Small RNA-seq analysis of exosomes from porcine uterine luminal fluid during peri-implantation

Abstract

Background The exosomes of uterine luminal fluid (ULF) mediate intrauterine communication between conceptus and uterus in pigs. The small RNAs of ULF exosomes are widely recognized as important factors that influence embryonic implantation in humans, mice, cattle and sheep. Therefore, it can be hypothesized that small RNAs of ULF exosomes are essential for implantation in pigs. Results Cup-shaped exosomes (diameters: 40–160 nm) of porcine ULF were isolated and characterized, and then PKH67 stained exosomes were taken up by endometrial epithelial cells and porcine trophectoderm cells. The expression of small RNAs in these exosomes on days 10 (D10), 13 (D13) and 18 (D18) of pregnancy was comprehensively profiled through sequencing. From nine libraries, a total of 152 known microRNAs (miRNAs), 43 novel miRNAs and 145 novel Piwi-interacting RNAs (piRNAs) were identified. Among these small RNAs, ssc-miR-21-5p, ssc-let-7a, ssc-let-7i-5p and ssc-let-7g were differentially and highly expressed during the three stages. Hierarchical cluster analysis and principal component analysis suggested that the expression profiles of small RNAs in D13 group were different from those in D10 and D18 groups. Bioinformatics analysis showed that the miRNAs differentially expressed in the comparisons between D10 and D13, D13 and D18, and D10 and D18 groups were involved in important molecular functions and pathways such as “G-protein coupled receptor activity” and “Insulin signaling pathway”. Conclusions Our results reveal that porcine ULF exosomes contain various small RNAs with potentially vital effects on implantation. This research also provides resources for studies of miRNAs and piRNAs in the cross-talk between embryo and endometrium.

Extracellular vesicles: Focus on peri‐implantation period of pregnancy in pigs

“…EVs transport a subset of small RNAs during the implantation cascade (Bidarimath et al, 2017;Hu et al, 2022;Hua et al, 2021;Szuszkiewicz, Myszczynski et al, 2022). In line with dynamic molecular changes occurring at the embryo-maternal interface, miRNA cargo composition also changes in uterine-derived EVs after maternal recognition of pregnancy.…”

“…RNA cargo analysis of porcine uterine-derived EVs at the time of maternal recognition showed enrichment in small biotypes (Hua et al, 2021;Szuszkiewicz, Myszczynski et al, 2022). Bioinformatic analysis of identified specific miRNAs, of possible endometrial and embryonic origin, showed their potential to target genes involved in endometrial receptivity and embryo development by regulating processes such as cell differentiation and cell adhesion, but also processes of embryo-maternal communication-molecular transport and signal exchange between cells (Hu et al, 2022;Hua et al, 2021;Szuszkiewicz, Myszczynski et al, 2022). Other studies showed that the effect of uterine-derived EVs on porcine trophoblast cells depends on their concentration and varies between Days 9 and 12 of pregnancy.…”

“…In recent years, despite numerous limitations in analyzing extracellular RNA (see reviews of Mateescu et al, 2017;Théry et al, 2018), most known noncoding RNA types, such as ribosomal RNA, transfer RNA, Y-RNA, large intergenic noncoding RNA (lincRNA), small nuclear RNAs, small nucleolar RNAs (snoRNAs), lncRNAs, PIWIinteracting RNAs (piRNAs), mitochondrial RNAs, and vault RNAs were found in EVs (Hua et al, 2021;Huang et al, 2013;C. C. Li et al, 2013;Nolte-'t hoen et al, 2012).…”

“…Hierarchical clustering of miRNAs detected in uterine-derived EVs pointed to a characteristic subset of miRNAs abundant on Day 13 of pregnancy, different from those increased on Days 10 and 18 (Hua et al, 2021). Among miRNAs repeatedly detected in uterine-derived EVs by several investigators are those characterized as important for endometrial receptivity (e.g., let-7a and let 7g in humans and mice (Q.…”

Early steps of embryo implantation are regulated by exchange of extracellular vesicles between the embryo and the endometrium

“…EVs have already gained attention among reproductive biologists, providing increasing data on their specific roles (17)(18)(19). To date, EVs have been identified as being released by embryos from the earliest days after fertilization (20)(21)(22), through the periimplantation phase (23)(24)(25)(26)(27), to the stages when placenta is already formed (28). EVs have also been studied in various reproductive pathophysiological conditions and diseases, including preeclampsia (29).…”