Protein disulfide isomerase 3 (PDIA3) is a chaperone protein that modulates the folding of newly synthesized glycoproteins, has isomerase and redox activity, and has been implicated in the pathogenesis of many diseases. However, the role of PDIA3 in pregnancy-associated diseases remains largely unknown. Our present study reveals a key role for PDIA3 in the biology of placental trophoblasts from women with preeclampsia (PE). Immunohistochemistry and Western blot analysis revealed that PDIA3 expression was decreased in villous trophoblasts from women with PE compared to normotensive pregnancies. Further, using a Cell Counting Kit-8 assay, flow cytometry, and 5-ethynyl-2’-deoxyuridine (EdU) staining, we found that siRNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR8/SVneo cell line, while overexpression of PDIA3 reversed these effects. Furthermore, RNA sequencing and Western blot analysis demonstrated that knockdown of PDIA3 inhibited MDM2 protein expression in HTR8 cells, concurrent with marked elevation of p53 and p21 expression. Conversely, overexpression of PDIA3 had the opposite effects. Immunohistochemistry and Western blot further revealed that MDM2 protein expression was downregulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies. Our findings indicate that PDIA3 expression is decreased in the trophoblasts of women with PE, and decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.
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Hui-Qin Mo, Fu-Ju Tian, Xiao-Ling Ma, Yu-Chen Zhang, Cheng-Xi Zhang, Wei-Hong Zeng, Yan Zhang, and Yi Lin
Mian Liu, Xia Chen, Qing-Xian Chang, Rui Hua, Yan-Xing Wei, Li-Ping Huang, Yi-xin Liao, Xiao-Jing Yue, Hao-Yue Hu, Fei Sun, Si-Jia Jiang, Song Quan, and Yan-Hong Yu
Small extracellular vesicles (sEVs) are important mediators of cell-to-cell communication involved in the successful establishment of a pregnancy. Human decidual stromal cells play a key role in regulating trophoblast invasion. Nevertheless, the regulatory functions of decidual stromal cells-derived sEVs in human trophoblast cells are still unclear. In this study, primary human decidual stromal cells were isolated, and immortalized human endometrial stromal cell line (HESCs) were decidualized into human decidual stromal cells (HDSCs) using hormonal cocktail containing medroxy progesterone 17-acetate (MPA), estrogen and cAMP analog. HDSC-sEVs were isolated from both primary human decidual stromal cells and immortal HDSCs, respectively, and identified by transmission electron microscopy and western blotting. EV uptake assay indicated that HDSC-sEVs could be uptaken by trophoblast cells. HDSC-sEVs could increase the invasiveness and the expression level of N-cadherin of trophoblast cells with elevated phosphorylation of SMAD2 and SMAD3 in the cells. Silencing of N-cadherin could block cell invasion induced by HDSC-sEVs, while knockdown of SMAD2 and SMAD3 could inhibit the upregulation of N-cadherin in trophoblast cells. Taken together, our results suggested a regulatory effect of HDSC-sEVs in the invasion of trophoblast cells, and HDSC-sEVs may be important mediators of trophoblasts during embryo implantation and placentation.
Hai-Yan Hou, Xi Wang, Qi Yu, Hong-Yi Li, Shao-Jie Li, Rui-Yi Tang, Zai-Xin Guo, Ya-Qiong Chen, Chun-Xiu Hu, Zhi-Juan Yang, Wen-ke Zhang, and Yan Qin
Decline in successful conception decreases more rapidly after 38 years of age owing to follicular depletion and decreased oocyte quality. However, limited information is available regarding the underlying mechanism and the useful treatment. This study aimed to evaluate the effects of growth hormone supplementation on oocyte maturation in vivo in aged and young mice and to determine its effect on mitochondrial function. The influence of three different doses of recombinant human growth hormone (rhGH) (0.4, 0.8 and 1.6 mg/kg/day) for 8 weeks before ovarian stimulation was analyzed. Superovulated oocytes were released from the oviduct of 12-week-old and 40-week-old female C57BL/6J mice 14–16 h after administration of human chorionic gonadotropin. Ovarian follicle and morphological analysis and oocyte maturation parameters were then evaluated. This study is the first, to our knowledge, to report that medium- and high-dose rhGH significantly increases antral follicles in aged mice but anti-Müllerian hormone (AMH) levels. Furthermore, derived oocytes, MII-stage oocyte rate, ATP levels, mitochondrial membrane potential and frequencies of homogeneous mitochondrial distribution increased. In contrast, in both aged and young mice, the mtDNA copy numbers per oocyte were similar before rhGH administration, and upon saline administration, they did not differ significantly. We conclude that medium-dose rhGH supplementation before standard ovarian stimulation regimens improves oocyte quality in aged mice, probably by enhancing mitochondrial functionality.
