Transcriptome profiling of OCT4 positive spermatogonia in wild type and Akt1s1 null mutant mice.. Transcriptome profiling of OCT4 positive spermatogonia in wild type and Akt1s1 null mutant mice.

In order to investigate the regulatory roles of AKT1S1 during mouse spermatogenesis, we analyzed gene expression changes that caused by the deletion of Akt1s1 gene in mouse male germ cells using RNA sequencing method. The transcriptome profiling analyses showed that OCT4+ spermatogonia from mice at different ages possess varied changes in gene expression due to the deletion of Akt1s1 gene, supporting the role of AKT1S1 plays in mediating AKT-mTORC1 signaling during mouse spermatogenesis. Overall design: Spermatogonia expressing OCT4 were isolated from mouse testes expressing green fluorescent protein (GFP) under the control of Pou5f1 (the gene encoding OCT4) promoter in either wild type or Akt1s1 null mutant background. OCT4+ cells were FACS isolated from enzyme-dispersed testicular cells using mice at three time points (3 days, 7 days and 2-3 months following birth) for both wild type control and Akt1s1 null mutants. Each time point was experimentally repeated 2-3 times, generating total 15 samples for the second generation RNA sequencing via Illumina HiSeq platform.

为探究AKT1S1在小鼠精子发生过程中的调控作用，我们采用RNA测序技术，分析了小鼠雄性生殖细胞中Akt1s1基因敲除所引发的基因表达变化。转录组谱分析结果显示，不同周龄小鼠的OCT4阳性精原细胞，因Akt1s1基因敲除会呈现差异化的基因表达谱，这佐证了AKT1S1在小鼠精子发生过程中介导AKT-mTORC1信号通路的功能。本实验总体设计如下：分别在野生型与Akt1s1纯合敲除突变体背景下，从由Pou5f1（编码OCT4的基因）启动子调控绿色荧光蛋白（GFP）表达的小鼠睾丸中，分离表达OCT4的精原细胞。我们于小鼠出生后3天、7天以及2-3个月三个时间点，对野生型对照组与Akt1s1敲除突变体小鼠的酶解离睾丸细胞进行荧光激活细胞分选（FACS）以获取OCT4阳性细胞。每个时间点均设置2-3次生物学重复，最终共获得15个样本，通过Illumina HiSeq平台开展第二代RNA测序。