text:
Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk
Mallikarjun Bidarimath1,
Kasra Khalaj1,2,
Rami T. Kridli2,3,
Frederick W. K. Kan1,
Madhuri Koti1 &
Chandrakant Tayade1,2
Extracellular signalling molecules
Multivesicular bodies
Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.
Exosomes are membrane-bound bioactive nanovesicles (30–100 nm) of multivesicular body origin that can be released from the cell surface by exocytosis1,2. Most cell types secrete exosomes and often reflect aspects of the physiological state and function of the originating cells, including the placenta and endometrium3,4,5. Exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) can reach bodily fluids, such as plasma6, urine7, amniotic fluid8, semen9, milk10, saliva11 as well as uterine luminal fluid in sheep12 and pigs13. In addition, the release and content of the EVs can be influenced by the extracellular micro-environment14. The exosomal lipid bilayer made up of relatively high concentrations of cholesterol, sphingomyelin, ceramide and detergent resistant membrane domains making these vesicles very stable in extracellular space15. In addition, exosomes possess surface receptors/ligands of the original cells and have the potential to selectively interact with specific target cells16. Intracellular pathways can also be regulated by the exosomes which can sequester signaling molecules in the cytoplasm either by reducing their bioavailability or preventing their packaging and release via exocytosis17. Reports to date provide evidence that exosomes contain lipids, proteins, mRNA and numerous small non-coding RNAs (~22 nucleotides) such as miRNAs1,5,18,19. Exosomes can horizontally transfer mRNA and miRNAs to other cells. mRNAs can then be translated into functional proteins in the new location and miRNAs can exert gene silencing in the recipient cells20,21. For instance, exosomes can elicit biological effects, such as increased endothelial cell proliferation and migration at the implantation site22,23, that are important for conceptus development in pigs.
Embryo implantation in pigs is a complex process that requires a synchronized reciprocal dialogue between a receptive endometrium and developing blastocysts24,25. During the early implantation period in pigs (Days 4–15 of pregnancy), the developing conceptus (including embryo, trophectoderm and associated extra-embryonic membranes) undergoes rapid morphologic changes (from spherical to tubular to filamentous forms) and migrates freely through the entire lumen of the uterus24. During days 15–20 of pregnancy, the initiation of porcine placentation is characterized by the expression of a unique repertoire of adhesive molecules on the surface of both the trophectoderm and the uterine luminal epithelium enabling the firm attachment25. This follows the dramatic change in physiological processes including angiogenesis on the endometrial side26. Unlike humans and mice, placental tissues in pigs do not invade the endometrium but, instead, lay in close apposition leading to the establishment of non-invasive epitheliochorial placenta by days 26–30 of pregnancy27,28. Once established, adhered trophoblast-endometrial epithelial bilayer initiates to develop firm contact in order to facilitate nutrient exchange across maternal-fetal interface29,30.
Cellular communication at the maternal-fetal interface during early gestation is crucial and thus determines the fate of pregnancy. Successful placentation allows rapid exchange of biomolecules between the endometrium and the developing conceptus31. The growing conceptus has to communicate with endometrium via angiogenic signals in order to get enough supply of nutrients through developing vasculature. In return, cells of the uterine microenvironment could also send signals to trophectoderm to influence the growth of conceptus25,32. Two decades ago, electron microscopic studies of porcine fetal-maternal interface revealed abundant secretory vesicles that were distributed over the microvilli during the peri-implantation period33. However, it was not clearly known until the discovery of exosomes that they provide an alternate mode of cell-cell communication. It is now well established that exosomes secreted by placental cells can cross the maternal side to influence biological functions of the recipient cells4,22,34. However, there is a dearth of information on how exosomes containing numerous biomolecules including miRNAs can migrate bi-directionally to modulate the pregnancy related processes including angiogenesis in pigs. We hypothesize that EVs secreted by trophectoderm can be internalized by the endothelial cells of the developing vasculature of the endometrium and vice-versa.
We examined porcine endometrium and the chorioallantoic membrane (CAM) isolated at day 20 of pregnancy, as well as porcine trophectoderm cells (PTr2) for EV membrane marker protein, CD63. We then harvested and positively identified EVs released by endometrial and CAM tissues as well as PTr2 and porcine aortic endothelial cells (PAOEC) using CD63 marker and size analysis. Further, we profiled 14 selected miRNAs present in PTr2 and PAOEC cells and their EVs. Mass spectrometry based analysis identified the proteins present in the EVs and bioinformatically studied their implications in signaling pathways relevant to early events in porcine pregnancy. Importantly, we demonstrated the shuttling of PTr2-Exo into PAOECs and vice-versa. Finally, we assessed the effect of PTr2-Exo on endothelial cell proliferation and vice-versa. The present study provides novel insights into the current understanding of embryo-endometrial communication in a unique non-invasive placental type seen in pigs.
Extracellular vesicles are present and secreted by the porcine endometrium as well as chorioallantoic membrane
We first investigated whether porcine endometrium and CAM could release EVs into the extracellular space. As shown in the Fig. 1a–c, transmission electron microscopy revealed the presence of EVs in the extracellular space of endometrium. Similarly, ultrathin sections of CAM (Fig. 1d–f) revealed the presence of EVs lying in close proximity to the cell membrane with a round shaped morphology and presence of EV membrane. Most EVs were in the 36- to 147- nm range.
Figure 1: Extracellular vesicles released by porcine endometrium and chorioallantoic membrane (CAM) were identified by transmission electron microscopy (TEM) on representative ultrathin sections.
(a–f) Endometrial and CAM biopsies were isolated from the conceptus attachment site at gestation day 20. TEM revealed vesicles of size in the range of approximately 50–150 nm, consistent with EVs in both the endometrium and CAM. Endometrial EVs (black arrows) appear to be localized in the extracellular space (a–c) while EVs (black arrows) in CAM are localized in the close proximity of cell membrane (d–f). Data is derived from three independent experiments. Scale bar: 500 nm.
Characterization of extracellular vesicles derived from PTr2 and PAOEC cells
To determine whether PTr2 and PAOEC cells can secrete EVs, we isolated EVs from the cell supernatants and examined these via transmission electron microscopy. The diameter of EVs released by PTr2 (Fig. 2a) was found to be in the range of 26- to 125- nm with an average diameter of 86 ± 21 nm (Fig. 2b) and the diameter of EVs from PAOEC (Fig. 2c) was in the range of 26- to 150- nm with an average diameter of 99 ± 26 nm (Fig. 2d). Because EVs are known to express CD63, a well characterized exosome protein marker35, we performed western blotting for CD63. As shown in Fig. 2e,f, Western blotting detected CD63 in the EV fraction as well as cellular fraction derived from both the PTr2 and PAOEC, respectively (See also Supplementary Fig. S1 for blots). An endoplasmic reticular protein, Calnexin (Negative control) was absent in the EV preparation while it was detected in the cell lysates. Furthermore, we also performed immunolabelling on PTr2 derived EVs. Morphometric analysis of isolated EVs showed that CD63 is expressed on the membrane (Fig. 2g).
Figure 2: Characterization of EVs isolated from culture supernatants of PTr2 and PAOEC cells.
(a) Transmission electron microscopy (TEM) of PTr2 derived EV pellets that are negatively stained with uranyl acetate and lead citrate. (b) Histogram of the number of isolated PTr2 derived EVs diameters. The Y axis shows the relative number of vesicles (%), and the × axis shows the vesicle diameter (nm). The size of EVs was approximately in the range of 26- to 125- nm (diameter [mean ± SD], 86 ± 21 nm). (c) TEM analysis of PAOEC derived EVs. (d) PAOEC derived EVs measured approximately in the range of 26- to 150- nm (diameter [mean ± SD], 99 ± 26 nm). (e) Western blotting detected CD63, exosomal marker, in the EV fraction as well as cellular fraction derived from both the PTr2 and PAOEC (f; cropped blots are displayed), respectively. (See also full-length blots in the Supplementary Figure S1). Calnexin (CANX) was only detected in cell lysates of PTr2 and PAOEC cells. (g) Characterization of EVs isolated from culture supernatant of PTr2 cells using transmission immunoelectron microscopy. Negatively stained EVs are labelled with 12-nm colloidal gold particles that recognize CD63 (black arrows) on the exosomal membrane. Data is derived from three independent experiments. Scale bar = 200 nm.
Porcine endometrium, chorioallantoic membrane and PTr2 cells express CD63, exosome protein marker
We performed CD63 immunoflourescence on formalin-fixed and paraffin-embedded porcine endometrial (Fig. 3a–c) and CAM (Fig. 3d–f) biopsies obtained at day 20 of gestation. CD63 localized in the cytoplasm/cell membrane of CAM, indicating that placenta may secrete EVs. After demonstrating the presence of CD63 expression in vivo, we further investigated whether PTr2 cells express CD63 in vitro using immunocytochemistry. Our results confirm the expression of CD63 protein by the PTr2 cells (Supplementary Fig. S2).
Figure 3: Porcine endometrium and CAM expresses CD63, a well characterized exosome marker.
CD63 immunoflourescence on formalin-fixed, paraffin-embedded porcine endometrial (a–c) and CAM (d–f) biopsies isolated at day 20 of gestation. Nuclei are stained with DAPI (blue; a,d,g), CD63 is stained with Anti-Rabbit CD63, reactive against pig (Red; b,e,h) followed by merge (c,f,i) to demonstrate its localization. *Endometrial stroma; LE: luminal epithelial layer of the uterus; Tr: Trophoblast, Magnification: 400x.
PTr2 and PAOEC derived extracellular vesicles carry miRNA cargo
To investigate the possibility of presence of miRNAs in the EVs released by the PTr2 and PAOEC cells, we tested the presence of 14 select miRNAs that regulate angiogenesis and other physiological processes associated with placental development. PTr2 expressed all 14 miRNAs and miR-16, miR-17-5P, miR-15b, let-7f, and miR-20a were found in greater abundance (Fig. 4a). We also found that PAOEC express all miRNAs. Among these, miR-16, miR-17-5P, let-7f, miR-126-5P, and miR-296-5P were relatively abundant (Fig. 4b). After establishing the presence of miRNAs in the parent cells, we investigated whether EVs released by these cell types contain the same miRNAs in a proportional concentration. PTr2 derived EVs contain all 14 miRNAs; however, only miR-126-5P was relatively abundant compared to all other miRNAs (Fig. 4c). PAOEC derived EVs only contained 10 out of 14 miRNAs, with miR-126-5P being relatively abundant. miR-155-5P, miR-221-5P, let-7f, and miR-181c-1 were either absent or not detectable in our samples (Fig. 4d).
Figure 4: PTr2 and PAOEC derived EVs contain miRNAs.
(a) Real-time PCR confirmed the expression of literature curated 14 miRNAs that are involved in the angiogenesis regulation. In PTr2 cells, miR-16, miR-17-5P, miR-15b, let-7f, and miR-20a were relatively abundant. (b) PAOEC cells also expressed all miRNAs. Among these, miR-16, miR-17-5P, let-7f, miR-126-5P, and miR-296-5P were relatively abundant. (c) PTr2 derived EVs contain all 14 miRNAs; however, only miR-126-5P was relatively abundant compared to all other miRNAs. (d) PAOEC derived EVs only contained 10 out of 14 miRNAs while miR-126-5P being relatively abundant. miR-155-5P, miR-221-5P, let-7f, and miR-181c-1 were either absent or not detectable in the samples. Relative levels of miRNA expression normalized to RNU1A levels and data are presented as mean ± SEM. Data is derived from three independent experiments; n = 5.
Proteomic analysis of PTr2 and PAOEC derived extracellular vesicles
LCMS-MS/MS identified an average of 187 proteins in PTr2 derived EVs and 150 proteins in PAOEC derived EVs (Supplementary Data S1). The list of peptides from both PTr2 and PAOEC were subjected to gene ontology and pathway analysis using PANTHER and Gene ontology algorithms and subsequently classified based on biological process (PTr2: Fig. 5a and PAOEC: Fig. 5b) and molecular function (PTr2: Fig. 5c and PAOEC: Fig. 5d). In the biological process, the most clusters identified in PTr2 EVs were: cellular component organization, cellular process, developmental process, metabolic process and protein localization on cell membrane (Fig. 5a). In PAOEC derived EVs, similar clusters were identified but the enrichment was varied (Fig. 5b). Molecular functions of proteins enriched in PTr2 EVs including binding, catalytic activity, enzymatic activity, receptor and transport activity (Fig. 5c). Proteins present in PAOEC EVs were involved in similar activities as PTr2 EVs but the levels of enrichment were different (Fig. 5d). Finally, PANTHER pathway analysis provided potential pathways that are regulated by the proteins present in EVs (Supplementary Fig. S3). Out of these, the 23 most relevant pathways from PTr2 EV group as well as PAOEC EV group were chosen in order to determine the similarity between the functions of EVs secreted by two different cell types. Out of 10 common pathways, angiogenesis, VEGF signaling, inflammation pathway mediated by chemokines, T and B cell activation, Wnt and integrin signaling were the prominent pathways that are regulated by the proteins that are enriched in both PTr2 and PAOEC EVs.
Figure 5: Analysis of PTr2 and PAOEC derived-EV proteins identified by mass spectrometry using PANTHER software.
Extracellular vesicle proteins isolated from PTr2 and PAOEC cell supernatants were subjected to ontology and pathway analysis using PANTHER and Gene ontology algorithms and subsequently classified based on their (a) Biological Process and (b) Molecular function.
In vitro model of bidirectional trophoblast-endothelial cell communication
To demonstrate the functional miRNA containing EV transfer between trophoblasts and endothelial cells, we employed an in vitro model system using PTr2 and PAOEC. To confirm the possibility of this system serving as an in vitro model of cell-to-cell communication via EVs, we first examined the transfer of PTr2 derived EVs to PAOECs in a time dependent manner (Fig. 6). We treated the PAOECs grown in a 6-well cell culture plate in triplicates with a 20 μg/mL of fluorescently labelled PTr2-derived EVs and allowed it to incubate at 37 °C for 6 hrs (Fig. 6a–d; see also Supplementary Video S1 for detailed 3D view of EV uptake). Time dependent uptake of EVs was determined by incubating the culture dish for 12 hrs. (Fig. 6e–h; see also Supplementary Video S2 for detailed 3D view of EV uptake). PTr2 derived EVs were successfully internalized by the endothelial cells in a time dependent manner. Relative fluorescence emitted by the EVs was used to calculate the concentration of EV uptake (Fig. 6m). Our data suggest that there was a slight increase in the relative fluorescence indicating an increased uptake over a period of time.
Figure 6: In vitro model of trophoblast-endothelial cell communication.
(a–l) In vitro transfer system using PTr2 cells are donors and PAOEC cells as recipients. Extracellular vesicles were isolated from culture supernatants of PTr2 cells. PTr2 derived fluorescently labelled EVs (20 μg/mL) were then added to PAOECs grown in a 6-well cell culture plate and allowed to incubate at 37 °C for 6 hrs (a–d) and 12 hrs (e–h) in a two set of experiments. PTr2 derived EVs were successfully internalized by the endothelial cells in a time dependent manner. Relative fluorescence emitted by the EVs was calculated in order to measure the concentration of EV uptake. There was a slight increase in the relative fluorescence indicating the increased uptake over a period of time (m). Nuclei are stained with DAPI (blue; a,e,i), cytoplasm is stained with CellTrackerTM Green BODIFY® dye (Green; b,f,j), EVs were labelled with CM-Dil (CellTracker, C7000; Red; c,g,k) and followed by merge (d,h,l) to demonstrate their localization in the cells. Control wells received the same preparation (PBS+DMSO+CM-Dil) except EVs in triplicate. Data is derived from three independent experiments.
To test whether our observation with PAOECs occurs in reverse direction, we treated the PTr2 cells with a 20 μg/mL of fluorescently labelled PAOEC-derived EVs and allowed it to incubate at 37 °C for 6 hrs (Fig. 7a–d; see also Supplementary Video S3 for detailed 3D view of EV uptake) and 12 hrs (Fig. 7e–h; see also Supplementary Video S4 for detailed 3D view of EV uptake) as previously described. As expected, PTr2 cells were able to internalize PAOEC derived EVs. However, when we compared the concentration of EV uptake between 6 hrs and 12 hrs, there was a slight decrease in the relative fluorescence indicating the disappearance of EVs in PTr2 cells over a period of time (Fig. 7m). Further we tested whether PTr2 derived EVs can be taken up by PTr2 cells (Supplementary Fig. S4). Similarly, PAOEC derived EVs can be taken up by PAOEC cells using the same conditions (Supplementary Fig. S5). Both the cell types did not take up EVs as much as they were when we used vice-versa conditions.
Figure 7: In vitro model of endothelial-trophoblast cell communication.
(a–l) In vitro uptake of PAOEC derived EVs by PTr2 cells in a time dependent manner. PAOEC derived fluorescently labelled EVs (20 μg/mL) were then added to PTr2 cells grown in a 6-well cell culture plate and allowed to incubate at 37 °C for 6 hrs (a–d) and 12 hrs (e–h) in a two set of experiments. PTr2 cells were able to successfully uptake the PAOEC derived EVs in a time dependent manner. Concentration of EVs uptake was measured by calculating the relative fluorescence emitted by the PAOEC derived EVs. Slight decrease in the relative fluorescence was observed, indicating the disappearance of EVs in PTr2 cells over a period of time (m). Nuclei are stained with DAPI (blue; a,e,i), cytoplasm is stained with CellTrackerTM Green BODIFY® dye (Green; b,f,j), EVs were labelled with CM-Dil (CellTracker, C7000; Red; c,g,k) and followed by merge (d,h,l) to demonstrate their localization in the cells. Control wells received the same preparation (PBS+DMSO+CM-Dil) except EVs in triplicate. Data is derived from three independent experiments.
