Utilization of substrates for testosterone and estradiol-17β production by small follicles of the chicken ovary

Abstract

A study was conducted to characterize steroidogenesis in small ovarian follicles (1–10 mm in diameter) of the hen. The aims of our study were: 1) to determine basal estradiol-17β (E 2) production by different sizes of small follicles; 2) to determine the ability of intact small follicles to utilize exogenous substrates for testosterone (T) and E 2 production; and 3) to investigate the preferred steroidogenic pathway in small follicles. Small follicles which had not entered the hierarchy were isolated from ovaries obtained 2 hr after oviposition and divided into three groups: small white follicles (SWF; 1–2 mm in diameter), large white follicles (LWF; 2–4 mm in diameter), and small yellow follicles (SYF; 5–10 mm in diameter). Yolk and granulosa cells were removed from LWFs and SYFs and the remaining theca layer was called a follicle shell. Intact follicles or follicle shells (4/4 ml/tube) were incubated in avian Ringer's buffer supplemented with 10 mM HEPES and 0.1% BSA at 37°C for 3 hr with various treatments. Testosterone and E 2 were measured in the medium. The SYFs and their corresponding follicle shells produced the greatest amount of E 2 when E 2 production was expressed per follicle. Addition of 2 mM 8-Br-cAMP to the incubation medium stimulated E 2 production by all sizes of follicles and follicle shells. However, follicle shells produced lower basal- and 8-Br-cAMP-stimulated E 2 secretion compared to corresponding intact follicles. There was no significant difference in E 2 production in response to various concentrations of 25-hydroxycholesterol (25-OH-CHOL; 0–100 μM) by intact follicles and follicle shells. On the contrary, intact follicles and follicle shells produced T and E 2 in a dose-dependent manner in response to increasing concentrations (0–100 μM) of pregnenolone (P 5). Intact follicles also used progesterone (P 4) and dehydroepiandrosterone (DHEA) as substrates for T and E 2 production. DHEA was the preferred substrate for steroid production compared to P 4. In summary, we found that: 1) steroidogenesis in small follicles is regulated by a cAMP-dependent protein kinase A second messenger system; 2) adequate amounts of endogenous cholesterol are available for steroidogenesis; and 3) both Δ 5 and Δ 4 pathways are functional in small follicles and the Δ 5 pathway may be the preferred steroidogenic pathway. pathway.