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. Transmission electron microscopy was used during early pregnancy to visualize different populations of 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 at 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 in luminal epithelial cells. Moreover, miRNA-mediated rapid negative regulation of gene expression was abolished by delivered embryonic signals. Our findings demonstrated that embryonic signals are potent modulators of EV-mediated secretory activity of the endometrium during the critical stages of early pregnancy.

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.

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During mouse embryogenesis initial specification of the cell fates depends on the type of division during 8- to 16- and 16- to 32-cell stage transition. A conservative division of a blastomere creates two polar outer daughter cells, which are precursors of the trophectoderm (TE), whereas a differentiative division gives rise to a polar outer cell and an apolar inner (the presumptive inner cell mass - ICM) cell. We hypothesize that the type of division may depend on the interactions between blastomeres of the embryo. To investigate whether modification of these interactions influences divisions, we analyzed the pattern of blastomere division and cell lineage specification in chimeric embryos obtained by injection of a different number of mouse embryonic stem cells (ESCs) into 8-cell embryos. As the ESCs populate only the ICM of the resulting chimeric blastocysts, they emulated in our model additional inner cells. We found that introduction of ESCs decreased the number of inner, apolar blastomeres at the 8- to 16-cell stage transition and reduced the number of ICM cells of host embryo-origin during formation of the blastocyst. Moreover, we showed that the proportion of inner blastomeres and their fate (EPI or PE) in chimeric blastocysts was dependent on the number of ESCs injected. Our results suggest the existence of a regulative mechanism, which links number of inner cells with a proportion of conservative vs. differentiative blastomere divisions during the cleavage and thus dictates their developmental fate.

The establishment of cell‐to‐cell communication between the endometrium and the developing embryo is the most important step in successful mammalian pregnancy. Close interaction between the uterine luminal epithelium and trophoblast cells requires triggering timely molecular dialog for successful maternal recognition of pregnancy, embryo implantation, and placenta development. Quite recently, extracellular vesicles (EVs) carrying unique molecular cargo emerged as evolutionarily conserved mediators of cell‐to‐cell communication during early pregnancy. To date, the presence of EVs at the embryo–maternal interface has been demonstrated in numerous mammals, including domestic livestock, such as pigs. However, few studies have focused on revealing the mechanism of EV‐mediated crosstalk between developing early embryos and receptive endometrium. Over the past years, it has appeared that understanding the role of EVs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes current knowledge of EVs, specifically in relation to the peri‐implantation period in pigs, characterized by common features of embryo implantation and high embryonic mortality in mammals.

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.

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.