O-008 Sperm-derived centrioles play a key role in ensuring correct chromosome segregation in the first mitotic division of mammalian zygotes

Abstract

Abstract Study question What is the role of the centrosomes during mitotic division in the mammalian zygote? Does the atypical structure of sperm-derived centrioles affect their function? Summary answer Correct centrosome positioning guarantees spindle stability and correct chromosome segregation. Centrosome malposition or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. What is known already The centrosomes of somatic cells comprise two centrioles surrounded by pericentriolar material, and are major microtubule-organizing centres with an essential role in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and, although two centrioles are introduced by the spermatozoon at fertilization, they are structurally different to the canonical centrioles found in somatic cells. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, when they are often mal-positioned (not at the spindle poles). However, the causes and consequences of abnormal centrosome positioning in zygotes are not known. Study design, size, duration We combined systematic immunofluorescence of bovine zygotes, fixed at different stages, with real-time imaging of live zygotes to investigate the implications of centrosome mal-positioning during zygotic mitosis on the fidelity of chromosome segregation and cytokinesis. To study the functionality of the sperm-derived centrioles alone, we inhibited the formation of daughter centrioles using the PLK4 inhibitor (PLK4i), Centrinone. Bovine zygotes were used because the structure of bovine and human sperm-derived centrioles are very similar. Participants/materials, setting, methods 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. Main results and the role of chance Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malposition, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation. Centrosome malposition and inactivity induced chromosome segregation errors in 44% and 82% of the zygotes respectively. PLK4i treatment successfully inhibited centriole duplication, since the volume of core-centriole protein (POC5) in treated zygotes was half that in non-treated zygotes. 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 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 and resulted in rotational behavior of the zygote and an increase of cytokinesis failure from up to 47% (8/17). Therefore we conclude that centrosomes are likely to play a key role in correct chromosome segregation in mammalian zygotes. Limitations, reasons for caution For ethical reasons, we could not manipulate and study human zygotes. Instead, we chose an animal model with a rate of embryonic aneuploidy similar to man. It is, however, possible that zygotic centrosome function differs between these two species. Wider implications of the findings A better understanding of the key regulators of centrosome function might lead to the development of interventions to reduce the risk of malfunction, thereby reducing the incidence of embryonic aneuploidy. Moreover, our study unveiled an mechanism by which impaired PLK4 function induces aneuploidy in mammalian embryos. Trial registration number not applicable