Verapamil Reversibly Inhibits Spontaneous Parthenogenetic Activation in Aged Rat Eggs CulturedIn Vitro

Abstract

The present study was designed to investigate whether verapamil could inhibit spontaneous parthenogenetic activation in aged rat eggs cultured in vitro. Eggs collected from oviduct after 19 h post human chorionic gonadotropin (hCG) were arrested at the metaphase-II (M-II) stage and exhibited a first polar body. Culture of these aged eggs in calcium/magnesium (Ca(2+)/Mg(2+))-deficient and serum-free medium for 3 h induced exit from M-II, a morphological sign of spontaneous parthenogenetic activation in all eggs. However, verapamil reversibly inhibited spontaneous parthenogenetic activation in a dose-dependent manner. Further, lower doses (6.25, 12.5, and 25 muM) of verapamil induced egg survival, while higher doses (50 and 100 muM) were associated with the appearance of morphological apoptotic features such as shrinkage, membrane blebbing and cytoplasmic granulation prior to degeneration. The DNA fragmentation was induced [as evidenced by terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) positive staining] in eggs undergoing morphological apoptotic changes. On the other hand, caspase-3 inhibitor (1 muM) partially inhibited morphological apoptotic changes (44.34+/-3.53%) suggesting the involvement of both Ca(2+)and caspase-3-mediated apoptotic pathways. These findings suggest that verapamil reversibly inhibits spontaneous parthenogenetic activation and induces egg survival at lower doses, while higher doses induce cell death via apoptosis.