Two steroidogenic pathways present in the chicken ovary: theca layer prefers delta 5 pathway and granulosa layer...

It has been suggested that there is a shift in steroidogenic pathways of the theca layer from delta 5 to delta 4 during follicular maturation in the chicken ovary. However, the preferred steroid metabolic pathway in the theca and granulosa layers of small and preovulatory follicles has not been identified. Therefore, the goal of our study was to identify the preferred steroidogenic pathways used by these follicles. Previously, we found that small follicles that had not entered the follicular hierarchy did not use cholesterol but did use pregnenolone (P5) to produce testosterone and estradiol. For this reason, P5 was used as the substrate for steroid production in the present study. The theca layer of small follicles (2-4 mm in diameter), and the theca and granulosa layers of the fifth largest (F5), third largest (F3), and largest (F1) preovulatory follicles were isolated from the chicken ovary 22 hr before ovulation. The granulosa layer of small follicles was excluded from the study because it is steroidogenically incompetent. Tissues were incubated with [3H]-P5 for 1 hr at 37 degrees C. Production of steroid metabolites was determined on the basis of comparison with known steroid standards using two-dimensional thin layer chromatography (2D-TLC). Conversion of [3H]-P5 to [3H]-DHEA was greater than that of [3H]-P4 in the theca layer of small and preovulatory follicles. Even though the amount of [3H]-P4 was less than that of [3H]-DHEA, theca cells from F3 and F1 follicles did produce [3H]-P4. In contrast to the theca layer, conversion of [3H]-P4 was greater than that of [3H]-DHEA in the granulosa layer of preovulatory follicles. Conversion of [3H]-P5 to [3H]-DHEA by the granulosa layer gradually decreased as the follicle matured and approached ovulation. We conclude that both delta 5 and delta 4 pathways are functional in theca and granulosa cells. However, in contrast to earlier speculation, the theca layer preferentially metabolizes steroids via the delta 5 pathway regardless of the maturational stage of the follicle, whereas the granulosa layer of preovulatory follicles preferentially metabolizes steroids via the delta 4 pathway.