PTr2 derived extracellular vesicles promote endothelial cell proliferation
WST-1 cell proliferation assays were used as a standard endpoint to assess the proliferative effect of PTr2 derived EVs. We treated PAOECs grown in a 6-well cell culture plate with different concentrations (5, 10 or 20 μg protein/mL) of PTr2 derived EVs and allowed it to incubate at 37 °C for 24 hrs. PTr2 derived EVs significantly increased PAOEC proliferation in a dose-dependent manner (p < 0.05, n = 5; Fig. 8a). PAOECs treated with 20 μg/mL of PTr2 derived EVs had significantly higher proliferation compared to PAOECs that were treated with 5 and 10 μg/mL of EVs (p < 0.05). Similarly, PAOECs treated with 10 μg/mL of PTr2 derived EVs had significantly higher proliferation compared to PAOECs that were treated with 5 μg/mL of EVs (p < 0.05).
Figure 8: Effect of extracellular vesicles on cell proliferation.
(a) PTr2 derived EVs promote endothelial cell proliferation. PTr2 derived EVs in different concentrations (5, 10 or 20 μg protein/mL) were added to PAOECs grown in a 6-well cell culture plate and allowed to incubate at 37 °C for 24 hrs. PTr2 derived EVs significantly increased PAOEC proliferation in a dose-dependent manner (p < 0.05, n = 5). PAOECs treated with 10 μg/mL and 20 μg/mL of PTr2 derived EVs had significantly higher proliferation compared to other treatments. (b) PTr2 cells grown in cell culture plate were treated with PAOEC derived EVs in different concentrations (5, 10 or 20 μg protein/mL) and allowed to incubate at 37 °C for 24 hrs. PAOEC derived EVs had no significant effect on PTr2 cell proliferation. Similarly, (c) PAOEC derived EVs (5, 10 or 20 μg protein/mL) were added to PAOECs grown in a culture plate and allowed to incubate at 37 °C for 24 hrs. PAOEC derived EVs did not have significant effect on PAOEC cell proliferation. Finally, (d) PTr2 derived EVs did not have significant effect on PTr2 cell proliferation after 24 hrs of incubation at 37 °C. *p < 0.05, Data is presented as mean ± SEM and derived from three independent experiments.
We then investigated the effect of PAOEC derived EVs on PTr2 cell proliferation at different concentrations (5, 10 or 20 μg protein/mL). PAOEC derived EVs at different concentrations had no significant effect on the proliferation of PTr2 cells (p > 0.05, n = 5; Fig. 8b). When PAOEC cells were treated with PAOEC derived EVs (Fig. 8c) and PTr2 cells with PTr2 EVs (Fig. 8d) there was considerable variability in the absorbance levels between control PBS group and EV treated groups.
Recognition and maintenance of pregnancy depends on coordinated and precisely regulated cellular and molecular processes resulting in endometrial growth, conceptus development, implantation, and placenta formation that are accompanied by the establishment of unique conceptus-endometrial cross talk24. Recently, EVs have been shown to be vesicles of great potential that can participate in and facilitate embryo-uterine cross talk. Transfer of genetic material and proteins between cells at a distant site via exosomes/microvesicles is a relatively new concept for cell-cell communication36. Therefore, we aimed to characterize and investigate the role of EVs secreted by porcine endometrium and CAM/placenta in vivo as well as PAOEC and PTr2 cells in vitro. The current study sheds new light on the physiological relevance of secretory genomic information by EVs, and hints that EVs to be potentially new mediators of intercellular signaling during embryo-uterine dialogue.
Here, for the first time, we (1) identified that EVs are present in the endometrium and CAM as well as in the cell supernatants of PAOECs and PTr2 cell lines; (2) investigated the validity of these EVs by the presence of known surface marker, CD63 and size analysis; (3) showed that EVs contain miRNAs that can potentially regulate angiogenesis which is an important process in peri-attachment period; (4) we further characterized signaling molecules (proteins) present in the EVs; (5) importantly, demonstrated that EVs derived from PTr2 can be shuttled into PAOEC and vice versa; (6) Finally, examined the effects of EVs derived from PTr2 on PAOEC cell proliferation and vice versa.
A classical approach to any experiment seeking to elucidate the physiological or pathophysiological role of EVs is their specific isolation and characterization. Methods have already been established to accurately characterize EVs in any biological tissues or fluids37. Those methods are primarily based on presence of known surface markers and particle size. According to classification, exosomes are nanovesicles with a diameter of 30–100 nm and express characteristic membrane surface markers such as CD9, CD63 and CD81 along with heat shock proteins HSP70 and HSP90. Many studies involving humans5, mice38, and pigs39, have confirmed the presence of exosomes in various reproductive tissues. Very relevant to our study, we wanted to investigate the presence of EVs in the endometrium and CAM. As a first approach, using TEM, we were able to confirm the presence of EVs in the extracellular space of endometrial tissues and in the close vicinity of chorionic villi. Due to unavailability of pig specific exosome markers such as CD9 and CD81, testing CD63 expression in our tissues was the only feasible option. Nevertheless, both the endometrium (luminal epithelial layer) and the CAM expressed CD63, both intracellularly and along the cell surface. CD63 is primarily expressed on monocytes, macrophages, and activated platelets, and is also weakly expressed on granulocytes, T cell and B cells. However, using immunocytochemistry we confirmed the specific expression of CD63 in the representative porcine placental cell line, PTr2. Due to the focus of our investigation on feto-maternal cross-talk, we decided to use two representative cell lines, PTr2 (representing the fetal side) and PAOEC (representing the endometrial vasculature). We isolated EVs from the cell supernatants of PTr2 and PAOEC using a modified differential centrifugation and miRCURY exosome isolation kits. This method provided us with sufficient EVs to use in all downstream applications. Using TEM, we confirmed the negatively stained EVs. Particle size analysis revealed PTr2 and PAOEC cell derived EV pellet consisting mostly exosomes; however, few traces of microvesicles (100–200 nm) were found. Immunogold labelling of the PTr2 derived EVs specifically identified CD63 molecule on the characteristic exosomal membrane. Finally, the detection of CD63 protein in the cells and their EVs by Western blot analysis supports and confirms our other findings in the present study.
Recent studies involving cell-cell communication via exosomes have mostly focused on the content and their exchange between the distant cells to aid in transfer of signals34. Exosomes are now known to contain lipids, proteins, and numerous RNA species including mRNA and miRNAs. These biomolecules are shown to transfer from the cell of origin to adjacent or distant cells and influence biological functions of the recipient cells. Since Valadi et al. 2007 demonstrated the presence of miRNAs in exosomes, there has been growing interest in the role of exosomal miRNAs in cell-cell communication. We sought to determine whether selected pro- and anti-angiogenic miRNAs involved in modulation of angiogenesis at the maternal-fetal interface are present in the representative cell lines (PTr2 and PAOCs) and in EVs derived from them. As anticipated, pro- and anti-angiogenic miRNAs were expressed in the PAOEC cells but the same set of miRNAs were also present in the PTr2 cells. In addition, EVs released by PTr2 also contained these miRNAs including the relatively abundant pro-angiogenic miR-126-5P. These findings suggest that EVs potentially participate in the regulation of placental angiogenesis. However, a few miRNAs such as miR-155, miR-221, let-7f, miR-181C-1 were not detected in the EVs released by PAOEC and we are unable to speculate about their absence.
Similar to miRNAs, protein trafficking between cells can influence functions of recipient cells. The type and concentration of protein present in the exosome is highly dependent on the cell type and pre-conditioning18. One of the first studies to characterize the exosomal proteome was from mesothelioma cells, in which only 38 proteins of various types were identified. Studies involving isolation of exosomes from a human first trimester trophoblast cell line40 and placental mesenchymal stem cells23 identified numerous proteins that are involved in various cellular and molecular processes including: immune system regulation, reproduction, apoptosis, biological adhesion, cellular binding, receptor and enzymatic activity. Our EV proteome study identified various signaling pathways including angiogenesis and cellular communication that are relevant to early events during porcine pregnancy. Overall, our study provides the first proteomic analysis of EVs derived from PTr2 and PAOEC cells, shedding light on the influence of PTr2 derived EVs on placental angiogenesis and endometrial vasculature.
After investigating the content and potential functions that may be regulated by EVs, we evaluated whether miRNAs and protein loaded EVs can be transferred to recipient cells. In order to test this hypothesis, we established an in vitro model of bidirectional trophoblast-endothelial cell communication. The biological processes in the recipient cells are influenced by exosomes released by the cell of origin and its surrounding conditions. Currently, exosomes are thought to fuse with the plasma membrane of the recipient cell and release their contents into the target cell. Previous studies have proposed a mechanism that there is binding at the cell surface via surface receptors41, or that internalization of exosomes is by endocytosis42. In our study, PTr2 derived EVs were successfully internalized by PAOEC and vice versa. In order to test the specificity of EV uptake, we treated PTr2 cells with PTr2 derived EVs and PAOEC cells with PAOEC derived EVs. The recipient cells did not take up the EVs originated by the same cell supporting the idea that EVs derived from one cell type are more likely taken up by distant cells of different phenotype. However, we did not perform any experiments to determine specificity of EV uptake either by PTR2 or PAOC cells.
Finally, we assessed the effect of EVs on the recipient cells and their functions. We treated the PAOECs with EVs derived from PTr2 and vice versa. PTr2 derived EVs were able to stimulate the PAOEC proliferation in vitro. When we treated PAOEC cells with PAOEC derived EVs and PTr2 cells with PTr2 derived EVs, we found considerable variability in the control PBS groups compared to EV treated groups. This discrepancy could be attributed to the quality of cells as well as potential variability in the numbers of cells used in this experiment. Nevertheless, this experiment did not conclusively establish whether EVs of the same cell origin have any effect on the parent cell. Further experiments are needed to establish the specificity of PTr2 or PAOEC derived EVs on biological functions.
Importantly, these findings support the idea that EVs containing miRNA and proteins can elicit a biological response in the recipient cells leading to increased proliferation which is crucial during peri-implantation period. We were, however, unable to assign specific miRNAs or proteins involved in potentiation of proliferation. Future studies should be conducted by labelling specific miRNAs based on their abundance in exosomes to determine specific function.
In summary, this study supports the notion that EVs are present at the porcine maternal-fetal interface, contain specific miRNAs and several important proteins, and are capable of successfully delivering their cargo in vitro. Additionally, identification of specific miRNAs and proteins have enabled bioinformatic identification of pathways that could be influenced if the EVs are taken up by the endothelial cells or trophectoderm at the time of implantation. Taken together, this study provides critical insight into cell-cell communication at the porcine maternal-fetal interface that aids in successful conceptus-endometrial cross talk.
Endometrial and chorioallantoic membrane tissue collection
This study was approved and conducted in accordance with the recommendation by the Animal Care Committee of the University of Guelph (Animal Utilization Protocol Number 10R061). Endometrial and chorioallantoic membrane (CAM) tissues were collected as previously described43. Briefly, the reproductive tracts were collected immediately after euthanizing sows on gestation day 20 at the University of Guelph abattoir. All conceptuses were exposed for visual examination. Endometrial and CAM tissues were collected and formalin fixed and paraffin embedded for downstream applications. Similarly, tissues were also collected for transmission electron microscopy.
Porcine trophectoderm cells (PTr2) were kindly provided by Dr. Laurie A. Jaeger, College of Veterinary Medicine, Purdue University. PTr2 cells are established from dispersed cell culture of day 12, filamentous conceptus obtained from pigs. These cells have been previously characterized for SN1/38 positive, cytokeratin positive, vimentin negative, express fibronectin and many of the integrin subunits present in porcine trophectoderm in vivo. These also express ER alpha and PR via immunocytochemistry. Detailed isolation methods have been published44. Briefly, PTr2 cells were maintained in special medium containing DMEM-F12 (21041-025), Phenol Red-free, charcoal stripped sFBS (5%; F6765, Sigma-Aldrich, St. Louis, MO, USA), bovine insulin (0.1 Units/mL; 10516; Sigma-Aldrich, St. Louis, MO, USA), glutamine (1X; i.e., 2 mM), Penicillin-streptomycin (1X). Porcine aortic endothelial cells (PAOEC) were purchased from the Cell Applications, Inc., San Diego, CA, USA. PAOEC's were maintained according to manufacturer's instructions using the porcine endothelial cell medium (P211-500) and subcultured using subculture reagent kit (090 K; Cell Applications, Inc., San Diego, CA, USA). Supernatant from both the PTr2 and PAOEC cultures was collected for exosome isolation.
Isolation of extracellular vesicles from culture supernatants
Extracellular vesicles were isolated from the supernatant of both the PTr2 and PAOEC cell lines according to the combination of methods from Thery et al.37 and using miRCURY exosome isolation kit (300102; Exiqon, inc. Woburn, MA, USA). Briefly, culture supernatants were harvested and sequentially centrifuged at 4 °C at 300 × g for 10 min, 2000 × g for 10 min, and 10 000 × g for 30 min. This serial centrifugation step was used to eliminate cells, dead cells, and cell debris. The supernatants were filtered through a 0.22 μm filter and subsequently ultracentrifuged at 100 000 × g for 70 min at 4 °C and the top 2/3 portion of supernatant was discarded. The remaining 1/3rd portion was pooled and 2 mL of precipitation buffer was added to every 10 mL of supernatant and allowed to incubate overnight at 4 °C. The supernatant was centrifuged at 20 °C at 3200 × g for 30 min to pellet EVs. The EV pellet was resuspended in 200 μL of resuspension buffer. The total protein concentration of purified EVs was determined using a Pierce BCA protein assay kit (23225; Life Technologies Inc. Burlington, ON, Canada).
Transmission electron microscopy on endometrial and fetal tissues
To assess the presence of EVs in the endometrium and CAM, standard TEM procedure was used. Briefly, tissues were dehydrated in alcohol, embedded in epoxy resin, ultrasectioned, transferred to 300-mesh Formvar coated nickel grids (Electron Microscopy Sciences) and stained using 4% uranyl acetate and lead citrate according to previously published methods45. The grids were subsequently observed using a JEM-1010 transmission electron microscope (Jeol, Korea) at 100 kV. Images were obtained and processed using Gatan Microscopy Suite (GMS) 3 Software (Gatan, Inc. Pleasanton, CA, USA).
Immunoelectron microscopy analysis of extracellular vesicles
Extracellular vesicles isolated from PTr2 and PAOEC supernatants were mixed with equal quantities of freshly prepared 4% paraformaldehyde for 20 min. Samples were washed using PBS, pelleted by ultracentrifugation and then fixed in 2% glutaraldehyde for 5 min. After fixation, EVs were resuspended in PBS and 5 μL of the sample was transferred to 300-mesh Formvar coated nickel grids. The sample was allowed to settle by incubating at room temperature (RT) for 20 min. Grids were stained using 4% saturated aqueous uranyl acetate and lead citrate for approximately 10 min. The prepared grids were examined under a Hitachi H-7000 transmission electron microscope system (Hitachi High-Technologies) operated at 80 kV. Extracellular vesicles can be quantified using three available standard methods: (1) Transmission Electron microscopy; (2) nanotracking (nanoparticle) analysis using nanosight; (3) ExoELISA kit (System Biosciences Inc), which are specific to quantify exosomes/microvesicles. We chose transmission electron microscopy due to its easy access. Randomly selected regions (at least 6) were analyzed for the presence of EVs, photographed and individual EV size were measured using ImageJ software46,47 by adjusting the scale to TEM image.
Immunogold labelling of CD63 was performed using a rabbit polyclonal IgG (sc-15363; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) reactive against pigs. Extracellular vesicle samples were fixed using 4% paraformaldehyde as previously described and loaded onto 300-mesh Formvar coated nickel grids and allowed to settle for 20 min at RT. Extracellular vesicle bearing grids were blocked with 5% bovine serum albumin in PBS and subsequently incubated with anti-CD63 for 30 min at 37 °C. The grids were then incubated with goat anti-rabbit CD63 immunoglobulin G, pAb conjugated to 12-nm colloidal gold particles (H&L EM grade; Jackson ImmunoResearch laboratories, Inc. West Grove, PA, USA). Immunolabelled EVs were postfixed with 2% glutaraldehyde in PBS for 30 min at RT. The grids were negatively stained with 4% uranyl acetate. Control grids were treated with PBS instead of primary antibody and samples were examined under a Hitachi H-7000 TEM. Extracellular vesicles visualized under microscope were subjected to morphometric analysis. The size and specificity of the CD63 positive EVs were performed on electron micrographs using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Total protein was isolated from PTr2 and PAOEC cells as well as their secreted EVs using Total exosome RNA and protein isolation kit (#4478545; Life Technologies Inc. Burlington, ON, Canada). Briefly, exosomal pellets as well as freshly harvested cells were treated with 100–625 μL of ice-cold resuspension buffer. The cell lysate was vortexed and passed through 26 gauze needle and incubated on ice for 5–10 minutes to ensure complete cell lysis. Micro BCA protein assay kit (#23235; ThermoFisher Scientific, Burlington, ON, Canada) was used to measure the protein concentration. Equal amount of protein sample was loaded and separated on 4–20% pre-cast mini-PROTEAN TGX gels (Bio-Rad Laboratories, Mississauga, ON, Canada) and run at 140 V for 1 h. The proteins were transferred onto PVDF membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). Blots were incubated at 4 °C overnight with anti-pig CD63 rabbit polyclonal IgG at a concentration of 1:100. This was followed by incubation with secondary antibody (Goat anti-rabbit IgG H&L (HRP), ab97051, 1:10000). Electro-chemiluminescence detection was performed using Bio-Rad Immun-Star Clarity Chemiluminescent kit (Bio-Rad Laboratories, Mississauga, ON, Canada). The X-ray films were developed and analyzed using Image J software (NIH, Bethesda, MD, USA) to obtain densitometry values.
