Activation of Nrf2 might reduce oxidative stress in human granulosa cells.

Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress (OS). It is well documented that equilibration status of OS plays fundamental roles in human reproductive medicine, and the physiological role of Nrf2 in ovarian granulosa cells (GCs) has not been determined yet. Herein we aimed to study the function of Nrf2 in GCs. Human ovarian tissues were subjected to immunohistochemistry to localize Nrf2 and Keap1 and we detected the expression of Nrf2 and Keap1 in the human GCs. Human luteinized GCs were isolated and cultured, and hydrogen peroxide (H2O2) or Dimethylfumarates (DMF), an activator of Nrf2, were added to GCs to analyze the relationship between Nrf2 and antioxidants by quantitative RT-PCR. The mRNA levels of Nrf2, catalase, superoxide dismutase 1 (SOD1), and 8-Oxoguanine DNA glycosylase (OGG1) were elevated by H2O2, and DMF treatment showed similar but pronounced effects through activation of Nrf2. To determine the relationship of Nrf2 and the generation of antioxidants, siRNAs were used and quantitative RT-PCR were conducted. Decreased expression of Nrf2 resulted in a decreased level of these antioxidant mRNA. Intracellular levels of ROS were investigated by fluorescence of 8-hydroxy-2'-deoxyguanosine and fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate after H2O2 and/or DMF treatment, and DMF treatment quenched intracellular ROS generation by H2O2. These results show that activation of Nrf2 might lead to alleviate OS in human GCs, and this could provide novel insight to conquer the age-related fertility decline that is mainly attributed to the accumulation of aberrant OS.

The Emerging Role of FOXL2 in Regulating the Transcriptional Activation Function of Estrogen Receptor ß: An Insight Into Ovarian Folliculogenesis.

Germline mutations of the fork-head transcriptional factor forkhead box L2 (FOXL2) predispose embryos to autosomal-dominant blepharophimosis-ptosis-epicanthus inversus syndrome with primary ovarian insufficiency in female patients, but the mechanisms of FOXL2 in ovarian follicular development remain elusive. Estrogens produced by ovarian granulosa cells and estrogen receptor (ER) α and ERß play fundamental roles in ovarian pathophysiology, and a previous study revealed that ERα and ERß physically interact with FOXL2. However, the underlying functions of these interactions have not been investigated. Herein, we report an ERß-specific repressive function of FOXL2. Histological examination demonstrated that FOXL2 expression tends to be intense during early follicular development. Immunoprecipitation revealed that ERß and FOXL2 interact in a ligand-independent manner. In vitro pull-down assays revealed a direct interaction between FOXL2 and the activation function (AF)-1/2 domain of ERß. The expression of FOXL2 represses the ligand-dependent transcriptional activation of ERß, but FOXL2 does not influence the ligand-dependent transcriptional activation of ERα. Consistent with these results, RNA interference-mediated depletion of FOXL2 stimulates the expression of the ERß-downstream gene p450 aromatase. The convergence between FOXL2 functions and ERß-mediated transcription in the ovary suggests the putative mechanism of FOXL2 in early-phase follicular development, which may be partially attributed to the regulation of ERß-dependent gene expression.

HAND2-mediated proteolysis negatively regulates the function of estrogen receptor α.

A previous study demonstrated that the progesterone­inducible HAND2 gene product is a basic helix­loop­helix transcription factor and prevents mitogenic effects of estrogen receptor α (ERα) by inhibiting fibroblast growth factor signalling in mouse uteri. However, whether HAND2 directly affects the transcriptional activation function of ERα remains to be elucidated. In the present study, the physical interaction between HAND2 and ERα was investigating by performing an immunoprecipitation assay and an in vitro pull­down assay. The results demonstrated that HAND2 and ERα interacted in a ligand­independent manner. The in vitro pull­down assays revealed a direct interaction between HAND2 and the amino­terminus of ERα, termed the activation function­1 domain. To determine the physiological significance of this interaction, the role of HAND2 as a cofactor of ERα was investigated, which revealed that HAND2 inhibited the ligand­dependent transcriptional activation function of ERα. This result was further confirmed and the mRNA expression of vascular endothelial growth factor, an ERα­downstream factor, was decreased by the overexpression of HAND2. This inhibition of ligand­dependent transcriptional activation function of ERα was possibly attributed to the proteasomic degradation of ERα by HAND2. These results indicate a novel anti­tumorigenic function of HAND2 in regulating ERα­dependent gene expression. Considering that HAND2 is commonly hypermethylated and silenced in endometrial cancer, it is hypothesized that HAND2 may serve as a possible tumor suppressor, particularly in uterine tissue.

CCAR2 negatively regulates nuclear receptor LXRα by competing with SIRT1 deacetylase.

