Oestrogen has been reported to control the invasiveness of endometrial stromal cells in endometriosis. Notch signalling, a master regulator of cell invasion in tumours, is regulated by oestrogen in other diseases and hyperactivated in endometriotic stromal cells. Therefore, we hypothesized that an interaction between Notch signalling and oestrogen may exist in the regulation of endometrial stromal cell invasion, which is essential for the development of endometriosis. Western blot analysis of tissues showed that the expression levels of Notch components (JAG1 and NOTCH1) and Notch activity were markedly higher in ectopic endometria than in their eutopic and normal counterparts. Primary stromal cells obtained from normal endometria cultured with oestrogen presented significant increases in the expression of Notch components and Notch activity, the cytoplasmic and nuclear accumulation of NOTCH1 intracellular domain, the expression of matrix metallopeptidase 9 and vascular endothelial growth factor and cell invasiveness. Knockdown of NOTCH1 markedly alleviated oestrogen-induced matrix metallopeptidase 9 and vascular endothelial growth factor expression and cell invasion. ICI (an oestrogen receptor α antagonist) also blocked these oestrogenic effects. Oestrogen-responsive elements were found in the promoters of NOTCH1 and JAG1. A luciferase reporter analysis revealed that oestrogen regulated the expression of Notch components via oestrogen receptor alpha, which is bound to oestrogen-responsive elements in the JAG1 and NOTCH1 promoters. Collectively, our findings indicate that oestrogen engages in crosstalk with Notch signalling to regulate cell invasion in endometriosis via the activation of oestrogen receptor alpha and the enhancement of Notch activity. Notch signalling blockade may therefore be a novel therapeutic target for endometriosis.
Na Li, Ling Zhang, Qi Li, Yu Du, Hengwei Liu, Yi Liu, and Wenqian Xiong
Yu Du, Zhibing Zhang, Wenqian Xiong, Na Li, Hengwei Liu, Haitang He, Qi Li, Yi Liu, and Ling Zhang
Endometriosis is an estrogen-dependent benign gynecological disease that shares some common features of malignancy. Epithelial–mesenchymal transition (EMT) has been recognized as a core mechanism of endometriosis. MALAT1 is widely known as EMT promoter, while miR200 family members (miR200s) are considered as EMT inhibitors. Previous studies have reported that MALAT1 upregulation and miR200s downregulation are observed in endometriosis. MiR200c has been regarded as the strongest member of miR200s to interact with MALAT1. However, whether MALAT1/miR200c regulates EMT remains largely unclear. In this study, the roles of miR200s and MALAT1 in ectopic endometrium were investigated. Additionally, the effects of E2 on EMT and MALAT1/miR200s were examined in both EECs and Ishikawa cells. Notably, E2 could upregulate MALAT1 and downregulate miR200s expression levels and induce EMT in EECs and Ishikawa cells. PHTPP, an ERβ antagonist, could reverse the effect of E2. Overexpression of miR200c and knockdown of MALAT1 significantly inhibited E2-mediated EMT, suggesting that both miR200c and MALAT1 are involved in the E2-induced EMT process in endometriosis. In addition, a reciprocal inhibition was found between miR200s and MALAT1. Therefore, the role of MALAT1/miR200c in EMT is influenced by the presence of estrogen during endometriosis development.
Hengwei Liu, Zhibing Zhang, Wenqian Xiong, Ling Zhang, Yao Xiong, Na Li, Haitang He, Yu Du, and Yi Liu
Endometriosis is a benign gynecological disease that shares some characteristics with malignancy like migration and invasion. It has been reported that both hypoxia-inducible factor-1α (HIF-1α) and autophagy were upregulated in ectopic endometrium of patients with ovarian endometriosis. However, the crosstalk between HIF-1α and autophagy in the pathogenesis of endometriosis remains to be clarified. Accordingly, we investigated whether autophagy was regulated by HIF-1α, as well as whether the effect of HIF-1α on cell migration and invasion is mediated through autophagy upregulation. Here, we found that ectopic endometrium from patients with endometriosis highly expressed HIF-1α and autophagy-related protein LC3. In cultured human endometrial stromal cells (HESCs), autophagy was induced by hypoxia in a time-dependent manner and autophagy activation was dependent on HIF-1α. In addition, migration and invasion ability of HESCs were enhanced by hypoxia treatment, whereas knockdown of HIF-1α attenuated this effect. Furthermore, inhibiting autophagy with specific inhibitors and Beclin1 siRNA attenuated hypoxia triggered migration and invasion of HESCs. Taken together, these results suggest that HIF-1α promotes HESCs invasion and metastasis by upregulating autophagy. Thus, autophagy may be involved in the pathogenesis of endometriosis and inhibition of autophagy might be a novel therapeutic approach to the treatment of endometriosis.