Total RNA extraction and purification
Total RNA including miRNA from PTr2 and PAOEC cells was extracted using Total RNA extraction kit (Norgen BioTek Corp, Thorold, ON, Canada). Total RNA within EVs was isolated using Total exosome RNA and protein isolation kit (#4478545; Life Technologies Inc. Burlington, ON, Canada) according to manufacturer's instructions. The concentration and purity of isolated total RNA was assessed using a Nanodrop 2000 C UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C until further use.
Real-time PCR analysis
Real-time PCR for selected miRNAs was carried out using Qiagen miRNA Assays in a LightCycler real-time PCR system (LightCycler, Roche Diagnostics, Laval, QC, Canada) according to manufacturer's instructions. Briefly, miRNAs present in the total RNA samples were reverse transcribed using miscript II RT kit (Qiagen, Mississauga, ON, Canada). The resultant cDNA was assessed for the concentration and purity using Nanodrop 2000 C UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at −20 °C until required. miRNA expression relative to RNU1A reference genes. RNU1A is wet bench validated for PCR assays and its stable expression in the porcine tissues has been documented previously48. The focus of this investigation was to study the miRNAs that are involved in regulating the angiogenesis and therefore we conducted an extensive literature search which allowed us to list out 14 miRNAs for further investigation. The select miRNAs include: miR-16, miR-17-5P, miR-150, miR-20b, miR-155-5P, miR-15b, miR-222, miR-221-5P, let-7f, miR-20a, miR-126-5P, miR-296-3P, miR-181a-1, miR-181c-1. Porcine specific primer assays for selected miRNAs were custom designed using sequences available in the miRBase database49,50,51 version 19 (http://www.mirbase.org) and ordered from Qiagen (Qiagen, Mississauga, ON, Canada). The mature miRNA ID and accession numbers are listed in Supplementary Table S1. A melt curve analysis was conducted as an additional quality control after running each PCR plate.
Immunoflourescence
Formalin-fixed and paraffin-embedded porcine endometrial and CAM biopsies obtained at day 20 of gestation were sectioned (5 μm) and mounted on glass slides. CD63 immunoflourescence was performed as per the standard immunofluorescence protocol. Briefly, sections were deparaffinized, rehydrated, CD63 antigen was retrieved using 10 mM sodium citrate pH 6.0 at 95 °C for 30 min. Sections were blocked using 2% bovine serum albumin and then slides were incubated with primary antibody (CD63; rabbit polyclonal IgG; sc-15363; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) overnight at 4 °C. Secondary antibody, donkey anti-rabbit IgG H&L (Alexafluor® 594; ab150076; 1:10000) was applied and incubated for 1 hr at room temperature in the dark. After washing with PBS, slides were mounted with Prolong® Gold Antifade Reagent with DAPI (#8961; Thermofisher Scientific, Inc. Carlsbad, CA, USA). The slides were examined by epifluorescence microscopy and photographed with an AxioCam-equipped M1 imager (Zeiss, Toronto, ON, Canada) with Axiovision 4.8 software.
PTr2 cells were grown on coverslips for 24 hrs, fixed with 100% methanol (chilled at −20 °C) at RT for 5 min followed by ice-cold PBS wash three times for 5 min. Samples were permeabilized using PBS containing 0.1% Triton X-100 and washed with PBS three times for 5 min. Cells were blocked using 1% BSA, 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20) for 30 min and then incubated with diluted primary antibody (1:100; CD63, rabbit polyclonal IgG; sc-15363; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) in 1% BSA in PBST in a humidified chamber overnight at 4 °C. Cells were washed with PBS and secondary antibody was applied at a concentration of 1:10000. Slides were mounted with Prolong® Gold Antifade Reagent with DAPI and observed and photographed using epifluorescence microscopy and Axiovision 4.8 software.
Proteomic analysis of extracellular vesicles by mass spectrometry
Total protein from PAOEC and PTr2 cell derived EVs was extracted using Total exosome protein isolation kit (#4478545; Life Technologies Inc. Burlington, ON, Canada) according to manufacturer's instructions. The sample was desalted with a dialysis in 10 mM ammonium bicarbonate solution and subsequently digested with trypsin (20 μg) at 37 °C for 18 h. The peptide mixture was further analyzed by Liquid Chromotagraphy (LC)/Mass Spectrometry (MS) LC-MS/MS on a nLC-Orbitrap Velos Pro (Thermo Scientific) and MS/MS spectra were collected. The data were searched using Thermo Proteome Discoverer 1.4.0.288.
Extracellular vesicle proteome analysis
MS/MS identified proteins were analyzed by PANTHER (Protein Analysis Through Evolutionary Relationships; http://www.pantherdb.org). This software specialized in predictions and classification of proteins in order to facilitate high-throughput analysis. Using this software, the classified proteins were further classified according to their biological and molecular function. Protein IDs were also subjected to further classification using available protein database as a background. This process yielded families and subfamilies of proteins that were annotated with ontology terms and sequences were assigned to PANTHER pathways, cell signaling pathways.
Shuttling assays using confocal microscopy
Shuttling and internalization of EVs was performed using confocal microscopy. Briefly, freshly harvested EVs derived from either PAOEC or PTr2 were stained with CM-Dil (CellTracker, C7000), a fluorescent dye that labels the plasma membrane, according to the manufacturer's instructions. Next, fluorescently-labelled EVs were diluted in respective medium and were added into either PTr2 or PAOEC cell cultures grown on coverslips in 6 well cell culture plates. At the indicated time points, cells were fixed with 100% methanol (chilled at −20 °C) at RT for 5 min followed by ice-cold PBS wash three times for 5 min. CellTrackerTM Green BODIFY® dye (1:1000) was added to the cells and allowed to incubate at 37 °C for 30 min. Cells were briefly washed and coverslips were mounted with Prolong® Gold Antifade Reagent with DAPI. After 2 mins, the coverslips were examined under a confocal microscope and images were acquired for further processing ImageJ software.
Cell proliferation assays
Cell proliferation assay was performed as previously described52. Briefly, PAOEC and PTr2 cells were cultured at the rate of 1.25 × 104 cells/well in two separate 96 well plates for 24 hrs. Each well was treated with varying concentrations of EVs derived from either PTr2 or PAOEC (5, 10, and 20 μg/mL) in triplicates with PBS as control. After treating the wells with EV preparations, the rest of the wells were filled with medium to make up the volume to 100 μL. Plates were allowed to incubate in humidified chamber at 37 °C for 24 hours. 10 μL of WST-1 reagent was added to each well and incubated for 2 hrs at 37 °C. Absorbance in each plate was measured using microtitre plate reader at 450–690 nm with the reference wavelength of ~650 nm.
miRNA PCR data was analyzed using the ∆∆Ct method; gene expression levels of each miRNA was measured against RNU1A as a reference gene. Data are presented as mean ± SEM (n = 5 for each miRNA expression analysis). Comparison between multiple groups in the cell proliferation data was performed by means of analysis of variance (ANOVA) using GraphPad Prism 6.01 (GraphPad Software, La Jolla, CA, USA). If ANOVA demonstrated a significant interaction between variables, post hoc analyses were performed by the multiple-comparison Bonferroni correction test. P ≤ 0.05 value between groups was considered to be statistically significant. All experiments were conducted three times and in triplicate (i.e. biological replicates).
How to cite this article: Bidarimath, M. et al. Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk. Sci. Rep. 7, 40476; doi: 10.1038/srep40476 (2017).
Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).
Simpson, R. J., Jensen, S. S. & Lim, J. W. E. Proteomic profiling of exosomes: Current perspectives. Proteomics 8, 4083–4099 (2008).
Ng, Y. H. et al. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One 8, e58502 (2013).
Luo, S. S. et al. Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes. Biol. Reprod. 81, 717–729 (2009).
Donker, R. B. et al. The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes. Mol. Hum. Reprod. 18, 417–424 (2012).
Caby, M.-P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G. & Bonnerot, C. Exosomal-like vesicles are present in human blood plasma. Int. Immunol. 17, 879–887 (2005).
Pisitkun, T., Shen, R.-F. & Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. 101, 13368–13373 (2004).
Keller, S. et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int. 72, 1095–1102 (2007).
Poliakov, A., Spilman, M., Dokland, T., Amling, C. L. & Mobley, J. A. Structural heterogeneity and protein composition of exosome-like vesicles (prostasomes) in human semen. Prostate 69, 159–167 (2009).
Reinhardt, T. A., Lippolis, J. D., Nonnecke, B. J. & Sacco, R. E. Bovine milk exosome proteome. J. Proteomics 75, 1486–1492 (2012).
Ogawa, Y. et al. Proteomic analysis of two types of exosomes in human whole saliva. Biol. Pharm. Bull. 34, 13–23 (2011).
Burns, G. et al. Extracellular vesicles in luminal fluid of the ovine uterus. PLoS One 9 (2014).
Krawczynski, K., Najmula, J., Bauersachs, S. & Kaczmarek, M. M. MicroRNAome of porcine conceptuses and trophoblasts: expression profile of micrornas and their potential to regulate genes crucial for establishment of pregnancy. Biol. Reprod. 92, 21 (2015).
Théry, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579 (2002).
Sokolova, V. et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf. B. Biointerfaces 87, 146–50 (2011).
Rana, S., Yue, S., Stadel, D. & Zöller, M. Toward tailored exosomes: The exosomal tetraspanin web contributes to target cell selection. Int. J. Biochem. Cell Biol. 44, 1574–1584 (2012).
Verweij, F. J., Middeldorp, J. M. & Pegtel, D. M. Intracellular signaling controlled by the endosomal-exosomal pathway. Commun. Integr. Biol. 5, 88–93 (2012).
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476 (2008).
Denzer, K., Kleijmeer, M. J., Heijnen, H. F., Stoorvogel, W. & Geuze, H. J. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113 Pt 19, 3365–3374 (2000).
Hergenreider, E. et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat. Cell Biol. 14, 249–256 (2012).
Aliotta, J. M. et al. Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription. Exp. Hematol. 38, 233–245 (2010).
Salomon, C. et al. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration. PLoS One 9 (2014).
Salomon, C. et al. Exosomal Signaling during Hypoxia Mediates Microvascular Endothelial Cell Migration and Vasculogenesis. PLoS One 8 (2013).
Bazer, F. W. & Johnson, G. A. Pig blastocyst-uterine interactions. Differentiation. 87, 52–65 (2014).
Spencer, T. E. & Bazer, F. W. Uterine and placental factors regulating conceptus growth in domestic animals. J. Anim. Sci. 82 E-Suppl, E4–13 (2004).
Leiser, R. & Dantzer, V. Initial vascularisation in the pig placenta: II. Demonstration of gland and areola-gland subunits by histology and corrosion casts. Anat. Rec. 238, 326–334 (1994).
Dantzer, V. Electron microscopy of the initial stages of placentation in the pig. Anat. Embryol. (Berl). 172, 281–293 (1985).
Geisert, R. D. & SCHMITT, R. a M. Early embryonic survival in the pig: Can it be improved? J. Anim. Sci. 80, E54–E65 (2002).
Johansson, S., Dencker, L. & Dantzer, V. Immunohistochemical localization of retinoid binding proteins at the materno-fetal interface of the porcine epitheliochorial placenta. Biol. Reprod. 64, 60–8 (2001).
Liu, R. et al. The Expression Pattern of microRNAs and the Associated Pathways Involved in the Development of Porcine Placental Folds That Contribute to the Expansion of the Exchange Surface Area. Biol. Reprod. 93, 62 (2015).
Bazer, F. W. et al. Uterine biology in pigs and sheep. J. Anim. Sci. Biotechnol. 3, 23 (2012).
Wu, G. et al. Impacts of amino acid nutrition on pregnancy outcome in pigs: mechanisms and implications for swine production. J. Anim. Sci. 88, E195–204 (2010).
Keys, J. L. & King, G. J. Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy. Am. J. Anat. 188, 221–238 (1990).
Kambe, S. et al. Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells. Biol. Reprod. 91, 129 (2014).
Mittelbrunn, M. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).
Ouyang, Y., Mouillet, J. F., Coyne, C. B. & Sadovsky, Y. Review: placenta-specific microRNAs in exosomes - good things come in nano-packages. Placenta 35, S69–73 (2014).
Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3, Unit 3 22 (2006).
Montecalvo, A. et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 119, 756–766 (2012).
Eirin, A. et al. MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells. Gene 551, 55–64 (2014).
Atay, S., Gercel-Taylor, C., Kesimer, M. & Taylor, D. D. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells. Exp. Cell Res. 317, 1192–1202 (2011).
Segura, E. et al. CD8+ dendritic cells use LFA-1 to capture MHC-peptide complexes from exosomes in vivo . J. Immunol. 179, 1489–96 (2007).
Morelli, A. E. et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104, 3257–3266 (2004).
Bidarimath, M. et al. Distinct microRNA expression in endometrial lymphocytes, endometrium, and trophoblast during spontaneous porcine fetal loss. J. Reprod. Immunol. 107, 64–79 (2015).
Jaeger, L. a. et al. Functional effects of transforming growth factor beta on adhesive properties of porcine trophectoderm. Endocrinology 146, 3933–42 (2005).
Sakai, T., Saruwatari, T., Fukushima, O. & Saito, T. The covering method: An improved negative staining method for ultrathin cryo-sections of tissue. J. Electron Microsc. (Tokyo). 44, 479–484 (1995).
Rasband, W. ImageJ [Software]. U. S. Natl. Institutes Heal. Bethesda, Maryland, USA//imagej.nih.gov/ij/ (2015).
Abràmoff, M. D., Magalhães, P. J. & Ram, S. J. Image processing with imageJ. Biophotonics International 11, 36–41 (2004).
Wessels, J. M., Edwards, A. K., Zettler, C. & Tayade, C. Selection and validation of reference genes for miRNA expression studies during porcine pregnancy. PLoS One 6, e28940 (2011).
Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A. & Enright, A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34, D140–4 (2006).
Griffiths-Jones, S., Saini, H. K., van Dongen, S. & Enright, A. J. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36, D154–8 (2008).
Kozomara, A. & Griffiths-Jones, S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 39, D152–7 (2011).
Ahn, S. H. et al. IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors. J. Immunol. 195, 2591–600 (2015).
The authors thank Matt Gordon, Queen's University Biomedical imaging center; Oliver Jones, Queen's laboratory for molecular pathology, Department of Pathology and Molecular Medicine for their technical assistance with confocal microscopy and transmission electron microscopy; Technical assistance from Dr. Jiaxi Wang, Queen's Mass spectrometry facility, and Xiaohu Yan, Department of Biomedical and Molecular Sciences (DBMS) for their help with mass spectrometry and transmission electron microscopy is greatly appreciated. Authors would also like to thank Dr. Anne Croy for access to the fluorescence microscopy and Dr. Stephen C. Pang for access to the real-time PCR facilities in the DBMS, Queen's University. Support from technical staff at the Arkell Swine Research Station, and the Meat's Wing of Animal and Poultry Science at the University of Guelph is gratefully acknowledged. Generous Funding support from NSERC-CRD, Ontario Pork, and OMAFRA(CT), Queen's Graduate Award, Queen's University (MB) is greatly appreciated.
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Mallikarjun Bidarimath
, Kasra Khalaj
, Frederick W. K. Kan
, Madhuri Koti
& Chandrakant Tayade
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, N1G 2W1, Ontario, Canada
Kasra Khalaj
, Rami T. Kridli
Department of Animal Production, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan
Rami T. Kridli
Search for Mallikarjun Bidarimath in:
Search for Kasra Khalaj in:
Search for Rami T. Kridli in:
Search for Frederick W. K. Kan in:
Search for Madhuri Koti in:
Search for Chandrakant Tayade in:
Conceived and designed the experiments: M.B. and C.T. Performed the experiments: M.B. Assisted in Western blots: K.K. Analyzed the data: M.B. Contributed reagents/materials/analysis tools: C.T. and F.W.K. Wrote the manuscript: M.B. and C.T. Reviewed the Manuscript: M.B., C.T., M.K., K.K., R.K. and F.W.K.
Correspondence to Chandrakant Tayade.
Supplementary Information (PDF 1448 kb)
Supplementary Video S1 (AVI 292276 kb)
Supplementary Dataset S1 (XLS 335 kb)
Bidarimath, M., Khalaj, K., Kridli, R. et al. Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk. Sci Rep 7, 40476 (2017) doi:10.1038/srep40476
Therapeutic effects of human umbilical cord mesenchymal stem cell-derived microvesicles on premature ovarian insufficiency in mice
Ziling Yang
, Xin Du
, Cunli Wang
, Jin Zhang
, Conghui Liu
, Yu Li
& Hong Jiang
Stem Cell Research & Therapy (2019)
Classification of fetal resilience to porcine reproductive and respiratory syndrome (PRRS) based on temporal viral load in late gestation maternal tissues and fetuses
Carolina M. Malgarin
, Roman Nosach
, Predrag Novakovic
, Muhammad Suleman
, Andrea Ladinig
, Susan E. Detmer
, Daniel J. MacPhee
& John C.S. Harding
Virus Research (2019)
Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri-implantation embryos
Abu Musa Md Talimur Reza
, Yun-Jung Choi
, Sung Gu Han
, Hyuk Song
, Chankyu Park
, Kwonho Hong
& Jin-Hoi Kim
Biological Reviews (2019)
Exosomes and Female Infertility
Xiaoyan Sun
, Xiaoling Ma
, Xia Yang
& Xuehong Zhang
Current Drug Metabolism (2019)
A review on protein markers of exosome from different bio-resources and the antibodies used for characterization
Fengyan Deng
& Josh Miller
Journal of Histotechnology (2019)


