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To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis. A quantitative and comparative method was developed to estimate how known pathways were disturbed by each substance. This pathway-driven analysis revealed a strong alteration of steroidogenesis and an impairment of meiosis in all cases, albeit the initial molecular events were different for both substances. The ex vivo cytogenetic analysis confirmed that both fungicides alter the course of the first meiotic prophase. In addition, the mixture of both substances triggered effects greater than the sum of their cumulative effects and compromised future sperm motility after a shorter time of exposure compared with the fungicides tested separately. The alliance of an ex vivo culture with "omics" strategies complemented with a physiological examination is a powerful combination of tools for testing substances, separately or in a mixture, for their testicular toxicity. In particular, proteomics allowed the identification of systematically differentially expressed proteins in the secretomes of exposed cultures, such as FUCO and PEBP1, two proteins linked with the motility and fertilizing ability of spermatozoa, respectively. These proteins may be potential biomarkers of testicular dysfunction and infertility.

Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture