ToxSci Advance Access originally published online on January 19, 2005
Toxicological Sciences 2005 84(2):378-393; doi:10.1093/toxsci/kfi085
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Essential Role of Extracellular Matrix (ECM) Overlay in Establishing the Functional Integrity of Primary Neonatal Rat Sertoli Cell/Gonocyte Co-cultures: An Improved In Vitro Model for Assessment of Male Reproductive Toxicity
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105
Received October 19, 2004; accepted January 4, 2005
The development of in vitro models for testicular toxicity may provide important tools for investigating specific mechanisms of toxicity in the testis. Although various systems have been reported, their application in toxicological studies has been limited by the poor ability to replicate the complex biochemical, molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved Sertoli cell/gonocyte co-culture (SGC) system that employs a 3-dimensional extracellular matrix Matrigel (ECM) applied as an overlay instead of a substratum. We explored the dose- and time-dependent effects of the addition of such an ECM overlay on cytoskeletal and morphological changes in the SGC system, and the resulting effects on cellular integrity. Furthermore, we correlated the latter effects with the ECM-dependent modulation of stress and survival signaling pathways and, most critically, the expression levels of the spermatogonia-specific protein, c-Kit. Finally, we applied this co-culture system to investigate the dose- and time-dependent effects on the morphology and induction of apoptosis of cadmium. We observed that the dose-dependent addition of an ECM overlay led to an enhanced attachment of Sertoli cells and facilitated the establishment of SGC communication and cytoskeletal structure, with a dramatic improvement in cell viability. The latter was consistent with the observed dose- and time-dependent modulation of both stress signaling pathways (SAPK/JNK) and survival signaling pathways (ERK and AKT) in the presence of the ECM overlay. Furthermore, the dose-dependent stabilization of c-Kit protein expression confirmed the functional integrity of this co-culture system. We conclude that this modified SGC system will provide investigators with a simple, efficient, and highly reproducible alternative in the screen for testicular cell-specific cytotoxicity and the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental toxicants.
Key Words: in vitro model; Sertoli cell/gonocyte co-culture; Matrigel (ECM) overlay; stress signaling; c-Kit; cadmium.
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