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Open access

Song Li, Qi Fan, Yanqiu Xie, Haiyan Lin, Qi Qiu, Yihua Liang, and Qingxue Zhang

In vitro activation of primordial follicles is becoming more essential in assisted reproductive technologies. Vasoactive intestinal peptide (VIP) is one of the members of the neurotrophin family which has demonstrated to have an impact on follicle development in recent years. This study aims to investigate the effect of VIP on the activation of primordial follicles in neonatal rat in an in vitro culture system and to determine the relevant molecular mechanism of their activation. Ovaries of 4-day-old rats were examined for the expression of VIP receptors and were cultured in mediums containing VIP with or without inhibitors of the ERK–mTOR signalling pathway. They were then collected for histological analysis or measurement of the molecular expression of this pathway. The receptors of VIP were found in granular cells and oocytes of primordial and early-growing follicles in neonatal ovary. The ratio of growing follicle increased in the presence VIP at different concentrations, with the highest level of increase being observed in the 10−7 mol/L VIP-treated group. The ratio of PCNA-positive granular cells was also increased, while that of the apoptotic oocytes were decreased, and protein analysis showed increased phosphorylation of ERK1/2, mTOR and RPS6 in the VIP-treated group. However, the effect of VIP on the activation of primordial follicle became insignificant with the addition of MEK inhibitor (U0126) or mTORC1 inhibitor (rapamycin). This study indicated that VIP could activate neonatal rat primordial follicle through the ERK-mTOR signalling pathway, suggesting a strategy for in vitro primordial follicle recruitment.

Free access

Zhuxia Zheng, Hongmei Li, Qinfen Zhang, Lele Yang, and Huayu Qi

Cell lineage determination during early embryogenesis has profound effects on adult animal development. Pre-patterning of embryos, such as that of Drosophila and Caenorhabditis elegans, is driven by asymmetrically localized maternal or zygotic factors, including mRNA species and RNA binding proteins. However, it is not clear how mammalian early embryogenesis is regulated and what the early cell fate determinants are. Here we show that, in mouse, mitochondrial ribosomal RNAs (mtrRNAs) are differentially distributed between 2-cell sister blastomeres. This distribution pattern is not related to the overall quantity or activity of mitochondria which appears equal between 2-cell sister blastomeres. Like in lower species, 16S mtrRNA is found to localize in the cytoplasm outside of mitochondria in mouse 2-cell embryos. Alterations of 16S mtrRNA levels in one of the 2-cell sister blastomere via microinjection of either sense or anti-sense RNAs drive its progeny into different cell lineages in blastocyst. These results indicate that mtrRNAs are differentially distributed among embryonic cells at the beginning of embryogenesis in mouse and they are functionally involved in the regulation of cell lineage allocations in blastocyst, suggesting an underlying molecular mechanism that regulates pre-implantation embryogenesis in mouse.

Free access

Na Li, Ling Zhang, Qi Li, Yu Du, Hengwei Liu, Yi Liu, and Wenqian Xiong

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.

Free access

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.

Free access

Sha Peng, Jing Li, Chenglin Miao, Liwei Jia, Zeng Hu, Ping Zhao, Juxue Li, Ying Zhang, Qi Chen, and Enkui Duan

Dickkopf-1 (Dkk1) is one of the secreted antagonists in the canonical Wnt signaling pathway. It plays important roles in diverse developmental processes. However, the role of Dkk1 in trophoblast cell invasion during placentation remains unclear. In this study, we found that Dkk1 was mainly expressed in maternal decidual tissue but trivially in ectoplacental cones (EPCs) in day 8 post coitum (p.c.) pregnant mouse uterus and that the efficiency of EPC attachment and outgrowth was increased when co-cultured with decidual cells, which secreted Dkk1, and this enhancement was abolished by pretreating decidual cells with Dkk1 blocking antibody before co-culture experiment. This indicates that Dkk1 secreted by decidual cells plays an important role in trophoblast cell invasion. Indeed, when recombinant mouse Dkk1 was added to EPCs in vitro, the efficiency of attachment and outgrowth was increased. Migration of EPCs toward the decidua was retarded when antisense Dkk1 oligonucleotide (ODN) was administered via intrauterine injection in vivo. Furthermore, the active β-catenin nuclear location was lost when we treated cultured EPCs with recombinant mouse Dkk1, and the efficiency of EPCs attachment and outgrowth was obviously increased when we treated cultured EPCs with antisense β-catenin ODN. Taken together, Dkk1 secreted by decidual cells may induce trophoblast cell invasion in the mouse and β-catenin may be involved in such functions of Dkk1.

Free access

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.

Open access

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.

