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Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk.

ABSTRACT: 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.

SUBMITTER: Bidarimath M

PROVIDER: S-EPMC5228034 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk.

Bidarimath Mallikarjun M Khalaj Kasra K Kridli Rami T RT Kan Frederick W K FW Koti Madhuri M Tayade Chandrakant C

Scientific reports 20170112

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 demonstrat ...[more]

PMID: 28079186

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Project description:Exosomes are nanoparticles (?100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100-300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50-150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.

Project description:BackgroundEmbryo implantation is a complex, synchronized process that requires establishment of a reciprocal dialogue between a receptive endometrium and developing blastocysts. Recently, microRNAs (miRNAs), known to modulate gene expression through post-transcriptional mechanisms, were implicated in regulation of early pregnancy events including maternal recognition of pregnancy and implantation. To characterize complex transcriptomic changes, expression of miRNAs in pregnant and cyclic endometria collected on days 12, 16 and 20 was analyzed using Illumina deep sequencing and analyzed with bioinformatic pipeline. Moreover, expression profiles of ten genes related to miRNA synthesis and transport such as DROSHA, DGCR8, XPO5, DICER, TARBP2, TNRC6A, and AGO1-4 were determined.ResultsAmong genes involved in miRNA transport and synthesis DROSHA, XPO5, DICER1, TARBP, and AGO1 expression was affected by the reproductive status. Moreover, DICER1 and AGO2 proteins were localized in luminal and glandular epithelium with immunofluorescence staining. Several hundred mature, canonical and non-canonical miRNAs were found to be expressed in the endometrial samples. Detailed analysis revealed that miRNA length variants, isomiRs, accounted for the vast majority of defined sequences. Both miRNA and isomiR of miR-140-3p were shown to affect expression of putative targets in endometrial stromal cells in vitro. Computational analysis of putative target genes for miRNAs differentially expressed (DE) between pregnant and cyclic animals resulted in lists of biological processes and regulatory pathways indicating their role in cellular development, cell cycle, immunological response and organismal development. Among predicted target genes for DE miRNAs, vascular endothelial growth factor (VEGF), progesterone and estradiol receptors (PGR, ESR1) and leukemia inhibitory factor (LIF) were found.ConclusionsThis research revealed a repertoire of pregnancy-related miRNAs in porcine endometrium during initial stages of conceptus implantation and during the estrous cycle, and sheds light on mechanisms regulating miRNA-mediated gene expression at the maternal-conceptus interface.

Dataset Information

Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk.

Publications

Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk.

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