text:
Solunska fronta je poimenovanje za zavezniško fronto proti centralnim silam v prvi svetovni vojni na Balkanu. Ime je dobila po grškem mestu Solun.
Fronta je bila odprta avgusta leta 1916, ko so sile srbske in francoske vojske začele napad in po hudih spopadih zasedle strateško pomembna Kajmakčalan in Bitolo.

Frontna črta je potem obmirovala do preboja zaveznikov. Vsesplošna ofenziva na bolgarsko-nemške položaje se je začela 15. septembra 1918 (bitka pri Dobrem polju). Glavnino napada sta nosili srbska vojska, ki se ji je po preživeti »Golgoti« preko Albanije mudilo v domovino, in dve diviziji francoskih sil.
Bolgarija je kmalu spoznala, da ne bo zdržala pritiska in je zato zaprosila za premirje (30. septembra so ji ga zavezniki zagotovili), nemške enote pa so se umikale na sever. Srbska vojska je pričela naglo napredovati (v samo nekaj dneh so napredovali za 170 kilometrov). Tako so njene enote že 1. novembra 1918 vkorakale v Beograd. Nekaj dni kasneje so se spopadi končali s prodorom srbskih in francoskih enot preko Donave.
Na strani srbske vojske se je borilo tudi veliko Slovencev, t. i. dobrovoljcev.

