Hormonal Regulation of Microrna-221 and Its Effect on Bovine Ovarian Theca Cell Function

Abstract

Development of ovarian follicles in cattle is controlled by systemic and locally produced hormones acting at the molecular level controlling numerous gene transcripts which spatial-temporal expression leads to one follicle ovulating and other follicles undergoing regression. MicroRNA-221 (miRNA-221) is increased in granulosa and theca cells of subordinate follicles compared with dominant follicles on day 3 of the estrous cycle in cattle. The objectives of this study were to investigate the hormonal regulation of miRNA-221 expression in theca cells and its possible role in regulating follicular function. Bovine ovaries were collected from a local abattoir and theca cells were obtained from large (8 to 22 mm) follicles, cultured for 2 to 7 days in 10% fetal calf serum (FCS), and treated with various hormones in serum-free medium for an additional 24 or 48 h in five experiments. Medium was collected for analysis of progesterone and androstenedione concentrations via radioimmunoassay, or cellular RNA was collected for gene expression analysis of miRNA-221 via real-time PCR. In Exp. 1, FGF9 increased (P = 0.08) abundance of miRNA-221 2.0-fold after 12 h and 2.4-fold after 24 h compared with controls. In Exp. 2, forskolin and dibutyryl cyclic adenosine monophosphate (dbcAMP) had no effect (P > 0.10) on miRNA-221 expression in bovine theca cells, but FGF9 treatments increased (P < 0.05) miRNA-221 abundance 1.94-fold. In Exp. 3, IGF1 had no effect (P > 0.10) on basal or FGF9- induced miRNA-221 expression (3.3-fold increase) in bovine theca cells, however, 10% FCS increased (P < 0.05) miRNA-221 abundance by 3-fold greater than control cultures; the combined treatment of FGF9 and 10% FCS did not differ (P > 0.10) from either treatment alone. In Exp. 4, estradiol, androstenedione, and phytoestrogens had no effect (P > 0.10) on miRNA-221 abundance. In Exp. 5, neither miRNA-221 mimic nor inhibitor affected cell numbers; nonetheless FGF9 stimulated an increase in cell proliferation (P < 0.01). Similarly, neither miRNA-221 mimic nor inhibitor affected steroidogenesis whereas FGF9 inhibited (P < 0.01) IGF1-induced production of androstenedione and progesterone. In summary, exposure of bovine theca cells to FGF9 in vitro increased expression of miRNA-221, however miRNA-221 was not regulated by steroids or cAMP. The role of miRNA-221 in follicular function will require further study.