Stimulation of Xenopus laevis oocyte maturation by methyl-β -cyclodextrin

Abstract

The cholesterol-depleting drug methyl-β-cyclodextrin (Me-β-CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5-50 mM Me-β-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-β-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [ 3H]cholesterol, 30-90 min of treatment with 5-50 mM Me-β-CD removed 50%-70% of cell-associated label, and cholesterol depletion was not observed with α-cyclodextrin. After long-term (20-23 h) labeling of oocytes with [3H]cholesterol, Me-β-CD treatment resulted in dose-dependent cholesterol depletion in the 5-50 mM range, and 50 mM Me-β-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5-50 mM Me-β-CD also decreased recovery of low-density membrane by detergent-free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.