Glej tudi
 Balkansko bojišče

Prva svetovna vojna
Zgodovina Jugovzhodne Evrope
Bitke prve svetovne vojne

redpajama_set_name: RedPajamaWikipedia


Francisco Fern´ andez de Vega and Erick Cantu- ´ Paz The growing success of biologically inspired algorithms in solving large and complex problems has spawned many interesting areas of research. Over the years, one of the mainstays in bio-inspired research has been the exploi- tion of parallel and distributed environments to speedup computations and to enrich the algorithms. From the early days of research on bio-inspired algorithms, their inherently parallel nature was recognized and di?erent p- allelization approaches have been explored. Parallel algorithms promise - ductions in execution time and open the door to solve increasingly larger problems. But parallel platforms also inspire new bio-inspired parallel al- rithms that, while similar to their sequential counterparts, explore search spaces di?erently and o?er improvements in solution quality. Our objective in editing this book was to assemble a sample of the best work in parallel and distributed biologically inspired algorithms. We invited researchers in di?erent domains to submit their work. We aimed to include diverse topics to appeal to a wide audience. Some of the chapters sum- rize work that has been ongoing for several years, while others describe more recent exploratory work. Collectively, these works o?er a global snapshot of the most recent e?orts of bioinspired algorithms' researchers aiming at pr- iting from parallel and distributed computer architectures—including GPUs, Clusters, Grids, volunteer computing and p2p networks as well as multi-core processors.


