In the present study, we examined the in vivo effects of estradiol (E2) and progesterone on cyclooxygenase (COX) 2, prostaglandin F synthase (PTGFS, also known as PGFS), and membrane-associated prostaglandin E synthase 1 (mPTGES1) expression at both mRNA and protein levels using a nonpregnant ovariectomized (OVX) sheep model. Sixteen ewes were OVX shortly after ovulation. After 40 days, ewes were treated with saline (Cont, n=5), or E2 infused intravenously for 2 days (50 μg/day, n=5) or intravaginal progesterone (P) sponges for 10 days (0.3 g P, n=6). Cervical COX2, PTGFS, and mPTGES1 mRNA and protein were quantified by northern and western blot analyses respectively. In situ hybridization and/or immunocytochemistry were used to localize the cellular distribution of COX2, PTGFS, and mPTGES1 mRNAs and proteins. COX2 mRNA abundance increased significantly in the cervix after E2 treatment (P<0.05). However, progesterone was a more potent stimulator than E2 of COX2 mRNA and protein abundance in the cervix (P<0.01). In contrast, PTGFS and mPTGES1 mRNA and protein concentrations did not change after E2 or progesterone treatment (P>0.05). COX2, PTGFS, and mPTGES1 mRNA and protein were only localized in cervical glandular epithelial cells. This study shows that increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E2 and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E2. The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was confined in the cervical glandular epithelial cells of nonpregnant sheep.
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Qi Zhang, Valta Collins, Kaushik Chakrabarty, James C Rose, and Wen Xuan Wu
Xiaohui Deng, Hua Zheng, Xuan Yu, Hongling Yu, Chengmei Zhang, Lan Chao, Ruichang Li, and Wenjun Liu
The functional longevity of cryopreserved ovarian grafts is one of the most challenging questions regarding ovarian transplantation at present. This study used a rat ovarian grafting model to investigate whether ovarian tissues from adult rats, which had been cryopreserved by vitrification and followed by heterotopic transplantation, could establish long-term hormone secretion and follicle development. Fresh and cryopreserved ovarian tissues were autologously transplanted under the kidney capsule. One-third of the animals in each group (sham-operated, fresh autografts, cryopreserved autografts, or castrated) were killed 5, 8, or 10 months after transplantation. Vaginal cytology, serum estradiol (E2), progesterone, and the morphology of the reproductive tract were used to assess ovarian function. Both fresh and cryopreserved ovarian grafts survived well in all the animal models with comparable proportion of follicles at each stage of folliculogenesis at all three time points. The serum E2 and progesterone concentrations in the groups with fresh or cryopreserved grafts remained comparable with those in sham-operated controls at all investigated time points. However, a loss of grafts and primordial follicles following heterotopic transplantation was noted. In conclusion, the heterotopic autotransplantation of vitrified ovarian tissues from adult rat without vascular anastomosis can maintain long-term ovarian function and exert endocrine function in target organs, in spite of the reduction in follicle pool.
Guo-Min Zhang, Ming-Tian Deng, Zhi-Hai Lei, Yong-Jie Wan, Hai-Tao Nie, Zi-Yu Wang, Yi-Xuan Fan, Feng Wang, and Yan-Li Zhang
During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.
Xuan-Tong Liu, Hui-Ting Sun, Zhong-Fang Zhang, Ru-Xia Shi, Li-Bing Liu, Jia-Jun Yu, Wen-Jie Zhou, Chun-Jie Gu, Shao-Liang Yang, Yu-Kai Liu, Hui-Li Yang, Feng-Xuan Xu, and Ming-Qing Li
It has been reported that the impaired cytotoxicity of natural killer (NK) cells and abnormal cytokines that are changed by the interaction between ectopic endometrial cells and immune cells is indispensable for the initiation and development of endometriosis (EMS). However, the mechanism of NK cells dysfunction in EMS remains largely unclear. Here, we found that NK cells in peritoneal fluid from women with EMS highly expressed indoleamine 2,3-dioxygenase (IDO). Furthermore, IDO+NK cells possessed lower NKp46 and NKG2D but higher IL-10 than that of IDO-NK. Co-culture with endometrial stromal cells (nESCs) from healthy control or ectopic ESCs (eESCs) from women with EMS led to a significant increase in the IDO level in NK cells from peripheral blood, particularly eESCs, and an anti-TGF-β neutralizing antibody suppressed these effects in vitro. NK cells co-cultured with ESC more preferentially inhibited the viability of nESCs than eESCs did, and pretreating with 1-methyl-tryptophan (1-MT), an IDO inhibitor, reversed the inhibitory effect of NK cells on eESC viability. These data suggest that ESCs induce IDO+NK cells differentiation partly by TGF-β and that IDO further restricts the cytotoxicity of NK cells in response to eESCs, which provides a potential therapeutic strategy for EMS patients, particularly those with a high number of impaired cytotoxic IDO+NK cells.
