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.

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.

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.

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.

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.

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.

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.

The effect of bta-miR-26b in intrauterine extracellular vesicles on maternal immune system during the implantation period.

Extracellular vesicles (EVs) in utero play a role in cellular interactions between endometrium-conceptuses (embryo plus extraembryonic membranes) during peri-implantation periods. However, how intrauterine EVs function on endometrium have not been well characterized. In our previous study, bta-miR-98 found in intrauterine EVs from uterine flushing fluids (UFs) on pregnant day 20 (a half day after initial conceptus attachment, P20) could regulate the maternal immune system and collaborate with other miRNAs and/or components of EVs for conceptus implantation. We, therefore, hypothesized that in addition to bta-miR-98, other miRNAs present in bovine intrauterine EVs may regulate the maternal immune system in the endometrial epithelium. A global analysis of differentially expressed proteins between EVs from P17 and P20 UFs revealed that components of intrauterine P20 EVs had the effect on the down-regulation of "neutrophil activation involved in immune response" and "neutrophil mediated immunity". In silico analyses predicted bta-miR-26b as one of potential miRNA to regulate maternal immune system. In our cell culture experiments, bta-miR-26b negatively regulated several immune system-related genes PSMC6, CD40, and IER3 in bovine endometrial epithelial cells. Our findings revealed that intrauterine EV-derived bta-miR-26b contributes to the down-regulation of the maternal immune system, allowing conceptus implantation to the uterine endometrium. Furthermore, our results suggest that intrauterine EVs extracted from P20 UFs could regulate neutrophils, the first line of immunological defense, to modulate endometrial immune and inflammatory responses for implanting conceptuses.

Senolytic treatment modulates decidualization in human endometrial stromal cells.

Decidualization - the differentiation of endometrial stromal cells (ESCs) into decidual cells - is a crucial step for successful embryo implantation and placentation that is initiated in the secretory phase of the menstrual cycle. During decidualization, ESCs undergo proliferation arrest and secrete inflammatory mediators, including senescence-associated secretory phenotype (SASP). Although several senolytic agents improve age-related diseases, their effects on cellular senescence in decidualizing ESCs has not been explored. To do this, we treated decidualized ESCs with the senolytic agents Quercetin (Que), Dasatinib (Das), and BPTES. Que decreased the number of senescence-associated ß-galactosidase (SA-ß-Gal) positive cells and expression of senescence markers in ESCs treated with the decidual stimulus (dibutyryl-cAMP plus progesterone: DP). Concomitantly, Que markedly increased the expression of the decidualization markers IGFBP1, PRL, and FOXO1, in decidualizing ESCs. Similar to Que, Das also stimulated decidualization. Treatment with a combination of Que and Das synergistically increased the expression of decidualization markers and senescence markers compared with treatment with Que or Das alone. However, BPTES did not enhance the expression of decidualization markers. These results imply that treatment with Que and/or Das can remove senescent decidual cells and enhance the decidualization of the rest of ESCs. Thus, senolytic modulation of abnormal ESC decidualization could alleviate infertility caused by dysfunctions of endometrial receptivity and embryo implantation.

Neutrophils recognize and amplify IFNT signals derived from day 7 bovine embryo for stimulation of ISGs expression in vitro: A possible implication for the early maternal recognition of pregnancy.

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

Heat stress induces oxidative stress and activates the KEAP1-NFE2L2-ARE pathway in bovine endometrial epithelial cells†.

Heat stress adversely affects the reproductive function in cows. Although a relationship between heat stress and oxidative stress has been suggested, it has not been sufficiently verified in bovine endometrial epithelial cells. Here, we investigated whether oxidative stress is induced by heat stress in bovine endometrial epithelial cells under high temperature. Luciferase reporter assays showed that the reporter activity of heat shock element and antioxidant responsive element was increased in endometrial epithelial cells cultured under high temperature compared to that in cells cultured under basal (thermoneutral) temperature. Also, nuclear factor, erythroid 2 like 2 (NFE2L2), a master regulator of cellular environmental stress response, stabilized and the expression levels of antioxidant enzyme genes increased under high temperature. Immunostaining confirmed the nuclear localization of NFE2L2 in endometrial epithelial cells cultured under high temperature. Quantitative polymerase chain reaction analysis showed that the expression levels of representative inflammatory cytokine genes, such as prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin 8, were significantly decreased in endometrial epithelial cells cultured under high temperature compared to those in cells cultured under basal temperature. Thus, our results suggest that heat stress induces oxidative stress, whereas NFE2L2 plays a protective role in bovine endometrial epithelial cells cultured under heat stress conditions.

