GSE172171
Transcriptome analysis of early and late pachytene spermatocytes in WT and Zfp541-KO mice (Smart-Seq2)
The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes. Overall design: To identify differentially expressed genes (DEGs) in Zfp541-KO spematocytes, single-cell RNA-seq was performed on synchronized spermatocytes using a modified Smart-Seq2 method (Chen et al., 2018). Briefly, 2-dpp mice were pipette fed WIN 18,446 (MP) for 7 consecutive days. At Day 8 of WIN 18,446 treatments, these animals were received an injection of RA (Sigma), and then left to recover. We collected testes enriched for early (260 hours after retinoic acid treatment) and late pachytene (360 hours after retinoic acid treatment) cells from WT and Zfp541-KO mice.