Nan Meng, Xinyue Wang, Yan Shi, Yanyan Mao, Qian Yang, Baohui Ju, Qianxi Zhu, Tingting Zhang, Yan Gu, and Xuan Zhang
Decidualization is essential for the successful establishment of pregnancy, and the dysregulated decidualization may lead to early pregnancy loss. It was previously reported by us that miR-3074-5p could promote apoptosis but inhibit invasion of human extravillous trophoblast (EVT) cells in vitro, and the expression level of miR-3074-5p in villus tissues of recurrent miscarriage (RM) patients was significantly increased. The aim of this study was to preliminarily explore the role of miR-3074-5p played in the decidualization of human endometrial stromal cells (ESCs). It was found that the decidual expression level of miR-3074-5p in RM patients was remarkably higher than that in the control group. The overexpression of miR-3074-5p in the immortalized human ESC line, T-HESCs, showed suppressive effects not only on the cell proliferation, as well as the intracellular expression levels of cyclin B1 (CCNB1), CCND1 and CCNE1 but also on the in vitro-induced decidualization. CLN8 mRNA, encoding an endoplasmic reticulum (ER)-associated membrane protein, was validated to be directly targeted by miR-3074-5p. And, the expression level of CLN8 was continuously increased along with the decidualization process, whereas down-regulated CLN8 expression could inhibit the decidualization of T-HESCs in vitro. Furthermore, contrary to the increased expression level of miR-3074-5p, a significantly decreased CLN8 expression was observed in decidual tissues of RM patients. Collectively, these data suggested that an increased miR-3074-5p expression in ESCs might cause early pregnancy failure by disturbing decidualization of ESCs via the miR-3074-5p/CLN8 pathway, providing a potential diagnostic and therapeutic target for RM.
Wen-Wen Gu, Long Yang, Xing-Xing Zhen, Yan Gu, Hua Xu, Miao Liu, Qian Yang, Xuan Zhang, and Jian Wang
The invasion of maternal decidua by extravillous trophoblast (EVT) is essential for the establishment and maintenance of pregnancy, and abnormal trophoblast invasion could lead to placenta-associated pathologies including early pregnancy loss and preeclampsia. SEC5, a component of the exocyst complex, plays important roles in cell survival and migration, but its role in early pregnancy has not been reported. Thus, the present study was performed to explore the functions of SEC5 in trophoblast cells. The results showed that SEC5 expression in human placental villi at first trimester was significantly higher than it was at the third trimester, and it was abundantly localized in the cytotrophoblast (CTB) and the trophoblastic column. SEC5 knockdown was accompanied by reduced migration and invasion in HTR-8/SVneo cells. In addition, the expression and plasma membrane distribution of integrin β1 was also decreased. Furthermore, shRNA-mediated knockdown of SEC5 inhibited the outgrowth of first trimester placental explants. SEC5 and InsP3R were colocalized in the cytoplasm of HTR-8/SVneo cells, and the cell-permeant calcium chelator BAPTA-AM could significantly inhibit HTR-8/SVneo cell invasion. The Ca2+ imaging results showed that the 10% fetal bovine serum-stimulated cytosolic calcium concentration ([Ca2+]c) was not only reduced by downregulated SEC5 but also was blocked by the InsP3R inhibitor. Furthermore, either the [Ca2+]c was buffered by BAPTA-AM or the knockdown of SEC5 disrupted HTR-8/SVneo cell F-actin stress fibers and caused cytoskeleton derangement. Taken together, our results suggest that SEC5 might be involved in regulating trophoblast cell migration and invasion through the integrin/Ca2+ signal pathway to induce cytoskeletal rearrangement.
Xinyi Li, Jiaxin Shi, Weijie Zhao, Xixi Huang, Liyuan Cui, Lu Liu, Xueling Jin, Djin Li, Xuan Zhang, and Meirong Du
Decidual stromal cells (DSCs) modulate the function of trophoblasts through various factors. Wnt signaling pathway is active at the maternal–fetal interface. Here, we isolated endometrial stromal cells (ESCs) from women of reproductive ages and DSCs from normal pregnancy during the first trimester (6–10 weeks). Real-time quantitative PCR and western blotting were used to screen out the most variable WNT ligands between ESCs and DSCs, which turned out to be WNT16. Both culture mediums from DSCs and recombinant protein of human WNT16 enhanced the survival and invasion of HTR8/SVneo trophoblastic cells. Furthermore, the regulation of DSCs on trophoblast was partly blockaded after we knocked down WNT16 in DSCs. Treating HTR8/SVneo trophoblastic cells with small molecular inhibitors and small interfering RNA (siRNA), we found that the activity of AKT/beta-catenin (CTNNB1) correlated with the effect of WNT16. The crosstalk of WNT16/AKT/beta-catenin between DSCs and trophoblasts was determined to be downregulated in unexplained recurrent spontaneous abortion. This study suggests that WNT16 from DSCs promotes HTR8/SVneo trophoblastic cells invasion and survival via AKT/beta-catenin pathway at the maternal–fetal interface in human early pregnancy. The disturbance of this crosstalk between DSCs and trophoblasts might cause pregnancy failure.