Age-associated increase in aneuploidy and changes in gene expression in mouse eggs

Abstract

An increase in the incidence of aneuploidy is well documented with increasing maternal age, in particular in human females. Remarkably, little is known regarding the underlying molecular basis for the age-associated increase in aneuploidy, which is a major source of decreased fertility in humans. Using mouse as a model system we find that eggs obtained from old mice (60-70 weeks of age) display a 6-fold increase in the incidence of hyperploidy as assessed by chromosome spreads. Expression profiling of transcripts in oocytes and eggs obtained from young and old mice reveals that approximately 5% of the transcripts are differentially expressed in oocytes obtained from old females when compared to oocytes obtained from young females (6-12 weeks of age) and that this fraction increases to approximately 33% in eggs. The latter finding indicates that the normal pattern of degradation of maternal mRNAs that occurs during oocyte maturation is dramatically altered in eggs obtained from old mice and could therefore be a contributing source to the decline in fertility. Analysis of the differentially expressed transcripts also indicated that the strength of the spindle assembly checkpoint is weakened and that higher errors of microtubule-kinetochore interactions constitute part of molecular basis for the age-associated increase in aneuploidy in females. Last, BRCA1 expression is reduced in oocytes obtained from old females and RNAi-mediated reduction of BRCA1 in oocytes obtained from young females results in perturbing spindle formation and chromosome congression following maturation.

Figure 1

Chromosome spread derived from old oocyte. Shown is an example of a hyperploid metaphase II egg that matured in vivo.

Figure 2

Morphology of MII spindle in young and old eggs. Eggs from young (A) and old (B) mice were processed for immunocytochemical detection of DNA (green) and tubulin (green). Shown are examples that represent more extreme cases. Also note in B the presence of an unattached chromosome, a condition that would inevitably lead to production of an aneuploid egg. A total of 50 and 40 spindles in young and old eggs was analyzed, respectively. The bar in panel A represents 10 µm and the magnification in panel B is similar.

Figure 3

Hierarchical cluster analysis of transcripts expressed in oocytes and eggs obtained from young and old mice. GV, oocyte; MII, metaphase II-arrested egg. 6w and 66w refer to age of females from which oocytes and eggs were collected. In pseudocolor scheme, red represents high level of expression and blue low level.

Figure 4

ATRX and BRCA1 expression in young and old oocytes/eggs. ATRX expression was visualized in GV-intact oocytes in young (A) and old (B) oocytes. The staining is nuclear and clearly reduced in old oocytes. (C) BRCA1 expression (red) was assessed in young (C) and old (D) MII eggs , where it associates with the spindle poles. BRCA1 staining on the spindle poles is reduced; DNA is in green. The bar in panel A represents 10 µm and the magnification in the other panels is similar.

Figure 5

(A) RNAi-mediated reduction of Brca1 mRNA. Oocytes were injected with either Egfp dsRNA or Brca1 dsRNA and the amount of Brca1 mRNA relative to that present in the Egfp-injected oocytes was determined by qRT-PCR 7 h and 16 h following injection. The experiment was performed three times and the data are presented as mean ± SEM. Immunocytochemical detection of BRCA1 in Egfp dsRNA- (B) and Brca1 dsRNA-injected (C) oocyte that was then matured to MII. A total of 410 oocytes were injected and shown is a representative example. The arrow points to the region where the MII spindle is present.

Figure 6

Immunocytochemical analysis of spindle morphology and chromosome congression in BRCA1-depleted oocytes matured to MII. (A) Control oocyte injected with Egfp dsRNA. (B–D) Oocytes injected with Brca1 dsRNA. In B, a spindle didn't form but the chromosomes condensed into a hollow ball-like structure. In C, a disorganized spindle formed with chromosomes poorly congressed on the metaphase plate. In D, an unattched chromosome is present. The bar in panel A represents 10 µm and the magnification in the other panels is similar.