DIRECT ACTIONS OF KIT-LIGAND ON THECA CELL-GROWTH AND DIFFERENTIATIONDURING FOLLICLE DEVELOPMENT

The direct actions of kit-ligand/stem cell factor (KL) in developing o varian follicles were investigated. Previous studies have shown that g ranulosa cells express KL that can support oocyte development. The cur rent study demonstrates that KL can also act directly on theca cells t o promote cellular growth and differentiation. Through RT-PCR analysis it was shown that bovine granulosa cells express KL, and theca cells express the receptor c-kit. Bovine theca interna cells were isolated a nd cultured in serum-free conditions to study KZ actions. KL stimulate d theca cell growth in a dose-dependent manner as measured by [H-3]thy midine incorporation into DNA when cells were cultured under subconflu ent conditions. KL had no effect on theca cell androstenedione or prog esterone production under these growth-permissive conditions. In contr ast, KL stimulated theca cell androstenedione production but had no ef fect on progesterone production when theca cells were cultured under c onfluent (non-growth-permissive) conditions. Estradiol (10(-7) M) and human CG (100 ng/ml) were used as controls and regulated theca cell st eroid production at any cell density. These results demonstrate that K L can directly stimulate theca cell growth and steroid production duri ng follicular development. The observation that KL stimulated androste nedione production but not progesterone production suggests that KL pr omotes a follicular phase differentiated state in theca cells. The pot ential reg ulation of KL and c-kit expression during follicular develo pment was studied using a specific quantitative RT-PCR procedure. Tota l RNA from granulosa cells (for KL) and theca cells (for c-kit) was ex amined from small (<5 mm), medium (5-10 mm), and large (>10 mm) size f ollicles. Steady state levels of KL messenger RNA were highest in gran ulosa cells from large size follicles and lowest in small and medium s ize follicles. No differences were observed in the steady state levels of c-bit messenger RNA in theca cells from small, medium, or large si ze follicles. The observation that KL expression is highest in large s ize follicles suggests that KL may be important for increased growth a nd steroid production in large and dominant follicles. Observations de monstrate that KL can dramatically alter theca cell function and suppo rt the hypothesis that local granulosa-theca cell interactions play an important role in regulating cellular function within ovarian follicl es. This study identifies KL as the first granulosa cell-derived growt h factor that can directly stimulate theca cell growth and androstened ione production in the absence of gonadotropins.