Zhenghong Zhang

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1α mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1α and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1α and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1α directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1α-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

Expression and clinical significance of the HIF-1a/ET-2 signaling pathway during the development and treatment of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a major health problem in reproductive-aged women worldwide, but the precise pathogenesis of PCOS remains unclear. Our previous study revealed that hypoxia-inducible factor (HIF)-1a mediated endothelin (ET)-2 signaling plays an important role in ovulation in rats. Therefore, the present study used a PCOS rat model to test the hypotheses that HIF-1a signaling is expressed and inhibited in ovaries during PCOS formation and that the HIF-1a/ET-2 signaling pathway is a target of dimethyldiguanide (DMBG) in the clinical treatment of PCOS. First, the development of a PCOS model and the effect of DMBG treatment were examined through ovarian histology and serum hormone levels, which were consistent with previous reports. Second, HIF-1a and ET-2 expression were detected by immunohistochemistry and western blot. The results showed decreased HIF-1a/ET-2 expression in the ovaries of PCOS rats, whereas DMBG treatment reversed the protein decreases and improved the PCOS symptoms. Third, to understand the molecular mechanism, HIF-1a/ET-2 mRNA expression was also examined. Interestingly, HIF-1a mRNA increased in the ovaries of PCOS rats, while ET-2 mRNA decreased, indicating that HIF-1a protein degradation may be involved in POCS development and treatment. Finally, HIF prolyl hydroxylase (PHD) activity was examined to further clarify the contribution of HIF-1a signaling to the development and treatment of PCOS. The results suggested that the inhibition of HIF-1a/ET-2 signaling may be caused by increased PHD activity in PCOS. DMBG-treated PCOS may further activate HIF-1a signaling at least partly through inhibiting PHD activity. Taken together, these results indicate that HIF-1a signaling is inhibited in a PCOS rat model through increasing PHD activity. DMBG treatment improved PCOS by rescuing this pathway, suggesting that HIF-1a signaling plays an important role in the development and treatment of PCOS. This HIF-1a-mediated ET-2 signaling pathway may be an important mechanism regulating PCOS formation and treatment in mammalian ovaries in vivo and should be a new clinical target for PCOS prevention and treatment in the future.

High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development

The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovaries in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27(Kip1) and p21(Cip1), were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure.

Expression and contribution of the HIF‑1α/VEGF signaling pathway to luteal development and function in pregnant rats

Vascular endothelial growth factor (VEGF) is vital in normal and abnormal angiogenesis in the ovary, particularly during the early development of the corpus luteum in the ovary. However, the molecular regulation of the expression VEGF during luteal development in vivo remains to be fully elucidated. As the expression of VEGF is mediated by hypoxia‑inducible factor (HIF)‑1α in luteal cells cultured in vitro, determined in our previous study, the present study was performed to confirm the hypothesis that HIF‑1α is induced and then regulates the expression of VEGF and VEGF‑dependent luteal development/function in vivo. This was investigated using a pregnant rat model treated with a small‑molecule inhibitor of HIF‑1α, echinomycin (Ech). The development of the corpus luteum in the pregnant rat ovary was identified via performing assays of the serum progesterone, testosterone and estradiol concentrations by radioimmunoassay, accompanied with determination of the changes in the expression levels of HIF‑1α and VEGF by reverse transcription‑quantitative polymerase chain reaction at different days of the developmental process. On day 5, serum progesterone levels were markedly increased, whereas serum levels of testosterone and estradiol did not change significantly. On day 17, the highest level of serum progesterone was observed, however, this was not the case for testosterone and estradiol. Further analysis of the expression levels of HIF‑1α and VEGF revealed that their changes were consistent with the changes in serum levels of progesterone, which occurred in the development of the corpus luteum in the ovaries of pregnant rats. Further investigation demonstrated that Ech inhibited luteal development through inhibiting the expression of VEGF, mediated by HIF‑1α, and subsequent luteal function, which was determined by detecting changes in serum progesterone on days 8 and 14. Taken together, these results demonstrated that HIF‑1α‑mediated expression of VEGF may be one of the important mechanisms regulating ovarian luteal development in mammals in vivo, which may provide novel strategies in treatment for fertility control and for certain types of ovarian dysfunction, including polycystic ovarian syndrome, ovarian hyperstimulation syndrome and ovarian neoplasia.

Effect of HIF-1a/VEGF signaling pathway on plasma progesterone and ovarian prostaglandin F₂a secretion during luteal development of pseudopregnant rats.

