Spermatogenesis is regulated by a complex interplay between endocrine signals and local interactions of tubules with the interstitial tissue, mainly androgen producing Leydig cells (LC). We hypothesized that the functional phenotype of LC reflects the developmental environment in which these cells grow and interact with other testicular cells, including both somatic and germ cells. As experimental model, we used the organotypic culture of 2.5dpp mouse testicular fragments in the presence of 10% KSR (Knockout Serum Replecement), which is essential to maintain the entire spermatogenetic process from spermatogonia to spermatids in vitro up to 5-6 weeks. At different times we analyzed: 1) testicular morphology, to assess germ cell differentiation; 2) LC proliferation by KI67 immunostaining; and 3) LC maturation, by measuring androgens and INSL3 secreted into the medium. LC were identified by immunostaining of specific markers, including 3βHSD, c-Kit and INSL3. In the presence of KSR, testosterone significantly increased from the first to the second week and was maintained up to the fifth week, in association with germ cell differentiation up to haploid elongated spermatids. On the contrary, in the absence of KSR, testosterone markedly declined during the fourth week, together with an altered spermatogenesis. These results indicate that LC undergo normal development and function in vitro, provided that culture conditions maintained spermatogenesis. We also addressed the regulation of LC function in humans, using fresh and cryopreserved testicular biopsies obtained from beating heart organ donors. Having assessed several culture conditions, we set up a new system biology model allowing to successfully in vitro culture testicular fragments for three hours and to analyze the differential LC response to LH and hCG in terms of testosterone secretion. The study of the age-dependent LC response is in progress, comparing young (15-40 years) and elderly (60-85 years) donors
