A novel method for purifying dispersed porcine theca cells, with less than 3% granulosa cell contamination, was developed by the repeated use of mechanical and enzymatic procedures. The steroidogenic criteria used for the identification and purity evaluation of both theca and granulosa cells were also improved. Purified theca and granulosa cells from medium-sized follicles displayed steroidogenic differences when they were cultured in the presence of 10% fetal bovine serum: (1) the theca cells synthesized oestradiol (239.1 ± 35.1 pg ml⁻¹ per 2.5 × 10⁵ cells in 40 h), but the granulosa cells did not synthesize it unless aromatizable androgens were added; (2) theca cells synthesized androstenedione (73.2 ± 14.4 ng ml⁻¹ per 2.5 × 10⁵ cells in 40 h), but granulosa cells did not; (3) FSH did not affect progesterone production in theca cells; (4) the theca cells secreted androstenedione for up to 48 h; and (5) FSH significantly stimulated progesterone production in granulosa cells during a culture for 40 h (P < 0.05), but not during culture for 12 h. The lack of response to FSH was used as a reliable, functional indicator of the purity of porcine theca cells. However, this criterion proved not to be useful for cells cultured for 12 h; porcine FSH had no effect on the progesterone production of theca cells co-cultured for this time with as many as 20% granulosa cells. However, after co-culturing for 40 h, this criterion resulted in the detection of only 3% granulosa cell contamination. Lack of response to FSH is a sensitive and reliable criterion for evaluating the purity of porcine theca cells, as long as FSH responsiveness of granulosa cells is fully confirmed.

The activity of GnSI/AF was measured in pig follicular fluid (pFF) from 58 individual follicles of various sizes, by bioassay using rat pituitary cells, to investigate the relationship between gonadotrophin surge inhibiting/attenuating factor (GnSI/AF) activity and follicular development. In addition, the correlation between GnSI/AF and inhibin activities and the content of sex steroids (oestradiol, progesterone and testosterone) of follicles was examined. The activity of GnSI/AF in pFF varied significantly (0.155–1.69 U μl⁻¹) with size of the follicle. The activities (mean ± sem) were intermediate and constant in follicles with diameters from 3 to 5 mm (0.583 ± 0.080 U μl⁻¹, n = 24), were higher and reached the highest value in follicles with diameters between 6 and 8 mm (0.863 ± 0.068 U μl⁻¹, n = 21), and were lower, reaching the lowest value in follicles with diameters of 9 and 10 mm (0.401 ± 0.089 U μl⁻¹, n = 13). In contrast, inhibin activity was almost constant during the development of follicles, although individual values varied from 0.9 to 2.5 U μl⁻¹. For follicles with diameters of 4–8 mm, inhibin activity was 1.754 ± 0.042 U μl⁻¹ (n = 39); activity was higher in the smallest follicles with diameters of 3 mm (2.063 ± 0.015 U μl⁻¹, n = 6) and was lower in follicles with diameter of 9 mm, reaching the lowest value in follicles with diameter of 10 mm (1.176 ± 0.068 U μl⁻¹, n = 7); inhibin activity was not significantly correlated with GnSI/AF activity. Steroid concentration increased in a similar pattern to that of GnSI/AF activity, but no marked decrease was noticed in the large follicles. These characteristic changes of the activity of GnSI/AF of follicles during development, especially in the preovulatory stage, suggest that GnSI/AF plays a role in the ovulatory process as an ovarian regulator for the timely occurrence of the LH surge.