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, polys permy, parthenogenesis, and nuclear transfer is examined with the goal of understanding microtubule activity and the manner in which the cen trosome 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 see n 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, wh ich is anastral, fusiform, and often located eccentrically. During ana phase, asters assemble at each spindle pole, After telophase, these as ters develop into the interphase array of microtubules in the daughter blastomeres. During polyspermy, an aster forms from a site between ea ch incorporated sperm head and tail. Multiple mitotic spindles are obs erved in polyspermic zygotes; multipolar spindles are not seen. Parthe nogenetic activation with 5 mu m ionomycin followed by a 4-hr incubati on in 1.9 mM dimethylaminopurine results in >80% activation, and antit ubulin immunofluorescence microscopy demonstrates that initially disar rayed microtubules are observed and the some microtubules extend from the remnants of the second meiotic spindle. At the time normal for cel l division, these parthenogenotes form anastral, barrel-shaped bipolar mitotic spindies. Asters form at the spindle poles at anaphase and th e parthenogenotes divide from one to two blastomeres. Nuclear fusion o f a morula-derived blastomere to a parthenogenetically activated oocyt e results in either a single microtubule aster or at times, two asters , formed in association with the donated nucleus. These results demons trate that fertilization in the cow is a blending of paternally and ma ternally derived centrosomal material. (C) 1994 Academic Press, Inc.