Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Low proviral load in the Kumamoto strain of Japanese Brown cattle infected with the bovine leukemia virus.

BACKGROUND: The Kumamoto strain of Japanese Brown (JBRK) cattle is a sub-breed of Wagyu and has a different genetic background than that of Japanese Black (JB) cattle. Bovine leukemia virus (BLV) is the pathogen causing enzootic bovine leukosis (EBL), the predominant type of bovine leukosis (BL). EBL is one of the most common bovine infectious diseases in dairy countries, including Japan. Some host genetic factors, including the bovine leukocyte antigen (BoLA)-DRB3 gene, have been associated with the proviral load (PVL) of BLV and/or onset of EBL. Here, we determined the number of BL cases by analyzing prefectural case records in detail. We measured the PVL of BLV-infected JBRK cattle and compared it with that obtained for other major breeds, JB and Holstein-Friesian (HF) cattle. Finally, the relationship between PVL levels and BoLA-DRB3 haplotypes was investigated in BLV-infected JBRK cattle. RESULTS: We determined the number of BL cases recorded over the past ten years in Kumamoto Prefecture by cattle breed. A limited number of BL cases was observed in JBRK cattle. The proportion of BL cases in the JBRK was lower than that in JB and HF. The PVL was significantly lower in BLV-infected JBRK cattle than that in the JB and HF breeds. Finally, in BLV-infected JBRK cattle, the PVL was not significantly affected by BoLA-DRB3 alleles and haplotypes. BoLA-DRB3 allelic frequency did not differ between BLV-infected JBRK cattle with low PVL and high PVL. CONCLUSIONS: To our knowledge, this is the first report showing that BL occurred less in the JBRK population of Kumamoto Prefecture. After BLV-infection, the PVL was significantly lower in JBRK cattle than that in JB and HF breeds. The genetic factors implicated in maintaining a low PVL have yet to be elucidated, but the BoLA-DRB3 haplotypes are likely not involved.

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.

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.

Epithelial-mesenchymal transition process during embryo implantation.

The epithelial to mesenchymal transition (EMT) in endometrial epithelial and trophectoderm cells is essential for the progression of embryo implantation and its impairment could cause implantation failure. Therefore, EMT should be tightly regulated in both embryonic and endometrial cells during implantation. Studies reported the involvement of numerous factors in EMT regulation, including hormones, growth factors, transcription factors, microRNAs, aquaporins (AQPs), and ion channels. These factors act through different signaling pathways to affect the expression of epithelial and mesenchymal markers as well as the cellular cytoskeleton. Although the mechanisms involved in cancer cell EMT have been well studied, little is known about EMT during embryo implantation. Therefore, we comprehensively reviewed different factors that regulate the EMT, a key event required for the conceptus implantation to the endometrium.Summary sentence: Abnormal epithelial-mesenchymal transition (EMT) process within endometrial epithelial cells (EECs) or trophoblast cells can cause implantation failure. This process is regulated by various factors. Thus, the objective of this review was to summarize the effective factors on the EMT process during implantation.

The vaginal and uterine blood flow changes during the ovsynch program and its impact on the pregnancy rates in Holstein dairy cows.

