Maternal age-dependent APC/C-mediated decrease in securin causes premature sister chromatid separation in meiosis II

Abstract

Sister chromatid attachment during meiosis II (MII) is maintained by securin-mediated inhibition of separase. In maternal ageing, oocytes show increased inter-sister kinetochore distance and premature sister chromatid separation (PSCS), suggesting aberrant separase activity. Here, we find that MII oocytes from aged mice have less securin than oocytes from young mice and that this reduction is mediated by increased destruction by the anaphase promoting complex/cyclosome (APC/C) during meiosis I (MI) exit. Inhibition of the spindle assembly checkpoint (SAC) kinase, Mps1, during MI exit in young oocytes replicates this phenotype. Further, over-expression of securin or Mps1 protects against the age-related increase in inter-sister kinetochore distance and PSCS. These findings show that maternal ageing compromises the oocyte SAC-APC/C axis leading to a decrease in securin that ultimately causes sister chromatid cohesion loss. Manipulating this axis and/or increasing securin may provide novel therapeutic approaches to alleviating the risk of oocyte aneuploidy in maternal ageing.

Figure 1. Chromosome misalignment and reduced sister chromatid cohesion in MII eggs from aged MF1 mice.

Representative images of immunostaining for DNA in blue and tubulin in red (a), and quantification of chromosome misalignment (b) in MII eggs from young (1 month) and aged (>1 year) mice. The number of eggs used is shown in parentheses. The arrow points towards misaligned DNA. (c) The mean distance between sister kinetochores in MII eggs from 1 month (n=14 eggs) and >1 year (n=20 eggs) old mice. The number of kinetochores measured is shown in parentheses. In b,c, the results are mean±s.e.m. ***P<0.001. P values were calculated with one-sided Student's t-test. (d) Rates of PSCS in MII eggs from 1 month- versus >1 year old mice. The number of eggs used is shown in parentheses. (e) Representative example of the chromosome spreads assay demonstrating MII eggs from >1 year- (top panel) and 1 month old (bottom panel) mice. DNA is shown in blue and CREST-labelled kinetochores are shown in red. The arrows point towards two-separated sister chromatids, and the insets show the difference in the inter-sister kinetochore distance between eggs from 1 month- and >1 year old mice. Scale bars, 50 μm. Results are from three to four independent experiments involving two to five mice per experimental group.

Figure 2. Age-related decrease in securin stability during MII but not MI.

Western blots (a,c,e) and densitometric analysis (b,d,f) of oocytes from 1 month- and >1 year old mice for securin during MII arrest (14 h post release from GV, 50 oocytes per lane), GV arrest (20 oocytes per lane) and MI (8 h post release from GV, 20 oocytes per lane). Actin was used as a loading control. Results are mean±s.e.m. ^NSP>0.05 and **P<0.01. P values were calculated with one-sided Student's t-test. All western blots were repeated three times using oocytes from two to six mice per experimental group.

Figure 3. Compromised SAC and higher APC/C-mediated securin destruction at the MI exit in oocytes from aged mice.

Time-lapse fluorescence readings (a,c) and rates of securin-GFP destruction (b,d) in oocytes from 1 month- and >1 year old mice in the absence (a,b) or presence (c,d) of the Mps1 inhibitor (AZ3146), which was added to the imaging medium at 7.5 h post release from GV arrest (arrow). Number of oocytes used is shown in parentheses. (e–g) Re-analysis of the data in a,c. The timing of onset of securin-GFP destruction is synchronized by normalizing time 0 to the peak of the curves. Western blot and (h) densitometric analysis (i) of MII oocytes (100 oocytes per lane, n=3) from 1 month old mice for securin. All oocytes were collected at 14 h post release from GV arrest. The AZ3146-treated oocytes were incubated in the presence of the drug from 7.5 h post release from GV arrest. Actin was used as a loading control. All results are mean±s.e.m. ^NSP>0.05 and *P<0.05. P values were calculated with one-sided Student's t-test. Results are representative of two to three independent experiments involving two to five mice per experimental group.

Figure 4. Rescue of the age-related increase in inter-sister kinetochore distances and PSCS frequency by over-expression of Mps1 or securin.

(a) Mean inter-sister kinetochore distance in MII eggs from 1 month old mice in the absence (n=21 eggs) or presence (n=20 eggs) of AZ3146. The AZ3146-treated oocytes were incubated with the drug from 7.5 h post release from GV arrest. (b) Representative example of the chromosome spreads demonstrating the increase in inter-sister kinetochore distances (inset) in the AZ3146-treated MII oocytes. DNA is shown in blue and CREST-labelled kinetochores are shown in red. Scale bar, 10 μm. (c) Mean inter-sister kinetochore distances in MII eggs from 1 month old mice in the absence (n=12 eggs) or presence (n=23 eggs) of Mps1-GFP. (d) Mean inter-sister kinetochore distances in MII eggs from >1 year old mice in the absence (n=20 eggs) or presence of either Mps1-GFP (n=20 eggs) or securin-GFP (n=27 eggs). (e) Mean inter-sister kinetochore distances in MII eggs from 1 month old mice in the absence (n=21 eggs) or presence (n=29 eggs) of securin-GFP. All MII eggs were fixed at 14 h post release from GV arrest. Mps1-GFP and securin-GFP cRNA were microinjected at the GV stage. The number of sister-kinetochores measured is shown in parentheses. (f) Rates of PSCS in MII eggs from >1 year old mice in the presence or absence of securin. The number of eggs used is shown in parentheses. All results are mean±s.e.m. ^NSP>0.05, *P<0.05 and ***P<0.001. P values were calculated with one-sided Student's t-test. Results are representative of two to four independent experiments involving two to five mice per experimental group.