Oocyte Membrane Components Preventing Trypsin-Induced Germinal Vesicle Breakdown in the Surf Clam Oocytes

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Maturation of surf clam oocytes can be induced with sperm, KCl, serotonin or calcium ionophore. We now report that the oocytes treated with trypsin can also induce germinal vesicle breakdown (GVBD). Oocytes incubated in 0.1% trypsin solution for 0.5 to 1 h, subsequently washed and incubated in control medium for 1 to 2 h underwent maturation. Treatment with glycosidase, DNase and RNase did not induce GVBD. The optimal condition for the induction of maturation was to incubate oocytes in 0.1 % trypsin solution for 1 h and to transfer the oocytes to a medium composed of 0.4 M Tris-HCl, pH 7.4, 20 mM CaCl2. The occurrence of spontaneous GVBD was dependent upon the presence of Ca2+ in the suspending medium. The optimal concentration of Ca2+ was 20 mM. To show that a membrane component is involved in sustaining maturation arrest, oocytes were incubated in a medium containing 1 M urea, 5 mM EDTA, 10 mM Tris-HCl, pH 7.4, for 30 min. The extracted membrane protein at final concentrations of 0.4 to 0.8 mg/ml blocked GVBD of trypsin treated oocytes. To identify the active component, the extracted membrane protein preparations were fractionated by gel filtration through a Sephadex G-100 column. The active principle had an estimated Mr of 18 kD based on elution position of standard proteins on gel filtration. The partially purified product was active at a concentration of 0.1-0.2 mg/ml. The present findings suggest that maturation arrest is sustained by an oocyte membrane protein and the resumption of oocyte maturation results from the hydrolysis of this substance(s) by a protease in the presence of Ca2+.
日本繁殖生物学会の論文

Oocyte Membrane Components Preventing Trypsin-Induced Germinal Vesicle Breakdown in the Surf Clam Oocytes

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