Wenbo Yan, Joseph Chen, Anne A Wiley, Bethany D Crean-Harris, Frank F Bartol, and Carol A Bagnell
The porcine female reproductive tract undergoes estrogen receptor (ER) α-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERα, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17β (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERα increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERα and VEGF expression may be important for neonatal reproductive tract development.
Yanfei Yin, Jiajia Ma, Xiaofang Lu, Saina Yan, Qianqian Jiang, Dazhi Wu, Bin Chen, Bo Weng, and MaoLiang Ran
The appropriate growth and functions of Sertoli cells are crucial to testis development and spermatogenesis in mammals. This study reveals a novel mechanism of follicle-stimulating hormone in immature porcine Sertoli cell proliferation.
Follicle-stimulating hormone (FSH) is a major Sertoli cell mitogen that binds to the FSH receptor. Sertoli cells are indispensable for testis development and spermatogenesis. However, the regulatory mechanisms of FSH in immature Sertoli cell proliferation have not been determined, particularly in domestic animals. In the present study, we identified the regulatory mechanisms of FSH during immature porcine Sertoli cell proliferation. Transcriptome analysis revealed 114 differentially expressed genes that were induced by FSH treatment, which contains 68 upregulated and 46 downregulated genes. These differentially expressed genes were enriched in multiple pathways, including the Ras signaling pathway. Knockdown of the CC-chemokine receptor 7 (CCR7) gene, which was upregulated by FSH, inhibited cell cycle progression by arresting cells in the G1 phase and reduced the cell proliferation and ERK1/2 phosphorylation. In addition, Kobe0065 inhibited Ras signaling in a similar manner as CCR7 knockdown. Furthermore, FSH abolished the effects of Ras signaling pathway inhibition and CCR7 knockdown. Collectively, FSH promotes immature porcine Sertoli cell proliferation by activating the CCR7/Ras-ERK signaling axis. Our results provide novel insights into the regulatory mechanism of FSH in porcine testis development and spermatogenesis by deciding the fate of immature porcine Sertoli cells.
Yingying Zhou, Yangying Peng, Qingqing Xia, Dewen Yan, Huiping Zhang, Lingmin Zhang, Ying Chen, Xiumin Zhao, and Jie Li
Indian hedgehog (Ihh) signaling regulates endometrial receptivity and is an indispensable mediator of embryonic implantation. Hedgehog signaling is known to regulate autophagy, and aberrant regulation of autophagy is critically implicated in the pathogenesis of endometriosis and adenomyosis. However, potential dysregulation of Ihh signaling and its role in autophagy modulation in these diseases remain obscure. In this study, we found that components of Ihh signaling were significantly decreased, whereas the autophagy marker protein, LC3BII, was significantly increased in endometrial tissues of women with endometriosis or adenomyosis. Inhibition of Ihh signaling with the small-molecule inhibitor GANT61 or Gli1 silencing in primary endometrial stromal cells increased autophagic activity, as measured by LC3 turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy. Furthermore, we observed that GANT61 treatment significantly attenuated hydrogen peroxide-induced cell death, whereas disruption of autophagy with chloroquine diminished this effect. Collectively, these findings reveal that Ihh signaling is suppressed in endometrial tissues of patients with endometriosis or adenomyosis. This abnormal decrease may contribute to endometrial autophagy activation, which may promote aberrant survival of endometrial cells in ectopic sites in these two gynecological diseases.
