Longitudinal Analysis of Male Fertility Using an Acr‐Luc Knock‐In Mouse Model: A Preclinical Platform for Reproductive Toxicity Testing

ABSTRACT Reproductive toxicity testing is essential for evaluating whether xenobiotics, including pharmaceuticals, environmental chemicals, or ionizing radiation, adversely affect reproductive function. However, conventional assessments rely on mating outcomes or histopathology, which are labor‐intensive, variable, and require large numbers of animals. Acrosin, a serine protease encoded by the Acr gene and localized in the acrosome of spermatozoa, plays a critical role in sperm penetration of the zona pellucida. To exploit this germ cell‐specific expression, we generated a genetically engineered mouse model in which the Luciferase ( Luc ) reporter gene is driven by the Acr promoter. This Acr‐Luc knock‐in (KI) model enables longitudinal and quantitative imaging of spermatogenesis using bioluminescence. We demonstrate that this platform captures radiation‐induced impairments in male fertility in real time, eliminating the need for terminal analyses. By allowing repeated evaluation within the same individuals, our approach reduces interindividual variability and enables a substantial reduction in animal use, aligning with the “Reduction” principle of the 3Rs. Moreover, it reveals both the onset and recovery phases of spermatogenic disruption with high temporal resolution. The Acr‐Luc KI model provides a reliable preclinical platform for reproductive toxicity testing and offers broad utility for studies in reproductive biology, toxicology, and oncofertility research.