Liver X receptors (LXRs) monitor endogenous sterol levels to maintain whole-body cholesterol levels and regulate inflammatory responses. Recent studies have demonstrated that LXRs may inhibit cellular proliferation, but the underlying mechanism remains unclear. Cell cycle and apoptosis regulator 2 (CCAR2), previously known as DBC1/KIAA1967, is a transcriptional regulator that regulates cellular proliferation and energy metabolism by inhibiting sirtuin 1 (SIRT1) deacetylase. Based on the findings that CCAR2 regulates several nuclear receptors, including the estrogen receptors and androgen receptor, we aimed to identify the underlying mechanism of CCAR2 regulation of LXRα. We found that CCAR2 formed a complex with LXRα in a ligand-independent manner in HepG2 cells, and in vitro pull-down assays, it revealed a direct interaction between the amino terminus of CCAR2 and the AF-2 domain of LXRα. Thereby, CCAR2 attenuates the ligand-dependent transcriptional activation function of LXRα. RNA interference-mediated depletion of endogenous CCAR2 potentiated the expression of the LXRα target genes ATP-binding cassette transporter A1 and G1, and the abrogation of CCAR2 resulted in decreased cellular proliferation. Moreover, competitive immunoprecipitation studies revealed that the LXRα downregulation involves the inhibition of SIRT1-LXRα complex formation. Therefore, these results clearly indicate a novel mechanism in which CCAR2 may regulate the transcriptional activation function of LXRα due to its specific inhibition of SIRT1 and serve to regulate cellular proliferation.

SIRT3 positively regulates the expression of folliculogenesis- and luteinization-related genes and progesterone secretion by manipulating oxidative stress in human luteinized granulosa cells.

SIRT3 is a member of the sirtuin family and has recently emerged as a vital molecule in controlling the generation of reactive oxygen species (ROS) in oocytes. Appropriate levels of ROS play pivotal roles in human reproductive medicine. The aim of the present study was to investigate SIRT3 expression and analyze the SIRT3-mediated oxidative response in human luteinized granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemical analysis to localize SIRT3 expression. Hydrogen peroxide and human chorionic gonadotropin were used to analyze the relationship between ROS and SIRT3 by quantitative RT-PCR and Western blotting. Intracellular levels of ROS were investigated by fluorescence after small interfering RNA-mediated knockdown of SIRT3 in human GCs. To uncover the role of SIRT3 in folliculogenesis and luteinization, mRNA levels of related genes and the progesterone concentration were analyzed by quantitative RT-PCR and immunoassays, respectively. We detected the expression of SIRT3 in the GCs of the human ovary. The mRNA levels of SIRT3, catalase, and superoxide dismutase 1 were up-regulated by hydrogen peroxide in both COV434 cells and human GCs and down-regulated by human chorionic gonadotropin. Knockdown of SIRT3 markedly elevated ROS generation in human GCs. In addition, SIRT3 depletion resulted in decreased mRNA expression of aromatase, 17ß-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in GCs and thus resulted in decreased progesterone secretion. These results have the important clinical implication that SIRT3 might play a positive role in the folliculogenesis and luteinization processes in GCs, possibly by sensing and regulating the generation of ROS. Activation of SIRT3 function might help to sustain human reproduction by maintaining GCs as well as oocytes.

Acupuncture on the Endometrial Morphology, the Serum Estradiol and Progesterone Levels, and the Expression of Endometrial Leukaemia-inhibitor Factor and Osteopontin in Rats.

Although it is well known that acupuncture has beneficial effects on a variety of medical conditions especially in pain relief, nausea, and vomiting, it remains controversial whether it has positive impact on the female reproduction. The present study aimed to evaluate whether the following endometrial receptivity factors: the endometrial morphology, the hormone concentrations, and the protein expression of endometrial leukaemia-inhibitory factor (LIF) and osteopontin (OPN) could be improved by the acupuncture in clomiphene citrate(CC)-induced rat model during implantation period. Results showed that, compared with the CC group, glandular development advanced, the serum estradiol levels decreased significantly, and the glandular area and endometrial LIF and OPN expression were significantly higher in acupuncture group. There were no significant differences in serum progesterone levels, endometrial thickness, and stromal area between groups. These results suggest that acupuncture can improve certain aspects of endometrial receptivity in CC-induced rat model during implantation period, which might result in endometrial state better to female reproduction.

Protective effect of magnesium isoglycyrrhizinate on ethanol-induced testicular injuries in mice.

OBJECTIVE: Ethanol treatment induces an increase in oxidative stress. As licorice compounds are potent antioxidants, our aim was to examine whether magnesium isoglycyrrhizinate attenuated lipid peroxidation, the major end-point of oxidative damage resulting from ethanol administration. METHODS: Four groups(18 animals in each group) of male Kunming mice were used. The first group served as control and received 0.4 ml normal saline daily for 18 days orally. The second group of mice was given 56% ethanol at 16 ml/kg body weight per day for 18 days orally. The third group was given the same dose of ethanol and administrated magnesium isoglycyrrhizinate (15 mg/kg.d, i.p.) for 18 days. The fourth group was given the same dose of ethanol and administrated with magnesium isoglycyrrhizinate (45 mg/kg.d, i.p.) for 18 days. Twenty four hours after 9 days or 18 days of treatment the mice were sacrificed using 10% chloral hydrate. Sperm counts and motility in the epididymis were assessed. The lipid peroxidation and antioxidants of testicular mitochondria were also determined. The pathological changes of testicle tissue of the mice were observed by light microscopy. RESULTS: Magnesium isoglycyrrhizinate effectively prevented the ethanol-induced seminiferous epithelium disorganization and degeneration of Sertoli cells and germ cells. Sperm counts and motility of the magnesium isoglycyrrhizinate treated groups were higher than those of the alcohol treated group, but were lower than those of the control group. The drug exhibited an ability to counteract ethanol induced oxidative challenge as it effectively reduced testicular malondialdehyde (MDA) and increased the activities of superoxide dismutase and glutathione peroxidase. CONCLUSION: Magnesium isoglycyrrhizinate is able to inhibit the ethanol-induced lipid peroxidation and has a protective effect against testicular oxidative injury.