Cells of the zona fasciculata and zona reticularis lack aldosterone synthase (CYP11B2) that converts corticosterone to aldosterone, and thus these tissues produce only the weak mineralocorticoid corticosterone. However, both these zones do contain the CYP17A1 missing in zona glomerulosa and thus produce the major glucocorticoid, cortisol. Zona fasciculata and zona reticularis cells also contain CYP17A1, whose 17,20-lyase activity is responsible for producing the androgens, dehydroepiandosterone (DHEA) and androstenedione. Thus, fasciculata and reticularis cells can make corticosteroids and the adrenal androgens, but not aldosterone.
Granulosa cells were suspended in mM Tris-HCl, pH , 6 M urea, 10% (v/v) glycerol, 2% (w/v) SDS, % (w/v) bromophenol blue, and freshly added 5% (v/v) β -mercaptoethanol and subjected to sonication on ice. Total protein content was quantified using Bradford assay (Bio-Rad Bradford Solution, USA). 20 μ g protein was loaded on 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, along with prestained molecular weight markers. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (GE Healthcare) by a wet method (Bio-Rad, USA). The transfer was performed at a constant voltage (100 V) for 90 min in a buffer consisting of 25 mM Tris, 192 mM glycine, and 20% methanol. The membrane was then incubated for 1 h at room temperature in blocking buffer (TBS-containing 5% skimmed milk and % Tween-20). Then, the membranes were incubated overnight at 4°C with appropriate antibody at a dilution of 1 : 1000 in TBS-containing 5% skimmed milk and % Tween-20. They were washed in PBS-% Tween-20 and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (final dilution 1 : 5000; Bangalore Genei) in TBS-containing 5% skimmed milk and % Tween-20. The signal was detected by ECL (enhanced chemiluminescence, Millipore Inc., USA).
Glucocorticoids (GCs) play an important role in the regulation of the immune system. Because of their anti-inflammatory activity GCs are used to treat diseases caused by an overactive immune system, such as allergies, asthma, autoimmune diseases and sepsis. There is accumulating evidence suggesting that GCs can also promote the pathogenesis of allergic diseases by enhancing T-cell pro-allergic differentiation of CD4+T cells to Th2 and Th17, by amplifying immune responses in steroid-insensitive CD8+ T cells and by inhibiting Th1 cytokine production.