UTILIZATION OF SUBSTRATES FOR TESTOSTERONE AND ESTRADIOL-17-BETA PRODUCTION BY SMALL FOLLICLES OF THE CHICKEN OVARY

A study was conducted to characterize steroidogenesis in small ovarian follicles (1-10 mm in diameter) of the hen. The aims of our study wer e: 1) to determine basal estradiol-17 beta (E(2)) production by differ ent sizes of small follicles; 2) to determine the ability of intact sm all follicles to utilize exogenous substrates for testosterone (T) and E(2) production; and 3) to investigate the preferred steroidogenic pa thway in small follicles. Small follicles which had not entered the hi erarchy were isolated from ovaries obtained 2 hr after oviposition and divided into three groups: small white follicles (SWF; 1-2 mm in diam eter), large white follicles (LWF; 2-4 mm in diameter), and small yell ow 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) wer e incubated in avian Ringer's buffer supplemented with 10 mM HEPES and 0.1% BSA at 37 degrees C for 3 hr with various treatments. Testostero ne and E(2) were measured in the medium. The SYFs and their correspond ing follicle shells produced the greatest amount of E(2) when E(2) pro duction 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- a nd 8-Br-cAMP-stimulated E(2) secretion compared to corresponding intac t follicles. There was no significant difference in E(2) production in response to various concentrations of 25-hydroxycholesterol (25-OH-CH OL; 0-100 mu M) by intact follicles and follicle shells. On the contra ry, intact follicles and follicle shells produced T and E(2) in a dose -dependent manner in response to increasing concentrations (0-100 mu M ) of pregnenolone (P-4). Intact follicles also used progesterone (P,) and dehydroepiandrosterone (DHEA) as substrates for T and E(2) product ion. DHEA was the preferred substrate for steroid production compared to P-4. In summary, we found that: 1) steroidogenesis in small follicl es is regulated by a cAMP-dependent protein kinase A second messenger system; 2) adequate amounts of endogenous cholesterol are available fo r steroidogenesis; and 3) both Delta(5) and Delta(4) pathways are func tional in small follicles and the Delta(5) pathway may be the preferre d steroidogenic pathway.