Effect of Calcium Ion on the Maturation of Cumulus-enclosed Pig Follicular Oocytes Isolated from Medium-sized Graafian Follicles1

Abstract

Porcine follicular oocytes from medium-sized follicles (3-5 mm in diameter) were cultured in modified Hank''s balanced salts solution (MHBS) to which pyruvate, lactate and glucose were added as an energy source. Bovine serum albumin (0.4%) was added as a protein source and the oocytes were cultured for 42 h at 37.degree. C in 5% CO2 in air. In this medium porcine oocytes underwent 80-90% nuclear maturation after 42 h. Oocytes were cultured in MHBS with various amounts of CaCl2 as well as in the presence of verapamil, a Ca2+ channel blocker and the divalent cationophore A23187 [calcimycin]. The lowest concentration of Ca2+ required for oocyte maturation was .apprx. 0.0265-0.053 mM. Such a requirement for Ca2+ in the culture medium extended through metaphase II. If Ca2+ was omitted during the final 4 h of culture, the metaphase II chromosomes appeared extremely condensed or degenerated. Verapamil at a level of 0.2 mM inhibited germinal vesicle breakdown or resulted in degeneration; lower concentrations did not affect oocyte maturation. In the presence of 0.02 mM verapamil, the maturation of cumulus-enclosed oocytes ws not affected; at the same dose of verapamil the maturation of denuded oocytes was inhibited. Less than 3.8 .times. 10-7 M divalent cationophore did not inhibit oocyte maturation. Maturation was inhibited by 3.8 .times. 10-7 and 3.8 .times. 10-6 M divalent cationophore. Maintenance of oocytes in a nondegenerated state also requires the constant presence of Ca2+ in the culture medium.