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Presence of IFNT in UFs and exosomes from days 15 and 17 pregnant ewes and the effect of those exosomes on EECs. (A) The presence of IFNT in C15, C17, P15, or P17 UFs and exosomes was examined by western blot analysis (n = 3 each day), and IFNT was detected in P15 and P17 UFs. A representative of three independent experiments is shown. (B) Effects of exosomes on EECs. EECs were treated with or without C15, C17, P15, or P17 exosome (10 μg each), or P17 UF (10 μg proteins) for 48 h. RNA was extracted from EECs and subjected to real-time PCR analysis for STAT1, STAT2, BST2, MX1, MX2, and ISG15 transcript levels. ACTB and GAPDH mRNA were used as internal controls for RNA integrity. Values were from three independent experiments, each containing duplicates. Values represent mean ± SEM. *Statistically significant difference in mRNA levels vs. control (Ctrl) without exosomes or UFs treatment (P<0.05). doi:10.1371/journal.pone.0158278.g005
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Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial...
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Context 1
... with the study by Ruiz-González et al. [12], P15 and P17 UFs contained IFNT, and exosomes isolated from P15 and P17 UFs contained IFNT (Fig 5A). To study potential func- tions of these exosomes, UFs or isolated exosomes were added to the cultured EECs, from which RNA was extracted and subjected to qPCR analysis to determine changes in ISGs, The presence of IFNT in C15, C17, P15, or P17 UFs and exosomes was examined by western blot analysis (n = 3 each day), and IFNT was detected in P15 and P17 UFs. ...
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... STAT2, MX1, MX2, BST2, and ISG15 transcripts. The results revealed that ISG tran- scripts in EECs treated with P17 UFs, and P15 or P17 exosomes were up-regulated (Fig 5B). These results indicated that up-regulation of ISGs expression in EECs could result from the IFNT-containing exosomes released from the conceptuses. ...
Citations
... Ovine endometrial epithelial EVs are enriched with endogenous retroviral mRNA, which can stimulate IFN-τ secretion via Tolllike receptors (TLRs) in the trophectoderm. Similarly, bovine embryonic EVs from uterine flushings, rich in IFN-τ, upregulate apoptosis-related genes and adhesion molecules in endometrial epithelial cells, suggesting the involvement of EV-mediated communication in animals with similar placentation patterns [34][35][36][37] . In species like pigs, characterized by epitheliochorial placentation without embryo invasion, EVs play a pivotal role in recruiting natural killer (NK) cells and T-cells to the uterine microenvironment, maintaining a proinflammatory status 38 . ...
Mammalian reproduction relies on precise maternal-fetal communication, wherein immune modifications foster tolerance toward the semi-allogeneic embryo. Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as crucial mediators, transporting molecules like microRNAs securely. EVs influence various reproductive stages, from gamete maturation to implantation, and impact pathologies like pregnancy loss. In the embryo-maternal dialogue, EVs notably affect oviductal interactions, gene expression, and the embryo-endometrial interface, crucial for successful implantation. Key queries persist about EV uptake, cargo delivery, and the specific biomolecules driving communication. Their potential in diagnostics, therapeutics, and understanding environmental impacts on fertility signals an exciting future, reliant on collaborative efforts for transformative strides in reproductive health.
... This result may be due to the fact that lipoxygenase and cyclooxygenase enzymes interfere with the complement cascade, and the regulation of proinflammatory cytokines all contribute to the anti-inflammatory properties of RA (Barile and Vassalli 2017). Exosomes can be effective in adhering the embryo to the uterine wall and helping to secrete progesterone, thereby the activation of interferon tau (IFN-s) during implantation, in addition to the antiinflammatory role of RA in controlling inflammation (Nakamura et al. 2016). Upon RA loading in Figure 5. Progesterone levels of blood serum (ng/mL). ...