text:
Hite's colorful history recounted in library lecture
A vehicle drives to Hite on the old road.
By: Phil Clark - Updated: 2 months ago
Posted Nov 21, 2022
Page resident Cindy Stafford gave a presentation on the history of Hite, Utah, at Page Public Library on Nov. 15. The event was part of the Glen Canyon Conservancy's monthly lecture series.
Many locals know Hite – located at the northernmost extent of Lake Powell – as a place where commercial raft trips take out after floating through Canyonlands National Park, where there used to be a marina. Few knew Hite when there were no bridges, no paved highways and no lake. Stafford's presentation featured historic photographs and documents from a time when many in the audience had not yet been born.
Stafford visited Page and Hite in 1989 on a pleasure trip. In 2005, she started thinking of working in the Southwest. When she discovered the Page Public Library, she realized that a town that cared enough to build such a great library was a place where she wanted to live. Stafford got a job at Glen Canyon NRA in the interpretation division and volunteered for the cultural resources division.
Stafford's presentation, entitled "Submerged Memories: Dick Sprang Recalls Hite Ferry," included many historic photographs describing the original settlement and the ferry that transported cars across the Colorado River before there was a lake or a paved road. Hite is 167 miles upstream along the river from Lees Ferry.
As plans were being made for construction of dams on the Colorado River, surveyors documented the topography of the area in the early 1950s. In the interest of efficiency, the survey was limited to a maximum elevation of 3,900 feet, and many canyons simply didn't show up on the survey drawings. The maximum designed level for Lake Powell is 3,700, so as far as the survey, little mattered for the topography above 3,900 feet.
At the time of the survey, the only way to reach Hite was by driving down a treacherous road in North Wash, also known as Crescent Wash. A sign along the road read, "Danger Flash Flood Area. 60 Wash Crossings Next 18 Miles. Do Not Cross Wash When In Flood." If the sign didn't give a traveler pause, the actual wash crossings did, as many of them showed no hint of any sort of road. Maps of the area had the note, "Travel in this area not recommended without a guide."
Gus Scott floated the river as a young man in 1955. Scott and his friends were anxious to explore the area, as they knew that the dams would come and the chance to explore would be lost. At the time of their trip, the river flowed some 1,500 cubic feet per second. They were the first non-Natives to explore Three Warrior Ruins, which later became known as Defiance House.
Cass Hite (1834-1914) was the first white settler in the area known as Hite. He was a gold prospector and explorer who arrived in southern Utah around 1880. Hite spun tales to the national press about mining for gold in Glen Canyon. These stories helped trigger a rush of miners to the area.
Prospectors soon found that the ore was too small and dusty to extract. The Salt Lake Herald described Hite as a "genial old miner who has found more gold and made less money than any man." C. Gregory Crampton, a historian at the University of Utah who is best known for documenting cultures and artifacts in Glen Canyon before it was flooded by the Glen Canyon Dam, said of Hite that he was "a strong believer in the rule that it is easier to mine gold out of an investor's pocket than it is out of the ground."
Arthur "Arth" Chaffin (1884-1979), an avid hiker and explorer, settled in the area. He knew the area well and was reluctant to share information about it with others, knowing that once the beauty became known, more and more people would arrive. Chaffin coined the term "canyonitis" – "a humorous affliction, but I know of no old riverman who doesn't seriously carry this disease," a condition in which one does not disclose too much information about canyon country. The nearest railroad was about 120 miles away, and Chaffin's nearest Colorado River neighbor was at Lees ferry, almost 170 miles downstream along the river.
The original crossing of the Colorado River was called Dandy Ford, about 5 miles downstream from what would later be the Hite Marina in Glen Canyon National Recreation Area. The ford would allow crossing the river when its flow wasn't too high.
In 1933, every Utah politician voted for the establishment of Capitol Reef National Monument, and bridges were built across the Colorado and Dirty Devil rivers. The governor of Utah at the time was the first to cross the new bridges, which were still approached by unpaved gravel roads.
There wasn't enough funding for both the road and the bridges, so the bridges were built first. At the time of the governor's visit, the bridges were first dedicated as a "marriage" among San Juan, Garfield and Kane counties.
The bridges were described by author John V. Young as standing "a few miles apart in the middle of a vast and silent wilderness, with nothing at either end but a 50-mile stretch of gravel track stretching east and west across a nearly waterless desert."
The Hite Ferry served from 1946 to 1964 and ceased operations when the paved highway was complete.
Almost 60 years after the completion of Glen Canyon Dam, the area is once again coming out of the watery depths. All that remains of the water-based facilities is a boat ramp, now a slab of concrete in the tamarisk flats. For many in the audience, last week's lecture was a chance to learn more about an area that they never saw. Scott was in the audience and posed in front of the photo of himself in 1955. He later became a cartoonist for the Batman and Robin comic books.
The schedule for future Glen canyon Conservancy lectures at Page Public Library is available in the Copper Newsletter, a publication by the City of Page. To subscribe free to the newsletter, send a request to [email protected]
Young artists wanted for Page Attacks Trash logo contest
Water reductions may be just the beginning, experts say
BOR completes project at Glen Canyon Dam to protect Page water supply
ZenniHome looks to overcome supply chain hurdles
We see you are enjoying our articles!
If you wish view the full all article, watch this short video or subscribe now for full access.
Subscribe Watch Video No, thanks! I will stick to the free stuff

redpajama_set_name: RedPajamaCommonCrawl


When it comes to producing leaders, AFL-CIO president Richard Trumka told AFT convention delegates on July 10, the AFT moves to the head of the class.
That's because our union doesn't complain about problems but uses our expertise to solve them. And that's why the AFT has grown to 1.5 million members and why Trumka expressed his gratitude for the AFT's leadership on behalf of union workers everywhere.
Case in point: The third-generation coal miner who went on to college and law school told how he'd seen AFT president Randi Weingarten "school our friends in the Obama administration" when they initially applauded teacher firings last spring in Central Falls, R.I.
That day, Vice President Joe Biden came to visit union leaders. "Now, usually at these meetings we try to be respectful," Trumka said, but on that day, Randi Weingarten was angry.
"The toughest thing to do as a leader is to tell your friends when they're wrong," he said. "Joe Biden is a friend to this union and a friend to the labor movement. But Randi stood up and told our nation's vice president that the administration was wrong."
Wrong to celebrate what happened, he added. Wrong to hold up mass firings as an answer to education's problems.
Weingarten was articulate, Trumka said, but also "a tad heated. The Secret Service guys started to look a little jumpy when Randi put her finger to the veep's chest to emphasize her point."
To a loud ovation, Trumka then asked: "You know what happened after that exchange? The conversation changed." Instead of seeing the firings as a bold action to be celebrated, officials started seeing them as a problem to be solved. The AFT, Trumka repeated, solves problems.
Now, working families across America are facing a triple whammy, he said: soaring unemployment, budget crises and crushing new demands on public services. With 15 million people out of work, even those lucky enough to have jobs live in fear of pink slips, medical emergencies and foreclosure.
And because Americans are so anxious and angry, they're hunting for scapegoats. But while some people want punching bags, Trumka said, the AFT looks for solutions. Until the nation gets back on its feet, he continued, lawmakers need to head off reductions in force and extend unemployment benefits and healthcare assistance.
"We need a jobs bill with a scale and scope as serious as the crises we face," the union leader said to applause and cheering. "We absolutely must provide emergency assistance. … We need 'Pink Hearts, Not Pink Slips,' as your pins say. I'm proud to wear one."
Finally, the nation must invest in "brilliant new technologies of the future," sparking a green economy of good jobs.
To those who raise concerns about the federal deficit, Trumka acknowledged the deficit as a legitimate concern but pointed out that it's also a long-term problem. Right now, we're millions of jobs in the hole; if we fail to invest in education and other public services, our nation may never dig out of deficits in skills, technology and competitiveness.
To bring this point home, Trumka called on AFT members to give elected officials "the report cards they deserve" in November. The AFT, he said, has always understood that the challenges of poverty find their way into classrooms, and that the best weapon against poverty is a strong labor movement. He called on members to renew support for the Employee Free Choice Act, federal legislation that would permit all workers who want a union to have one.


Mensagem: What sort of music do you listen to?
Mensagem: How much does the job pay?
Mensagem: Do you know each other?
Mensagem: How do I get an outside line?
Mensagem: Could you tell me the dialing code for ?
Mensagem: Where do you come from?
Mensagem: What do you want to do when you've finished?
Mensagem: Do you know what extension he's on?
Mensagem: How much notice do you have to give?
Mensagem: Could I have , please?


The Finnish designer Armi Ratia presented the Finnish homewares company iittala with her colour palette from her design company Marimekko. With Marimekko's spectacular design principles and iittala specialising in creating wonderful coloured glass for the home, a new Mariskooli was born.
Ratia's garden parties in the 1960's were famous for dripping with decadence and colour because she used these fabulous bowls to serve up all her delicious treats. They're a great way to serve ice cream and sweets at parties and they also make for charming display pieces.


Graphic Design and Illustrations. T-shirt and Gifts Design Ideas.
Are you strapped for cash at the moment and unsure how you'll be able to afford gifts for the next special occasion on the calendar? Are you not sure what to give to the person who has everything? If so, why not use your creative skills to put together a special and unique gift that you know your friends and family will love.
Some people think that home-made gifts are cheap and tacky, but it's likely that their memories are tainted by the sort of things that they made while they were at school. Now that you're an adult, you can let your imagination run wild and make some amazing home-made gifts, even if you aren't a talented artist.
If you have a talent for cooking, why not make some jam, cakes, or alcoholic truffles (depending on how well you know the recipient), and wrap them up using off-cuts of floral fabrics. Complete the design with a cute bow, and you'll have a low cost but luxurious looking gift that could have come from a country boutique.
If you have a knack for graphic design, pick up an inexpensive, plan cushion, and sew a felt motif onto it. There are some high-end designers making "geek-chic" cushions that are designed to look like Apple iOS logos or traditional keyboard keys. You could recreate this look quite easily with a little felt and some careful sewing.
Talented cross-stitch lovers and people that are skilled in painting, photography or embroidery have a lot of other options open to them for sewing and design. Try to figure out what your own talents are, and take some time to experiment with new designs. You can use communities such as Pinterest and Deviant Art to get some inspiration. If your attempt is a failure, you can always rush to the supermarket and buy a box of chocolates instead!


text:
DEADPOOL 3 – FIRST TRAILER (2023) Marvel Studios & Disney+
The inspiration behind this video:
Deadpool 3 is happening in the MCU, and Marvel's Secret Wars could be the key to explaining where the talkative mercenary has been all this time. Following Disney's purchase of Fox, Deadpool and the mutants are coming to Marvel Studios to have a major role in the next phases of the MCU.
After a period of speculation about what the future of the Deadpool franchise would be now under the Disney umbrella, a Deadpool film in the MCU has been confirmed with Ryan Reynolds set to return and The Adam Project's Shawn Levy as the director – but with still no indication as to how and when the character will be introduced.
Like the X-Men and the Fantastic Four, Deadpool's arrival in the MCU is challenging in terms of storytelling. The MCU will soon have to decide as to when and how such important parts of the Marvel universe will be integrated into the already established franchise.
Between showing those characters' origins again or saying that they have always been there, options abound to explain the arrival of big names like Doctor Doom and Deadpool – each with its pros and cons.
Thank You So Much For Watching!
Stay Tuned! Stay Buzzed!


text:
Gottfried August Bürger (Molmerswende – ma Mansfeld része – , Szász-Anhalt, Németország), 1747. december 31. – Göttingen, Német-római Birodalom, 1794. június 8.) német író, költő, a Münchhausen báró kalandjai szerzője.