Formation of fibrin at sights of conceptus adhesion in the ewe.

In ruminants, various molecules are involved in regulating conceptus attachment and adhesion; however, molecules that maintain the conceptus adhesion have not been well characterized. We hypothesized that conceptus must produce a molecule(s), yet uncharacterized or overlooked, which maintain conceptus adhesion to the uterine epithelium. In this study, we aimed to identify new candidate(s) in conceptus secretory proteins responsible for maintaining conceptus adhesion in sheep. We performed RNA-sequence analysis with ovine conceptuses, followed by endometria obtained from pregnant animals on day 15 (P15: pre-attachment), 17 (P17: right after attachment), and 21 (P21: post-attachment; adhesion) and iTRAQ analysis of uterine flushing on P15 and P17. To identify the proteins secreted from conceptuses, we cross-referenced the transcriptome and proteome data. These analyses identified 16 and 26 proteins as conceptus secretory proteins on P15 and P17, respectively. Gene ontology analysis revealed that the conceptus secretory proteins were enriched in those categorized to fibrinolysis and coagulation. RT-qPCR analysis verified that the expression levels of transcripts in conceptuses encoding coagulation factors, fibrinogen subunits, and fibrinolysis factors were significantly higher on P21 than on P15 or P17, which were supported by those through in situ hybridization, Western blotting and immunohistochemistry. Histology analysis confirmed that fibrin protein was present at the conceptus adhesion region on P21. These results suggest that in addition to the numerous adhesion molecules so far characterized, fibrin is a new candidate molecule for maintaining conceptus adhesion for pregnancy continuation in ruminants.

Alpha-1 Antitrypsin-Induced Endoplasmic Reticulum Stress Promotes Invasion by Extravillous Trophoblasts.

Alpha-1 antitrypsin (A1AT) is a glycoprotein that has been shown to protect tissues from proteolytic damage under various inflammatory conditions. Several studies show that A1AT may be associated with pre-eclampsia. However, the role of A1AT expression in placental physiology is not fully understood. In the present study, we aim to characterize the expression and function of placental A1AT. A1AT knockdown is found to reduce the expression of the serine protease HTRA1 in a trophoblast cell line. In addition, A1AT overexpression (A1AT-OE) increases the expression of HTRA1, IL6, CXCL8, and several markers of endoplasmic reticulum (ER) stress. Treatment with tunicamycin or thapsigargin, which induces ER stress, increases HTRA1 expression. Furthermore, immunohistochemistry reveals that HTRA1 is expressed in trophoblasts and the endometrial decidual cells of human placentas. An invasion assay shows that A1AT and HTRA1 stimulate cell invasion, but treatment with the ER stress inhibitors reduces the expression of HTRA1 and ER stress markers and prevents cell invasion in A1AT-OE trophoblasts. These results suggest that endogenous A1AT regulates inflammatory cytokine expression and HTRA1-induced trophoblast invasion via the induction of ER stress. It is concluded that an imbalance in the functional link between A1AT and ER stress at the maternal-fetal interface might cause abnormal placental development.

Possible Roles of Calreticulin in Uterine Decidualization and Receptivity in Rats and Humans.

Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17ß in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.

IFNT-independent effects of intrauterine extracellular vesicles (EVs) in cattle.

Extracellular vesicles (EVs) present in uterine lumen are involved in conceptus-endometrial interactions during the pre-implantation period. Despite numerous studies conducted on interferon tau (IFNT), a major protein of maternal recognition of pregnancy, the effect of intrauterine EVs on the endometrium during pre-implantation periods has not been well-characterized. To characterize conceptus-derived intrauterine EVs independent of IFNT, transcripts found from RNA-seq analysis in RNAs extracted from primary bovine endometrial epithelial cells (EECs) treated with cyclic day 17 (C17) EVs, pregnant day 17 (P17) EVs or IFNT were analyzed. These analyses identified 82 transcripts uniquely induced by IFNT-independent P17 EVs, of which a large number of transcripts were associated with 'the TNF signaling pathway' and 'Inflammatory response'. Moreover, high expression of CD40L, a member of the TNF superfamily, and its receptor CD40 were found in P17 EVs and in EECs, respectively. Furthermore, the expression of TNF signaling pathway-related genes was up-regulated by the treatment with P17 EVs, but these increases were down-regulated by NF-kB signaling inhibitor. These findings suggest that P17 EVs could induce a pro-inflammatory response in the endometrium, independent of IFNT, to regulate uterine receptivity, facilitating conceptus implantation.

