Involvement of ferroptosis in eribulin-induced cytotoxicity in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a unique clinicopathological subtype of epithelial ovarian cancer that is resistant to standard chemotherapy. Eribulin, a microtubule dynamics inhibitor of halichondrin class, has unique effects in the cancer microenvironment such as induction of epithelization and reduction in metastatic potential in breast cancer cells; however, nothing is known about the effect of eribulin and the detailed mechanisms in OCCC. This study aimed to investigate the involvement of ferroptosis and its mechanism in the antitumor activity of eribulin in OCCC cells and a mouse xenograft model. We found that eribulin-induced cell death was reduced by ferroptosis inhibitors; deferoxamine, an iron chelator and ferrostatin-1, a lipid peroxidation inhibitor. Eribulin increased the levels of intracellular iron, reactive oxygen species (ROS), and lipid peroxides, and increased the mitochondrial membrane potential. Eribulin downregulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), and superoxide dismutase (SOD) activity. The combination of eribulin and ML210, a glutathione peroxidase 4-inhibiting ferroptosis inducer, had a synergistic effect on ferroptosis. Taken together, our findings show firstly that eribulin triggers ferroptosis in OCCC and this effect occurs via the suppression of the Nrf2-HO-1 signaling pathway, SOD activity and the promotion of lipid peroxidation. These findings suggest that eribulin-induced ferroptosis is associated with its anti-tumor effect and also could be a potential therapeutic target in OCCC.

Quercetin stimulates trophoblast fusion via the mitochondrial function.

The fusion of mononuclear trophoblasts into multinucleate syncytiotrophoblasts is the critical event in the process of syncytialization, and its dysregulation can lead to pregnancy complications, notably hypertensive disorders of pregnancy (HDP). Oxidative stress may disrupt trophoblast syncytialization in HDP. Specifically, placentas with HDP exhibit impaired mitochondria, giving rise to the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Quercetin, a bioflavonoid known for its antioxidant and anti-aging properties, has the potential to mitigate oxidative stress during trophoblast syncytialization. However, the precise mechanism underlying the action of quercetin in these processes remains to be elucidated. To explore the impact of quercetin on syncytialization, mitochondrial function, and ROS generation, cyclic AMP-stimulated BeWo cells were treated with quercetin. The expression of markers associated with cell fusion, mitochondrial function, and oxidative stress was determined using qPCR and western blotting. Additionally, morphological syncytialization and mitophagy (mitochondrial degradation) were assessed by immunofluorescence analysis. Our results revealed that quercetin increased the expression of syncytialization markers and promoted cell fusion. Furthermore, this compound also upregulated markers associated with mitophagy and mitochondrial fusion, which are corroborated by visual evidence of mitophagy through the fluorescence microscope. Cell fusion naturally stimulated ROS generation, which was attenuated by quercetin. Quercetin downregulated the expression of NRF2 and HO-1 during syncytialization, while increasing the expression of sirtuin1/3/6, which are known to play essential roles in antioxidant responses. In conclusion, quercetin effectively regulates mitochondrial function through its antioxidant properties and the suppression of ROS generation, ultimately promoting trophoblast fusion, suggesting that the flavonoid has the potential to ameliorate pregnancy-related disorder stemming from placental dysplasia.

Downregulation of PGRMC1 accelerates differentiation and fusion of a human trophoblast cell line.

Mononuclear cytotrophoblasts (CTs) differentiate and fuse to form multinuclear syncytiotrophoblasts (STs), which produce human chorionic gonadotropin (hCG) and progesterone to maintain pregnancy. Impaired differentiation and fusion of CTs to form STs are associated with hypertensive disorders of pregnancy and fetal growth restriction. Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional single transmembrane heme-binding protein. We previously demonstrated that downregulation of PGRMC1 promotes endometrial stromal cell differentiation (decidualization). Here, we explored the role of PGRMC1 in trophoblast differentiation and fusion. PGRMC1 expression was lower in STs than in CTs of first-trimester placental tissues. PGRMC1 expression in BeWo cells (a trophoblast-derived choriocarcinoma cell line) decreased upon dibutyryl-cAMP (db-cAMP)-induced differentiation. Both inhibition and knockdown of PGRMC1 stimulated hCG production in the presence of db-cAMP. Furthermore, a quantitative cell fusion assay we developed revealed that inhibition and knockdown of PGRMC1 enhanced db-cAMP-stimulated cell fusion. Peroxisome proliferator-activated receptor γ (PPARγ) agonists decreased PGRMC1 expression and stimulated the cell fusion in BeWo cells. These findings suggest that downregulation of PGRMC1 expression in part through activation of PPARγ during trophoblast differentiation promotes hCG production and cell fusion for formation and maintenance of placental villi during pregnancy.