Endometritis is an inflammatory and histopathologic disease in uterine tissues that interferes with the proper decidualization and implantation of the embryo. In this study, rosmarinic acid (RA) is used as an anti-inflammatory agent that encapsulates in exosomes and is used to attenuate lipopolysaccharide (LPS)-induced endometritis and improve implantation. For this purpose, exosomes were loaded with RA and then administrated into the animal groups, including RA, exosome, RA plus exosome (RA þ Exo), and RA-loaded exosomes (RALExo) groups. The concentrations of RA or exosomes used in this study were 10mg/kg, and the compounds were injected into the uterine horn 24h following the induction of endometritis. Upon the presence of inflammation detected by the histopathological method, the most proper groups were mated with male mice. The effect of the treatment group on the implantation rate, progesterone levels, and gene expressions were assessed by Chicago Blue staining, enzyme-linked immunosorbent assay (ELISA), and Quantitative PCR (qPCR), respectively. Results showed RALExo10 and RA10 þ Exo10 groups improved pathological alterations, enhanced progesterone levels, increased implantation rate, as well as heightened expression levels of Leukemia inhibitory factor (LIF) and Mucin-16 (MUC-16) genes. Besides, the expression levels of inflammatory cytokines, including Transforming growth factor-b (TGF-b), Interlukine-10 (IL-10), Interlukine-15 (IL-15), and Interlukine-18 (IL-18), were regu- lated. Our findings indicated that the expression of LIF, Muc-16 genes as well as IL-18, were significantly correlated with serum progesterone concentrations and the implantation rate in the treatment groups. The RALExo10 and RA10 þ Exo10 groups showed ameliorated implantation rates in experimental groups.
... The way EVs transmit signals between cells provides a new mechanism for intercellular communication in addition to contactdependent and autocrine, paracrine, or endocrine signals [28]. EVs have been demonstrated to be secreted by the uterus and embryo, playing a crucial functional role in embryo-endometrial dynamic communication [14,15,27,[42][43][44][45][46][47][48][49]. Therefore, it is important to explore the role and mechanism of EVs in endometrial receptivity during embryo implantation. ...
... Furthermore, Nakamura et al. assessed the potential role of EVs containing interferon tau (IFNT) on primary uterine EECs. The EVs secreted by the blastocyst after hatching from the zona pellucida regulate genes and maintain progesterone production for the successful establishment of pregnancy [47]. ...
Endometrial receptivity has been widely understood as the capacity of the endometrium to receive implantable embryos. The establishment of endometrial receptivity involves multiple biological processes including decidualization, tissue remodeling, angiogenesis, immune regulation, and oxidative metabolism. Extracellular vesicles (EVs) are lipid-bilayer-membrane nanosized vesicles mediating cell-to-cell communication. Recently, EVs and their cargo have been proven as functional factors in the establishment of endometrial receptivity. In this review, we comprehensively summarized the alteration of endometrium/embryo-derived EVs during the receptive phase and retrospected the current findings which revealed the pivotal role and potential mechanism of EVs to promote successful implantation. Furthermore, we highlight the potentiality and limitations of EVs being translated into clinical applications such as biomarkers of endometrial receptivity or reproductive therapeutic mediators, and point out the direction for further research.
... IFNT-mediated expression of interferon-stimulated genes (ISGs) occurs in uterine tissues (Ott et al. 1998, Hicks et al. 2003 including the endometrium of the uterine horn ipsilateral to the ovulation side (Sponchiado et al. 2017) as well as other tissues such as the corpus luteum (Yang et al. 2010), liver (Meyerholz et al. 2016) thymus tissues (Zhang et al. 2020b), and mucosal membranes of the cervix (Kunii et al. 2018), vagina (Kunii et al. 2021), and peripheral blood leukocytes (Yankey et al. 2001). Indeed, IFNT was detected in exosomes of the uterine flushing media from pregnant ewes, which stimulated various ISGs in uterine epithelial cells (Nakamura et al. 2016). Measurement of expression of ISGs in peripheral blood leukocytes (PBLs) is a minimally invasive method for detecting early pregnancy (Han et al. 2006, Green et al. 2010). ...
In brief
Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle.
Abstract
IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein ( LAMP) 1, 2 , CTSB and CTSK . Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.
... 31 Similarly, microarray analyses of ovine endometrium revealed the molecular actions of progesterone and identified a list of IFNT-stimulated genes involved in pregnancy recognition. [32][33][34] Using RNA sequencing, transcriptomic profiles of conceptuses and uterine endometrial tissues at the critical period of development have been generated, [35][36][37] and detailed mining of these datasets led to the identification of new components in embryo-uterus communication, such as extracellular vesicle, 38 exosome, 39 and the luminal fluid proteome. 40 Nevertheless, because of the cellular heterogeneity and complexity in this process, cell fate decisions and lineage-specific gene expression must be considered to comprehensively understand the molecular control of conceptus elongation and implantation. ...
Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.
... Moreover, previous studies show that epalrestat, which is an inhibitor of AKR1B1, can be used as an inhibitor of metastasis in different types of tumors (52). Considering that AKR1B1 leads to negative regulation of lysosome trafficking to endosomes and increases the formation of multivesicular bodies (MVBs) in cancer cells (53), the application of drugs such as epalrestat leads to a decrease in metastasis and reduces the amount of exosome production from cancer cells. Therefore, it can be concluded that the production of exosomes by PCCs can be a potential pathway to stimulate metastasis-related mechanisms. ...
