Kinetochore microtubule establishment is defective in oocytes from aged mice.

Report Report Cell Cycle 13:7, 1171–1179; April 1, 2014; © 2014 Landes Bioscience Kinetochore microtubule establishment is defective in oocytes from aged mice Maria Shomper, Christina Lappa, and Greg FitzHarris* Cell and Developmental Biology; University College London; London, UK Keywords: oocyte aging, spindle, chromosome segregation, kinetochore, cohesin, aneuploidy Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible for age-related oocyte aneuploidy is unknown. Here we report that oocytes from naturally aged mice exhibit substantially increased chromosome misalignment, and fewer kinetochore pairs that make stable end-on attachments to the appropriate spindle poles compared with younger oocytes. The profile of misattachments exhibited is consistent with the types of chromosome segregation error observed in aged oocytes. Loss of chromosome cohesion, which is a feature of oocytes from older females, causes altered kinetochore geometry in meiosisI. However, this has only a minor impact upon MT attachment, indicating that cohesion loss is not the primary cause of aneuploidy in meiosis-I. In meiosis-II, on the other hand, age-related cohesion loss plays a direct role in errors, since prematurely individualized sister chromatids misalign and misattach to spindle MTs. Thus, whereas cohesion loss leading to precocious sister chromatid separation is a direct cause of errors in meiosis-II, cohesion loss plays a more minor role in the etiology of aneuploidy in meiosis-I. Our data introduce altered MT-kinetochore interactions as a lesion that explains aneuploidy in meiosis-I in older females. Introduction Ensuring that daughter cells inherit the correct chromosomes at the time of cell division is essential for maintaining cellular health. Whereas chromosome mis-segregation is unusual in most mammalian cell types, errors are relatively common in mammalian oocytes, resulting in aneuploid oocytes that are developmentally compromised. Oocyte aneuploidy is particularly common in older females and is thus a major cause of age-related infertility in humans.1,2 Why oocytes from older females mis-segregate chromosomes more frequently than those from younger females is poorly understood. Chromosome segregation is executed by the spindle, a dynamic transient organelle assembled from microtubules (MTs).3,4 Chromosomes interact with spindle microtubules via kinetochores, complex proteinaceous structures that assemble on centromeric DNA.5 In mitosis, accurate chromosome segregation requires that kinetochores of sister chromatids (“sister kinetochores”) bind to microtubules from opposite poles of the spindle, such that shortening of kinetochore-bound MTs (kMTs) in anaphase causes one sister chromatid to be inherited by each daughter cell.6 Failure to establish kMTs correctly can lead to chromosome mis-segregation. For example, kinetochore attachment to both spindle poles simultaneously causes chromosomes to experience pulling forces from both poles in anaphase, resulting in “lagging” chromosomes that might be mis-segregated.7 This scenario, termed merotelic attachment, is particularly hazardous, as it is not detected by the spindle assembly checkpoint (SAC) that surveys and prevents other errors.8-10 Merotelic attachments are thus considered a key cause of aneuploidy in somatic cells. Whereas in mitosis sister chromatids are separated, in meiosisI sister chromatids remain attached and co-segregate in order to separate homologous chromosomes (see Fig. 1A). Essential for this is that sister chromatid cohesion is maintained during meiosis-I, and sister kinetochores serve together as a single microtubule-binding unit to enable co-segregation.11 Sister cohesion is subsequently lost at anaphase of meiosis-II, generating a haploid genome that forms the maternal contribution at fertilization (Fig. 1A). Chromosome segregation errors increase with maternal age both in meiosis-I and meiosis-II. However, the nature of the age-related defect remains mysterious. Laboratory mice aged 1–2 years exhibit increased chromosome mis-segregation and aneuploidy analogous to age-related oocyte aneuploidy in humans, and are thus an invaluable model to examine the cause of errors.12 Using this approach, several groups have found that chromosome cohesion is weakened in oocytes from older females.13-16 This conclusion is supported by *Correspondence to Greg FitzHarris; Email: Submitted: 12/09/2013; Revised: 01/27/2014; Accepted: 01/29/2014; Published Online: 02/11/2014 http://dx.doi.org/10.4161/cc.28046 www.landesbioscience.com Cell Cycle 1171 a reduced abundance of cohesin component proteins on chromosome arms,13,14 a reduced threshold for sister individualization by separase,17 and an increased distance between sister kinetochores in metaphase-II (Met-II) eggs.13-15 As a result of cohesion loss, sister pairs can be prematurely individualized in Met-II in older mice.14 Importantly, similar to mice, chromosome cohesion is lost with increasing age in human oocytes,18 and it has thus been widely suggested that cohesion loss might be a fundamental cause of age-related aneuploidy. Although the evidence that chromosome cohesion is lost with advancing age is strong, a critical unanswered question is: why should cohesion loss cause chromosome mis-segregation (dyad gain or loss) in meiosis-I? A prevalent hypothesis is that a loosening of sister chromosome cohesion in meiosis-I could change the size and geometry of the effective microtubule-binding area, which might, in turn, promote erroneous kinetochore–microtubule interactions, thereby causing mis-segregation.8,17,19-21 In support of this hypothesis, oocytes from old mothers frequently exhibit lagging anaphase chromosomes, which in mitosis are symptomatic of merotelic attachment errors.13,14,22 However, whether age-related cohesion loss has a direct impact upon chromosome segregation in meiosis-I is yet to be formally examined. Specifically, whether sister kinetochore geometry is indeed altered in aged oocytes in meiosis-I, when MT–kinetochore attachments are being formed, and whether this can affect the fidelity of MT binding are unknown. In any cellular setting, the occurrence of aneuploidy must be explained in terms of the failure of spindle microtubules to accurately dispatch chromosomes to the forming daughter cells. Here we present a detailed examination of kinetochore–microtubule interactions and chromosome positioning and report substantial differences in the profile of MT attachments between oocytes from young and natur (...truncated)