Élete

Korai évek és tanulmányai
Bürgert tizenkét esztendős korában gyakorlatilag az anyai nagyapja, Bauer fogadta örökbe, Ascherslebenben. A kisvárosban a Pädagogium at Halle-hoz küldték. 1764-től a teológia szakon tanult tovább. Azonban nem sokkal később elhagyta a teológiát, és a jogtudomány hallgatójává vált. Itt Christian Adolph Klotz (1738-1771) német filológus befolyása alá került. Az egyetemet itt nem sikerült elvégeznie, ezért 1768-ban átiratkozott a Göttingeni Egyetemre, itt újra a jogtudomány hallgatójaként tanult tovább.

Hamarosan az irodalommal kezdett foglakozni, és igen rendetlen, kicsapongó életet élt. Legelső költeményei 1771-ben a Göttinger Musenalmanach című évkönyvben jelentek meg. 1772-ben körjegyző lett a hannoveri Altengleichenben. Lenore című költeménye, mely 1774-ben jelent meg a Gött. Musenalmanachban, egész Németországban ismertté tette nevét. Ugyanebben az évben nőül vette Leonhard niedecki körjegyző leányát, Dorottyát, de a lakodalom után nemsokára szenvedélyesen megszerette felesége húgát, Augusztát, akit költeményeiben Molly néven ünnepelt. Ebből szertelen viszályok keletkeztek, melyek nemcsak családi életét, hanem jó hírnevét is tönkre tették. Ezekhez még pénzbeli zavarok és hivatali bajok is járultak.

1784-ben felesége meghalt, ő lemondott hivataláról és Göttingenbe ment egyetemi magántanárnak. 1785 júliusában feleségül vette Augusztát, aki már 1786. június 6-án meghalt. 1790 őszén újra megházasodott; felesége Elise Hahn lett, aki egy költeményben nyilvánosan ajánlotta fel neki a kezét. De ez a házasság is áldatlan volt; felesége feslett életet élt, és emiatt Bürger általános gúny és üldözés tárgya lett. Végül 1792 márciusában törvényesen elvált Elise Hahntól. De a költő többé nem szedhette össze magát. Egészséges kedélye tönkre volt téve, ezenkívül végtelenül megbántotta őt Schillernek a jénai Allgemeine Litteraturzeitungban közölt túlságosan szigorú ítélete; harmadik feleségének pazarlása folytán pedig anyagilag is úgy tönkrement, hogy koldusbotra jutott. Fordításokkal tengette életét, ismerősei elfordultak tőle. Így halt meg tuberkulózisban.

Költeményeinek első gyűjteménye (Gedichte) 1778-ban jelent meg Göttingenben, és azóta számtalan kiadást ért meg. Összes műveit K. Reinhard adta ki 1823-24-ben 7 kötetben és Ed. Grisebach 1872-ben 2 kötetben. Legszebb balladáit magyarázta Heinrich Gusztáv, Német balladák és románcok I, 2. kiadás 1887.

Legismertebb műve a Münchhausen báró kalandjai (1786), egy nagyotmondó katonanemes képtelen és mulatságos anekdotái, históriái. A világhírű történetfüzérnek számos újramesélése, megfilmesítése stb. létezik szerte a világon.

Magyarul megjelent művei

1919-ig
 Lenore és más balladák

 Báró de Mánx lengyel orsz. confed generálisának a tengeren és szárazon tett utazásai. Öszve-szedettek és magyar nyelven ki-adattak hív unokája által; Landerer, Pozsony–Pest, 1805 (Téli és nyári könyvtár)
 Báró de Mánx a tengeren és szárazon tett utazásai és tsudálkozásra méltó történetei. Öszve-szed. és magyar nyelven ki-ad. hív unokája által; Landerer, Pozsony–Pest, 1813
 Valóságos mesterség úgy hazudni, hogy érdemes légyen kinyomtatni, vagy Báró Nyargalótzinak tsudálatos utazása; Trattner Mátyás, Pest, 1813
 Báró de Manx vizen, szárazon és a levegőben, gyalog és lóháton, háborúban és békében való egyedül igaz élményei. Meséli ő maga; Lampel, Pest, 1859
 Báró Münchausen csodálatos kalandjai, csinjai, binjai amint azokat ő maga elbeszélni szokta; ifjúsági ford., átdolg. Lauka Gusztáv; Lauffer és Stolp, Pest, 1863 (Pesti képeskönyvek)
 Münchhausen báró kalandjai; G. A. Bürger eredetije után átdolg., előszó Hoffmann Ferenc, ford. Győri József; Nagel, Bp., 1882
 Münchausen báró csodálatos kalandjai és utazásai; ifjúsági átdolg. Sándor A.; Nagel, Bp., 1888
 Münchhausen báró csodálatos utazásai és kalandjai vízen és szárazon; az ifjúság számára Oskar Höcker nyomán ford. Koltai Virgil; Révai-Salamon Ny., Bp., 1900 k.
Münchhausen, az igazmondó német báró csodálatos kalandjai; elbeszélte ő maga, a magyar ifjúságnak elmeséli Gaal Mózes; Lampel, Bp., 1903
Münchhausen báró csodálatos kalandjai; átdolg. Baráth Dániel; Eisler, Bp., 1906
Münchhausen báró vidám kalandjai szárazon és vízen; az ifjúságnak elbeszéli Vágó Ferenc; Athenaeum, Bp., 1906
Münchhausen báró csodálatos kalandjai szárazon és vízen; a magyar ifjúság számára átdolg. Baróti Lajos, ill. Gustav Doré; Rozsnyai, Bp., 1911
Forray Dezső: Egy kocsis fia vagy A forgalom jutalma / Münchhausen báró csodálatos kalandjai és utazásai; ford. Sárosi Bella / Sas Ede: Együtt maradunk, Éjszaka a Balatonon stb.; Magyar Kereskedelmi Közlöny, Bp., 1919

1920–1944
Münchhausen vidám kalandjai; Tolnai, Bp., 1920 k.
Münchhausen báró világraszóló utazásai és kalandjai; ifjúsági átdolg. kiad.; Hajnal, Bp., 1924
Münchhausen báró újabb kalandjai; Kellner, Bp., 1925 (Mesekönyvtár)
Münchausen báró csodálatos kalandjai; Pallas Ny., Bp., 1927 (Tündérvásár könyvtára)
Münchhausen csodálatos kalandjai; a magyar ifjúság számára átdolg. Z. Tábori Piroska, ill. Bortnyik Sándor; Dante, Bp., 1931
Münchhausen báró utazásai és kalandjai; ifjúsági átdolg. Kertész Erzsébet, ill. Pályi Jenő; Nova, Bp., 1943
Münchausen báró csodálatos kalandjai; az ifjúságnak elbeszéli Gálszécsi István, címlap Weidlinger Júlia, ill. Korcsmáros Pál; Péchyné, Bp., 1943
Münchhausen csodálatos utazásai szárazon és vizen. Önvallomásai vándorlásairól és vidám kalandjairól; ford. Kuthy Pál, ill. Gustav Doré; Fővárosi, Bp., 1943
Münchhausen báró kalandjai; átdolg. Ajtai kálmán, ill. Andor Loránt; Szt. István Társulat, Bp., 1943
Münchhausen báró kalandjai; Mándy Iván átírásában, ill. Fery Antal; Forrás, Bp., 1944

1945–1989
Münchausen báró kalandjai, 1-2.; ill. Zórád Ernő; Ifjúsági Lapkiadó Vállalat–Junge Welt, Bp.–Berlin, 1957 (ifjúsági képregény)
G. A. Bürger–Karl Immermann: Münchhausen báró; ford., átdolg., utószó Homoródy József, ill. Würtz Ádám; Magvető, Bp., 1957 (Vidám könyvek)
Münchhausen vidám kalandjai; G. A. Bürger nyomán feldolg. Elena Chmelová, szlovákból ford. Tordon Ákos, ill. Vincent Hloznik; Móra, Bp., 1975
G. A. Bürger: Münchhausen báró csodálatos utazásai szárazon és vizen, haditettei és vidám kalandjai, ahogyan kancsó mellett, baráti körében maga mesélte el; ford. Homoródi József, ill. Gustave Doré; Helikon, Bp., 1983
Münchhausen báró csodálatos kalandjai, 1-2.; szerk., Bürger műve nyomán szöveg Erdei D. András, ill. Zórád Ernő; Lapkiadó Vállalat, Bp., 1986

1990–
Erich Kästner mesél / Till Eulenspiegel / Münchhausen / Don Quijote / A schildai polgárok / Gulliver utazásai; ford. Rónaszegi Éva, ill. Walter Trier, Horst Lemke; Móra, Bp., 1992
Gottfried August Bürger: Münchhausen báró kalandjai / Charles Perrault: Lúdanyó meséi; ifjúsági átdolg. és ford. Tordon Ákos, Rónay György, Szűcs János, összeáll., szerk. Tóth Emese, utószó Kovács Ágnes, ill. Gustave Doré; Kossuth, Bp., 1994
Münchhausen báró barangolásai a nagyvilágban. Átdolgozás G. A. Bürger műve, a Münchhausen báró kalandjai (1786) nyomán; ford. Csire Gabriella, ill. Felszegi Stefánia; Erdélyi Gondolat, Székelyudvarhely, 2001
Báró Münchhausen csodálatos kalandjai, csinjai, binjai, cselei és bohoczságai, amint azokat barátai körében ö maga elbeszélni szokta; ford., átdolg. Lauka Gusztáv [1863]; hasonmás kiadás; Pytheas, Bp., 2003
Münchhausen. Münchhausen báró csodálatos utazásai és kalandjai szárazon és vízen. Nyolc történet; ill. Walter Trier, ford. Rónaszegi Éva; Móra, Bp., 2014 (Erich Kästner mesél)
Rudolf Erich Raspe–Gottfried August Bürger: Münchhausen báró vidám kalandjai; ford., átdolg. Szögi Mihály, ill. Olekszandr Krutik; Elektra, Érd, 2017

Jegyzetek

Fordítás

Források

 Tolnai Vilmos: Báró de Manx és Münchhausen; in: Irodalomtörténet, 1934/1-2.