Progressive Exaptation of Endogenous Retroviruses in Placental Evolution in Cattle.

Viviparity is made possible by the placenta, a structure acquired relatively recently in the evolutionary history of eutherian mammals. Compared to oviparity, it increases the survival rate of the fetus, owing to the eutherian placenta. Questions such as "How was the placenta acquired?" and "Why is there diversity in placental morphology among mammalian species?" remain largely unsolved. Our present understanding of the molecules regulating placental development remains unclear, owing in no small part to the persistent obscurity surrounding the molecular mechanisms underlying placental acquisition. Numerous genes associated with the development of eutherian placental morphology likely evolved to function at the fetal-maternal interface in conjunction with those participating in embryogenesis. Therefore, identifying these genes, how they were acquired, and how they came to be expressed specifically at the fetal-maternal interface will shed light on some crucial molecular mechanisms underlying placental evolution. Exhaustive studies support the hypothesis that endogenous retroviruses (ERVs) could be evolutional driving forces for trophoblast cell fusion and placental structure in mammalian placentas including those of the bovine species. This review focuses on bovine ERVs (BERVs) and their expression and function in the placenta.

Regulatory action of PGRMC1 on cyclic AMP-mediated COX2 expression in human endometrial cells.

Human endometrial stromal cells (ESCs) undergo differentiation, known as decidualization, and endometrial epithelial cells mature around the embryo implantation stage. In the uterus, cyclooxygenase 2 (COX2), the rate-limiting enzyme that produces prostaglandin E2, is expressed in endometrial stromal and epithelial cells, and promotes decidualization of the former cells. Our recent study demonstrated that progesterone receptor membrane component 1 (PGRMC1) is downregulated during decidualization and may be involved in cellular senescence associated with decidualization via the transcription factor forkhead box protein O1 (FOXO1). Therefore, we investigated the role of PGRMC1 in COX2 expression during differentiation and maturation of endometrial stromal and epithelial cells. Inhibition or knockdown of PGRMC1 significantly enhanced differentiation stimuli-induced COX2 expression in both cell types. However, this COX2 expression was suppressed by FOXO1 knockdown or nuclear factor-kappa B (NF-κB) inhibition. Silencing of COX2 expression inhibited PGRMC1 knockdown-induced expression of decidual markers in ESCs. Thus, PGRMC1 may be linked to FOXO1- and NF-κB-mediated COX2 expression in endometrial cells. Taken together, our data suggest that downregulation of PGRMC1 expression facilitates differentiation of endometrial cells, i.e., decidualization and glandular maturation, via upregulation of COX2 expression.

Small GTP-binding protein Rap1 mediates EGF and HB-EGF signaling and modulates EGF receptor expression in HTR-8/SVneo extravillous trophoblast cells.

Purpose: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion. Methods: Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Results: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression. Conclusion: Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.

Global analyses and potential effects of extracellular vesicles on the establishment of conceptus implantation during the peri-implantation period.

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of "Developmental Biology" and "morphogenesis of an endothelium" compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of "mitochondrial calcium ion homeostasis" and "Viral mRNA Translation" compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with "type I interferon signaling pathway". Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

Expression of NFIL3 and CEBPA regulated by IFNT induced-PGE2 in bovine endometrial stromal cells during the pre-implantation period.

