Transcription profiling of mouse oocytes to investigate age-associated increase in aneuploidy and changes in gene expression

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 Experiment Overall Design: We profiled the global gene expression in GV & MII oocyte by maternal aging, and identified the genes differentially expressed.母体年龄增长与非整倍体(aneuploidy)发生率升高的关联已有充分文献记载，这一现象在人类女性中尤为显著。值得注意的是，与年龄相关的非整倍体发生率升高的潜在分子机制仍鲜为人知，而该现象正是人类生育力下降的主要诱因之一。本研究以小鼠为模型系统，结果显示：通过染色体铺展(chromosome spreads)技术检测，老龄小鼠（60~70周龄）所获卵子的超倍体(hyperploidy)发生率较对照升高6倍。对年轻与老龄小鼠所获卵母细胞(oocytes)及卵子的转录本(transcripts)进行表达谱分析后发现：与6~12周龄年轻雌性小鼠的卵母细胞相比，老龄雌性小鼠卵母细胞中约5%的转录本存在差异表达；而在卵子样本中，这一比例升至约33%。上述后者结果表明，老龄小鼠卵子中，卵母细胞成熟过程中正常的母源mRNA降解模式发生了显著改变，这或为生育力下降的潜在诱因之一。对差异表达转录本的分析还显示：纺锤体组装检验点(spindle assembly checkpoint)的功能强度减弱，以及微管-动粒相互作用(microtubule-kinetochore interactions)错误率升高，共同构成了女性年龄相关性非整倍体发生率升高的部分分子机制。最后，老龄雌性小鼠卵母细胞中BRCA1的表达水平下调；而通过RNA干扰(RNAi)技术下调年轻雌性小鼠卵母细胞中的BRCA1表达，会破坏成熟过程中的纺锤体形成与染色体整列。实验整体设计：本研究针对母体衰老过程中的生发泡期(GV)与第二次减数分裂中期(MII)卵母细胞进行全局基因表达谱分析，以鉴定差异表达基因。