Dehydroepiandrosterone-induced polycystic ovary syndrome mouse model requires continous treatments to maintain reproductive phenotypes.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy associated with infertility and metabolic disorder in women of reproductive age. Animal models have been developed and used as tools to unravel the pathogenesis of PCOS, among which most postnatal models employ continuing experimental manipulations. However, the persistence and stability of these animals after modeling is unknown. Dehydroepiandrosterone (DHEA)-induced PCOS mouse model is commonly used in PCOS studies. Thus the aim of the present study was to investigate the reproductive features of DHEA-induced PCOS mice fed a normal chow or an high-fat diet (HFD) with treatment withdrawal or consecutive treatments after PCOS mouse models were established. METHODS: Prepubertal C57BL/6 J mice (age 25 days) were injected (s.c.) daily with DHEA on a normal chow or a 60% HFD for 20 consecutive days to induce PCOS mouse models. Mice injected with the vehicle sesame oil were used as controls. After 20 days, mice were divided into 2 groups, namely "Continue dosing group" and "Stop dosing group". The animals were consecutively treated with DHEA or DHEA + HFD, or housed without any treatment for 2 or 4 weeks. Estrous cycles were evaluated during this period. At the end of the experiment, serum testosterone (T) levels were measured and the morphology of ovaries was evaluated. RESULTS: The mice in Continue dosing groups maintained reproductive phenotypes of PCOS mouse models. In contrast, 2 or 4 weeks after PCOS models were established, the mice with treatment withdrawal in Stop dosing groups exhibited normal serum testosterone levels, regular estrous cycle, and relatively normal ovarian morphology. In addition, even with consecutive treatments, there was no marked difference in body weight between DHEA mice on the normal chow or an HFD in Continue dosing groups and the control animals 3 weeks after modeling. CONCLUSIONS: After PCOS mice were induced with DHEA or DHEA + HFD, the mice still need consecutive treatments to maintain reproductive phenotypes to be regarded as PCOS mice that meet the diagnostic criteria of PCOS defined by the 2003 Rotterdam criteria.

Effects of a ketogenic diet on reproductive and metabolic phenotypes in mice with polycystic ovary syndrome†.

Polycystic ovary syndrome (PCOS) is one of the most common female reproductive and metabolic disorders. The ketogenic diet (KD) is a diet high in fat and low in carbohydrate. The beneficial effects of KD intervention have been demonstrated in obese women with PCOS. The underlying mechanisms, however, remain unknown. The aim of the present study was to investigate the effects of a KD on both reproductive and metabolic phenotypes of dehydroepiandrosterone (DHEA)-induced PCOS mice. Female C57BL/6 mice were divided into three groups, designated Control, DHEA, and DHEA+KD groups. Mice of both Control and DHEA groups were fed the control diet, whereas DHEA+KD mice were fed a KD with 89%(kcal) fat for 1 or 3 weeks after PCOS mouse model was completed. At the end of the experiment, both reproductive and metabolic characteristics were assessed. Our data show that KD treatment significantly increased blood ketone levels, reduced body weight and random and fasting blood glucose levels in DHEA+KD mice compared with DHEA mice. Glucose tolerance, however, was impaired in DHEA+KD mice. Ovarian functions were improved in some DHEAmice after KD feeding, especially in mice treated with KD for 3 weeks. In addition, inflammation and cell apoptosis were inhibited in the ovaries of DHEA+KD mice. Results from in vitro experiments showed that the main ketone body ß-hydroxybutyrate reduced inflammation and cell apoptosis in DHEA-treated KGN cells. These findings support the therapeutic effects of KD and reveal a possible mechanism by which KD improves ovarian functions in PCOS mice.

Oxidative Stress as a Contributor to Insulin Resistance in the Skeletal Muscles of Mice with Polycystic Ovary Syndrome.

