The purpose of this experiment was to establish a proper system for the use of frozed-thawed chicken ovarian granulosa cells, for in vitro culture in order to study the progesterone (P4) secretion pattern of granulosa cells, and to compare their responses to stimulation. After dissection, the granulosa layers were separated from theca and yolk, digested and then either cultured immediately or stored frozen until next use. The main experiments included time course and dose response studies. The results showed: 1. The viability of fresh digested cells and frozen-thawed cells were between 75~100% and 67~98%, respectively. However, if granulosa cells from each preovulatory follicles were digested separately according to their size, viability of cells post thawing were obtained much lower (0~10%) of. 2. The ability of chicken granulosa cells to secrete P4 did not change in the freeze-thawing process. 3. The frozen- thawed granulosa cells respomed to LH, FSH, hCG and cAMP as the fresh cells did except that the degree of responsiveness was approximatiely half fold. 4. The continuity of follicular granulosa cells in in vitro culture system to secrete P4 was different from their in vivo situation. F4 granulosa cells secreted P4 only for one day, while P4 secretion from F1 granulosa cells was maintained continuously for 3 days in the culture. Giving LH to granulosa cells would prolong the secretion of P4. In conclusion, it is suggested that the use of frozen-thawed chicken granulosa cells for subsequent culture study system is worth while particularly when a large quantity of cells are required. However, it is not suitable to use this sytem when changes of events related to granulosa cells of individual follicle is of intrest. Moreover, the interaction between preovulatory follicles may affect their survival after freezng and thawing, and a further investigation is required.
