Abstract
The role of calcium (Ca^+ +^) during spontaneous meiotic maturation of pig oocytes was examined. The hypothesis that elevations of endogenously derived intracellular Ca^+ +^ are prerequisite for germinal vesicle breakdown (GVBD) and progression through meiosis was tested. In addition, investigations were carried out to determine whether GVBD and meiotic progression were dependent upon extracellular Ca^+ +^ influx. Elevation of endogenously derived Ca^+ +^ was inhibited directly by loading cells with BAPTA, a specific Ca^+ +^ chelator, or indirectly with neomycin. Extracellular Ca^+ +^ influx was prevented by culturing oocytes in Ca^+ +^โdeficient medium, with EGTA, or in the presence of the Ca^+ +^ channel blocker verapamil. Pretreatment with BAPTA/AM and subsequent culture in the absence of added exogenous Ca^+ +^ resulted in a similar inhibition of GVBD (1 ฮผM BAPTA/AM vs. untreated, P < 0.01). After 4 h following follicular release, oocytes were no longer sensitive to BAPTA/AM treatment. Neomycin also significantly inhibited GVBD (0.5 mM neomycin vs. untreated, P < 0.05) as well as meiotic progression past metaphase I (0.25 mM neomycin vs. untreated, P < 0.05). The incidence of GVBD was not significantly affected when oocytes were simply cultured in Ca^+ +^ deficient medium or when cultured in the presence of EGTA or verapamil. However, progression of meiosis past GVBD to metaphase II was suppressed by reducing levels of Ca^+ +^ in the culture medium (0.68 mM Ca^+ +^ vs. 1.7 mM Ca^+ +^, P < 0.05) and by treatment with verapamil (0.25 mM verapamil vs. untreated, P < 0.05). Meiotic progression was completely blocked at metaphase I in the presence of 0.85 mM EGTA. These results implicate a central role for the mobilisation of intracellular Ca^+ +^ for the initiation of GVBD, and suggest that external Ca^+ +^ influx is not a direct requirement for GVBD, although it may be required for development past metaphase I in pig oocytes. ยฉ 1993 WileyโLiss, Inc.