THE RESPONSE OF BOVINE GRANULOSA-CELLS TO DIFFERENT GONADOTROPINS IN CULTURE

Previous studies with bovine granulosa cells cultured in vitro indicat ed that follicle-stimulating hormone (FSH) stimulated differentiation and progesterone production of granulosa cells in a dose-dependent man ner; this was due mainly to an increase in the number of differentiate d cells. The objectives of the present study were to investigate (1) w hether the response of bovine granulosa cells in culture to luteinisin g hormone (LH) and equine chorionic gonadotrophin (eCG) was similar to the response to FSH, and (2) whether granulosa cells derived from dif ferent cattle breeds responded similarly to gonadotrophin stimulation. Pairs of ovaries were recovered post-mortem from Charolais (38) and H ereford (41) crossbred post-pubertal heifers, and granulosa cells were aspirated from 5-8 mm follicles. In two simultaneous experiments, gra nulosa cells (2-3 x 10(5) viable cells) were cultured with different g onadotrophins (oFSH or oLH in Experiment 1; oFSH or eCG in Experiment 2). Cell culture was for 4 days at 37 degrees C in a humidified atmosp here of 5% CO2 in air in 1 mi of serum-free culture medium. Progestero ne production, total DNA and the protein content of granulosa cells on Day 4 of culture were determined. Log(10) data were analyzed by analy sis of variance and multiple linear regression. In Experiment 1, both FSH and LH stimulated progesterone production (ng mu g(-1) DNA) and pr otein content (mu g mu g(-1) DNA) of granulosa cells in a dose-depende nt manner (P < 0.01). The relative potencies of FSH to LH (mu u/mu u) were found not to be different from unity. In Experiment 2, progestero ne production and the protein content of granulosa cells were stimulat ed by both FSH and eCG in a dose-dependent manner(P < 0.001), The prog esterone response curves (log/log) were linear up to 1-10 mu u FSH and 1-10 iu eCG, and were Y = 1.67 + 0.093 FSH and Y = 1.60 + 0.091 eCG f or progesterone production. Calculated on a mu u/iu basis, FSH was fou nd to be 5.8 times more potent than eCG (P < 0.05) in terms of stimula ting progesterone production. Granulosa cells derived from Hereford cr osses were more sensitive (P < 0.001) than those from Charolais crosse s to gonadotrophin stimulation (31 and 42 times for FSH and eCG, respe ctively, in terms of progesterone production, and 4.8 and 3.1 times fo r FSH and eCG, respectively, in terms of protein content). The respons e curves for both FSH and eCG were similar within each breed. The slop es of the progesterone response curves, and the protein responses were similar for all the gonadotrophins. In conclusion, these results impl y that FSH, LH and eCG have similar effects on the differentiation and progesterone production of bovine granulosa cells from 5-8 mm follicl es cultured in vitro. Furthermore, granulosa cells from different bree ds cultured in vitro had different sensitivities to gonadotrophin stim ulation.