Polycystic ovarian syndrome (PCOS) is a reproductive, endocrine, and metabolic disorder. Circulating markers of oxidative stress are abnormal in women with PCOS. There is a close relationship between oxidative stress and insulin resistance (IR). However, little information is available about oxidative stress in the skeletal muscles of those affected by PCOS. In this study, PCOS was induced in prepubertal C57BL/6J mice by injection with dehydroepiandrosterone. Oxidative stress biomarkers were then measured in both serum and skeletal muscles. The underlying mechanisms were investigated in C2C12 myotubes treated with testosterone (T). We discovered increased oxidative biomarkers, increased ROS production, and damaged insulin sensitivity in the skeletal muscles of mice with PCOS. High levels of T caused mitochondrial dysfunction and increased ROS levels through the androgen receptor (AR)-nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) signaling pathway in C2C12 cells. Treatment of C2C12 cells with an antioxidant N-acetylcysteine (NAC) decreased T-induced ROS production, improved mitochondrial function, and reversed IR. Administration of NAC to mice with PCOS improved insulin sensitivity in the skeletal muscles of the animals. Hyperandrogenism caused mitochondrial dysfunction and redox imbalance in the skeletal muscles of mice with PCOS. We discovered that oxidative stress contributed to skeletal muscle IR in PCOS. Reducing ROS levels may improve the insulin sensitivity of skeletal muscles in patients with PCOS.

Bisphenol A attenuates testosterone production in Leydig cells via the inhibition of NR1D1 signaling.

Bisphenol A (BPA) is an endocrine-disrupting compound that impairs testosterone synthesis in male mammals. A circadian clock gene deficiency leads to diminished fertility and even infertility in male mice. However, whether circadian clock signaling pathways mediate the suppressive effect of BPA on testosterone synthesis in Leydig cells (LCs) remains unknown. The present study aims to detect the effect of BPA on cellular circadian clock and testosterone synthesis in mouse LCs, and examine the mechanisms underlying NR1D1 signaling. BPA treatment significantly attenuated the transcription levels of Nr1d1 and steroidogenic genes (Hsd3b2 and Hsd17b3) in TM3 cells, but increased other circadian clock gene levels (Per2 and Dbp). BPA treatment also significantly downregulated NR1D1 and StAR protein expression, but upregulated BMAL1 protein expression in TM3 cells. Furthermore, there was a marked decline in testosterone production in BPA-treated TM3 cells. Intraperitoneal injection of BPA profoundly reduced NR1D1 and StAR protein levels and steroidogenic gene transcription levels (Cyp11a1, Hsd3b2, and Hsd17b3), while enhancing BMAL1 protein and other circadian clock gene (Per2 and Dbp) levels in mouse testes. Notably, serum testosterone levels were also drastically reduced in BPA-treated mice. Moreover, SR9009, an NR1D1 agonist, augmented testosterone production in TM3 cells via elevated expression of steroidogenic genes (StAR, Cyp11a1 and Hsd17b3). Conversely, Nr1d1 knockdown inhibited testosterone accumulation and attenuated steroidogenic gene expression. Moreover, treatment with SR9009 partially reversed the BPA effect on the circadian clock and testosterone production. Taken together, our study demonstrates that BPA perturbs testosterone production, at least partially, via inhibiting NR1D1 signaling in LCs.

Expression and clinical significance of Nectin-4 in hepatocellular carcinoma.

