FOLLICLE-STIMULATING-HORMONE STIMULATED DIFFERENTIATION AND PROGESTERONE PRODUCTION OF BOVINE GRANULOSA-CELLS IN CULTURE

Progesterone production of granulosa cells cultured in vitro is stimul ated and cell differentiation increased, by follicle-stimulating hormo ne (FSH). This study examined whether the increased progesterone produ ction observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is s timulated. Viable bovine granulosa cells (2-3 X 10(5)) from follicles 5-8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 m u u FSH (1 mu u = 1 mu g NIH-FSH-S1) for 6 days at 37 degrees C in a h umidified atmosphere of 5% CO2 in air in 1 mi of a 1:1 mixture of Dulb ecco's modified Eagle medium: Ham's F10 medium supplemented with 365 m u g ml(-1) L-glutamine, 100 U ml(-1) penicillin and 100 mu g ml(-1) st reptomycin. Progesterone production, total DNA and protein, and cell d iameter were determined sequentially over the culture period. The incr eases in progesterone production(ng mu g(-1) DNA per 24 h), cytoplasmi c:nuclear ratio (mu g protein mu g(-1) DNA) and cell diameter (mu m) o ver 6 days culture indicated that granulosa cells underwent differenti ation in the presence of FSH. Progesterone production of undifferentia ted granulosa cells (diameter 14 mu m or less) was stimulated by FSH ( P < 0.01) in a dose dependent manner (1.0 +/- 0.2, 2.9 +/- 0.3, 3.7 +/ - 0.3 and 4.9 +/- 0.4 ng mu g(-1) DNA per 24 h for 0, 1, 10 and 100 mu u ml(-1) FSH respectively) but remained constant within dose (P > 0.0 5) during a 6 day culture period. FSH stimulated (P < 0.05) the rate o f granulosa cell differentiation (10 +/- 3%, 53 +/- 13%, 74 +/- 21% an d 82 +/- 10% differentiating cells per well for 0 mu u, 1 mu u, 10 mu u and 100 mu u ml(-1) FSH respectively) but did not stimulate (P > 0.0 5) progesterone production by differentiating granulosa cells (8.7 +/- 0.5 ng mu g(-1) DNA per 24 h). In conclusion, the increase in progest erone production of FSH-stimulated granulosa cells cultured in vitro a ppears to be mainly due to an increase in the number of differentiatin g cells with a constant rather than an increasing progesterone product ion per cell.