Introduction: The interaction of gametes and embryo with the maternal environment has a crucial impact on gametes maturation, embryonic development and subsequent pregnancy success. Recent studies have recognised extracellular vesicles (EVs) as a potent vehicles for intercellular communication. Defining the type of EVs which are produced by different reproductive cells will help us to understand how these structures can influence reproductive processes. The aim of the current investigation is to characterise EVs secreted by Porcine oviductal epithelial cells (POECs) in vitro in conditioned medium (CM).
Methods: EVs were purified from CM by size exclusion chromatography (SEC) using Sepharose CL-2B SEC columns. Twenty eluted fractions each 500 μl were collected and analysed by Zetaview nanoparticle tracking analyser (Particle Metrix GmbH, Meerbusch, Germany) to measure EVs concentration and size distribution. Bicinchoninic acid protein assay was used to determine the efficacy of SEC in separating vesicles from soluble CM proteins. One-dimensional gel electrophoresis was used to visualize the protein profile of the purified EVs. Collected fractions were investigated for the presence of tetraspanin CD63 protein (cell surface marker for EVs) using western blot analysis.
Results and discussion: Size particle analysis confirmed the presence of particles of 70150 nm with the concentration 1×106 to 1×107 particles/ml from fraction 4 to 12. Negligible soluble protein was detected until fraction 12. The bulk of protein started to elute from fraction 13 onwards. The presence of CD63 was detected in SEC fraction 4 to fraction 12. In conclusions, SEC methodology efficiently isolated EVs from POEC CM with low levels of soluble proteomic contaminants. The obtained data will enhance our knowledge of periconception environment and the early stages of communication between maternal tract, gametes and embryo.
11 Jul 2016 - 11 Jul 2016