OBJECTIVE: Nectin-4 is a member of the Nectin family of four Ca(+)-independent immunoglobulin-like cell adhesion molecules and implicated in cell adhesion, movement, proliferation, differentiation, polarization, and survival. The aberrant expression of Nectin-4 has been found in a variety of tumors; however, its expression in hepatocellular carcinoma (HCC) is still poorly understood. This study was to investigate the expression of Nectin-4 and its clinical significance in the patients with HCC. METHODS: The expression of Nectin-4 was assessed at mRNA and protein levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting assays in 20 HCC specimens and adjacent non-tumor live tissues. Furthermore, the clinical significance of Nectin-4 in 87 cases of HCC was confirmed by immunohistochemistry. RESULTS: The mRNA and protein levels of Nectin-4 were higher in HCC tumor tissues than in the matched non-tumor tissues. Nectin-4 was located in the cytoplasm of tumor cells and over-expressed in 67.82% (59/87) HCC tissues by immunohistochemical staining. Positive Nectin-4 expression was significantly correlated with tumor size (P=0.029), status of metastasis (P=0.023), vascular invasion (P=0.018) and tumor-node-metastasis stage (P=0.003). In addition, Kaplan-Meier survival analysis indicated that positive Nectin-4 expression was associated with worse recurrence-free survival (RFS) and overall survival (OS) (P=0.006 and P=0.005, respectively). In multivariate analysis, Nectin-4 was an independent prognostic factor for RFS and OS in the patients with HCC. CONCLUSION: Nectin-4 is upregulated in HCC and may be a novel prognostic biomarker for the patients after surgical resection.

Prognostic significance and clinicopathological features of hypoxic inducible factor-2alpha expression in hepatocellular carcinoma.

OBJECTIVES: To assess the association between hypoxic inducible factor-2alpha (HIF-2α) and hepatocellular carcinoma (HCC) by meta-analysis. METHODS: This study was carried out at Anhui Province Hospital, Hefei, Anhui, China in February 2014. We searched various databases for studies published in English or Chinese up to February 28, 2014. The hazard ratio for overall survival and analyzed odds ratio were combined to evaluate the clinicopathological features of HIF-2α expression in HCC. RESULTS: A total of 7 eligible studies comprising 1066 patients with HCC were identified after our full assessment according to inclusion criteria. All of the patients came from China. The results indicated that the association between HIF-2α expression and prognostic values in HCC was inconspicuous, while the expression of HIF-2α was significantly associated with capsule infiltration, vein invasion, and histological grade. CONCLUSION: Expression of HIF-2α was associated with invasion and metastasis in HCC, but did not have a distinct significance in prognosis, according to the limited evidence. However, high quality, large sample size, and controlled trials are required.

Overexpression of HOXA13 as a potential marker for diagnosis and poor prognosis of hepatocellular carcinoma.

HOXA13 is a member of homeobox genes that encode transcription factors regulating embryonic development and cell fate. Abnormal HOXA13 expression was reported in hepatocellular carcinoma (HCC), but its correlation with tumor angiogenesis and prognosis still remain unclear. This study was aimed to uncover the expression, diagnostic and prognostic significance of HOXA13 in HCC. Immunohistochemistry was performed to detect HOXA13 expression in HCC and corresponding paracarcinomatous tissues from 90 patients. Enzyme-linked immunosorbent assay was used to detect serum HOXA13 in 90 HCC patients and 20 healthy volunteers. Receiver operating characteristics was analyzed to calculate diagnostic accuracy of serum HOXA13, alpha-fetoprotein (AFP) and their combination. Immunoreactivity of HOXA13 was detected in 72.2% of HCC, and 12.2% of adjacent non-cancerous samples. HOXA13 expression was significantly associated with tumor size, microvascular invasion, pathological grade, tumor capsula status, AFP level, tumor-node-metastasis stage and positively correlated with VEGF (p < 0.001) and microvessel density (p < 0.001). The combination of serum HOXA13 and AFP had a markedly higher area under the curve than HOXA13 alone. HOXA13 expression was associated with unfavorable overall survival (OS) (p < 0.001) and disease-free survival (DFS) (p < 0.001). Multivariate analysis indicated that patients with HOXA13-expressing tumors had a significantly shorter OS (p = 0.030) and DFS (p = 0.005) than those with HOXA13-negative tumors. Thus, HOXA13 expression possibly plays an important role in tumor angiogenesis, progression and prognosis of HCC. Moreover, we demonstrate that serum HOXA13 may serve as a biomarker for early HCC diagnosing and predicting outcome.