Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

Abstract

Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy.

Exosomes, endogenous retroviruses and toll-like receptors: pregnancy recognition in ewes

“…Of particular interest is the finding of mRNAs and protein for enJSRV, which supports results published by Burns et al (2014). It is known that the conceptus trophectoderm contains enJSRV around day 12 (Dunlap et al 2006) due to the incorporation of viral particles shed from the endometrium (Black et al 2010).…”

“…Indeed, Racicot et al (2012) demonstrated liberation of exosomes by ovine uterine cells in vitro and described the expression of IFNT-related genes in their cargo. Afterwards, another study described the presence of these microvesicles in ovine uterine flushings recovered from day 14 of the estrous cycle and day 14 of pregnancy (Burns et al 2014). The results presented here are the first to demonstrate that membrane-bound organelles matching features previously described for exosomes (Gyö rgy et al 2011, Vlassov et al 2012 are present in ovine uterine flushings as early as day 10 post-estrus/mating, which provide strong support for a role for exosomes in early events responsible for the onset of IFNT secretion and establishment of pregnancy in ewes.…”

Progesterone and conceptus-derived factors important for conceptus survival and growth

“…Uterine luminal fluid (ULF) contains embryotrophic substances, collectively termed histotroph, that drive elongation of the conceptus via effects on trophectoderm proliferation and migration as well as attachment and adhesion to the endometrial luminal epithelium (LE; Gray et al, 2001;Spencer et al, 2008;Bazer et al, 2010;Forde et al, 2014a). The ULF is derived primarily from transport and (or) synthesis and secretion of substances by the endometrial LE and glandular epithelium (GE), but also by the conceptus , and it is a complex and rather undefined mixture of proteins, lipids, amino acids, sugars (glucose, fructose), ions, and exosomes/microvesicles (Bazer, 1975;Gray et al, 2001;Bazer et al, 2012;Burns et al, 2014;Forde et al, 2014b). P4 induces the expression of a number of genes in the endometrial epithelium that are then further stimulated by factors from the conceptus (e.g., IFNT and PGs) and the endometrium itself Brooks et al, 2014;Lonergan and Forde, 2014).…”

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles

“…Cellsecreted vesicles containing bioactive molecules such as miRNA, mRNA and proteins have been shown to mediate cell communication among different cell types (BURNS et al, 2014;. Recently, an experiment demonstrated the transfer of cell-secreted vesicles from parthenogenetic embryos to cloned embryos, providing a new paradigm for embryo-to-embryo communication in vitro (SAADELDIN et al, 2014).…”

“…EXO described in uterine fluid contained miRNA, mRNA and proteins such as CD63 and HSP70 (exosome markers). Different mRNAs were identified in EXO from pregnant and cyclic ewes, among them the endogenous beta retrovirus (enJSRVs), enJSRVs mRNA is produced in the endometrium and is important for throphectoderm differentiation, thus suggesting that EXOs are mediating this process (BURNS et al, 2014).…”