Localization of 3β-hydroxysteroid dehydrogenase in the chicken ovarian follicle shifts from the theca layer to granulosa layer with follicular maturation

Abstract

3β-Hydroxysteroid dehydrogenase (3β-HSD) catalyzes the conversion of pregnenolone to progesterone in the Δ4-3-ketosteroid metabolic pathway and dehydroepiandrosterone to androstenedione in the Δ5-3β-hydroxysteroid pathway. It has been suggested that small follicles of the chicken ovary that have not entered the follicular hierarchy metabolize steroids via the Δ5- 3β-hydroxysteroid pathway, whereas preovulatory follicles that have entered the hierarchy metabolize steroids via the Δ4-3-ketosteroid pathway. Our objective was to localize 3β-HSD in follicles of the chicken ovary by immunocytochemistry using an anti-human placental 3β-HSD polyclonal antiserum to identify steroidogenic cells that convert pregnenolone to progesterone and/or dehydroepiandrosterone to androstenedione. Three groups of follicles of different maturities were examined: small follicles (1-10 mm in diameter and that have not entered the hierarchy), preovulatory follicles (10-35 mm in diameter and that have entered the hierarchy), and the most recent postovulatory follicle. Chicken ovaries were obtained 2 h after oviposition and fixed with Bouin's solution. Tissues were dehydrated with a series of ethanol, embedded in Paraplast (Brunswick Company, St. Louis, MO), and sectioned. Sections (4 μm) were immunostained for 3β-HSD with a Rabbit ExtrAvidin Staining Kit (Sigma Chemical Co., St. Louis, MO). 3β-HSD was localized in the single theca layer of cortical follicles (approximately 1 mm in diameter), which are still embedded in the stromal tissue, and in the theca interna and externa of other small follicles (< 10 mm in diameter). No immunoreactivity was observed in the granulosa layer of the majority of small follicles. Weak staining of 3β-HSD was occasionally present in the granulosa layer of mature small yellow follicles, which are the next follicles to enter the hierarchy. 3β-HSD was present in the theca interna of all preovulatory follicles; however, the intensity of the staining decreased as preovulatory follicles matured. In contrast, the staining of 3β-HSD in the granulosa layer became more intense as preovulatory follicles increased in size. Finally, the granulosa layer of the most recent postovulatory follicle obtained 2 h after oviposition stained intensively for 3β-HSD. These results based upon the immunolocalization of 3β-HSD in the chicken ovarian follicles suggest the following: 1) the theca layer can convert pregnenolone to progesterone and/or dehydroepiandrosterone to androstenedione, whereas the granulosa layer in the majority of small follicles lacks this ability; 2) both granulosa and theca layers of preovulatory follicles are production sites for progesterone and/or androstenedione; 3) the granulosa layer becomes more steroidogenically active for progesterone and/or androstenedione production, whereas the theca layer loses its steroidogenic activity as preovulatory follicles mature; and 4) the granulosa layer retains its steroidogenic capability after ovulation. We conclude that the principal sites of progesterone and androstenedione shift during follicular maturation from the theca layer of small follicles to the granulosa layer of preovulatory follicles and postovulatory follicles.