Prostaglandin E2 (PGE2) is considered as a luteoprotective factor, influencing the corpus luteum during the early pregnant period in the bovine species. Cyclic AMP (cAMP) is activated in response to PGE2 and plays a role in many physiological processes. The maternal recognition signal, interferon τ (IFNT), induces PGE2 secretion from the endometrial epithelial cells, the function of which in stroma cells has not been completely understood. In this study, PGE2 was found to activate cAMP in the bovine endometrial stromal cells (STRs). STRs were then treated with forskolin to activate the cAMP signaling, from which RNA extracted was subjected to global expression analysis. Transcripts related to transcription regulatory region nucleic acid binding of molecular function, nucleus of cellular component, and mitotic spindle organization of biological processes were up-regulated in cAMP-activated bovine STRs. An increase in the transcription factors, NFIL3, CEBPA, and HIF1A via the cAMP/PKA/CREB signaling pathway in the bovine STRs was also found by qPCR. Knockdown of NFIL3, CEBPA, or HIF1A blocked forskolin-induced PTGS1/2 and IGFBP1/3 expression. Moreover, NFIL3 and CEBPA were localized in endometrial stroma on pregnant day 17 (day 0 = estrous cycle), but not on cyclic day 17. These observations indicated that uterine PGE2 induced by conceptus IFNT is involved in the early pregnancy-related gene expression in endometrial stromal cells, which could facilitate pregnancy establishment in the bovine.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells.

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ ß-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

Endoplasmic reticulum stress-regulated high temperature requirement A1 (HTRA1) modulates invasion and angiogenesis-related genes in human trophoblasts.

The pathogenesis of hypertensive disorder of pregnancy (HDP), which affects about 10% of pregnant women, is still incompletely understood. Our previous study showed that endoplasmic reticulum (ER) stress influences high-temperature requirement A serine peptidase 1 (HTRA1) expression and trophoblast invasion. However, the involvement of ER stress in the regulation of HTRA subtype expression and pathophysiology of HDP has not been characterized in extravillous trophoblasts (EVTs). To investigate this, HTR8/SVneo EVTs cell line was treated with the ER stress inducers Thapsigargin (Thap) or Tunicamycin (Tuni). Treatment with either Thap or Tuni inhibited trophoblast invasion, reduced HTRA1 and HTRA3 expression, but did not alter HTRA2 or HTRA4 expression. Knockdown of HTRA1 or HTRA3 also inhibited trophoblast invasion. Furthermore, treatment with either ER stress inducer or HTRA1 silencing increased the ratio of soluble fms-like tyrosine kinase-1/placental growth factor (sFLT1/PlGF), which is a marker of HDP. Immunohistochemical analysis revealed that HTRA1 is localized to EVTs and the endometrial decidua in the placenta of patients with HDP. These results suggest that factors that cause ER stress could result in the inhibition of EVTs invasion via HTRA1.

The Impact of Eribulin on Stathmin Dynamics and Paclitaxel Sensitivity in Ovarian Cancer Cells.

Eribulin, an inhibitor of microtubule dynamics, is used for treating breast cancers and sarcomas. The microtubule-destabilizing protein stathmin may modulate the antiproliferative activity of eribulin on breast cancer cells and leiomyosarcoma cells. The antitumor activity of eribulin in ovarian cancers has not been fully explored, so the present study aimed to determine the antitumor efficacy of eribulin and the involvement of stathmin in ovarian cancers. In a xenograft model of ovarian cancer, eribulin treatment reduced the tumor weight, which was accompanied by an increased level of phosphorylated stathmin. Eribulin stimulated the phosphorylation of stathmin in cultured cancer cell lines. The eribulin-induced phosphorylation of stathmin was inhibited by treatment with FTY720, an activator of protein phosphatase 2A (PP2A), and eribulin downregulated the expression of PP2A subunits. Furthermore, stathmin knockdown abrogated the inhibitory effects of eribulin on cell viability. Eribulin enhanced the antiproliferative effects of paclitaxel and concomitantly decreased stathmin expression. These results suggest that eribulin-induced phosphorylation of stathmin, mediated in part by PP2A downregulation, reduces stathmin activity and enhances the antiproliferative effects of paclitaxel in ovarian cancer. Collectively, the results of this study indicate that eribulin may suppress the proliferation of ovarian cancer cells partly by regulating the activity of stathmin.

Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells.

Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. Previously, we found decreased SERPINA1 (alpha-1 antitrypsin) expression in endometriosis-like lesions in a mouse model of endometriosis, suggesting that it exacerbated inflammation in these lesions. However, the molecular mechanism(s) by which SERPINA1 affects expression of inflammatory factors and development of endometriotic lesions have not been fully characterized. To investigate the role of intracellular SERPINA1 in endometrial stromal cells (ESCs), we performed RNA sequence analysis using RNA extracted from ESCs in which SERPINA1 was knocked down. The analysis identified several toll-like receptor (TLR)-related factors as being upregulated. Silencing of SERPINA1 increased expression of TLR3 and TLR4 in ESCs, as well as several TLR signaling pathway components, including MYD88, IRAK1/4, interleukin (IL)-1ß, and interferon (IFN)-ß. TLR3 or TLR4 agonists increased expression of inflammatory factors in SERPINA1-knockdown ESCs, whereas TLR3 or TLR4 inhibitors decreased expression. In addition, treatment with recombinant IL-1ß or IFN-ß increased expression of MYD88 and inflammatory factors in ESCs. Immunohistochemical analysis of endometriotic tissues showed that TLR3, TLR4, and MYD88 were localized in endometriosis lesions. Taken together, the data suggest that reduced expression of SERPINA1 induces expression of inflammatory factors by ESCs, which in turn are associated with TLR3/4, IL-1ß, and IFN-ß signaling. Regulation of intracellular SERPINA1 levels in ESCs may be a strategy to inhibit inflammatory responses in endometriotic lesions.

PGRMC1 Regulates Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal Cells.

The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated ß-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase.

Origination of LTR Retroelement-Derived NYNRIN Coincides with Therian Placental Emergence.

The emergence of the placenta is a revolutionary event in the evolution of therian mammals, to which some LTR retroelement-derived genes, such as PEG10, RTL1, and syncytin, are known to contribute. However, therian genomes contain many more LTR retroelement-derived genes that may also have contributed to placental evolution. We conducted large-scale evolutionary genomic and transcriptomic analyses to comprehensively search for LTR retroelement-derived genes whose origination coincided with therian placental emergence and that became consistently expressed in therian placentae. We identified NYNRIN as another Ty3/Gypsy LTR retroelement-derived gene likely to contribute to placental emergence in the therian stem lineage. NYNRIN knockdown inhibited the invasion of HTR8/SVneo invasive-type trophoblasts, whereas the knockdown of its nonretroelement-derived homolog KHNYN did not. Functional enrichment analyses suggested that NYNRIN modulates trophoblast invasion by regulating epithelial-mesenchymal transition and extracellular matrix remodeling and that the ubiquitin-proteasome system is responsible for the functional differences between NYNRIN and KHNYN. These findings extend our knowledge of the roles of LTR retroelement-derived genes in the evolution of therian mammals.

Endogenous Retroviruses and Placental Evolution, Development, and Diversity.

The main roles of placentas include physical protection, nutrient and oxygen import, export of gasses and fetal waste products, and endocrinological regulation. In addition to physical protection of the fetus, the placentas must provide immune protection throughout gestation. These basic functions are well-conserved; however, placentas are undoubtedly recent evolving organs with structural and cellular diversities. These differences have been explained for the last two decades through co-opting genes and gene control elements derived from transposable elements, including endogenous retroviruses (ERVs). However, the differences in placental structures have not been explained or characterized. This manuscript addresses the sorting of ERVs and their integration into the mammalian genomes and provides new ways to explain why placental structures have diverged.

Intrauterine infusion of low levels of interferon-tau on day-8 post-estrus stimulates the bovine endometrium to secrete apolipoprotein-A1: A possible implication for early embryo tolerance.