További információk
 Munkái a zeno.org honlapon
 Munkái a Project Gutenberg-ben
 Gottfried August Bürger: Münchhausen vidám kalandjai (MEK)

Német költők
Német írók
1747-ben született személyek
1794-ben elhunyt személyek


Special relationship between the Fire and Rescue service and the RSPCA?
No mention of the self proclaimed emergency service for animals, the RSPCA. No question of where their trained seal experts had disappeared to, or why the money the public donates appears to have failed to produce an emergency RSPCA response service that can act without the need for police officers or members of the fire service to do the job for them?
There have been many criticisms of the special relationship between the RSPCA and the police.
For instance, the Wooler Report took on board the SHG submission and noted that the RSPCA 'piggy backing' on police powers by passes the important safeguards imposed on both local authorities and the police. Each has internal complaints procedures that culminate in independent external bodies, the IPCC and the local government ombudsman.
But what of the special relationship between the Fire Service and the RSPCA?
The Chief Fire Officers' Association (CFOA) has a Memorandum of Understanding with the RSPCA. The CFOA has committed to sharing the MoU, and encouraging its adoption, with every member of the Association who sit within every fire and rescue service within the UK.
Although the CFOA has no authority to ensure Fire and Rescue services compliance with the MoU it is clear that they are expected to adopt it.
Adherence to the MoU, which details how the Fire and Rescue Service can, if they deem it appropriate, charge members of the public for their services rescuing animals, but should never charge the RSPCA the full rate, have led to confusion and complaints that the Fire and Rescue service is wasting public money and resources by attending animal rescues at the behest of the RSPCA. On one occasion the Fire and rescue service sent two fire engines to provide light so that the RSPCA could continue an investigation in the dark. No-one thought to ask why an RSPCA investigation team had no lights or torches with them.
Some Fire and Rescue services are refusing to turn out to rescue animals unless they are asked by the RSPCA. Others say that the reason they undertake animal rescues is to prevent members of the public from attempting a rescue and putting themselves in danger.
All will turn out without being asked by the RSPCA if a member of the public is already attempting a rescue that could endanger them or is already in difficulties.
This raises an important question. Who should be responsible for Fire and Rescue Service policy in any local area? Unelected charities like the RSPCA and the CFOA deciding who will respond to calls for help from the public and who will pay? Or should local government make the decision?
Is this really the way the policies that govern our public services should be determined?
Slaughter for food? Only OK if the RSPCA do it.
How do I prove ownership of my animal?
Thanks – amazing really. An incredible quantum of chutzpah, combined with a narrative that people believe, and then…. the entire world is at their feet!!!!!! to be used and exploited and trampled on.


text:
Q: WindowsFormsHost in WPF MDI on Aero main form (C#) My crazy problem: I'm using a docking system (AvalonDock) on a WPF form with aero glass style background (what looks really nice). In one dockable MDI I need a WinForms custom control, so I just used the WindowsFormsHost that loads my control successfully. But there is one problem:
The WindowsFormsHost shows the glass background of the main form for the amount of black for all colors used in the custom control.
Changing the MDI background doesn't help because the WindowsFormsHost cuts a hole through everything in the WPF MDI.
Concluding neither trying to have white text on a black background nor the other way round makes the textbox in my custom control clearly readable.
Any suggestions?

redpajama_set_name: RedPajamaStackExchange


text:
Q: Render pixelated sprite from sprite sheet in React Native without crashing the springboard I'd like to render a non animated pixel art sprite in an image with nearest neighbour upscaling.
My image looks like this and at the moment it renders the entire sprite sheet:
<Image
  source={require("../assets/sheet.png")}
  style={{
    width: 30,
    height: 30
  }}

In CSS this would be simple to do with background-size, background-position and image-rendering: pixelated.


Pewter Tea Leaf – Hand leafed background wallpaper.
Gracie Studio Pewter Tea Leaf – Hand leafed background wallpaper.
Handpainted wallpaper on a gold tea leaf ground, inspired by an 18th century Japanese screen of wave and blossom design.
Gracie Studio Handpainted wallpaper on a gold tea leaf ground, inspired by an 18th century Japanese screen of wave and blossom design.
We take on where Consumer Reports and Yelp leave off. Join today and find and leave reviews of your favorite luxury home products and appliances!


text:
Ireland & Scotland 2010
Ireland & Scotland 2010 - Page Text Content
S: Charlie and Erin go to Ireland & Scotland
FC: May 2010 | Ireland & Scotland
1: "May your blessings outnumber The shamrocks that grow, And may trouble avoid you Wherever you go." ~Irish Blessing
2: In November of 2009, it became official... the tickets were purchased and Erin and Charlie were set to go to Ireland & Scotland in May of 2010! Charlie had never traveled outside of the United States, and Erin was dying to visit Ireland again. But... In April, the Eyafjallajkull volcano in Iceland erupted. Because of all the ash, it threatened to ruin our trip. Flights were continuously canceled. And so we sat for an entire month watching the news daily in hopes that our flight would still be a go. And when the day came for our trip to begin... | WE WERE ABLE TO TAKE OFF!! YAY!!
3: We landed in Dublin, and had 2 days to take in the city by ourselves. After that, we took part in a tour that was put together by CIE tours. | Our Trip | Day 1: Tour of Dublin Day 2: Visited the Guinness Factory, had dinner at the Merry Ploughboys Day 3: Stop in Killarney, See the Rock of Cashel, visit Blarney Castle, and kiss the stone of eloquence Day 4: Embarked on the Ring of Kerry, 100 miles visited Dingle Peninsula, stopped at Kells to watch sheep herding, and learned about Christian monks with the Skellig experience. Day 5: Took a ferry across the Shannon River, visited the Cliffs of Moher, and Bunratty castle Day 6: Medieval dinner at Bunratty Castle Day 7: Departed with CIE tours, stayed at a B&B in Shanon overnight and prepared for our trip to Scotland.
4: Erin found chocolate | funny spelling | At Heathrow Airport | In the Air
5: The Bus | Charlie & Palm Trees | Dublin | Sunrise, Dublin
6: St. Patrick's Day is an enchanted time - a day to begin transforming winter's dreams into summer's magic. ~Adrienne Cook
7: The White House of Ireland | Molly Malone | The Park | Bunratty Hotel
8: The Merry Ploughboys
9: May luck be our companion May friends stand by our side May history remind us all Of Ireland's faith and pride. May God bless us with happiness May love and faith abide. ~Irish Blessing | Blarney Castle, Co. Cork
10: Kissing the Blarney Stone
11: Climbing up to kiss the Blarney Stone. Who knew it was such a climb up? And who knew that Charlie was afraid of heights? The climb was great, the scenery was beautiful. And kissing the rock didn't take much skill. | (Below) The Rock of Cashel
12: Stuck in Funeral | Kilkenny | Downtown
13: Tree in Swords
14: Skellig Experience
15: The Ring of Kerry, Dingle Peninsula
16: Crossing the Shannon River
17: Cliffs of Moher
18: A beautiful day on top of the Cliffs of Moher
19: Drinking Buddies | Telefon
20: Muckross Gardens
21: Erin
22: Relaxing in Flowers
24: Charlie's best moment in Ireland... Drunker than Drunk at Bunratty's Castle & Medieval Dinner
27: We had... | Lots of Laughs & Lots of Fun
28: After leaving Ireland, Charlie and Erin decided to take off for Edinburgh, Scotland.
29: High Street, Edinburgh
30: They met up with an old friend and former exchange student, Pooh from Thailand who was finishing her masters there at the university. | She showed us around the city, we walked down the royal mile, took a tour of an art gallery and The Royal Botanical Gardens, walked the campus, went to a Japanese restaurant, checked out an old cemetery, and got to eat at Pooh's apartment. | It's good to know that one can remain friends for so long and even though they haven't seen each other for years, they can still pick up where they left off as if they had never left. | It was great getting to see Pooh again, as she hadn't changed a bit. And, even though she was in Scotland... she didn't pick up a Scottish brogue!
32: Enjoying our time with Pooh, at this beautiful place. There were tons of flowers, and gorgeous weather!
33: We even found Erin's initials! LoL! There were tons of them on the ceiling of the inside of one of the buildings at the botanical gardens.
36: Visiting the Cemetery... Eerily beautiful
38: After visiting with Pooh, Charlie and Erin did some wandering around Edinburgh by themselves before hitting up a Rabbies Tour to the highlands.
39: She's in the Guinness Book of World Records | Leave it up to us to find the lady with the most piercings in the world... | And we managed to find her by just wandering... We should wander more often...
41: Who knew climbing Arthur's Seat would be so much fun and lead to us finding Rome! | You can see all of Edinburgh from Arthur's Seat!
43: IRN BRU! | To the left: Scotts Monuement
44: The many things Charlie will not forget about Edinburgh... eating at Imans almost every night, trying haggis, and finding a new drink that he loves and is unable to get back in the U.S.
45: One night, we decided to go on a haunted tour of Edinburgh's underground city. We went on a tour that was put on by Auld Reekie's. It was an interesting experience... Cold, damp, tunnels everywhere... a heaviness in the air, and... a poltergeist room! | On this trip, Erin got to walk into her first poltergeist room. Men and women were to stand on opposite sides of the rooms in fear that the spirits may attack! Thank goodness I survived and I didn't run into this guy ------>
46: Inside St. Giles Cathedral High Street, Edinburgh Built in 1416
47: One of the prettiest pipe organs I've ever seen!
48: Finally! The Highlands!
49: Town of Callendar
50: We traveled up towards Loch Ness, and stopped at some interesting places along the way. Drove all the way up to Inverness, and had lots of fun!
51: The castle from Monty Python and the Holy Grail
53: William Wallace, Braveheart, Monuement
54: Loch Ness
55: WE FOUND NESSIE!
57: Pictures from Edinburgh, Loch Ness, and the town of Calendar
59: And so, Erin and Charlie ate their way through Ireland & Scotland. They had a lot of fun, saw many beautiful places, and met many interesting people along the way. Although their trip is over, they promise to return again. Slán agus beannacht leat!
60: May luck be our companion May friends stand by our side May history remind us all Of Ireland's faith and pride. May God bless us with happiness May love and faith abide. ~Irish Blessing | And now... a blast from the past! 2006
61: Blarney | Blarney
62: The things I will always remember... the 4 seasons all at once! Mom and I managed to have snow on our trip!
64: Rock of Cashel and Blarney
65: Rock of Cashel, Tree in Swords, and what looks to be the Isle of Mann
66: Cows on the side of the road.. they were sooo interested in us! The cute pony!
67: Hanging out with the Conways!
70: (Above) The Shannon River (To the Right) A rainbow over Bunratty Castle | Cliffs of Moher
71: Near the Cliffs of Moher
72: By the Burrens
74: Me with a Guinness Mom excited about our luggage!
75: Up by Skye Road in Clifden, Ireland
77: Taking photos out of the car window
79: Lough Gur, Co. Limerick
Sizes: mini|medium|large|jumbo
By: Erin R.
Title: Ireland & Scotland 2010
Despite the fact that in April the Eyjafjallajokull volcano in Iceland almost put an end to a trip before it even began... In May of 2010, Erin took Charlie on his first trip overseas. They traveled to Ireland and then Scotland.


Watch full Naruto Shippuden Season 20 Episode 31 English Dubbed full HD online. Naruto Shippuden Episode 444 English Dubbed online for free in HD.
Sasuke knocks Izumo and Kotetsu out on his way out of Konoha. He is intercepted by Kakashi, who asks what he is doing. Sasuke thanks him for his training, but explains he is unsatisfied with his progress, revealing he is going to train with Orochimaru, and incapacitates Kakashi with genjutsu. Upon waking up, Kakashi relays the information to Hiruzen, Minato, and Tsunade, herself informing the others about Jiraiya's intel regarding Orochimaru's imminent need for a new vessel. Sakura overhears their conversation. Minato wants to handle the situation internally as discreetly as possible, to avoid worsening the already delicate situation with the Uchiha, and orders a lockdown. The Konoha 11 meet up at the Academy at night, where Sakura and Shikamaru informs the others about Sasuke's departure. Naruto and Shikamaru sneak out of the village through a tunnel created by Kiba, and Naruto promises Sakura to bring Sasuke back. Neji notices someone else sneaking out as well. Kushina informs Minato that Naruto and Shikamaru have escaped the lockdown. Naruto and Shikamaru are attacked by Root Anbu sent by Danzō to ensure no one interferes with Sasuke reaching Orochimaru. Team Guy intercepts the attack. As the fight goes on, Shikamaru realises these are not regular Anbu. Sasuke continues on his way to Orochimaru.