Pancreatic cancer (PC) is one of the most dangerous diseases that threaten human life, and investigating the details affecting its progression or regression is particularly important. Exosomes are one of the derivatives produced from different cells, including tumor cells and other cells such as Tregs, M2 macrophages, and MDSCs, and can help tumor growth. These exosomes perform their actions by affecting the cells in the tumor microenvironment, such as pancreatic stellate cells (PSCs) that produce extracellular matrix (ECM) components and immune cells that are responsible for killing tumor cells. It has also been shown that pancreatic cancer cell (PCC)-derived exosomes at different stages carry molecules. Checking the presence of these molecules in the blood and other body fluids can help us in the early stage diagnosis and monitoring of PC. However, immune system cell-derived exosomes (IEXs) and mesenchymal stem cell (MSC)-derived exosomes can contribute to PC treatment. Immune cells produce exosomes as part of the mechanisms involved in the immune surveillance and tumor cell-killing phenomenon. Exosomes can be modified in such a way that their antitumor properties are enhanced. One of these methods is drug loading in exosomes, which can significantly increase the effectiveness of chemotherapy drugs. In general, exosomes form a complex intercellular communication network that plays a role in developing, progressing, diagnosing, monitoring, and treating pancreatic cancer.
... EVs derived from elongated sheep conceptuses are internalized by the uterine luminal and glandular epithelial cells [30]. Additionally, in sheep conceptuses, derived EVs contain IFNT and induce pathways associated with IFNT signaling in endometrial cells [31]. Bridi [32] found that bovine embryos secreted EVs containing miRNAs from Day 7 up to Day 9. ...
The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro-and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.
... A proteomic analysis confirmed that exosomes derived from uterine fluid of fertile women versus infertile women carry known predictors of embryo implantation (PRDX2 and IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU and ANXA2) and implantation success (CAT, YWHAE and PPIA) [88]. At the same time, embryo-derived exosomes are also detected in the uterine fluid of ewes during the implantation stage which traverse through the ZP and then are released into the surrounding culture medium [89]. Exosomes are detected in the cultured fluid of embryonic stem cells and in vitro cultured embryos from various species [80,[90][91][92]. ...
... A subsequent study showed that endometrial-derived exosomes regulate the apoptosis and adhesion of blastula (Transporting FBlN1, CYR61, CD55, HSPG2, miR-30d, PRDX2, IDHC et al.), influence uterine physiology (Transporting ICM, VEGF, JAK-STAT, TLR, S100A4, ANXA2 et al.), promote the proliferation of trophoblast cells and increase the phosphorylation of focal adhesion kinase and the production of fibronexin. At the same time, embryo-derived exosomes interact with integrns and stimulate trophoblast (JNK and FAK transportation) and regulate the immune system of uterus (CTCS, IL6, CASP4, IKBKE and miR-98 transportation)[89]. ...
Exosomes are a subtype of extracellular vesicles (EVs) with a size range between 30 and 150 nm, which can be released by the majority of cell types and circulate in body fluid. They function as a long-distance cell-to-cell communication mechanism that modulates the gene expression profile and fate of target cells. Increasing evidence has indicated exosomes' central role in regulating various complex reproductive processes. However, to our knowledge, a review that focally and vividly describes the role of exosomes in reproductive development is still lacking. This review highlights our knowledge about the contribution of exosomes to early mammalian reproduction, such as gametogenesis, fertilization, early embryonic development, implantation, placentation and pregnancy. The discussion is primarily drawn from literature pertaining to the mammalian lineage with emphasis on the roles of exosomes in human reproduction and laboratory and livestock models.
... Several signaling proteins, such as hormones or enzymes were found to be carried by EVs. In the context of early pregnancy, even proteins as crucial as interferon tau (IFNT), the pregnancy recognition signal in ruminants, were shown to be secreted in EVs by ovine conceptuses during the peri-implantation period (Nakamura et al., 2016). As part of the maternal-embryo dialog, ovine endometrial epithelial cells, in response to IFNT, release EVs containing MX1-a protein, which regulates the secretory activity of uterine glandular epithelial cells during early pregnancy (Racicot et al., 2012). ...
