Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension

Abstract

SUMMARYHomologous chromosome segregation errors during meiosis I are common and generate aneuploid embryos. Here, we provide a reason for this susceptibility to mis-segregation by live cell imaging of mouse oocytes. Our results show that stable kinetochoremicrotubule attachments form in mid-prometaphase, 3-4 hours before anaphase. This coincided with the loss of Mad2 from kinetochores and with the start of anaphase-promoting complex/cyclosome (APC/C)-mediated cyclin B1 destruction. Therefore, the spindle assembly checkpoint (SAC) ceased to inhibit the APC/C from mid-prometaphase. This timing did not coincide with bivalent congression in one-third of all oocytes examined. Non-aligned bivalents were weakly positive for Mad2, under less tension than congressed bivalents and, by live-cell imaging, appeared to be in the process of establishing correct bi-orientation. The time from when the APC/C became active until anaphase onset was affected by the rate of loss of CDK1 activity, rather than by these non-aligned bivalents, which occasionally persisted until anaphase, resulting in homolog non-disjunction. We conclude that, in oocytes, a few erroneous attachments of bivalent kinetochores to microtubules do not generate a sufficient SAC 'wait anaphase' signal to inhibit the APC/C.

Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice

“…This procedure causes complete dissolution of all microtubule fibers and a re-engagement of Mad2 with all unoccupied kinetochores. 22 When the same immunofluorescence analysis was performed for centromere-associated pAurora C, we similarly observed a significant fall in aged oocytes to a level about half that in young mice (Fig. 2C).…”

“…This analysis was performed at 5 h after GVBD, a timepoint that would correspond to when error correction is active and needed. 22,29 These data imply that with age, the centromere-kinetochore of a bivalent possesses reduced ability to recruit or retain both Mad2 and pAurora C. Indeed, this phenomenon of reduced levels may be more general, given that immunostaining with anticentromeric antibody (ACA), which detects centromeric protein epitopes, was also found to be 61% lower in aged oocytes when compared with young ( Fig. 2D).…”

“…We assessed Mad2 on kinetochores at 3 h after resumption of MI, marked by germinal vesicle breakdown (GVBD), when the SAC is active, 22 and found a significant ~30% reduction in mean levels in oocytes from aged mice ( Fig. 2A).…”

“…The SAC is certainly active in mouse oocytes, as its loss can raise aneuploidy rates considerably. 1,[15][16][17][18] However, the SAC is not as stringent in mouse oocytes as it is in somatic cells, [19][20][21][22][23] and in human and mouse oocytes, aging is associated with a reduction in levels of SAC components such as Mad2. [24][25][26][27] Although these data offer credence for a SAC basis to the maternal aging phenomenon, there are other studies demonstrating the SAC is active in aged oocytes.…”

“…Second, the length of MI, which would be much shorter in the absence of the SAC, is observed to be no different in aged oocytes, and, further, there is no association between aneuploid eggs and the time taken to transit through MI, as there should be if the SAC were being bypassed, resulting in chromosome mis-segregation. 28 Given recent work establishing the SAC is not so stringent in MI, [19][20][21][22][23] and that the establishment of correct KT-MT for chromosomes (bivalents) is very poor, 29 we decided to reexamine changes in the SAC with age. We used C57Bl6/J mice, because it is the standard laboratory mouse used in genome sequencing.…”