Effect of ovarian storage time at 4 degrees C on cumulus cell apoptosis in porcine antral follicles.

Abstract

The present study was conducted to investigate the effect of cold storage time on apoptosis of cumulus cells (CCs) from porcine ovaries, and to compare the sensitivity of four apoptosis-detection methods. Porcine ovaries were stored in physiological saline solution at 4°C for 0, 7, 24 and 48hr, and then cumulus cells or granulosa cells (GCs) in antral follicles were retrieved to detect cell apoptosis. Cumulus cells isolated from stored ovaries for 24hr presented obvious apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labeling (TUNEL) assay. A typical DNA ladder pattern of apoptosis was observed in GCs 24hr post storage treatment. The mean Olive Tail Moment of CCs was significantly increased after 24hr using comet assay; however, the mean tail migration and mean tail DNA increased gradually after 7hr of storage. In addition, annexin V/PI staining assay showed an obvious increase in apoptotic CCs (Annexin V positive, PI negative) 7hr after treatment, and the apoptotic rate reached to a peak at 24hr followed by a decline after 48hr of storage to the level at 7hr. In conclusion, cold storage of porcine ovary in physiological saline solution induced a time-dependent increase in apoptosis of cumulus cells, and annexin V/PI staining combined with comet assay provided a sensitive and reliable method to detect early damages in cumulus cells induced by cold storage of ovary.