The establishment of cell-to-cell communication between the endometrium and the developing embryo is the most important step in successful mammalian pregnancy. Close interaction between the uterine luminal epithelium and trophoblast cells requires triggering timely molecular dialog for successful maternal recognition of pregnancy, embryo implantation, and placenta development. Quite recently, extracellular vesicles (EVs) carrying unique molecular cargo emerged as evolutionarily conserved mediators of cell-to-cell communication during early pregnancy. To date, the presence of EVs at the embryo-maternal interface has been demonstrated in numerous mammals, including domestic livestock, such as pigs. However, few studies have focused on revealing the mechanism of EV-mediated crosstalk between developing early embryos and receptive endometrium. Over the past years, it has appeared that understanding the role of EVs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes current knowledge of EVs, specifically in relation to the peri-implantation period in pigs, characterized by common features of embryo implantation and high embryonic mortality in mammals.
... During embryo implantation, aldehyde reductase (AKR1B1) and macrophage-capping protein (CAPG) were up-regulated in the embryos, which also existed in EO-EVs. Both AKR1B1 and CAPG have the ability to promote the EMT process (Nakamura et al., 2016). EMT is the process by which an embryo invades the endometrium and is detected by maternal autoimmunity. ...
... MiRNA-34c-5p in mouse EM-EVs can be used to determine the best time for embryo implantation Tan et al. (2020b) Progesterone-treated EO-EVs derived from bovine embryonic trophoblast cells could increase the expression of ITGAV, ITGA3, and WNT7A in endometrial epithelial cells while decreasing the expression of MUC1 Su et al. (2022) EO-EVs from RSA patients can transfer miRNA-196a-5p into macrophages, induce macrophage M1 polarization via the NF-κB pathway, and secrete TNF-α to inhibit trophoblast development EO-EV Zhang et al. (2022) EO-EVs can assess the viability of embryo implantation Capalbo et al. (2016) Trophoblast EVs can promote EMT and migration of endometrial epithelial cells, recruit monocytes in a dose-dependent manner, promote the secretion of IL-1β, G-CSF, TNF-α, and IL-6, and enhance the ability of angiogenesis to promote embryo implantation, which may be the role of miRNA-1290 Shi et al. (2021) CAPG and AKR1B1 are both expressed in bovine EO-EVs and contribute to EMT. Nakamura et al. (2016) EVs produced by trophoblast cells were able to recruit monocytes and promote the secretion of IL-1β, G-CSF, TNF-α, and IL-6 in a dosedependent manner Atay et al. (2011) EVs secreted by embryonic trophoblast cells can recruit mesenchymal stem cells in the endometrium and peripheral blood, potentially weakening the embryo's maternal immune rejection Calle et al. (2021b) SP-EVs can promote decidualization of endometrial stromal cells in vitro, which may be due to the proteins they contain activating the IL-11 signaling pathway SP-EV George et al. (2020) Endometrial epithelial cells were treated with SP-EVs, which increased the expression of genes involved in immunity and inflammation while decreasing the expression of genes involved in steroid biosynthesis, metabolism, and T cell differentiation Bai et al. (2018) SP-EVs can stimulate endometrial epithelial cell production of IL-6 and IL- 8 Paktinat et al. (2019) Endometrial epithelial cells treated with SP-EVs induced macrophages to secrete IL-1α and IL-6, while IL-10 secretion was suppressed Paktinat et al. (2021) In aged mice, the implantation rate of embryos formed by semen and ovum after fertilization is reduced, which may be due to the weakened inhibitory effect of SP-EVs on middle dendritic cells in the uterus Wang et al. (2021b) The number of M1 macrophages increased in RSA patients' decidua. EVs released by M1 macrophages can up-regulate CDH1 expression while inhibiting CDH2 and VIM expression to prevent trophoblast cell EMT, which may be caused by miRNA-146a-5p and miRNA-146b-5p contained in EVs, which down-regulate TRAF6 expression Decidua-EV Ding et al. (2021) EVs produced by decidual stromal cells can increase trophoblast cell invasion, which may be caused by increased expression of SMAD2, SMAD3, and CDH1 Liu et al. (2020b) Frontiers in Cell and Developmental Biology frontiersin.org ...
Extracellular vesicles (EVs) have become a research hotspot in recent years because they act as messengers between cells in the physiological and pathological processes of the human body. It can be produced by the follicle, prostate, embryo, uterus, and oviduct in the reproductive field and exists in the extracellular environment as follicular fluid, semen, uterine cavity fluid, and oviduct fluid. Because extracellular vesicles are more stable at transmitting information, it allows all cells involved in the physiological processes of embryo formation, development, and implantation to communicate with one another. Extracellular vesicles carried miRNAs and proteins as mail, and when the messenger delivers the mail to the recipient cell, the recipient cell undergoes a series of changes. Current research begins with intercepting and decoding the information carried by extracellular vesicles. This information may help us gain a better understanding of the secrets of reproduction, as well as assist reproductive technology as an emerging marker and treatment.
