Luteinizing Hormone Contributes to Androstenedione Production by Regulating Nuclear Movement of Steroidogenic Factor 1 (SF-1) in Bovine Theca Cells.

Abstract

During follicular development in bovine ovary, androstenedione (A4) that is essential for estrogen production in granulosa cells is produced in theca cells. A4 production in theca cells is regulated by steroidogenic acute regulatory protein (StAR) and CYP17. In addition, steroidogenic factor 1 (SF-1) and DAX-1 transcription factors contribute to the expression of genes associated with A4 production. Although luteinizing hormone (LH) plays an important role in A4 production in theca cells, whether LH involves in the increase in A4 production by enhancing StAR and CYP17 via SF-1 is unknown well yet. Therefore, we examined whether LH affect the manner of intra-cellular SF-1 when theca cell produced A4 at high levels by LH. Theca cells were isolated from large follicle (10-15 mm diameter) in bovine ovary obtained slaughterhouse. Theca cells were seeded in 12-well or 6-well culture plate for 1×105 cell/well or 4×105 cell/well, respectively, in medium containing 5% FCS, and cultured for 24 h at 37°C in 5% CO2, 95% air. After 24 h of culture, the culture medium was replaced with serum-free medium supplemented with 0 or 50 ng/ml LH (high LH), and the culture performed for 24h. After culture, the media was replaced with fresh medium (1% FCS) supplemented with 0 and 2.5 ng/ml (low LH) every 48 h for 6 days. We divided culture treatment into four groups as follows; 1) control, 2) low LH alone group, 3) high LH alone group, and 4) high LH + low LH group. The concentration of progesterone (P4) and A4 in culture medium each 48 h were analyzed by enzyme immunoassay (EIA). Total RNA was extracted from theca cells and the mRNA expressions of StAR and CYP17 were analyzed by the real-time PCR. The total protein expressions of SF-1 and DAX-1, and those expressions in cytoplasm and nuclear fractions of theca cell were analyzed by Western blotting. Intra-cellular localization of SF-1 and DAX-1 were visualized by using confocal microscopy with fluorescence immunostaining. The interaction between SF-1, and StAR and CYP17 promoter regions were shown by chromatin immunoprecipitation (ChiP assay). A4 production in theca cells at 48 h of culture were stimulated by low LH alone. The expression of StAR and CYP17 genes in theca cells with treated low LH alone were significantly higher than in control group, and the high levels maintained throughout culture period. The total protein expression of SF-1 and DAX-1 in theca cell in all groups showed a constant level throughout culture period. In nuclear fractions of theca cell, protein expression of DAX-1 did not change by low LH alone, whereas those of SF-1 are stimulated by low LH alone but not high LH alone and high LH + low LH group. Low LH alone enhanced the binding of SF-1 to the promoter regions for star and CYP17 than in other groups. These results suggested that low LH alone induces the movement of SF-1 to nuclear of theca cell, and consequently the expression of StAR and CYP17 genes were induced in theca cells. Such sequential mechanism is associated with the increase in A4 production in theca cells. Taken together, our data indicated that low LH, but not high LH, may be a necessary factor for A4 production in theca cells during follicular development in bovine ovary. (poster)