The corpus luteum is a temporary endocrine structure in mammals that plays an important role in the female reproductive cycle and is formed from a ruptured and ovulated follicle with rapid angiogenesis. Vascular endothelial growth factor (VEGF) is thought to be vital in normal and abnormal angiogenesis in the ovary, but the molecular regulation of luteal VEGF expression during corpus luteum development in vivo is still poorly understood at present. Therefore, we examined whether hypoxia-inducible factor-1a (HIF-1a) is induced and regulates VEGF expression and luteal function in vivo using a pseudopregnant rat model treated with a small-molecule inhibitor of HIF-1a, echinomycin. Corpus luteum development in the pseudopregnant rat ovary was determined after measuring plasma progesterone concentration and ovarian prostaglandin F2a content to reflect changes in HIF-1a and VEGF on different days of this developmental process. At day 7, the corpus luteum was formed and the expression of HIF- 1a/VEGF reached a maximum, while a significant decrease in HIF-1a/ VEGF expression was observed when luteolysis occurred at day 13. Additionally, echinomycin blocked luteal development by inhibiting VEGF expression mediated by HIF-1a and following luteal function by detecting the progesterone changes at day 7. These results demonstrated that HIF-1a-mediated VEGF expression might be an important mechanism regulating ovarian luteal development in mammals in vivo, which may provide new strategies for fertility control and for treating some types of ovarian dysfunction, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, and ovarian neoplasia.

Expression of hypoxia-inducible factor-1α during ovarian follicular growth and development in Sprague-Dawley rats.

Hypoxia-inducible factor-1α (HIF-1α) has been identified as a transcription factor that is involved in diverse physiological and pathological processes in the ovary. In this study, we examined whether HIF-1α is expressed in a cell- and stage-specific manner during follicular growth and development in the mammalian ovaries. Using immunohistochemistry and Western blot analysis, HIF-1α expression was observed in granulosa cells specifically and was significantly increased during the follicular growth and development of postnatal rats. Furthermore, pregnant mare serum gonadotropin also induced HIF-1α expression in granulosa cells and ovaries during the follicular development of immature rats primed with gonadotropin. Moreover, we also examined proliferation cell nuclear antigen, a cell proliferation marker, during follicular growth and development and found that its expression pattern was similar to that of HIF-1α protein. Granulosa cell culture experiments revealed that proliferation cell nuclear antigen expression may be regulated by HIF-1α. These results indicated that HIF-1α plays an important role in the follicular growth and development of these 2 rat models. The HIF-1α-mediated signaling pathway may be an important mechanism regulating follicular growth and development in mammalian ovaries in vivo.

Defective insulin signaling and the protective effects of dimethyldiguanide during follicular development in the ovaries of polycystic ovary syndrome

It is established that the physiological effects of insulin are primarily mediated by the insulin signaling pathway. However, a defective insulin signaling is closely associated with the clinical manifestations of polycystic ovary syndrome (PCOS), which include excess androgen levels, insulin resistance and anovulation, and is involved in the pathophysiology of PCOS at the molecular level. Dimethyldiguanide (DMBG) has been widely employed to alleviate reproduction dysfunction in women with PCOS, however, the exact mechanism of this effect remains unclear. The objective of the present study was to investigate the effects of DMBG on the expression of the insulin signaling pathway in the ovaries of rats with PCOS, and to identify the potential underlying molecular mechanisms of these effects in PCOS. In the present study, a PCOS rat model was induced by letrozole, and successful establishment of the model was confirmed by examining ovarian histology and determining serum testosterone levels, by hematoxylin and eosin staining and ELISA, respectively. Subsequently, the expression of two key elements of insulin signaling, insulin receptor substrate (IRS)-2 and phosphatidylinositol 3-kinase (PI3K), was determined by immunohistochemistry and western blot analysis. The results demonstrated that IRS-2 and PI3K expression was markedly decreased in PCOS ovaries, which was rescued by DMBG treatment. These results indicate that IRS-2/PI3K signaling may be involved in the development of PCOS and the therapeutic effects of DMBG on PCOS. To further confirm the effects of DMBG on insulin signaling expression during this process, the expression of an additional two downstream proteins, phosphoinositide-dependent kinase-1 (PDK-1) and the mammalian target of rapamycin (mTOR), was also investigated in the present study, and the results demonstrated that the expression of PDK-1 and mTOR was significantly reduced in PCOS ovaries and increased following DMBG treatment, further indicating that altered insulin signaling may have an important role in the development and treatment of PCOS. In conclusion, the results of the present study indicate that the reduced expression of proteins involved in insulin signaling may contribute to the development of the clinical features of PCOS, and DMBG reverses reduced expression of insulin signaling components, by a mechanism that is yet to be determined, to attenuate certain symptoms of PCOS, such as obesity. To the best of our knowledge, the present study is the first to provide data regarding the detailed changes of insulin signaling during the development and treatment of PCOS, and may provide an important reference for clinical PCOS treatment.

