Low molecular mass factors from follicular fluid inhibit steroidogenesis in bovine granulosa cells

Gonadotropins are required for follicular growth and differentiation, but i ncreasing amounts of evidence indicate that intrafollicular factors modulat e their effects at granulosa cell level. In order to study the effect of fa ctors present in bovine follicular fluid, a partial purification of low mol ecular mass factors from fluids collected from small (< 5 mm), medium (5-8 mm) and large (> 8mm) follicles was performed and the biological activity o f these peptides on steroidogenesis of granulosa cells from small and large follicles was examined. The purification was carried out by filtration thr ough membranes in 25 and 10kDa molecular weight cutoffs. After filtration, samples were analysed by polyacrylamide gel electrophoresis and protein con centration was measured by spectrophotometric analysis. Granulosa cells fro m small and large follicles were cultured in serum-fret DMEM/Ham's F12 (1:1 ) plus transferrin (5mg/l) and selenium (5 mug/l) for 2days. At the end of the culture period, media were renewed and follicular extracts from two dif ferent preparations (< 25 and < 10 kDa) were added at the concentrations of 1-10-100-1000 ng/ml. After 24 h the media were collected and stored until estradiol 17 beta (E2) and progesterone (P4) determination by validated rad io-immunoassays. Basal P4 production was 18.3 +/- 1.4 (mean +/- SEM) and 9. 8 +/- 1.8 ng/24h per 3 x 10(4) cells from small and large follicles, respec tively. Both <10 and <25 kDa extracts reduced P4 production by cells from b oth the types of follicles (p < 0.05). Basal E2 release was 671.8+/-21.4 an d 5500 +/- 800pg/24h per 3 x 104 cells from small and large follicles, resp ectively. Both extracts reduced E2 production by either cells from small an d large follicles (p < 0.05). No differences were observed in the inhibitio n of steroidogenesis by purifications obtained from large, medium or small follicles. Results of this study indicate that factors present in bovine fo llicular fluid can reduce steroidogenesis in granulosa cells in vitro.