부산대학교 도서관

The centrosomes are the major microtubule-organizing centers in somatic cells and have an essential role in spindle assembly and chromosome segregation. Centrioles are also found in the zygotes, but they are introduced by the spermatozoon at fertilization and they are structurally different to the canonical centrioles. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, but its role in chromosome segregation remains unknown. The aim of this study is to investigate if centrosome malpositioning or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. Bovine oocytes were retrieved from slaughterhouse ovaries and matured in vitro. Matured oocytes were fertilized in vitro with semen from a bull of proven fertility and, either fixed at 27-29h post-fertilization, or used for time-lapse light-sheet imaging after injection of mRNA for microtubule and chromatin reporters. Fixed bovine zygotes were immunostained for chromatin, alpha-tubulin, centriolar and pericentriolar components, and imaged with a confocal microscope. Microtubule nucleation ability was evaluated using a microtubule regrowth assay. Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malpositioned, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation, whereas 44% of zygotes with abnormal centriole positioning showed lagging chromosomes and the formation of micronuclei. When centriole duplication was prevented, the sperm-derived centrioles showed a similar ability to recruit pericentrosomal material (NEDD1) and nucleate microtubules. However, live imaging showed that in 82% (14/17) of PLK4i treated zygotes the spindle, although bipolar, was unusually elongated and chromosomes either failed to align correctly on the metaphase plate or showed anaphase lag. Moreover, in treated zygotes where both centrioles were positioned at the same spindle pole, the spindle failed to anchor correctly to the cell membrane, resulting in rotational behavior of the zygote and an increase in cytokinesis failure of up to 47% (8/17). These results show how correct centrosome positioning guarantees spindle stability and correct chromosome segregation. On the other hand, when centrosomes are malpositioned or inactive, there is an increase in chromosome segregation errors and failure of cytokinesis. [ABSTRACT FROM AUTHOR]