In addition, it has been shown that IGF has a direct anti apoptotic effect and is selectively expressed in healthy follicles compared with small atretic follicles. The Akt and Erk pathways are considered the principle sig nalling pathways that mediate the effects of IGF. We have previously shown higher levels of total and phos phorylated Akt and Erk in dominant follicles compared with subordinate follicles. The objectives of the studies reported here were to examine the interactions of the gonadotrophins and IGF with the Akt and Erk signal ling pathways in theca and granulosa cells in vitro and to describe their functional significance for ovarian follicle growth in vivo. Materials and methods Experimental design Experiment 1 The aim was to test the hypothesis that FSH and IGF acti vate Akt and Erk pathways in bovine granulosa cells cul tured in vitro.

This was done using granulosa cells collected from 4 to 6 mm follicles from animals after slaughter using a validated granulosa cell culture system that maintains FSH responsiveness, oestradiol secretion and minimizes luteinization. Granulosa cells were {informative post| kinase inhibitor|selleckchem|selelck kinase inhibitor|ML323 cultured in serum free conditions for 144 h with conditioned medium collected and replaced with fresh media antibiotic antimycotic solution, 10 ng ml bovine insulin, 2 mM L glutamine, 10 mM HEPES, 5 g ml apotransferrin, 5 ng ml sodium selenite, 0. 1% BSA and 10 7M androstenedione treatments every 48 hours as described by Glister et al. Cells were seeded at a density of 0. 5 × 106 viable cells per well in 24 well plates and cultured in a 1 ml volume of media treatments.

Treatment groups were as follows untreated controls, 0. 33 ng ml FSH, 10 ng ml IGF, 0. 33 ng ml FSH and 10 ng ml IGF. These treatments have been shown previously to stimulate {this content| selleck chemicals|selelck kinase inhibitor|selleckchem|LDC000067 1073485-20-7 cell proliferation survival and hormone secretion by bovine granulosa cells over a 144 h treatment period. The more potent LR3 IGF I analogue was used rather than IGF I or IGF II because its action is not compromised by association with endogenous IGF BPs produced by the cells. At the end of culture, condi tioned media were collected and stored at 20 C until assayed for oestradiol, progesterone, inhibin A, activin A and follistatin. Cells were scraped off the culture plates in 1 ml of phosphate buffered saline and a small aliquot of cell suspension was taken and processed for via ble cell number by neutral red dye uptake as described previously. The remaining cell suspension was spun at 800 g and the cell pellet washed twice before snap freez ing the cell pellet and storing at 80 C until processed for Western blots. Western blot analysis was used to deter mine the levels of Akt and Erk and their phosphorylated proteins p Akt and p Erk in total protein extracted from cells at the end of culture.