Male germ cell-specific deletion of eukaryotic translation initiation factor 5 causes the apoptosis of mouse progenitor spermatogonia by excessive endoplasmic reticulum stress and defective DNA repair

To determine whether eIF5 plays a crucial role in spermatogenesis, we generated germ cell conditional Eif5 knockout mice by crossing Eif5fl/fl mice with Stra8-Cre mice. For ribosome profiling (Ribo-seq), the testis samples of cKO and control mice at P10 were used to analyze. Each sample contains testes from three mice. Ribosomes were extracted from these samples and analyzed by NeoRibo Biotechnology Co., Ltd. QEZ-seq® 1.0 kit (1012S, NeoRibo, China) was used to deliver high-resolution mapping of ribosome-protected mRNA fragments. Adapter sequences were removed from raw sequencing data using the cutadapt software. Meanwhile, reads with lengths between 25 and 35 bp were kept for downstream analysis. Then, reads were aligned to rRNA sequences to remove rRNA reads using Bowtie software, and the remaining reads were used to align to GRCm38 using STAR. The trinucleotide periodicity of ribosomes and codon usage frequency were estimated using the revised riboWaltz package. Read counts were calculated using the featureCounts software. Raw counts were further normalized in the DESeq2 package. Translational efficiencies were determined by the ratio (normalized abundance determined by ribosome profiling/normalized abundance determined by RNA-seq) as previously reported (Duan et al., 2022; Su et al., 2022). Different translational efficiencies were estimated using the DESeq2 package, and genes with fold changes≥1.5 and P-value<0.05 were considered significant. GO and KEGG analyses were performed using the cluster Profiler package in R software.