The volume of each treat ment injection was about 10% of follicle volume, which resulted in a final follicular fluid concentration of 50 M of the inhibitors, and 50 M of the DMSO. Concentrations of the inhibitors were based on the treatments used in vitro in Experiment 2. The ewes recovered from surgery and 48 h after treatment were euthanized, the two follicles were identified from drawings of the ovaries made at surgery and dissected out of the ovaries, measured and follicular fluid was aspirated. The follicles were cut open and the theca and adherent granulosa cells peeled from the stroma. The granulosa cells were then gently scraped from the theca and the granulosa and theca cells were snap fro zen in liquid nitrogen and stored at 80 C.

All exper imental procedures involving live animals were sanctioned by the UCD Animal Research Ethics Commit tee and licensed by the Department of Health and Chil dren, Ireland, in accordance with the cruelty to animals read full report act and European Community Directive 86 609 EC. Immunoassays Inhibin A concentrations were measured by a two site IRMA described by Knight and Muttukrishna which has a detection limit of 250 pg ml. Oestradiol con centrations were determined by RIA as described previ ously with a detection limit of 1. 5 pg ml. Progesterone concentrations were determined using an ELISA with a detection limit of 20 pg ml. Concentra tions of both activin A and follistatin were measured using ELISA. The inter and intra assay coefficients for all assays were under 11%.

Whole cell protein extract preparation Tissue samples were thawed on ice, homogenised in cold RIPA buffer and agitated on a shaker for 15 mins at 4 C. The homogenate was then centrifuged at 1400 rpm for 15 mins at 4 C. The selleck Etizolam resultant supernatant was snap frozen in liquid nitrogen and stored at 80 C. Protein concentra tions of the sample extracts were determined by spectro photometric assay using the Bio Rad protein assay dye reagent concentrate. Immunoblotting Levels of Akt and Erk and their phosphorylated forms were determined as we have previously described. Proteins from granulosa were resolved on 10% SDS poly acrylamide gels and then electrophoretically transferred onto nitrocellulose. The protein transfer was performed at 200 V for 1. 5 h at 4 C. Ponceau S stain solution was used to visually assess the equal transfer of the proteins from the gel to the mem brane.

TBS Tween was used to destain the membrane, which was then blocked in 5% Marvel in TBS Tween for 1 2 h. The blocking solution was removed with a brief rinse of TBS Tween and the membrane was incubated overnight for 14 16 h with the appropriate antibody diluted in 5% BSA in TBS Tween at 4 C. The antibodies were all rabbit anti mouse IgG. After incubation with the primary antibody, the mem brane was washed twice for 10 min in TBS Tween and then incubated for a further 1.