Proliferation and Differentiation of Theca-Like Cells from Granulosa Cells During In Vitro Follicle Culture.

Abstract

The acquisition of a functional theca cell layer is an important landmark in the development of a follicle. Theca cells may arise from fibroblast-like precursor cells within the ovarian stroma and functionally induced by contact with granulosa cells. An in vitro follicle maturation system was used to investigate theca cells development and differentiation in the ovarian follicles grown in vitro. Here we show that both stromal cells and granulosa cells can function like theca cells providing a new model to study the development of this critical cell type. Mouse preantral follicles (150-180 μm) were isolated from prepubertal mouse ovaries by either mechanically or enzymatically and encapsulated in 0.5% alginate and cultured up to 8 days. Follicles were harvested at different time points and were either fixed for immunofluoresence or frozen for real-time PCR. Follicles were stained with either 17α-hydroxylase (CYP17a1) or inhibin-α, as either theca or granulosa cells markers, and evaluated by confocal microscopy. Steroids profiles (androstenedione, estradiol and progesterone) were measured with spent media, and specific genes expression changes (17α-hydroxylase, aromatase (CYP19a1), steroidogenic acute regulatory (StAR) and inhibin α-subunit) were characterized. Mechanically isolated follicles (Mech-FL) presented intact theca-interstitial tissues, as indicated by AKP positive staining, while collagenase treated follicles (Enzy-FL) were free of theca-interstitial tissues. Mech-FL grew faster than Enzy-FL in any given time through the culture, and produced significant more androstenedione, estradiol and progesterone than Enzy-FL. Cyp17a1 and Star had significantly more expression in Mech-FL than Enzy-FL. Mech-FL cultured in vitro phenocopied in vivo pattern, with theca cells in the outer layer and granulosa cell in the middle, as monitored by CYP17a1 and inhibin-α staining. Interestingly, the outer most granulosa cell layer in Enzy-FL expressed CYP17a1, the theca cells marker from day 4 onwards. Nevertheless, these CYP17a1 positive and inhibin-α negative cells still displayed the granulose-like cuboidal nucleus. Both theca-intact (Mech-FL) and theca-free (Enzy-FL) follicles can be cultured in vitro in 3-dimensional alginate system. While the Mech-FL follicles phenocopied in vivo growth patterns, the Enzy-FL follicles grow more slowly and express theca cells markers later in culture. These newly formed theca cells produce low but measurable amounts of steroid. Taken together, these data are consistent with the conclusion that in vitro granulosa cells have the potential to differentiate into androgenic cells and compensate for theca cell function. Whether the inner and outer theca cell layers differentiate in a similar manner during in vivo development is under further investigation. This work was supported by Award Number U54HD041857 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. (poster)