Accumulating evidence indicates that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, increases in intracellular reactive oxygen species (ROS), and abnormal Ca2 oscillations. The objective of the present study was to characterize the relationships between intracellular ROS, Ca2 homeostasis of endoplasmic reticulum (ER), and fragmentation in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in elevated intracellular ROS level, impaired ER Ca2 homeostasis (i.e., Ca2 storage, Ca2 rising patterns after electroactivation, and the cluster distribution of ER), and increased fragmentation rates. However, when the porcine oocytes were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular Ca2 chelator, the fragmentation was significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit, respectively, the intracellular ROS levels in aged porcine oocytes during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca2 regulation of ER, leading to a high proportion of fragmented oocytes. In contrast, CHX, an intracellular inhibitor of ROS generation, prevented both increase of ROS level and damage of the ER Ca2 homeostasis in porcine oocytes during aging, resulting in low fragmentation rate. We conclude that the increased intracellular ROS damaged the ER clusters and ER Ca2 homeostasis, resulting in a disorder in ooplasmic free Ca2 , which caused the fragmentations seen in porcine MII oocytes during aging.
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Vol. 89 • No. 5