Development of canine testicular organoid for assessing the effects of environmental exposure

Modeling the male reproductive system in vitro remains challenging due to its complex structure and function such as spermatogenesis. To enhance translational relevance and adhere to the 3 R principles -Replacement, Reduction, Refinement, we developed a three-dimensional (3D) canine testicular organoid model using testes ethically obtained from routine neutering procedures. Testicular cells were isolated via enzymatic digestion, seeded into agarose micro-molds, embedded in extracellular matrix, and cultured for up to 21 days. Organoids increased in diameter (500-1072 μm) and developed complex branching morphologies. Immunofluorescence confirmed the presence of key testicular cell types, including germ cells (GCNA1), Sertoli cells (SOX9), and Leydig cells (HSD3B1), as well as germ cell populations expressing stage-specific differentiation markers, SALL4, DPPA3, PRMT7, and SCP3. Temporal gene expression analysis revealed dynamic regulation of markers involved in testicular function and spermatogenesis, including significant upregulation of GATA4. To evaluate toxicological responsiveness, organoids were exposed to cadmium chloride (CdCl₂). Treatment with 1 μM CdCl₂ significantly reduced cell viability, and 5 μM exposure induced γ-H2AX expression, indicating DNA damage and cellular stress. These findings demonstrate the successful generation and functional validation of a canine testicular organoid model that supports germ cell maintenance and enables mechanistic assessment of reproductive toxicants. This system represents a scientifically robust and ethically sourced alternative to traditional in vivo approaches for evaluating male reproductive toxicity.