We previously reported that interferon-tau (IFNT), derived from day-7 blastocyst, generates anti-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. However, the real in vivo impact of early embryo-derived IFNT on the uterine proteomic profile is mostly unknown. This study aimed to investigate proteomic changes of uterine flush (UF) when infused with a low physiological level of IFNT without embryo on day-8 post-estrus and its possible impact on the uterine immunological microenvironment. First, a fresh medium was infused into the uterine lumen on day-6, from which UF was obtained 24 h later, and this procedure was repeated on day-7 (control UF). On day-8, this procedure was done with a medium containing recombinant bovine IFNT (100 pg/ml) (IFNT-supplemented UF). Control and IFNT-supplemented UF were tested for immune responses in peripheral blood mononuclear cells (PBMCs). Real-time PCR results revealed that IFNT-supplemented UF downregulated pro-inflammatory cytokines (TNFA, IL1B) and upregulated anti-inflammatory cytokine (TGFB1) and PTGES in PBMCs. Through 2-D PAGE, followed by TOF/TOF mass spectrometer, apolipoprotein-A1 (Apo-A1) protein was identified in the IFNT-supplemented UF, which was confirmed by ELISA analysis. Proteomic analysis revealed again that the in vitro stimulation of BEECs by IFNT upregulated Apo-A1 expression. Further, stimulation of PBMCs with recombinant bovine Apo-A1 downregulated TNFA and NFKB and upregulated TGFB1 and PTGES in PBMCs. Altogether, our results suggest that minute amounts of IFNT alone, normally secreted from bovine blastocyst, stimulate Apo-A1 secretion from the endometrial epithelium in the absence of embryo that initiates an anti-inflammatory environment, which could pave the way for the acceptance of early embryo in the uterus.

Characterization of Serum Metabolome and Proteome Profiles Identifies SNX5 Specific for Pregnancy Failure in Holstein Heifers.

Pregnancy loss predominantly occurs during the first 3-4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers.

Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression.

The serine protease inhibitor alpha1-antitrypsin (A1AT) may possess protective functions of impaired organs in a manner independent of its protease inhibitor activity. A1AT expression has been shown to fluctuate in patients with pregnancy-induced hypertension, which suggests that A1AT may play a role in the syncytialization of villous trophoblasts. A1AT expression was knocked down in primary trophoblasts. RNA was extracted from these cells and subjected to RNA-sequencing analysis to determine the levels of expression of markers of syncytialization and inflammation. In addition, A1AT protein was localized in trophoblastic cells in placental tissues. Knockdown of A1AT upregulated the expression of FOSL1 and markers of syncytialization, as well as cell fusion, whereas overexpression of A1AT had the opposite effects. FOSL1 overexpression stimulated syncytialization, similar to the effects of A1AT knock down. Inhibitors of p38MAPK and JNK reduce the expression of inflammatory factors, whereas a p38MAPK inhibitor suppressed FOSL1 expression. Collectively, these findings indicated A1AT may negatively regulate inflammatory responses by controlling the activation of p38MAPK and JNK, and that p38MAPK mediates trophoblast syncytialization by altering FOSL1 expression. Therefore, a dysfunction in A1AT could be responsible for abnormal placental formation and pregnancy-associated disorders.

Characterization of lncRNA functioning in ovine conceptuses and endometria during the peri-implantation period.

In ruminants, RNA-sequence analyses have revealed many characteristics of transcripts expressed in conceptuses (embryo and extraembryonic membrane) during peri-implantation periods; however, lncRNA profiles are yet characterized. In this study, we aimed to characterize the lncRNA expression profile in conceptuses during peri-implantation periods in sheep. We analyzed the RNA-sequence data of ovine conceptuses and endometria obtained from pregnant animals on days 15, 17, 19 and 21 (day 0 = day of estrus, n = 3 or 4/day). We predicted the protein coding ability of the assembled transcripts to identify the lncRNA candidates. This analysis identified 8808 lncRNAs, 3423 of which were novel lncRNAs. Gene ontology analysis revealed that lncRNA target genes were enriched for biological processes involved in the respiratory electron transport chain (RETC). qPCR analysis demonstrated that the expression levels on transcripts encoding RETC such as mitochondrially encoded cytochrome c oxidase II (MTCO2) and mitochondria DNA copy number in conceptuses were not increased on P21, although western blotting analysis and immunohistochemistry demonstrated that MTCO2 protein in conceptuses was increased on P21. NAD/NADH assay revealed that NADH level in conceptuses was increased on P21. These results indicate that lncRNAs could regulate the RETC through post-transcriptional levels in the conceptuses. Therefore, lncRNA is a potential new regulator in ovine conceptus development during peri-implantation periods.

PGE2 and Thrombin Induce Myofibroblast Transdifferentiation via Activin A and CTGF in Endometrial Stromal Cells.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.