AIM: OvSynch is a hormonal protocol for synchronization of estrus and use of artificial insemination (AI) at an optimal time without adverse effects on the ovaries or uterus. This study investigated the use of noninvasive color Doppler ultrasound to assess changes in uterine and vaginal blood flow during the Ovsynch program for synchronization of estrus and its relation to the pregnancy rates in Holstein cows. MATERIALS AND METHODS: The experimental cows received an intramuscular dose of 10 µg of a GnRH analogue (G1), followed 7 days later with an intramuscular injection of synthetic prostaglandin F2α (P: PGF2α) analogue (500 µg cloprostenol sodium), and given a 10 µg, injection of the GnRH analogue (G2) i.m. 48 h after the PGF2α treatment, and the cows were bred 14-16 h after. Uterine and vaginal perfusion were investigated by performing transrectal Doppler ultrasonography of both the uterine and vaginal arteries in Holstein cows at different time points during the Ovsynch program to determine: peak systolic velocity (PSV), time-averaged maximum velocity (TAMV), the volume of blood flow (BFV), pulsatility index (PI), resistance index (RI), resistance impedance (S/D) and diameters of uterine (UA) and vaginal (VA) arteries. Steroid hormones were also assayed. Transrectal ultrasonography (TUS) was performed at 32 and 60 days to confirm the pregnancy per artificial insemination (P/AI). RESULTS: The uterine PSV, TAMV, and PV were greater at the time of the cloprostenol sodium and second GnRH injections (p<0.05) than at the time of the first GnRH injection. The vaginal PSV, PV were greater at the time of the cloprostenol sodium than at the time of the first and second GnRH injections (p<0.05). The receiver operating characteristic curve (ROC curve) indicated a high correlation between the uterine and vaginal blood flow and the rate of the pregnancy (p<0.05). The area under the ROC curve was 0.920 and 0.87 (p<0.05) for vaginal and uterine arteries respectively at time of G2. The serum levels of progesterone, estrogen and cortisol were correlated with the P/AI (p<0.05). The P/AI significantly decreased from 43.9 % at 32 d to 35.37 % at 60 d. CONCLUSION: These results indicate that noninvasive Doppler ultrasonography is a valid method to evaluate changes in the characteristics of uterine and vaginal blood flow in cows during the Ovsynch protocol. Furthermore, vaginal and uterine blood flow are two determinant factors for the higher conception rates in Holstein dairy cows.

Recent progress of interferon-tau research and potential direction beyond pregnancy recognition.

Since the discovery of interferon-tau (IFNT) over 30 years ago as the trophectodermal cytokine responsible for the maintenance of the maternal corpus luteum (CL) in ruminants, exhaustive studies have been conducted to identify genes and gene products related to CL maintenance. Recent studies have provided evidence that although CL maintenance, with the up- and down-regulation of IFNT, is important, its regulatory role in the endometrial expression of interferon-stimulated genes (ISGs) is far more important for conditioning the uterine environment for successful conceptus implantation and thereafter. This review initially describes the mammalian implantation process, briefly but focuses on recent findings, as there appears to be a common phenomenon during early to mid-pregnancy among mammalian species.

Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period.

Epithelial-mesenchymal transition (EMT), which is common in cancer metastasis, is also observed during developmental processes such as embryo implantation into the maternal endometrium in humans and rodents. However, this process has not been well characterized in the non-invasive type of implantation that occurs in ruminants. To understand whether EMT occurs in ruminant ungulates, ovine conceptuses (embryo plus extraembryonic membranes) from days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment, day 0 = day of estrus) were evaluated. RNA-seq analysis revealed that the expression of EMT-related transcripts increased on P21. Real-time PCR and western blotting analyses indicated that levels of transcripts and proteins indicative of mesenchyme-related molecules increased on P21, but a minor expression of epithelium-related molecules remained. Immunohistochemical analysis revealed that E-cadherin (CDH1) was localized in the elongated trophectoderm on P15 and P17. On P21, CDH1 was localized to the trophectoderm and on the conceptus cells undergoing differentiation. Vimentin (VIM) was localized in the uterine stroma on P15 and P17, and its expression was observed at the edge of elongating trophoblast on P21. Further, it was found that some bi-nucleated trophoblast cells were present on P17; however, numerous bi- and multi-nucleated trophoblast cells on the uterine epithelium or next to the uterine stroma were found on P21. A minor expression of pregnancy-associated glycoprotein (PAG) transcripts was found on P15 and P17, but a definitive expression of PAGs, transcripts, and proteins was found on P21. Although further investigation is required, these observations indicate that bi-nucleated trophoblast cell formation begins on the day conceptus implantation to the maternal endometrium is initiated, followed by EMT in trophoblast cells. These results suggest that these sequential events are required if pregnancy is to be established in ruminants.

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.

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.

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.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

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.

Promoting Roles of Embryonic Signals in Embryo Implantation and Placentation in Cooperation with Endocrine and Immune Systems.

