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 t he effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mi xed cells or populations enriched in astrocytes or oligodendrocytes. C ell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubat ed with 10% male rat serum, both total DNA and protein content increas ed after 6 days of culture. By contrast, neither of these parameters w ere altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activ ity paralleled changes in protein content. Graded concentrations of pr egnant serum (5-20%) added to mixed cell cultures produced consistentl y lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes an d oligodendrocytes incubated with graded concentrations of pregnant se ra when compared with similar concentrations of non-pregnant sera. Whe n potential effects of these different sera on the cell cycle were exa mined, 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 gr owth 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 t his inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of I GF-II by cerebellar cells. Production of an endogenous source of IGF-I I was apparent using an RNAse protection assay and was noted using Slo t Blot analysis of mRNA extracted at sequential times during cell incu bation. 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 pr ior 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 eit her a continuing production of endogenous growth factors or to the pot ential emergence of resistant cells due to prolonged tissue culture in cubation. 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 bra in development.