Effect of media substitutes on bovine granulosa cell function and proliferation during in vitro culture.

The development of a serum-free culture system for bovine granulosa cells that would allow for cellular proliferation without induction of steroidogenesis would provide researchers with an important in vitro tool for determining differentiation mechanisms during folliculogenesis. The objective of the present study was to determine the effect of a commercially prepared serum substitute and a medium supplement on proliferation and progesterone production by bovine granulosa cells. Granulosa cells were obtained by aspirating the follicular fluid of follicles 2 to 8 mm in diameter. For each experiment, growth curves to determine the proliferative and steroidogenic response of granulosa cells to several different medium additions were constructed. Cells were counted on d 1, 2, 4, 6, and 8 of culture to determine cell concentration and the media harvested to determine progesterone content. In Exp. 1, granulosa cells were seeded at an initial rate of 5.0 x 10(5) for 48 h in serum-supplemented medium then allotted to one of five treatments including medium alone or medium containing fetal bovine serum (FBS; 1%), Gibco BRL media supplement-x (GMS-X; 1%), fatty acid-free bovine serum albumin (FAF-BSA; 4 mg/mL), or SerXtend (5%). For Exp. 2 and 3, granulosa cells were plated in serum-supplemented medium for either 48 or 24 h and seeded at either 5.0 x 10(5) or 2.5 x 10(5) cells/flask, respectively, and cultured in different concentrations of SerXtend. All treatment media supported granulosa cell proliferation to some extent; the SerXtend treatment provided the highest proliferation rate at all concentrations above .3125%. Also, during the proliferative stage of the growth curve, cells in the SerXtend treatment produced lower amounts of progesterone compared with cells in the other treatments. In summary, granulosa cells may be propagated in vitro in a serum-free environment without inducing progesterone production.