Embryo implantation in the uterus is an essential process for successful pregnancy in mammals. In general, the endocrine system induces sufficient embryo receptivity in the endometrium, where adhesion-promoting molecules increase and adhesion-inhibitory molecules decrease. Although the precise mechanisms remain unknown, it is widely accepted that maternal-embryo communications, including embryonic signals, improve the receptive ability of the sex steroid hormone-primed endometrium. The embryo may utilize repulsive forces produced by an Eph-ephrin system for its timely attachment to and subsequent invasion through the endometrial epithelial layer. Importantly, the embryonic signals are considered to act on maternal immune cells to induce immune tolerance. They also elicit local inflammation that promotes endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation. Additional clarification of the immune control mechanisms by embryonic signals, such as human chorionic gonadotropin, pre-implantation factor, zona pellucida degradation products, and laeverin, will aid in the further development of immunotherapy to minimize implantation failure in the future.

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.

Potential roles of metalloproteinases of endometrium-derived exosomes in embryo-maternal crosstalk during implantation.

During embryo implantation, crosstalk between the endometrial epithelium and the blastocyst, especially the trophoblasts, is a prerequisite for successful implantation. During this crosstalk, various molecular and functional changes occur to promote synchrony between the embryo and the endometrium as well as the uterine cavity microenvironment. In the past few years, growing evidence has shown that endometrium-derived exosomes play pivotal roles in the embryonic-maternal crosstalk during implantation, although the exact mechanism of this crosstalk has yet to be determined. The presence of metalloproteinases has been reported in endometrium-derived exosomes, implying the importance of these enzymes in exosome-based crosstalk. Thus, in this review, we describe the potential roles of the metalloproteinases of endometrium-derived exosomes in promoting embryo attachment and implantation. This study could provide a better understanding of the potential roles of exosomal metalloproteinases in embryo implantation and pave the way for developing novel exosome-based regulatory agents to support early pregnancy.

Regulation of human trophoblast cell syncytialization by transcription factors STAT5B and NR4A3.

In human trophoblast cells, cyclic AMP or its inducer forskolin (FSK) activates two downstream signaling molecules, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), both of which induce syncytialization, cell fusion, and the production of human chorionic gonadotropin (hCG) and progesterone. However, a transcription factor other than GCM1 and molecular mechanisms associated with these events have not been well characterized. To identify novel transcription factors involved in syncytialization of cAMP-stimulated human choriocarcinoma BeWo cells, the microarray analysis was performed with RNAs extracted from PKA- or EPAC-selective cAMP analog-stimulated BeWo cells, from which two up-regulated transcription factors, STAT5 and NR4A3, were found. The knockdown of STAT5B decreased FSK-induced cell fusion and the expression of syncytialization markers, CGB, syncytin1, syncytin2, GCM1, and OVOL1, but NR4A3 knockdown increased FSK-induced cell fusion and the expression of CGB and syncytin2. These findings indicated that cAMP-PKA up-regulated STAT5B, followed by increase in syncytin2 expression through GCM1 and OVOL1, resulting in cell fusion and hCG production, while cAMP-PKA-up-regulated NR4A3 could decrease syncytin2 expression, and suggested that both positive and negative effects of STAT5B and NR4A3, respectively, are required to control the degree of syncytialization in human trophoblast cells.

Intrauterine exosomes are required for bovine conceptus implantation.

Exosomes, extracellular vesicles, are present in uterine flushing fluids (UFs), which are involved in conceptus-endometrial interactions during peri-implantation periods. Despite several studies on intrauterine exosomes conducted, the roles conceptus and endometrial exosomes play during peri-implantation periods have not been well characterized. To investigate the effect of bovine intrauterine exosomes on conceptus implantation, exosomes isolated from bovine UFs during peri-implantation periods were subjected to global protein analysis. The analysis detected 596 exosomal proteins, including ruminants' pregnancy recognition factor IFNT, and 172 differentially expressed proteins with more than 1.5-fold changes in UFs on days 17, 20 and 22 pregnancy (day of conceptus implantation is initiated on days 19-19.5). Treatment of primary bovine endometrial epithelial cells with exosomes from day 17 UFs up-regulated the expression of apoptosis-related genes, and treatment with exosomes from day 20 and 22 UFs up-regulated the expression of adhesion molecule. Based on these findings, intrauterine exosomes should be considered as an essential constituent for successful implantation.