Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes

The juvenile onset of spermatogenesis in mice is analyzed by combining cytological and transcriptomic data in a novel computational analysis, resulting in meiotic substage-specific transcriptomes and the discovery of a transcription factor network that regulates the substages of meiosis. Germ cells from testes of individual mice were obtained at two-day intervals from 8 to 18 days post-partum (dpp), with five biological replicates at each age (samples 8_1 through 18_5). Eight stages were discriminated cytologically by combinatorial antibody labeling, and RNA-seq was performed on the same samples. A novel permutation-based maximum covariance analysis (PMCA) method was developed to deconvolve genes into meiotic substages. To verify PMCA derived pachytene/diplotene substage-specific genes, we isolated enriched populations of adult pachytene germ cells (samples rep1 through rep4), followed the same RNA-seq protocol, and compared the PMCA derived substage-specific gene lists to the genes expressed in the pachytene/diplotene enriched germ cells.本研究通过整合细胞学与转录组学数据，采用全新的计算分析方法，对小鼠精子发生的幼年启动过程展开解析，成功获得了减数分裂各亚阶段特异性转录组，并揭示了调控减数分裂亚阶段的转录因子网络。实验采集了产后8至18天（days post-partum，dpp）每隔两天的小鼠睾丸生殖细胞，每个时间点设置5个生物学重复样本（样本编号为8_1至18_5）。通过组合抗体标记的细胞学方法区分出8个发育阶段，并对同一批样本进行RNA测序（RNA-seq）。本研究开发了一种基于置换的最大协方差分析（permutation-based maximum covariance analysis，PMCA）方法，可将基因解卷积至对应减数分裂亚阶段。为验证PMCA预测的粗线期/双线期（pachytene/diplotene）亚阶段特异性基因，本研究分离了富集的成年粗线期生殖细胞（样本编号为rep1至rep4），采用相同的RNA-seq实验流程进行测序，并将PMCA得到的亚阶段特异性基因列表与该富集生殖细胞中表达的基因进行对比。