The experiment was done on 4 separate occasions with 6 wells included per treatment per replicate. Experiment 2 The aim was to test the hypothesis that pharmacological inhibition of the activation of the Akt and Erk pathways would inhibit the actions of FSH and IGF on bovine gran ulosa cells in vitro. Granulosa cells were cultured as described above with one of four possible culture media, control medium, FSH, IGF or FSH plus IGF in combination. Additionally each of the above treatments was given in combination with either PD98059, a specific inhibitor of the Erk activating enzyme MEK or LY294002, a specific inhibitor of Akt activation or a combination of both inhibitors resulting in a total of 16 treatments. Both PD98059 and LY294002 were initially dissolved in DMSO and were diluted to a final concentration of 50 M in vitro.

Control media also contained DMSO at a final concentration of 0. 005% in all treatment groups. Experiment 3 Theca interna cells were isolated from the same sets of fol licles used in experiment 2 as described by Glister et al. Theca cells were plated out and cultured using the same serum free conditions as described above FR 180204 structure for granu losa cells except that androstenedione was omitted from the culture medium. Cells were cultured for 144 h with control media, media with LH and the same treatments in combination with PD98059 and or LY294002. The dose level of LH used here was shown previously to promote optimal secretion of androstenedione by bovine theca cells cultured under these conditions. Media were changed and treatments replenished every 48 h.

At the end of culture, conditioned media were collected and stored at 20 C until assayed for androstenedione and progesterone. Viable cell number was determined by neu tral red {you can find out more| kinase inhibitor|selleck chemical|selleckchem|ML323 molecular weight dye uptake. The experiment was done on 4 sepa rate occasions with 6 wells included per treatment per replicate. Experiment 4 The aim was to test the hypothesis that inhibition of the activation of the Akt and Erk pathways would decrease fol licle growth and oestradiol production by ovine ovarian follicles in vivo. The oestrous cycles of eighteen ewes were synchronised using a progestagen sponge and on Day 3 of the oestrous cycle the two largest follicles were identified, measured, follicular fluid sampled and all other follicles ablated.

This stage of the cycle was chosen as it is during the first follicle wave and at a time when the follicles are large enough to treat but also early enough that the follicles are still growing and producing oestra diol. In each animal the largest of the two remaining fol licles was treated and the second follicle served as an untreated control follicle. Ewes were assigned to one of four groups and the largest follicle treated with control medium, Akt inhibitor, Erk inhibitor or Akt Erk inhibitor.