STEROIDOGENESIS BY EQUINE PREOVULATORY FOLLICLES - RELATIVE ROLES OF THECA INTERNA AND GRANULOSA-CELLS

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17-beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5-mu-M testosterone, and culture media were assayed for progesterone, androstenedione, and 17-beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P < 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P < 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17-beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P < 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17-beta-estradiol biosynthesis derives primarily from the theca interna layer. However, our results clearly suggest that interactions between thecal and granulosa cells are required for estradiol synthesis in the preovulatory follicle of the mare, with androgens of thecal origin aromatized to estradiol by granulosa cells.