In the ovary, glucocorticoids are metabolised by 11βHSD enzymes. The bi-directional 11βHSD1 enzyme usually acts as an NADPH-dependent 11-ketoreductase to regenerate cortisol (F) from circulating cortisone (E), while 11βHSD 2 inactivates F using NAD+ as cofactor. Having isolated endogenous inhibitors of 11βHSD1 from porcine follicular fluid (pFF) and ovarian cysts, the aims of this study were to establish whether 11βHSD enzymes interconvert F and E in porcine granulosa cells (GCs) and, if so, whether this changes during follicle growth.
Porcine GCs were isolated from small, medium and large antral follicles (24 mm, 48 mm and >8 mm in diameter, respectively), and from spontaneous ovarian cysts (2540 mm diameter) (n=5 in each group). 11βHSD activities were measured over 4 h at 37.5 °C in primary cell cultures and in GC homogenates using radiometric conversion assays. Intact GCs were incubated with either 3H-F or 3H-E (100 nM) in serum-free medium supplemented with 10 ng/ml insulin, 10 ng/ml IGF-1, 5 μg/ml transferrin, 0.04 ng/ml sodium selenite, 100 nM androstenedione and 1 ng/ml FSH. Cell homogenates were incubated either with 100 nM 3H-F plus 4 mM NADP+/NAD+, or 100 nM 3H-E plus 4 mM NADPH ±10 mM glucose-6-phosphate.
In intact GCs, net oxidation of F increased with follicle diameter (from 0.8±0.3 pmol in GCs from small follicles to 2.1±0.5 pmol in dominant follicles) but was significantly decreased in ovarian cysts (0.7±0.1 pmol; P<0.05 versus large antral GCs). In GC homogenates, addition of both NADP+ and NAD+ increased F metabolism, and both activities increased with follicle diameter. There was no significant metabolism of 3H-E either in intact cells or GC homogenates, irrespective of follicle size or added cofactors. We conclude that porcine GCs can inactivate F using both NADP+-dependent 11βHSD1 and NAD+-dependent 11βHSD2 enzymes, but that 11βHSD1 appears to lack any 11-ketosteroid reductase activity in these ovarian cells. Furthermore, rates of F oxidation are lowest in rapidly growing small antral follicles and in ovarian cysts.
This work was supported by a BBSRC-Sygen CASE studentship.
01 - 05 Apr 2006
European Society of Endocrinology