Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

Abstract

Background: The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo.

Methods: Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later.

Results: We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo.

Conclusion: These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.

Figure 1

Effect of treating bovine granulosa cells in vitro with FSH (0.33 ng/ml), IGF-I (10 ng/ml) or FSH plus IGF-I on cell number and secretion of oestradiol, progesterone, inhibin-A, activin-A and follistatin. Treatment effects were highly significant (P < 0.0001) in all cases (4 replicates with 6 wells included per treatment per replicate). Bars with no common superscript are different (P < 0.05).

Figure 2

Representative Western blots and mean levels (± S.E.M) of (A) Akt, (B) p-Akt, (C) Erk and (D) p-Erk in granulosa cells (n = 4) treated with control medium, FSH (0.33 ng/ml), IGF (10 ng/ml) or FSH+IGF in combination in vitro. Bars with no common superscript are different (P < 0.05). The units represent the intensity of bands after background subtraction and are relative to white (value 0) and black (value 256). The blots each show a single band for Akt and p-Akt at about 60 kDa and each show a double band for Erk and p-Erk at about 44 and 42 kDa.

Figure 3

Effect of treating granulosa cells in vitro with control medium, FSH (0.33 ng/ml), IGF (10 ng/ml) or FSH+IGF in combination with PD98059 (Erk inhibitor) and/or LY2924002 (Akt inhibitor) on cell number and the secretion of oestradiol, progesterone, inhibin-A activin-A and follistatin (N = 3 replicates with 6 wells included per treatment per replicate). Bars with no common superscript are different (P < 0.05) within each treatment group.

Figure 4

Effects of treating bovine theca cells in vitro with control medium or LH (160 pg/ml) in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) on cell number and secretion of androstenedione and progesterone (n = 4 replicates). Bars with no common superscript are different (P < 0.05) within each treatment group.

Figure 5

Follicle diameter (mean ± sem) in ewes in which the largest follicle was treated in vivo with control solution with DMSO (DMSO n = 5), PD98059 (PD, n = 5), LY294002 (LY, n = 4) or PD98059 plus LY294002 (PD+LY, n = 4). The second follicle in each animal served as an untreated control control (n = 18). All other follicles were ablated via electrocautery. Follicle diameter was measured at the time of surgery (via laparotomy) and 48 h later after the ovaries were recovered. * indicates differences (P < 0.05) between diameters at surgery and recovery.

Figure 6

Follicular fluid oestradiol concentrations (mean ± sem) in ewes in which the largest follicle was treated in vivo with control solution with DMSO (DMSO n = 5), PD98059 (PD, n = 5), LY294002 (LY, n = 4) or PD98059 plus LY294002 (PD+LY, n = 4). The second follicle in each animal served as an untreated control (n = 18). All other follicles were ablated via electrocautery. Follicular fluid was sampled from follicles at the time of surgery (via laparotomy) and 48 h later after the ovaries were recovered. * indicates differences (P < 0.05) between concentrations at surgery and recovery.