Paternal ribosome biogenesis dysfunction caused by cyclophosphamide-busulfan chemotherapy contributes to early embryonic developmental impairment in mice

Combined cyclophosphamide (CTX) and busulfan (BUS) chemotherapy, while effective against malignancies, poses significant risks to male reproductive health. This study systematically investigates the proteomic basis underlying CTX + BUS-induced paternal reproductive toxicity and its impact on early embryonic development in mice. We utilized a mouse model of acute CTX + BUS exposure. Systemic germ cell apoptosis, sperm parameters (concentration, motility, morphology), and fertilization rates were assessed. Testicular and sperm proteomic profiling was performed. Embryonic developmental outcomes were evaluated following fertilization with sperm from treated males. Acute CTX + BUS exposure induced systemic germ cell apoptosis and profound sperm head teratozoospermia, despite preserved sperm concentration and motility. Proteomic profiling revealed testicular dysregulation of ribosome biogenesis, DNA replication, and cell cycle control, alongside sperm-specific depletion of ribosomal proteins. Sperm from CTX + BUS-treated mice exhibited a reduced fertilization rate and induced severe embryonic developmental defects. Molecularly, ribosomal insufficiency in sperm is linked to lower ribosomal protein levels in early embryos, potentially compromising the paternal role in translational activation. Our findings establish ribosome biogenesis defects as a novel pathway mediating paternal-derived embryotoxicity following CTX + BUS chemotherapy. They challenge the reliance on conventional semen parameters (motility, count) for fertility assessment, highlighting that significant molecular lesions in sperm can persist despite normal traditional metrics. These results advocate for incorporating protein biomarker analysis into clinical fertility risk stratification for cancer survivors. This work provides critical insights into the early embryonic consequences of chemotherapy and underscores the imperative to safeguard paternal genomic and epigenetic integrity in oncological care.