Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium

Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.