High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.