Activation and expression of urokinase-type plasminogen activator are modulated by freezing/thawing process through activation of redox signal pathway in primary porcine endometrial cells

Plasminogen activators (PAs) play a pivotal role in a variety of uterine physiologies, such as endometrial function, trophoblast invasion, and implantation process, but its alteration in expression or activity during cryopreservation of primary uterine cells has received little attention. In this study, we investigated whether PA expression and activity were modulated in first passage primary porcine uterus endometrial epithelium cells (PUEECs) treated with or without a freezing-thawing procedure. Western blotting and zymographic analysis showed that uPA expression and activity increased significantly in frozen-thawed PUEECs in a passage-dependent manner as compared to freshly prepared control cells. Moreover, intracellular reactive oxygen species (ROS) were increased by freezing-thawing and longer culturing, and were more prominent in frozen-thawed PUEECs than in control cells. However, the increase in both uPA expression and activity was greatly reduced or alleviated by treatment with either ROS scavenger N-acetylcysteine or extracellular signal-regulated kinase (ERK) inhibitor PD98059. These results suggest that ROS/ERK-mediated uPA activation may be an important factor in cryo-damage of primary uterine cells.