Effect of number of culture medium granulosa cells on gene expression of enzymes associated with synthesis of steroid hormones

Granuloca cells have a key role during estroeidogenesis. The purpose of this study was to determine the effect of number of culture medium granulosa cells on estradiol concentrations and mRNA codding estrogenic and progestagenic enzyme. Briefly, follicles between 2 and 5 mm diameter were dissected from the ovaries of adult cows and were collected by rinsing the follicle walls with Dulbecco Modified Eagle medium/F12 (DMEM/F12). The number of cells was counted with a haemocytometer and the viable cells were assessed by the dye exclusion method using 0.4% Trypan Blue. Treatments were 1) 500,000 cell/500 ml, 2) 250,000 cell/500 ml, 3) 500,000 cell/200 ml 4) 250,000 cell/ 200 ml. All data were analyzed by JMP (SAS). Low plating density increased E2 secretion and mRNA encoding LHR, FSHR and estrogenic enzymes (17βHSD, CYP19), whereas decreased mRNA encoding GADD45β. There were no differences among treatments for RNA and protein concentration. Low plating density also decreased protein amount but there was no difference among treatments for RNA amount. In conclusion, decreased cell density cause increase in mRNA encoding codding estrogenic enzyme gene expression and decrease in mRNA encoding progestagenic enzyme gene expression. Protein concentrations did not changed with decreased cell density therefore we can save cells against harmful effect of increasing cell density.