Temporal and spatial expression profiles of Frizzled 3 in the ovary during the estrous cycle.

Frizzled 3 is an important receptor in the Wnt/β-catenin pathway, a conserved signaling pathway that regulates gene expression and controls diverse developmental processes. However, the role of this protein during follicular development in the adult ovary is not known. The present study was designed to investigate the expression and localization of Frizzled 3 mRNA and protein during the estrous cycle in the mouse ovary through in situ hybridization (ISH), real-time quantitative polymerase chain reaction, immunohistochemistry and western blot. ISH results showed that in proestrus, high expression of Frizzled 3 was found in the granulosa and stroma with weak levels in the corpus luteum. In estrus and diestrus, the stroma had high Frizzled 3 expression, but levels were low in granulosa cells and corpus luteum. In the metestrus, moderate expression of Frizzled 3 was found in the stroma but low to no expression was found in luteal cells and follicles. The mRNA and protein levels of Frizzled 3 were found to be the highest in proestrus and diestrus compared to estrus and metestrus (P < 0.05), confirming the ISH results. During estrus and diestrus, high Frizzled 3 expression was observed in the stroma and moderate levels in granulosa cells, and during estrus and proestrus, low expression was seen in the oocyte cell membrane. The western blot results further confirmed this change during the estrous cycle. Together, these results indicate that Frizzled 3 is involved in regulating follicular development and oocyte maturation during the estrous cycle.

Improvement of Exercise Intervention on Vascular Endothelial Functions during Prediabetes Mellitus

Prediabetes is considered to be an at a risk state, with an annualized 5-10% conversion rate of diabetes and the similiar proportion converting back to normoglycaemia. The global incidence of prediabetes is increasing and will reach to 471 million patients in 2030. The elevation of blood sugar is a continuum and hence prediabetes cannot be considered an entirely benign status and present insulin resistance (IR) and β-cell dysfunction, so that the development of prediabetic complications and vascular endothelial dysfunction. Therefore, it is crucial to early recognition and treatment of prediabetic individuals. For prediabetic individuals, lifestyle modification is the cornerstone of diabetes prevention with evidence of a 40-70% relative risk reduction. In the review, we include the possible mechanism of IR-induced vascular endothelial dysfunction, and exercise intervention improving the vascular endothelial dysfunction. These results showed clearly positive effects of physical exercise intervention and provided a direct clinical evidence of the effectiveness of this approach for preventing early diabetes

Effects of dimethyl carbonate‑induced autophagic activation on follicular development in the mouse ovary

Dimethyl carbonate (DMC) is a widely used industrial chemical, which may be increasingly used in the future. However, its toxicity profile remains largely unknown. The present study was designed to investigate the effects of DMC exposure on the ovaries and the effect of autophagy activation on follicular development. Rats were randomly divided into a control group and low, medium and high dose DMC groups (all n=10). Histological analyses identified no marked differences in the rate of apoptosis between the control and low dose groups; however, marked apoptosis occurred in the medium and high dose groups. The expression of cleaved caspase-3 was significantly increased in the medium and high dose groups, which was consistent with changes observed in the expression of Bcl-2 and Bax. These results indicated that DMC exposure induces toxicity on ovarian function via the induction of apoptosis. The increased expression of the autophagy-related proteins light chain 3II, beclin-1 and p62 following exposure to DMC further indicated that autophagy was activated primarily in the granulosa cells of ovarian follicles in a dose-dependent manner. In addition, the changes in the expression of hypoxia inducible factor 1 α subunit (HIF-1α) and its target protein BCL2 interacting protein 3 (BNIP3) indicated that they may serve a role in the follicular development process induced by DMC. The results of the current study demonstrated that DMC exposure activated autophagy in the ovarian tissue. Furthermore, exposure to low doses of DMC may protect follicular development by activating the HIF-1α/BNIP3 signaling pathway. Taken together, these results indicate that exposure to medium and high doses of DMC induced follicular atresia by activating the apoptotic signaling pathway. This may be an important mechanism of regulating follicular development and ovarian function in mammals.