Proliferative growth of neonatal cerebellar cells in culture: Regulation by male and by maternal serum in late gestation

Neonatal cerebellar cells were utilized as a model system to examine the effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mixed cells or populations enriched in astrocytes or oligodendrocytes. Cell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubated with 10% male rat serum, both total DNA and protein content increased after 6 days of culture. By contrast, neither of these parameters were altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activity paralleled changes in protein content. Graded concentrations of pregnant serum (5–20%) added to mixed cell cultures produced consistently lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes and oligodendrocytes incubated with graded concentrations of pregnant sera when compared with similar concentrations of non-pregnant sera. When potential effects of these different sera on the cell cycle were examined, an increase in the number of cells in the S and G2/M phase was similar, and DNA doubling began to increase at 96 hrs in the presence of either male or 20 day pregnant sera. Thus the inhibition of cell growth by pregnant serum was not likely a result of either cytotoxicity or a delay of entry of cells into the cell cycle. To examine whether this inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of IGF-II by cerebellar cells. Production of an endogenous source of IGF-II was apparent using an RNAse protection assay and was noted using Slot Blot analysis of mRNA extracted at sequential times during cell incubation. Mixed cell cultures also secreted immunologically defined IGF-II. These observations are consistent with the previous demonstration that the fraction of pregnant serum which bound IGF-II also inhibited cell growth. The inhibitory effect of pregnant serum was diminished by preincubating aliquots of sera with graded concentrations of IGF-I prior to adding sera to tissue culture medium. Pregnant serum inhibition was also diminished by prolonging incubation times beyond 6 days. The blunting of pregnant serum inhibition may have been consequent to either a continuing production of endogenous growth factors or to the potential emergence of resistant cells due to prolonged tissue culture incubation. Since cells studied in a primary culture of limited duration may more accurately reflect the physiologic properties of this tissue, the model presented herein could provide a new approach to study brain development.West Side Medical CenterNorthwestern University medical School