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 ce lls that would allow for cellular proliferation without induction of s teroidogenesis would provide researchers with an important in vitro to ol for determining differentiation mechanisms during folliculogenesis. The objective of the present study was to determine the effect of a c ommercially prepared serum substitute and medium supplement on prolife ration and progesterone production by bovine granulosa cells. Granulos a 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 se veral 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, gran ulosa 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 incl uding medium alone or medium containing fetal bovine serum (FBS; 1%), Gibco BRL media supplement-x (GMS-X(R); 1%), fatty acid-free bovine se rum albumin (FAF-BSA; 4 mg/mL), or SerXtend(R) (5%). For Exp. 2 and 3, granulosa cells were plated in serum-supplemented medium for either 4 8 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. A ll treatment media supported granulosa cell proliferation to some exte nt; 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 am ounts of progesterone compared with cells in the other treatments. In summary, granulosa cells may be propagated in vitro in a serum-free en vironment without inducing progesterone production.