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 Cupshaped 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. BackgroundPrevious studies have indicated that the majority of pre-natal losses occur during the periimplantation period in pigs [1,2]. During this period, the porcine blastocyst expands from 0.5-1 mm in diameter at hatching to 2-mm spheres on day 10 of pregnancy (D10) and begins to rapidly elongate into tubular and filamentous shapes on day 11 or 12 [3,4]. The conceptus starts to attach to the surface of maternal endometrium at day 13 of pregnancy (D13) and the attachment is usually completed between days 18 (D18) and 24 of pregnancy [5,6]. The synchronous communication between conceptus and uterus exerts strong influence on embryo implantation [7]. Furthermore, the

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.…”

“…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).…”

“…Bidarimath et al (2017) further presented that the porcine trophoblast cell line established from Day 12 porcine trophoblast secretes EVs carrying proteins and miRNAs involved in angiogenesis, a process crucial during early pregnancy (Kaczmarek et al, 2008). 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).…”

“…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.…”

One Health challenges for pig reproduction

“…In this review we also wish to address certain comparative disease perspectives that concern pig reproduction, as examples of research areas that are associated with the One Health triad, namely the environment, animal health, and human health. Research around obesity (Oliviero et al, 2010), low-grade inflammation (Coleman et al, 2020), extracellular vesicles (Hua et al, 2021), and pre-and postnatal programming (Bagnell and Barton, 2019) all represent areas of comparative, translational reproductive health research that is interconnected within the One Health triad. Environmental toxins are another vital area of One Health research, especially regarding toxins with expected increases in future use, such as heavy metals.…”