Jun-Zuo Wang, Hong-Shu Sui, De-Qiang Miao, Na Liu, Ping Zhou, Li Ge, and Jing-He Tan
The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 °C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 °C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 °C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 °C cytoplasts. While oocytes reconstructed between 37 °C ooplasts and 37 or 40 °C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 °C ooplasts and 37 °C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 °C compared with oocytes matured at 37 °C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 °C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 °C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 °C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.
Qi Li, Na Li, Hengwei Liu, Yu Du, Haitang He, Ling Zhang, and Yi Liu
Endometriosis (EMs) is an estrogen (E2)-dependent inflammatory disorder. Although EMs is considered a benign disease, it presents with malignant characteristics, such as migration and invasion. An increasing number of studies have shown that aberrantly expressed circular RNAs (circRNAs) play an essential role in disease development and progression. However, the mechanisms by which circRNAs exert their pathological effects in EMs remain unclear. Hsa_circ_0001649, a novel cancer-associated circRNA, has been previously reported to be downregulated in several cancer types and related to cell migration and invasion. In the present study, real-time PCR (qRT-PCR) was carried out to measure hsa_circ_0001649 levels in human tissues, human primary endometrial stromal cells (ESCs) and a human endometrial stromal cell line (ThESCs). Matrix metalloproteinase 9 (MMP9) levels in ESCs and ThESCs were assessed by qRT-PCR and Western blotting, and the migration and invasion capacities of ThESCs were evaluated by transwell assay. As a result, hsa_circ_0001649 expression was significantly decreased in ectopic and eutopic endometrial samples compared with that in normal endometrial samples. E2 decreased hsa_circ_0001649 expression but increased MMP9 expression in ESCs and ThESCs. Furthermore, ThESCs were more invasive under E2 stimulation. However, these effects disappeared when ICI or hsa_circ_0001649 transfection was used. Collectively, our findings reveal that decreased hsa_circ_0001649 expression plays a role in E2-increased MMP9 expression through E2 receptors (ERs), which have critical functions in EMs.
Qiao-Song Zheng, Xiao-Na Wang, Qing Wen, Yan Zhang, Su-Ren Chen, Jun Zhang, Xi-Xia Li, Ri-Na Sha, Zhao-Yuan Hu, Fei Gao, and Yi-Xun Liu
Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1 −/flox; Cre-ER TM testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells' status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.
Rui-Song Ye, Meng Li, Chao-Yun Li, Qi-En Qi, Ting Chen, Xiao Cheng, Song-Bo Wang, Gang Shu, Li-Na Wang, Xiao-Tong Zhu, Qing-Yan Jiang, Qian-Yun Xi, and Yong-Liang Zhang
FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.
Kang Shan, Ma Xiao-Wei, Wang Na, Zhang Xiu-Feng, Wen Deng-Gui, Guo Wei, Zhang Zheng-Mao, and Li Yan
Endometriosis, one of the most frequent diseases in gynecology, is a benign but invasive and metastatic disease. The altered expression of E-cadherin may play an important role in developing endometriosis. In this paper, we discuss the association of three single nucleotide polymorphisms (SNPs) on the E-cadherin gene and risk of endometriosis. We examined the genotype frequency of three polymorphisms in 152 endometriosis patients and 189 control women. There was a significant difference in the frequency of the E-cadherin 3′-UTR C → T genotypes between endometriosis and controls (P = 0.01). The frequency of the C allele in patients (71.1%) was significantly higher than in the controls (63.8%; P = 0.04). When compared with the T/T + T/C genotypes, the C/C genotype had a significantly increased susceptibility to endometriosis, with an adjusted odds ratio of 1.79 (95% confidence interval = 1.17–2.76). No significant difference was found between endometriosis and control women on two polymorphisms (−160 C → A, −347 G → GA) at the gene promoter region of E-cadherin. The −160 C → A and −347 G → GA polymorphisms displayed linkage disequilibrium (D′ = 0.999). The −160 A/−347 GA haplotype was only detected in endometriosis patients (2%). These data show a relation between the E-cadherin 3′-UTR C → T polymorphism, the −160 A/−347 GA haplotype of two promoter polymorphisms and risk of endometriosis, suggesting a potential role in endometriosis development, at least in North Chinese women.
Na Liu, Yan-Guang Wu, Guo-Cheng Lan, Hong-Shu Sui, Li Ge, Jun-Zuo Wang, Yong Liu, Tian-Wu Qiao, and Jing-He Tan
Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus–oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.