary induces developmental disorders of primordial follicles and the differential expression of SCF/c-kit gene

Since the 1990s, the rising problem that gonad reproductive toxicity on adult female after exposing to cadmium (Cd), an environmental endocrine disruptor, has attracted high attention at home and abroad,and was systematically studied. Our research focuses on a further problem is that early cadmium exposure (during birth to before puberty) impact on development and function of ovarian cells and its possible mechanism. Our research focuses on the changes of ovarian cells growth and development after the newborn rat ovaries with cadmium exposure in vitro, and different expression of ovarian cells development-related factors, SCF/c-kit and changes of their DNA methylation status. We obtained ovaries from 4-day-old SD rats and cultured them in DMEM/F12 mixed with α-MEM media in vitro. Different doses of cadmium were designed as control, 0.5, 5, 10 and 50μM, and then the constituent ratio of ovarian follicle and follicular oocytes diameter were observed with microscope after 4-h exposure. We found that the increased constituent ratio of original follicle and decreased diameter of all levels of follicular oocytes(compared with control, with statistically significant differences, P<0.01).After the measurement of expression of SCF/c-kit by qRT-PCR and Western Blotting, the mRNA and protein expression of SCF/c-kit in ovarian were both decreased. We further found that the increased constituent ratio of growth follicle and increased diameter of oocytes under the treatment of adding SCF in cell culture media. Finally, MALDI-TOF-MS method showed DNA-low methylation status of SCF/c-kit promoter region after Cd exposure. Overall, we concluded that the exposure of cadimium (5-50μM) on newborn rats ovaries could inhibit follicle development.SCF/c-kit system might mediate follicle development damage caused by cadmium, which is associated with DNA hypomethylation of SCF/c-kit promoter region may be worthy of further study.

Gestational N-hexane inhalation alters the expression of genes related to ovarian hormone production and DNA methylation states in adult female F1 rat offspring.

Research has revealed that n-hexane can disrupt adult female endocrine functions; however, few reports have focused on endocrine changes in adult F1 females after maternal exposure during gestation. In this study, female Wistar rats inhaled 100, 500, 2500, or 12,500 ppm n-hexane for 4 h daily during their initial 20 gestational days. The F1 female offspring exhibited abnormal oestrus cycles. Compared with the controls, the in vitro-cultured ovarian granulosa cells of the 12,500 ppm group showed significantly reduced in vitro progesterone and oestradiol secretion. Elevated progesterone secretion was observed in the 500 ppm group, and decreased and significantly upregulated mRNA expression of the Star, Cyp11a1, Cyp17a1, and Hsd3b genes was observed in the 12,500 ppm and 500 ppm groups, respectively. The protein expression levels were consistent with the mRNA expression levels. Methylation screening of the promoter regions of these genes was performed using MeDIP-chip and confirmed by methylation-sensitive high-resolution melting (MS-HRM), and the observed methylation state changes of the promoter regions were correlated with the gene expression levels. The results suggest that the hormone levels in the female offspring after gestational n-hexane inhalation correspond to the expression levels and DNA methylation states of the hormone production genes.

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.

Waist Circumference Coupled with Either HDL-C or TG Can Be Used as a Diagnostic Marker for Metabolic Syndrome in Chinese Women with Polycystic Ovary Syndrome

Although quite a few polycystic ovary syndrome (PCOS) patients suffering from metabolic syndrome (MS) have been reported in previous studies, no reliable and early diagnostic biomarkers for MS in PCOS patients have yet been identified. To identify early and reliable diagnostic biomarkers for MS in Chinese women with PCOS, a total of 401 patients (200 PCOS patients and 201 controls) were enrolled in our present study. All of the subjects were examined for anthropometric (weight, waist circumference, blood pressure, etc.) and biochemical (fasting glucose, serum lipid indices, total testosterone, etc.) parameters. Our results showed that the prevalence of MS in the PCOS patients (20.50%) was 6.8-fold higher (P < 0.05) than that in the controls (2.99%). Nearly 71.0% of the PCOS patients had at least one component of MS, of which dyslipidemia was the most prevalent. Furthermore, within the PCOS group, the prevalence of MS increased with increasing age and body mass index (BMI). Logistic analysis indicated that BMI, triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), hypertension, and fasting glucose were significantly associated with the presence of MS in PCOS patients. Analysis of the ability of the potential diagnostic biomarkers to indicate MS in PCOS patients showed that the PPV, NPV, specificity, sensitivity, and Youdens index for waist circumference (WC) coupled with HDL-C were 59.68%, 97.10%, 84.28%, 90.24%, and 74.52, respectively, and those for WC coupled with TG were 93.33%, 92.35%, 98.74%, 68.29%, and 67.03%, respectively. ROC curve analysis showed that the areas under the ROC curves (AUCs) for WC coupled with HDL-C and for WC coupled with TG were 0.882 and 0.901, respectively. Our present study demonstrates that WC coupled with either HDL-C or TG can be used as a relatively early and reliable diagnostic biomarker for MS in Chinese PCOS patients.

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.