RESUMO
The last two decades have seen an increasing search for in vitro models that can replace the use of animals for safety testing. We adapted the methods from a recent nonquantitative report of spermatogenesis occurring in ex vivo mouse testis explants and tried to develop them into a screening assay. The model consisted of small pieces of neonatal mouse testis (testis "chunks"), explanted and placed on pillars of agarose or chamber inserts, and cultured at the air-liquid interface. A peripheral torus-shaped zone in these explants would often contain tubules showing spermatogenesis, while the middle of each chunk was often necrotic, depending on the thickness of the tissue. The endpoint was histology: what proportion of tubules in the "permissive torus" actually contained healthy pachytene spermatocytes or spermatids? Extensive statistical modeling revealed that a useful predictive model required more than 60% of these tubules to show spermatogenesis. Separately, the logistics of running this as a predictive assay require that the controls consistently produce ≥ 60% tubules with pachytenes and round spermatids, and achieving this level of spermatogenesis reliably and consistently every week proved ultimately not possible. Extensive trials with various media additions and amendments proved incapable of maintaining the frequency of spermatogenic tubules at consistently ≥ 60%. Congruent with Schooler's "decline effect"; generally, the more often we ran these cultures, the worse the performance became. We hope that future efforts in this area may use our experience as a starting point on the way to a fully productive in vitro model of spermatogenesis.
