Purification of meiotic spindles and cytoplasmic asters from mouse oocytes

Abstract

The unfertilized mouse oocyte is arrested at second metaphase of meiosis with microtubules existing exclusively in the meiotic spindle. Multiple inactive cytoplasmic microtubule organizing centers (MTOCs) are also present. These MTOCs can be identified immunocytochemically with an autoimmune serum (No. 5051) directed against pericentriolar material (PCM) and also by their nucleating capacity in the presence of taxol which effectively lowers the critical concentration for tubulin polymerization. Taxol induces the formation of cytoplasmic microtubule asters around the PCM foci, a process which also occurs in untreated eggs after fertilization. The molecular characterization of these structures has not been undertaken previously, probably due to the very small amount of material available. We have developed a single-step purification procedure by which very clean preparations of meiotic spindles and cytoplasmic asters can be obtained, as judged by phase-contrast microscopy and transmission electron microscopy. The purified structures were shown to correspond to those observed in vivo: positive staining of the spindles was observed with anti-tubulin and anti-phosphoprotein (MPM2) antibodies, and positive staining of the MTOCs was observed with MPM2, No. 5051, and anti-calmodulin antibodies. As expected, tubulin was the major protein present in the preparations. Silver staining of SDS-PAGE also revealed the presence of a small number of other polypeptides ( M r of around 47, 35, and 25K). Amongst newly synthesized polypeptides associated with the preparation, two prominent high molecular weight proteins (>200K) were enriched in addition to tubulin and polypeptides with M r of around 52, 41, and 35K.