To evaluate the [P.sub.4] production by hen granulosa cells cultured for a long-term, granulosa cells (3 x [10.sup.5] cells/well) of the [F.sub.1] and [F.sub.3] cultured for 48 h in serum-free medium were cultured for an additional 48 h with serum-free medium containing one of the following materials: oLH and oFSH at the concentrations of 0.1, 1, 10, and 100 ng/mL, respectively, and cVIP, AVT, MT, T, [E.sub.2], E, NE, PGE1, and [PGF.sub.2[alpha]] at the concentrations of [10.sup.-9], [10.sup.-8], [10.sup.-7], and [10.sup.-6] M, respectively.
Experiment 4: Time course of [P.sub.4] production by granulosa cells with oLH, cVIP, T, and [E.sub.2]
In order to evaluate the time course of [P.sub.4] production in culture with oLH, cVIP, T, and [E.sub.2], after [F.sub.1] granulosa cells (3 x [10.sup.5] cells/well) were cultured for 48 h in serum-free medium, the cells were washed and cultured in serum-free medium with or without oLH (100 ng/mL), cVIP ([10.sup.-6] M), T ([10.sup.-6] M) and [E.sub.2] ([10.sup.-6] M) for 4, 6, 8, 12, 24, 36, or 48 h, respectively.
In both [F.sub.1] and [F.sub.3] granulosa cells, [P.sub.4] production was increased in a dose dependent manner by oLH, cVIP, and T (p<0.05) but not by other substances (p>0.05), without affecting the number of cells.
When the time course of [P.sub.4] production by oLH, cVIP, T, and [E.sub.2] in [F.sub.1] granulosa cells was examined for 48 h during 48 to 96 h of culture (Figure 8), although [E.sub.2] had no effect on [P.sub.4] production by granulosa cells of [F.sub.1] during the period from 48 until 96 h of the culture, the increase in [P.sub.4] production by oLH was found at 4 h of the culture with the maximal 9.14-fold level at 6 h.
Furthermore, LH, cVIP, and T, but not [E.sub.2], stimulated [P.sub.4] production without affecting the cell number in both [F.sub.1] and [F.sub.3] granulosa cells of hen granulosa cells with the treatment of these hormones for 48 h.