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Primary testicular cell coculture model has been used to evaluate testicular abnormalities during development, and was able to identify the testicular toxicity of phthalates. However, the primary testicular cell coculture model has disadvantages in employing animals for the isolation of testicular cells, and the complicated isolation procedure leads to inconsistent results. We developed an invitro testicular coculture model from rodent testicular cell lines, including spermatogonial cells, Sertoli cells, and Leydig cells with specified cell density and extracellular matrix (ECM) composition. Using comparative high-content analysis of F-actin cytoskeletal structure between the coculture and single cell culture models, we demonstrated a 3D structure of the coculture, which created an invivo-like niche, and maintained and supported germ cells within a 3D environment. We validated this model by discriminating between reproductive toxicants and nontoxicants among 32 compounds in comparison to the single cell culture models. Furthermore, we conducted a comparison between the invitro (IC50) and invivo reproductive toxicity testing (lowest observed adverse effect level on reproductive system). We found the invitro coculture model could classify the tested compounds into 4 clusters, and identify the most toxic reproductive substances, with high concordance, sensitivity, and specificity of 84%, 86.21%, and 100%, respectively. We observed a strong correlation of IC50 between the invitro coculture model and the invivo testing results. Our results suggest that this novel invitro coculture model may be useful for screening testicular toxicants and prioritize chemicals for further assessment in the future.

From the Cover: An Animal-Free In Vitro Three-Dimensional Testicular Cell Coculture Model for Evaluating Male Reproductive Toxicants