PREPARATION OF HIGHLY PURIFIED PORCINE THECA CELLS

A novel method for purifying dispersed porcine theca cells, with less than 3% granulosa cell contamination, was developed by the repeated us e of mechanical and enzymatic procedures. The steroidogenic criteria u sed for the identification and purity evaluation of both theca and gra nulosa cells were also improved. Purified theca and granulosa cells fr om medium-sized follicles displayed steroidogenic differences when the y were cultured in the presence of 10% fetal bovine serum: (1) the the ca cells synthesized oestradiol (239.1 +/- 35.1 pg ml(-1) per 2.5 x 10 (5) cells in 40 h), but the granulosa cells did not synthesize it unle ss aromatizable androgens were added; (2) theca cells synthesized andr ostenedione (73.2 +/- 14.4 ng ml(-1) per 2.5 x 10(5) cells in 40 h), b ut granulosa cells did not; (3) FSH did not affect progesterone produc tion in theca cells; (4) the theca cells secreted androstenedione for up to 48 h; and (5) FSH significantly stimulated progesterone producti on in granulosa cells during a culture for 40 h (P < 0.05), but not du ring culture for 12 h. The lack of response to FSH was used as a relia ble, functional indicator of the purity of porcine theca cells. Howeve r, this criterion proved not to be useful for cells cultured for 12 h; porcine FSH had no effect on the progesterone production of theca cel ls co-cultured for this time with as many as 20% granulosa cells. Howe ver, after co-culturing for 40 h, this criterion resulted in the detec tion of only 3% granulosa cell contamination. Lack of response to FSH is a sensitive and reliable criterion for evaluating the purity of por cine theca cells, as long as FSH responsiveness of granulosa cells is fully confirmed.