Huijuan Liao, Yan Chen, Yulong Li, Shaolong Xue, Mingfeng Liu, Ziyuan Lin, Yanyan Liu, Hsiao Chang Chan, Xiaohu Zhang, and Huaqin Sun
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affect fertility in both sexes. However, the involvement of CFTR in regulating germ cell development remains largely unknown. Here, we used zebrafish model to investigate the role of CFTR in primordial germ cells (PGCs) development. We generated a cftr frameshift mutant zebrafish line using CRISPR/Cas9 technique and investigated the migration of PGCs during early embryo development. Our results showed that loss of Cftr impairs the migration of PGCs from dome stages onward. The migration of PGCs was also perturbed by treatment of CFTRinh-172, a gating-speciﬁc CFTR channel inhibitor. Moreover, defected PGCs migration in cftr mutant embryos can be partially rescued by injection of WT but not other channel-defective mutant cftr mRNAs. Finally, we observed the elevation of cxcr4b, cxcl12a, rgs14a and ca15b, key factors involved in zebrafish PGCs migration, in cftr-mutant zebrafish embryos. Taken together, the present study revealed an important role of CFTR acting as an ion channel in regulating PGCs migration during early embryogenesis. Defect of which may impair germ cell development through elevation of key factors involved in cell motility and response to chemotactic gradient in PGCs.
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.
Yu-chen Zhang, Xiao-li Qin, Xiao-ling Ma, Hui-qin Mo, Shi Qin, Cheng-xi Zhang, Xiao-wei Wei, Xue-qing Liu, Yan Zhang, Fu-ju Tian, and Yi Lin
Preeclampsia is a gestational hypertensive disease; however, preeclampsia remains poorly understood. Bioinformatics analysis was applied to find novel genes involved in the pathogenesis of preeclampsia and identified CLDN1 as one of the most differentially expressed genes when comparing patients with preeclampsia and healthy controls. The results of the qRT-PCR, Western blotting and immunohistochemistry experiments demonstrated that CLDN1 was significantly downregulated in the chorionic villi in samples from patients with preeclampsia. Furthermore, knockdown of CLDN1 in HTR-8/SVneo cells resulted in the inhibition of proliferation and induction of apoptosis, and overexpression of CLDN1 reversed these effects. In addition, RNA-seq assays demonstrated that the gene BIRC3 is potentially downstream of CLDN1 and is involved in the regulation of apoptosis. Knockdown of CLDN1 confirmed that the expression level of BIRC3 was obviously decreased and was associated with a significant increase in cleaved PARP. Interestingly, the apoptotic effect in CLDN1 knockdown cells was rescued after BIRC3 overexpression. Overall, these results indicate that a decrease in CLDN1 inhibits BIRC3 expression and increases cleaved PARP levels thus participating in the pathogenesis of preeclampsia.
Muyun Wei, Ying Gao, Bingru Lu, Yulian Jiao, Xiaowen Liu, Bin Cui, Shengnan Hu, Linying Sun, Shaowei Mao, Jing Dong, Lei Yan, Zijiang Chen, and Yueran Zhao
Defective decidualization of human endometrial stromal cells (ESCs) has recently been highlighted as an underlying cause of implantation failure. FK-506-binding protein 51 (FKBP51) has been shown to participate in the steroid hormone response and the protein kinase B (AKT) regulation process, both of which are important pathways involved in decidualization. The objective of the present study was to investigate the potential effects and mechanisms of FKBP51 in the regulation of ESC decidualization. By performing immunohistochemical staining on an endometrial tissue microarray (TMA) derived from normal females, we found that FKBP51 expression was much higher in the luteal phase than in the follicular phase in ESCs. Primary ESCs were isolated from patients to build an in vitro decidualization model through co-culture with medroxyprogesterone acetate (MPA) and 8-bromoadenosine (cAMP). SC79, a specific AKT activator in various physiological and pathological conditions, and shRNA-FKBP51 were used to examine the roles of AKT and FKBP51 in decidualization. The Western blot and RT-PCR results showed that FKBP51, insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL) expression increased in ESCs treated with MPA + cAMP; meanwhile, the level of p-Ser473 AKT (p-S473 AKT) decreased and forkhead box protein O1 (FOXO1A) expression increased. Decidualization was inhibited by the AKT activator SC79 and the transfection of FKBP51-shRNA by affecting protein synthesis, cell morphology, cell growth and cell cycle. Furthermore, this inhibition was rescued by FKBP51-cDNA transfection. The results supported that FKBP51 promotes decidualization by reducing the Ser473 phosphorylation levels in AKT.