11BHSD1 is a NADP-dependant, bi-directional enzyme which inter-converts the glucocorticoid cortisol with its inactive metabolite cortisone. Follicular fluid (FF) from large antral ovarian follicles contains hydrophilic and hydrophobic compounds which stimulate and inhibit 11BHSD1 enzyme activities, respectively. The objective of this study was to assess whether levels of these enzyme modulators change in FF during development of porcine follicles. Pooled samples of FF, aspirated from small (2mm diameter), medium (6mm) and large (>8mm) antral follicles, were loaded onto C18 Sep-pak cartridges and sequentially eluted with 0-100% (v/v) methanol. For each size class of follicle, 3 independent pools of FF were evaluated. The effect of each column fraction on NADP-dependent oxidation of 100nM [3H]-cortisol was assessed over 1hr at 37degC using rat kidney homogenate as a source of 11BHSD1. Steroids were resolved by thin layer chromatography (TLC) and quantified using a radiochromatogramme scanner. FF aspirated from all 3 follicle size classes contained hydrophobic inhibitors of 11BHSD1 activity. Hydrophobic fractions of small, medium and large antral follicles inhibited NADP-dependent cortisol oxidation by 62 plus/minus 6% (P<0.01), 38 plus/minus 6% (P<0.01) and 45 plus/minus 3% (P<0.01), respectively. While small and medium sized follicles contained no stimuli of 11BHSD1, the hydrophilic fractions of FF eluted at <20% methanol from large antral follicles increased cortisol oxidation by 26 plus/minus 4%. We conclude that porcine ovarian follicles contain local modulators of 11BHSD1 activity, the profile of which changes with follicular growth. Specifically, during follicular growth, the level of 11BHSD1 inhibitors decreases with a concomitant increase in the level of 11BHSD stimulators.
Supported By: BBSRC-Sygen International Plc. CASE Studentship BBS/S/L/2003/10221
01 - 03 Nov 2004
Society for Endocrinology