This project will inform a systems biology platform for identifying adverse responses to environmental compounds on testicular development in mice, a commonly used model system in the field of Toxicology. In order to validate the organotypic culture system, we have investigated the adverse effects of the known testicular toxicant, cadmium. To initiate the culture model, we isolate and plate testicular cells from immature mice on postnatal day (PND) 9 in order to capture a developmental window of susceptibility. Based on a literature search derived developmental timeline, we chose to treat the culture on days in vitro (DIV) 2, 5 and 15 with cadmium and quantify effects 24 hours later. We hypothesized that dosing with cadmium during these time points would correspond to impacts on steroid regulation, proliferation, and spermatogenesis processes. In order to evaluate a dose response relationship in our testicular culture, we treated the testis cells with 2.5, 5.0 and 10 uM concentrations of cadmium. To quantify the effects of cadmium in our cell culture model we evaluated; total protein, testosterone production, cell type specific proteins with western blots, LDH cytotoxicity, 3 color assay for live and dead cells and immunofluorescence to capture morphology for DIV 3,7 and 16, 24 hours post treatment for all three doses. Our initial studies have observed that cadmium treatment for all three doses impacted testosterone production, germ cell morphology and proliferation on DIV 7 and 16.  

Multiple strains of rats and mice are used to evaluate differences in response after exposures to various toxicants at the molecular or cellular levels. However, neuronal toxicity in different strains of species has received much less attention. Therefore, the objective of our research was to compare and characterize fetal mouse micromass culture system using two different strains of mouse—C57BL/6 and A/J—which may be used to screen and prioritize variety of neurodevelopmental toxicants. Our mouse midbrain micromass culture system, a high-density in vitro cultures of primary midbrain cells, is an alternative method to in vivo neurodevelopmental testing because of the ability of cells within the cultures to differentiate in vitro without additional stimulants. Differentiation is purely driven by high cell density. For our 3D in vitro micromass culture system, we isolated the primary midbrain cells from fetus at gestational day 11 (for C57BL/6 mice) or 12 (for A/J mice). The embryos were surgically removed, and midbrain cells of fetal mice were digested into single neuronal cells. These primary in vitro neuronal cells were plated onto plates and cultured up to 22 days in vitro. When morphology was examined throughout 22 days in culture with a 400x phase microscope, cell proliferation, differentiation, and migration were observed for both strains of mice. Proliferation (PCNA, Nestin) and differentiation (B-tubulin, EN1, MAP-2) related protein expression levels were examined using western blot for both C57BL/6 and A/J mouse strains. Dynamics of protein expression levels in western blot indicated that proliferation may be at its highest point between DIV 4-6 while differentiation is between DIV 8-14. If midbrain cells from multiple mouse strains can be harvested at similar neurodevelopmental stages, differences in response due to genetic variability may be studied using our 3D fetal mouse micromass model.