(190) Endometrial Uptake of Intrauterine Extracellular Vesicles in Cows

Rei Ichikawa^１; Satoshi Ohkura^１; Sho Nakamura^１; Koji Kimura^２; Shuichi Matsuyama^１

^１Laboratory of Animal Production Science, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

^２Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan
 

Abstract Text:

Lack of uterine receptivity and decreased embryo viability are the major causes of pregnancy failure in cows. Bovine embryonic death often occurs before implantation. Intrauterine fluid components are responsible for the early embryonic development and survival in cows as pre-implantation embryos float in the uterine luminal fluid. Extracellular vesicles (EVs), known as intercellular communication mediators, are uterine luminal fluid components containing microRNAs (miRNAs). We previously demonstrated that intrauterine miRNAs contained in EVs recovered from low-fertility cows were taken up by pre-implantation embryos and reduced the expression of genes involved in embryonic development. Specifically, miR-1 is the most highly expressed miRNA in the uterine EVs of low-fertility cows. Its transfection into endometrial stromal cells via lipofection significantly decreases (p < 0.05) the transgelin 2 mRNA levels. As transgelin 2 is involved in cell proliferation and migration, which contribute to pre-implantation endometrial remodeling, miRNAs in intrauterine EVs possibly affect the endometrium, thereby decreasing the cow fertility. In this study, we aimed to further verify the uptake of EVs by the endometrial tissues of cows during the luteal phase. EVs were isolated from the uterine perfusate non-surgically collected from Japanese Black cows during the luteal phase using a balloon catheter. Subsequently, the EVs were fluorescently labeled and returned to the uterus of the same cow the following day with phosphate-buffered saline. The entire uterus was collected after 24 hours. The tissues were fixed with 10% formalin, cut into 6-μm sections using a cryostat, stained with 4',6-diamidino-2-phenylindole, and observed under a microscope. Green signals of labeled EVs were observed on the apical sides of the luminal and glandular epithelial cells and around the nuclei of some endometrial stromal cells. This result suggests that endometrial luminal and glandular epithelial cells, as well stromal cells as, are exposed to the uterine luminal EVs, which influence the survival and development of the embryos during the luteal phase. We are currently analyzing the mechanisms by which EVs in the uterine lumen pass through the epithelial cells to reach stromal cells in cows. Moreover, as EVs are also taken up by the luminal and glandular epithelial cells, we are analyzing the effects of the miRNAs in intrauterine EVs on these cells.