Free access

Da Li, Yue You, Fang-Fang Bi, Tie-Ning Zhang, Jiao Jiao, Tian-Ren Wang, Yi-Ming Zhou, Zi-Qi Shen, Xiu-Xia Wang, and Qing Yang

The importance of autophagy in polycystic ovary syndrome (PCOS)-related metabolic disorders is increasingly being recognized, but few studies have investigated the role of autophagy in PCOS. Here, transmission electron microscopy demonstrated that autophagy was enhanced in the ovarian tissue from both humans and rats with PCOS. Consistent with this, ovarian granulosa cells from PCOS rats showed increases in the autophagy marker protein light chain 3B (LC3B), whereas levels of the autophagy substrate SQSTM1/p62 were decreased. In addition, the ratio of LC3-II/LC3-I was markedly elevated in human PCOS ovarian tissue compared with normal ovarian tissue. Real-time PCR arrays indicated that 7 and 34 autophagy-related genes were down- and up-regulated in human PCOS , Signal-Net, and regression analysis suggested that there are a wide range of interactions among these 41 genes, and a potential network based on EGFR, ERBB2, FOXO1, MAPK1, NFKB1, IGF1, TP53 and MAPK9 may be responsible for autophagy activation in PCOS. Systematic functional analysis of 41 differential autophagy-related genes indicated that these genes are highly involved in specific cellular processes such as response to stress and stimulus, and are linked to four significant pathways, including the insulin, ERBB, mTOR signaling pathways and protein processing in the endoplasmic reticulum. This study provides evidence for a potential role of autophagy disorders in PCOS in which autophagy may be an important molecular event in the pathogenesis of PCOS.

Restricted access

Shengxian Li, Jia Qi, Yongzhen Tao, Qinling Zhu, Rong Huang, Yu Liao, Jiang Yue, Wei Liu, Hanting Zhao, Huiyong Yin, and Yun Sun

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women usually accompanied by lipid metabolic disorders. However, it remains unknown whether arachidonic acid (AA) and its metabolites in follicular fluid (FF) were altered in PCOS patients. This study was intended to measure the levels of AA and its metabolites in the FF of non-obese PCOS patients that underwent in vitro fertilization (IVF) and to explore the possible causes of the alterations. Thirty-nine non-obese women with PCOS and 30 non-obese women without PCOS were enrolled. AA and its metabolites were measured by liquid chromatography-mass spectrometry. The levels of AA metabolites generated via cyclooxygenase-2 (COX-2) pathway and cytochrome P450 epoxygenase pathway but not lipoxygenase (LOX) pathway were significantly higher in the FF of PCOS patients. The metabolites generated via COX-2 pathway were significantly correlated with levels of testosterone and fasting insulin in serum. The in vitro study further demonstrated that insulin but not testosterone could promote the IL-1β and hCG-induced COX-2 expression and prostaglandin E2 (PGE2) secretion in primary human granulosa cells. In conclusion, there was an elevation in AA metabolites in FF of PCOS patients. Insulin played a pivotal role in the increased AA metabolites generated via COX-2, which could be interpreted as another novel molecular pathophysiological mechanism of PCOS.

Restricted access

Hong-Jie Yuan, Zhi-Bin Li, Xin-Yue Zhao, Guang-Yi Sun, Guo-Liang Wang, Ying-Qi Zhao, Min Zhang, and Jing-He Tan

Mechanisms by which female stress and particularly glucocorticoids impair oocyte competence are largely unclear. Although one study demonstrated that glucocorticoids triggered apoptosis in ovarian cells and oocytes by activating the FasL/Fas system, other studies suggested that they might induce apoptosis through activating other signaling pathways as well. In this study, both in vivo and in vitro experiments were conducted to test the hypothesis that glucocorticoids might trigger apoptosis in oocytes and ovarian cells through activating the TNF-α system. The results showed that cortisol injection of female mice (1.) impaired oocyte developmental potential and mitochondrial membrane potential with increased oxidative stress; (2.) induced apoptosis in mural granulosa cells (MGCs) with increased oxidative stress in the ovary; and (3.) activated the TNF-α system in both ovaries and oocytes. Culture with corticosterone induced apoptosis and activated the TNF-α system in MGCs. Knockdown or knockout of TNF-α significantly ameliorated the pro-apoptotic effects of glucocorticoids on oocytes and MGCs. However, culture with corticosterone downregulated TNF-α expression significantly in oviductal epithelial cells. Together, the results demonstrated that glucocorticoids impaired oocyte competence and triggered apoptosis in ovarian cells through activating the TNF-α system and that the effect of glucocorticoids on TNF-α expression might vary between cell types.