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Endometriosis is an estrogen-dependent disease that involves the adhesion, invasion, and angiogenesis of endometrial tissues outside of the uterine cavity. We hypothesized that a link exists between estrogen and beta-catenin (β-catenin) signaling in the pathogenesis of endometriosis. Human endometrial stromal cells (HESCs) were separated from eutopic endometrial tissues that were obtained from patients with endometriosis. β-catenin expression and cells invasiveness ability were up-regulated by 17β-estradiol (E₂) in an estrogen receptor (ESR)-dependent manner, whereas β-catenin siRNA abrogated this phenomenon. Moreover, co-immunoprecipitation and dual immunofluorescence studies confirmed ESR1, β-catenin, and lymphoid enhancer factor 1/T cell factor 3 co-localization in the nucleus in HESCs after E₂ treatment. To determine the role of β-catenin signaling in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium from endometriosis patients into ovariectomized severe combined immunodeficiency mice. The implantation of the endometrium was suppressed by β-catenin siRNA. Collectively, studies regarding β-catenin signaling are critical for improving our understanding of the pathogenesis of estrogen-induced endometriosis, which can translate into the development of treatments and therapeutic strategies for endometriosis.

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.

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.

Summary. Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase. Progesterone production was assessed after short-term incubation of luteal cell suspensions. Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0·01 i.u./ml, and the response was 5 times higher than the basal production.

Oestradiol (1–100 μm) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production. The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 μm. The ED50 values were approximately 3 μm, 75 μm and 100 μm for oestradiol, AF-45 and anordrin respectively. Anordrin showed no significant effects on basal progesterone production. In addition, oestradiol markedly inhibited the activity of 3β-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low.

The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.

Keywords: A-nor steroid; oestradiol; luteal cells; progesterone; 3β-hydroxysteroid dehydrogenase; man

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.

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 μM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 × 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.