Transcriptome and translatome during different stages of mouse germ cell development.

Male germ cell development involves delayed translation during spermiogenesis but the contribution of translational machinary on gene expression control remains largely unexplored. Here, we report heterogeneity of ribosome profiles across mouse tissues and spermatogenic stages and show that male germ cells use a paralog ribosomal protein (RP) L39L instead of the core RPL39, and exclusively form the RPL39L-based RibosomeRPL39L. Deletion of RibosomeRPL39L causes spermiogenesis defects. We analyzed whether the protein expression changes identified by proteome analysis were caused by changes at the transcript level, translational activity, or protein stability. RNA-seq analysis showed that only 2 of the 465 differential proteins in elongated spermatids exhibited changes in corresponding mRNA levels in RibosomeRPL39L-/- testis, revealing little contribution of mRNA expression levels to the protein changes. The translational activity of mRNAs can be estimated based on the amount of ribosome-bound mRNAs. We therefore separated cytoplasmic ribosomes into different functional classes by sucrose gradient fractionation and performed RNA-seq analysis of mRNAs bound to purified 80S, 2-6 and 7+ polysomes (Ribosome-nascent chain complex (RNC)-RNA). Of the 465 proteins exhibiting differential expression in the proteome of elongated RibosomeRPL39L-/- spermatids, few exhibited changes in the corresponding RNC-RNA levels, with 10, 0 and 1 proteins showing corresponding RNA changes in the 80S, 2-6 and 7+ polysomes of RibosomeRPL39L-/- testis, respectively.To evaluate the properties of proteins regulated by RibosomeRPL39L exit tunnel, we investigated the mRNA and protein expression of proteins found to be downregulated in RibosomeRPL39L-/- elongated spermatids at different stages of germ cell development(SG, spermatogonia; PS ,pachytene spermatocytes; RS, round spermatids; ES, elongated spermatid), as many testis-specific proteins exhibited delayed translation. These differentially-expressed genes were classified into 4 clusters. In Clusters 1 and 2, the mRNA levels peaked in spermatocytes or round spermatids, while the protein levels peaked at elongated spermatids, showing apparent delayed translation. For Clusters 3 and 4, all mRNA expression levels peaked in elongated spermatids, consistent with the protein expression pattern. Therefore, the down-regulated testis-specific proteins in RibosomeRPL39L-/- elongated spermatids exhibited the highest translational activity in the elongated spermatids.