TRIM7-mediated autophagy-lysosomal signaling is essential for preimplantation development in mice

Background: Pre-implantation development in mammals requires precise coordination of zygote genome activation (ZGA), cleavage divisions, and early lineage specification. These events rely on tightly regulated transcriptional programs, proteostasis, and organelle quality control. TRIM7, a RING-type E3 ubiquitin ligase, is known to modulate immune signaling and cellular stress response; however, its role in early embryogenesis remains undefined. Methods: Mouse oocytes and pre-implantation embryos were collected at defined developmental stages. TRIM7 expression and localization were examined by immunofluorescence and confocal microscopy. Functional loss-of-function studies were performed by culturing zygotes with TRIM7-targeting Vivo-Morpholinos (2.5–10 µM). The TRIM7 function was independently validated by microinjection and electroporation of TRIM7-specific siRNA into mouse zygote embryos. Global transcriptional activity was assessed at the late 2-cell stage using 5-ethynyl uridine (EU) incorporation, followed by fluorescence-based detection. Single-cell RNA sequencing was conducted on late 2-cell and arrested 8-cell embryos treated with 3.5 µM Morpholino to identify early transcriptional defects, complemented by mass spectrometry-based proteomics. Key lysosomal transcripts were validated by qRT-PCR, and protein-level changes were confirmed by immunofluorescence. Autophagy flux was assessed by LC3B immunostaining and Western blot analysis. Rapamycin (5-7.5 µM) supplementation was used to evaluate functional rescue. Results: TRIM7 displayed dynamic spatiotemporal expression, peaking at the 4-cell stage and localizing to both the nucleus and cytoplasm, consistent with a regulatory role in early embryonic signaling. TRIM7 knockdown caused a severe, dose-dependent developmental arrest, with ~ 98% of embryos failing to progress beyond the 8-cell stage at 3.5 µM (p < 0.0001). TRIM7-specific siRNA delivery by both microinjection and electroporation consistently resulted in embryonic arrest at the 8-cell stage, confirming the specificity of TRIM7 depletion. Integrated scRNA-seq and proteomics revealed profound disruption of lysosomal-autophagic pathways, with early repression of lysosomal, autophagy, and spliceosome genes detectable as early as the late 2-cell stage, coinciding with ZGA onset. EU incorporation analysis showed no significant impairment of global transcriptional activation at the 2-cell stage, indicating that zygotic genome activation is largely preserved. Arrested 8-cell embryos exhibited marked downregulation of key lysosomal regulators and impaired autophagic flux, while proteomics indicated reduced levels of essential lysosomal proteins, including CTSL, HEXB, and GM2A. Rapamycin-mediated activation of autophagy resulted in partial but significant developmental rescue, restoring 40–50% blastocyst formation and normalizing the expression of several lysosomal and autophagy markers. LC3B analysis confirmed that TRIM7 depletion suppresses autophagy, whereas Rapamycin effectively reinstates autophagic flux. Conclusion: TRIM7 is essential for early mouse embryogenesis, acting independently of ZGA but critically supporting lysosomal-autophagic function required for progression beyond the 8-cell stage and successful blastocyst development. Impaired proteostasis underlies the TRIM7-deficient arrest, while Pharmacological activation of autophagy partially restores embryo viability, highlighting lysosomal-autophagic regulation as a central TRIM7-dependent developmental checkpoint.