Effect of Oxidative Stress on Extracellular Vesicles Secreted by Caruncular Epithelial Cells Isolated from Bovine Placenta During Pregnancy-Preliminary Results.

Maintaining the pro- and antioxidative balance during pregnancy is crucial. Oxidative stress (OS) can complicate pregnancy and disrupt maternal-foetal communication, which is partly mediated by extracellular vesicles (EVs). This study investigated how OS affects EV secretion by bovine caruncular epithelial cells isolated from the established bovine placenta (4th month of gestation). To induce OS, cells were treated for 48 h with 1% cigarette smoke extract (CSE), known to increase reactive oxygen species (ROS) levels, while controls remained untreated. EVs were isolated via ultracentrifugation (UC) and size-exclusion chromatography (SEC) and characterized using Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy (TEM), and Western blotting (WB). WB confirmed small-EV enrichment in fractions 5-8 of SEC by detecting CD9 and flotillin-1 markers. TRPS analysis demonstrated that OS increased EV concentration. In fractions 5-6, CSE treatment increased EV concentration from 1.79 × 109 (mode diameter 127 nm) to 3.12 × 109 particles/mL (122 nm). In fractions 7-8 of SEC, concentration rose from 4.48 × 1010 (84 nm) to 7.53 × 1010 particles/mL (80 nm). These data indicate that OS increases EV secretion by bovine maternal epithelial cells, providing a basis for future studies on the potential role of stress-induced EVs in foetal-maternal communication.