miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway

Abstract Intercellular communication is a critical process that ensures cooperation between distinct cell types at the embryo–maternal interface. Extracellular vesicles (EVs) are considered to be potent mediators of this communication by transferring biological information in their cargo (e.g., miRNAs) to the recipient cells. miRNAs are small non-coding RNAs that affect the function and fate of neighboring and distant cells by regulating gene expression. Focusing on the maternal side of the dialog, we recently revealed the impact of embryonic signals, including miRNAs, on EV-mediated cell-to-cell communication. In this study, we show the regulatory mechanism of the miR-125b-5p ESCRT-mediated EV biogenesis pathway and the further secretion of EVs by trophoblasts at the time when the crucial steps of implantation are taking place. To test the ability of miR-125b-5p to influence the expression of genes involved in the generation and release of EV subpopulations in porcine conceptuses, we used an ex vivo approach. Next, in silico and in vitro analyses were performed to confirm miRNA–mRNA interactions. Finally, EV trafficking and release were assessed using several imaging and particle analysis tools. Our results indicated that conceptus development and implantation are accompanied by changes in the abundance of EV biogenesis and trafficking machinery. ESCRT-dependent EV biogenesis and the further secretion of EVs were modulated by miR-125b-5p, specifically impacting the ESCRT-II complex (via VPS36) and EV trafficking in primary porcine trophoblast cells. The identified miRNA–ESCRT interplay led to the generation and secretion of specific subpopulations of EVs. miRNA present at the embryo–maternal interface governs EV-mediated communication between the mother and the developing conceptus, leading to the generation, trafficking, and release of characteristic subpopulations of EVs

MiRNAs in the Peri-Implantation Period: Contribution to Embryo–Maternal Communication in Pigs

MicroRNAs (miRNAs) constitute a large family of noncoding RNAs, approximately 22 nucleotides long, which function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathophysiological processes in animals. To date, the regulatory roles of miRNAs in reproduction, such as fertilization, embryo development, implantation, and placenta formation, among others, have been demonstrated in numerous mammalian species, including domestic livestock such as pigs. Over the past years, it appeared that understanding the functions of miRNAs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes the current knowledge on miRNAs, specifically in relation to the peri-implantation period when the majority of embryonic mortality occurs in pigs. To present a broader picture of crucial peri-implantation events, we focus on the role of miRNA-processing machinery and miRNA–mRNA infarctions during the maternal recognition of pregnancy, leading to maintenance of the corpus luteum function and further embryo implantation. Furthermore, we summarize the current knowledge on cell-to-cell communication involving extracellular vesicles at the embryo–maternal interface in pigs. Finally, we discuss the potential of circulating miRNAs to serve as indicators of ongoing embryo–maternal crosstalk

Embryonic signals mediate extracellular vesicle biogenesis and trafficking at the embryo–maternal interface

Abstract Background Extracellular vesicles (EVs) are membrane-coated nanoparticles secreted by almost all cell types in living organisms. EVs, as paracrine mediators, are involved in intercellular communication, immune response, and several reproductive events, including the maintenance of pregnancy. Using a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation, we focused on EV biogenesis pathway at the embryo–maternal interface, when the embryonic signaling occurs for maternal recognition and the maintenance of pregnancy. Results Transmission electron microscopy was used during early pregnancy to visualize EVs and apocrine and/or merocrine pathways of secretion. Immunofluorescent staining localized proteins responsible for EV biogenesis and cell polarization at the embryo–maternal interface. The expression profiles of genes involved in biogenesis and the secretion of EVs pointed to the possible modulation of endometrial expression by embryonic signals. Further in vitro studies showed that factors of embryonic origin can regulate the expression of the ESCRT-II complex and EV trafficking within endometrial luminal epithelial cells. Moreover, miRNA-mediated rapid negative regulation of gene expression was abolished by delivered embryonic signals. Conclusions Our findings demonstrated that embryonic signals are potent modulators of ESCRT-dependent EV-mediated secretory activity of the endometrium during the critical stages of early pregnancy. Video Abstrac

Additional file 1 of Embryonic signals mediate extracellular vesicle biogenesis and trafficking at the embryo–maternal interface

Additional file 1: Supplementary Figure 1. Control staining performed without primary antibodies. Supplementary Figure 2. mRNA expression in luminal epithelial cells. Supplementary Figure 3. Ultrastructural changes in luminal/glandular epithelium and trophectoderm on day 20 of pregnancy. Supplementary Figure 4. Overview of micrographs showing heterogeneous population of extracellular vesicles found in endometrial compartments on Day 12 and 16 of pregnancy. Supplementary Figure 5. CD63 appearance in the endocytic membrane transport pathway. Supplementary Figure 6. miRNA effect on target gene and protein abundance. Supplementary Table 1. List of primary and secondary antibodies used in immunofluorescent staining of endometrial/trophoblast tissue and luminal epithelial cells. Supplementary Table 2. List of mimics used in miRNA and hormone treatment experiment. Supplementary Table 3. List of genes and TaqMan gene expression assays used in real-time PCR