Inhi bition of the Erk pathway with PD98059 treatment sup pressed the FSH induced increase in activin A, oestradiol and progesterone secretion. Further more, PD98059 suppressed follistatin secretion from cells co stimulated with FSH and IGF and progesterone secre tion from cells treated with IGF alone or in combination with FSH. No effect of PD98059 was seen on either FSH or IGF stimulated inhibin A secretion or viable cell number. Inhibition of the Akt pathway with LY294002 dramati cally reduced FSH, IGF or FSH and IGF stimu lated inhibin A, activin A, oestradiol and progesterone secretion. Follistatin secretion was suppressed in cells treated with IGF alone or in combination with FSH by LY294002 compared to their respective control treatments without LY294002.

Experiment 3 Effects of LH in combination with PD98059 and or LY294002 on cell number and secretion of androstenedione and progesterone from theca cells Theca cells stimulated with LH showed an 8 fold increase in androstenedione BAPTA-AM secretion compared to the control treatment. Inhibition of the Erk path way with PD98059 treatment and the Akt pathway with LY294002 reduced both basal and LH induced androstenedione secretion compared to controls. Progesterone concentrations in media were not affected by LH stimulation but treatment with PD98059 LH stimulated an increase in progesterone con centrations compared to LH alone. Neither the Erk nor Akt inhibitors affected the number of viable theca cells at the end of culture. Experiment 4 Follicle diameters and follicular fluid oestradiol concen trations were not different among groups for the largest follicles or the second largest follicles before treatment.

However, both the diameter and follicular fluid oestradiol concentrations where inhibitor CORM-3 greater in the largest compared to the second largest follicles before treatment. Of the treated follicles, only the control follicles that were treated with DMSO increased in diameter between the time of injection and 48 h later when recov ered. The other follicles treated with PD98059, LY294002 or PD98059 plus LY294002 showed no increase in diameter over the same period. The untreated, second largest, control follicles also increased in diameter. Follicular fluid oestradiol concentrations were similar between the time of injection and recovery of the ovaries 48 h later in the control follicles treated with DMSO but decreased in follicles treated with PD98059, LY294002 and PD98059 LY294002.

Follicular fluid oestradiol concentrations also decreased in the second largest folli cles over the 48 h period. Discussion Findings from the present study indicate that inhibition of the Akt and Erk pathways inhibit the stimulatory actions of FSH and IGF on cultured bovine granulosa cells and LH on theca cells in vitro.