Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes

Abstract

Western blottingPre-cast 3-8% Tris-acetate gels (Invitrogen) and a mouse monoclonal anti-CENP-E antibody (Abcam) were used for CENP-E. BubR1, securin, GAPDH and actin immunoblotting were performed as described (Homer et al., 2009;Homer, 2011). HRP-conjugated antibodies were detected using ECL Plus (GE Healthcare) and protein bands were semi-quantitatively assayed (Homer et al., 2009 SUMMARYThe spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles.

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

“…17,44,45 One possibility not explored so far is that maternal aging may affect SAC activity, due to a lowering of Mad2 and pAurora C, but the reduction is not so dramatic as to compromise SAC function in the ways that have been measured previously. Oocytes proceed through MI with unattached and/or incorrectly attached bivalents [19][20][21][22][23] that from equivalent mitotic chromosome studies are predicted to activate a checkpoint, and yet complete microtubule depolymerization leads to a meiotic arrest. 17,45 These seemingly conflicting observations are resolved by the proposition that a small number of attachment errors are tolerated by the SAC, because it may not generate a sufficiently strong "wait-anaphase" signal, whereas when many bivalents are affected, the SAC is no longer weak and can act as a checkpoint.…”

“…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.…”

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

“…Several lines of evidence, notably from meiotic systems, suggest that chromosome positioning per se is not being monitored by the SAC (Gui and Homer 2012;Lane et al 2012;Winey et al 1995;Straight et al 1997;Hays et al 1982;Palevitz 1990). Yet, in many species, chromosomes do align at the cell equator, defining a state known as metaphase.…”

Mouse oocyte, a paradigm of cancer cell

“…It has been recently established that during mammalian meiosis I, the SAC has a surprisingly low threshold for satisfaction. [32][33][34][35] In mitosis, Mad2 is displaced from the chromosomes when they are bi-oriented at the metaphase plate via kinetochore-microtubule attachment. In meiosis I, Mad2 is lost from kinetochores 3 to 4 h before anaphase, even when all K-fibers are not assembled and in the presence of chromosomes that have not become aligned at the metaphase plate and that are not bi-oriented.…”