Genetic variation in Japanese Holstein cattle for EBL development.

BACKGROUND: Infection with bovine leukemia virus (BLV), the causative agent for enzootic bovine leukosis (EBL), is increasing in dairy farms of Japan. The tendency of tumor development following BLV infection in certain cow families and bull lines has previously been described. We therefore hypothesized the existence of a genetic component which differentiates cattle susceptibility to the disease. RESULTS: We analyzed routinely collected large-scale data including postmortem inspection data, which were combined with pedigree information and epidemiological data of BLV infection. A total of 6,022 postmortem inspection records of Holstein cattle, raised on 226 farms served by a regional abattoir over 10 years from 2004 to 2015, were analyzed for associations between sire information and EBL development. We then identified statistically the relative susceptibility to EBL development for the progeny of specific sires and paternal grandsires (PGSs). The heritability of EBL development was calculated as 0.19. Similarly, proviral loads (PVLs) of progeny from identified sires and PGSs were analyzed, but no significant differences were found. CONCLUSIONS: These observations suggest that because EBL development in our Holstein population is, at least in part, influenced by genetic factors independent of PVL levels, genetic improvement for lower incidence of EBL development in cattle notwithstanding BLV infection is possible.

Emerging Role of Extracellular Vesicles in Embryo-Maternal Communication throughout Implantation Processes.

In ruminants, the establishment of proper conceptus-endometrial communication is essential for conceptus implantation and subsequent successful placentation. Accumulated evidence supports the idea that extracellular vesicles (EVs) present in uterine lumen are involved in conceptus-endometrial interactions during the preimplantation period. EVs make up a new field of intercellular communicators, which transport a variety of bioactive molecules, including soluble and membrane-bound proteins, lipids, DNA, and RNAs. EVs thus regulate gene expression and elicit biological effects including increased cell proliferation, migration, and adhesion in recipient cells. Uterine EVs are interactive and coordinate with ovarian progesterone (P4), trophectoderm-derived interferon tau (IFNT) and/or prostaglandins (PGs) in the physiological or pathological microenvironment. In this review, we will focus on intrauterine EVs in embryo-maternal interactions during the early stage of pregnancy, especially the implantation period in ruminant ungulates.

Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.

Embryo implantation into the uterine endometrium is required for pregnancy establishment in most mammals. By using global expression analysis, we investigated the molecules that are related to epithelial-mesenchymal transition (EMT) in noninvasive bovine trophoblasts and found that the transcription factor, ovo-like zinc finger 2 ( OVOL2), which is essential for mesenchymal-epithelial transition in various cancers, was down-regulated after trophoblast attachment to the endometrial epithelium in utero. In cultured bovine trophoblast cells, OVOL2 down-regulation occurred only when cells were allowed to attach to bovine endometrial epithelial cells via the TEAD3/YAP signaling pathway. This resulted in the up-regulation of the EMT-associated transcription factors, ZEB1 and SNAI2, and the mesenchymal cell markers, N-cadherin ( CDH2) and vimentin ( VIM), whereas epithelial cell marker, E-cadherin ( CDH1), was down-regulated. In contrast, OVOL2 overexpression in bovine trophoblast cells exhibited a decrease in ZEB1 transcripts and an increase in E-cadherin. These observations revealed that ovo-like protein (OVOL)2 down-regulation occurred concurrently with conceptus implantation into the uterine endometrium via the YAP/TEAD3 signaling pathway, and suggest that the down-regulation of OVOL2 expression contributes to the up-regulation of EMT-related transcription factor expression, which enables EMT progression in the noninvasive bovine trophectoderm postimplantation.-Bai, R., Kusama, K., Nakamura, K., Sakurai, T., Kimura, K., Ideta, A., Aoyagi, Y., Imakawa, K. Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.