Eukaryotic translation elongation factor EEF1G orchestrates the translational program to ensure meiotic progression in transcriptionally silent spermatocytes [Ribo-Seq]

During meiotic prophase I, mammalian spermatocytes face a critical challenge that they need to synthesize recombination and synapsis proteins while global transcriptional suppression occurs at leptotene/zygotene stages. Here, we identify a spermatogenic cell highly expressed protein, eukaryotic translation elongation factor 1 gamma (EEF1G), as a master regulator of translational adaptation in mouse spermatocytes. Germ cell-specific Eef1g knockout causes complete male infertility due to the meiotic arrest at the zygotene stage, which is characterized by unresolved DNA double-strand breaks, failed homologous chromosome synapsis, and abolished crossover formation. Mechanistically, EEF1G can interact with ribosome proteins and RNA-binding factors. Ribosome profiling reveals that EEF1G deficiency reduces ribosome processivity, leading to ribosomal stalling and decreased protein production. Consequently, proteomic analyses show a specific decrease of synapsis (e.g., SYCP1/SYCE1) and recombination (e.g., MSH4/TEX11) proteins. This study establishes EEF1G as a critical coordinator of spatial translation in transcriptionally silent spermatocytes, bridging limited mRNA engagement to explosive protein synthesis for meiotic progression. Our work challenges traditional views of meiotic control by highlighting translation elongation, not just as a housekeeping process, but as a stage-specific regulator of germ cell development. Ribosome profiling (Ribo-seq) was carried out to investigate the translational regulation associated with EEF1G in early meiotic spermatocytes. Testes were collected from postnatal day 12 (PD12) mice, a developmental stage enriched for leptotene and zygotene spermatocytes. To obtain highly purified populations, spermatocytes at the leptotene (L) and zygotene (Z) stages were isolated by fluorescence-activated cell sorting (FACS). Both wild-type (WT) and Eef1g conditional knockout (cKO) groups were included in the design, and each group contained two independent biological replicates to ensure reproducibility and statistical robustness. Ribosome-protected fragments (RPFs) were prepared using the QEZ-seq kit (Hangzhou NeoRibo Biotechnology Co., Ltd., Cat. R1024L) following the manufacturer's instructions, and the resulting libraries were sequenced on an Illumina platform. This design allowed a direct comparison of translational profiles between WT and Eef1g cKO spermatocytes at the L and Z stages, thereby providing insights into the functional role of EEF1G during meiotic progression in male germ cells.