Prepubertal exposure to SARS-CoV-2 single-stranded RNA disrupts spermatogenesis and causes intergenerational effects in mice.

The COVID-19 pandemic has caused widespread morbidity and mortality worldwide, with emerging evidence documenting transient reproductive impairment in adult males following SARS-CoV-2 infection. However, prepubertal males appear especially vulnerable to infection-related testicular injury, yet the underlying mechanisms and long-term consequences remain poorly understood. We investigated the impact of systemic exposure to synthetic SARS-CoV-2 guanine-uracil (GU)-rich single-stranded RNA (ssRNA), a viral pathogen-associated molecular pattern (PAMP), on testicular integrity and reproductive outcomes in a juvenile mouse model. 4-week-old C57BL/6J males received systemic administration of GU-rich ssRNA, and we comprehensively assessed testicular architecture, molecular signatures, and offspring outcomes extending into adulthood. Exposure induced robust systemic and local inflammation that disrupted blood-testis barrier (BTB) integrity, disorganized seminiferous tubule architecture, and coordinately suppressed transcripts essential for spermatogonial maintenance (Kit, Csf1), germ cell survival (Bcl2, Bcl6), and meiotic progression (Spo11, Mlh1, Atm, Msh5). Paradoxically, despite significant upregulation of steroidogenic enzymes (Star, Hsd3b2, Cyp17a1, Hsd17b3), serum testosterone levels were markedly reduced, coinciding with profound downregulation of genes critical for ganglioside biosynthesis (B3galt4, St3gal5, St8sia1, B4galnt1), which are essential for maintaining lipid-raft-dependent junctional integrity and hormone transport. Paternal CoV2 GU-rich ssRNA exposure did not alter litter size or offspring sex distribution but was associated with reduced early postnatal offspring body weight, impaired glucose handling, and selective changes in metabolic tissue weights. Our findings reveal a critical developmental vulnerability window and provide mechanistic insights that support the need to monitor testicular function following systemic inflammation triggered by viral PAMPs in prepubertal males.