In vitro steroidogenesis by granulosa cells from equine pre-ovulatory follicles

Twenty-three follicles were collected from 14 mares on specific days and grouped to represent follicles from early (Group 1; n = 6), mid (Group 2; n = 11) and late (Group 3; n = 6) oestrus, as described previously (Tucker et al., 1988). Isolated granulosa cells (GC) from each follicle were cultured in multiwell plates containing either Eagle's Minimum Essential Medium (MEM) alone, eLH (300 ng/ml), eFSH (300 ng/ml) or eLH + eFSH (300 ng/ml each), in the presence or absence of 0.5 microM testosterone. Media were collected and replaced at 24 h of culture, and 24 h later, media were again collected and cultures terminated. Media were assayed for oestradiol-17 beta (E2), progesterone (P4), androstenedione (A) and testosterone (T) by radioimmunoassay (RIA). Media containing exogenous T were assayed for E2 and P4. There was no effect of exogenous gonadotrophins on steroid secretion by GC from any group, so these values were pooled and averaged. Steroid concentrations were expressed as ng/ml/1000 live cells seeded (mean +/- s.e.m.). Progesterone was the predominant steroid secreted by GC from all groups without added T after 24 h of culture, and was highest in media from Group 3 (7.86 +/- 1.19 ng/ml) 1000 cells; vs Group 1 (0.02 +/- 0.004 ng/ml/1000 cells) and Group 2 (0.15 +/- 0.03 ng/ml/1000 cells; P less than 0.001). Androstenedione was the predominant androgen secreted, but its levels did not change from group to group. Oestradiol and T levels were comparable in media from Group 1 cultures (E2: 0.003 +/- 0.0004 ng/ml/1000 cells; T: 0.003 +/- 0.0009 ng/ml/1000 cells), but E2 was highest in Group 2 media (0.17 +/- 0.11 ng/ml/1000 cells; P less than 0.001) and T levels were greatest in Group 3 (0.02 +/- 0.003 ng/ml/1000 cells; P less than 0.005). In the presence of exogenous T, E2 increased in media from all groups (P less than 0.0001). Oestradiol levels from Group 1 (0.52 +/- 0.12 ng/ml/1000 cells) and Group 3 (0.57 +/- 0.09 ng/ml/1000 cells) cultures were not different, but were highest in media from Group 2 (2.15 +/- 0.29 ng/ml/1000 cells; P less than 0.01). By 48 h, P4 secretion was higher in all groups (P less than 0.0001) and was the predominant steroid secreted by Group 3 GC. Progesterone levels were not affected by the addition of T. Androstenedione increased in Group 3 media (P less than 0.001), whereas E2 and T remained unchanged. These results suggest that the pattern of equine GC steroid secretion changes as follicular maturation proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)