An adverse outcome pathway-aligned ex vivo mouse testicular organ culture platform for mechanistic integration of multi-level endpoints with recovery assessment

Mechanistic understanding is important for improving the safety assessment of male reproductive toxicity; however, current evaluations still rely primarily on in vivo studies. Therefore, the development of new approach methodologies (NAMs) requires test systems capable of capturing key events organized within adverse outcome pathways (AOPs). The PDMS ceiling (PC) method is a testicular organ culture technique previously used to detect testicular toxicity. However, its applicability as an AOP-aligned platform for multi-level mechanistic assessment, including recovery evaluation, has not been fully examined. Here, we evaluated the utility of the PC-based organ culture system for AOP-aligned assessment of testicular toxicity. Methoxyacetic acid (MAA), a testicular toxicant with established AOPs, was used as a reference compound. Molecular, cellular, and histological endpoints were assessed under identical conditions, and a membrane-supported PC (msPC) configuration was introduced to enable controlled recovery assessment. Testes from Acro3-EGFP transgenic mice, in which EGFP expression driven by the acrosin promoter accumulates in developing acrosomes, were cultured and exposed to MAA. GFP fluorescence indicating spermatogenic progression decreased in a concentration-dependent manner. Histological and immunohistochemical analyses demonstrated selective loss of pachytene spermatocytes with apoptosis, whereas transcriptomic profiling suggested disruption of cell cycle- and meiosis-related pathways consistent with established AOP-defined key events. Recovery of GFP fluorescence and tissue morphology occurred earlier in the msPC system than in the conventional PC method. These findings support the PC-based organ culture system as an AOP-aligned platform within NAMs for evaluating testicular toxicity and recovery.