Microtubule organization in the cow during fertilization, polyspermy, parthenogenesis, and nuclear transfer: the role of the sperm aster.

Microtubule organization in bovine oocytes during fertilization, polyspermy, parthenogenesis, and nuclear transfer is examined with the goal of understanding microtubule activity and the manner in which the centrosome during fertilization and mitotic spindle poles are established. In the unfertilized bovine oocyte, microtubules are detected only in the metaphase-arrested second meiotic spindle; no cytoplasmic asters are observed. After insemination, a small aster of microtubules is seen adjacent to the incorporated sperm head. This aster enlarges and, at the time of pronuclear apposition, fills the cytoplasm. At prophase, the aster splits and forms the poles for the first mitotic spindle, which is anastral, fusiform, and often located eccentrically. During anaphase, asters assemble at each spindle pole. After telophase, these asters develop into the interphase array of microtubules in the daughter blastomeres. During polyspermy, an aster forms from a site between each incorporated sperm head and tail. Multiple mitotic spindles are observed in polyspermic zygotes; multipolar spindles are not seen. Parthenogenetic activation with 5 microns ionomycin followed by a 4-hr incubation in 1.9 mM dimethylaminopurine results in > 80% activation, and antitubulin immunofluorescence microscopy demonstrates that initially disarrayed microtubules are observed and the some microtubules extend from the remnants of the second meiotic spindle. At the time normal for cell division, these parthenogenotes form anastral, barrel-shaped bipolar mitotic spindles. Asters form at the spindle poles at anaphase and the parthenogenotes divide from one to two blastomeres. Nuclear fusion of a morula-derived blastomere to a parthenogenetically activated oocyte results in either a single microtubule aster or at times, two asters, formed in association with the donated nucleus. These results demonstrate that fertilization in the cow is a blending of paternally and maternally derived centrosomal material.