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. Furthermore, inhibition of the Akt and Erk pathways in vivo had a negative effect on follicular fluid oestradiol production and follicle growth in sheep. Taken together, these results suggest an important role for Akt and Erk signalling pathways in mediating the effects of the gonadotropins and IGF on follicle cell function and on follicular development.
The stimulation of inhibin-A, activin-A, follistatin, oestradiol, progesterone and cell number by FSH and IGF in granulosa cells in vitro agrees with earlier findings . However, the regulation of the Akt and Erk pathways in relation to these hormonal and proliferative changes has not been studied previously in the bovine model. Increases in Akt and Erk signalling proteins in response to FSH and IGF stimulation suggest a role for Akt and Erk signal transduction pathways in FSH and IGF mediated granulosa cell development as reflected by cell proliferation/survival and production of inhibin-A, activin-A, follistatin, oestradiol, and progesterone (Figure 1). The significant reductions in hormonal output as a result of inhibition of the Akt and Erk pathways further support a role for Akt and Erk in FSH- and IGF- mediated action in granulosa cells. However, there appear to be differences in the relative importance of each pathway with respect to the endpoints measured. Our findings suggest that Akt is important in mediating the effects of FSH on inhibin-A, activin-A, oestradiol and progesterone secretion and also important in mediating IGF-I stimulated inhibin-A, activin-A, follistatin, oestradiol and progesterone secretion by granulosa cells. In addition, the results also suggest that the Erk pathway is involved in mediating FSH-induced activin-A and oestradiol production, and progesterone secretion induced by both FSH and IGF-I stimulation of granulosa cells in vitro.
The regulation of activin-A secretion by FSH and IGF displayed a similar pattern to that of oestradiol with the Erk pathway only involved in FSH-stimulated production and the Akt pathway involved in both FSH- and IGF-stimulated production. Inhibition of the Erk pathway had no effect on inhibin-A concentrations. Only the Akt pathway was indicated in regulating the production of inhibin-A. However, this might be a simplistic view of what is happening. Activin is known to upregulate FSH receptors and aromatase gene expression, thus promoting production of oestradiol . Additionally, expression of the inhibin α-subunit is increased in response to activin-A . Previous work suggests that activin-A may mediate the effects of FSH stimulation on oestradiol and inhibin-A production  but this explanation remains to be proved. The observed differences in oestradiol and inhibin-A production in this present study might not relate directly to inhibition of the Akt and Erk pathways but rather the indirect effect of inhibition of these pathways on regulation of activin-A production/secretion.
Granulosa cell proliferation is a critical step in follicular development and both FSH and IGF are required for successful follicle development. Our results (Figure 1) confirmed other research showing that FSH and IGF promote proliferation/survival of granulosa cells [5, 34]. Despite the fact that FSH and IGF stimulated the Akt and Erk pathways (Figure 2) and that inhibition of these pathways markedly influenced hormone secretion, neither inhibitor affected FSH and IGF stimulated increases in cell number (Figure 3). It may be that additional signalling pathways activated by FSH and IGF, such as PKA , compensated for the block in Akt and Erk signalling. Our findings are not in agreement with others that found that FSH-stimulated porcine granulosa cell proliferation/survival was significantly reduced by treatment with PD98059 through a negative effect on cell cycle proteins and DNA synthesis [9, 35, 36].
In addition to FSH and IGF, LH is also important for follicle development and it has been shown that LH increases activation of Erk Akt in porcine and rat theca cells [10, 12]. As expected from previous studies on bovine theca cells , our results demonstrated a marked increase in androstenedione production by theca cells in response to LH (Figure 4). Moreover, this LH-induced increase was attenuated by inhibition of Erk and completely blocked by inhibition of the Akt pathway. Conversely, progesterone production increased in response to inhibition of the Erk pathway. This is in agreement with other recent findings that demonstrated that LH-induced Erk activation differentially regulates production of progesterone and androstenedione in bovine theca cells in vitro.
The results from Experiment 4 clearly indicate that treatment of follicles in vivo with inhibiters of the Akt and Erk pathways in the largest follicle in sheep had a negative effect on follicular oestradiol production and follicle growth, two key markers of follicle health and dominant follicle development. There was a difference between the largest and second largest follicles at the start of treatment with respect to diameter and oestradiol concentration, which agrees with previous findings that showed that ovine follicles exist in a hierarchy in relation to follicle diameter and oestradiol concentrations . Day 3 of the cycle was chosen as the day of treatment in the present study as follicles would be large enough to treat, be producing relatively high amounts of oestradiol and still be growing. Previous research indicated that between Days 1 and 3 of the cycle oestradiol concentrations increase; however, that they then start to decline on Day 4 . None-the-less, despite treating follicles relatively late in the follicle wave we still demonstrated an inhibitory effect on follicle growth and oestradiol production through blocking the activation of Akt and Erk pathways.
The significant decrease in oestradiol concentrations in follicles treated in vivo with Akt and Erk inhibitors agrees with the results from Experiments 1 and 2 where inhibition of the Erk pathway inhibited FSH-induced oestradiol production and inhibition of the Akt pathways inhibited both FSH- and IGF-induced oestradiol production in granulosa cells in vitro (Figure 3). Androstenedione secretion in cultured theca cells was also abrogated by inhibition of both the Akt and Erk pathways (Figure 4). In Experiment 3, the inhibitors were injected directly into the antral cavity and it is reasonable to suggest that granulosa cells would be first to be exposed to and affected by the inhibitors. However, it is possible that the inhibitors might have diffused through the granulosa layer of cells into the theca layer and affect signalling pathways there. Thus the significant reductions in follicular fluid oestradiol concentrations may be due to the effect of the Akt and Erk inhibitors on both granulosa and theca cells in combination.
In summary, this study demonstrates a role for the Akt and Erk pathways in mediating the actions of FSH and IGF on granulosa cells and LH on theca cells in vitro and their role in follicle growth and oestradiol secretion in vivo. While both pathways appear to be important for the actions of these hormones in both cell types, we conclude that the actions of the Akt pathway are more pronounced than the Erk pathway in granulosa cells and vice versa in the in theca cells. None the less, administration of inhibitors of these pathways in vivo inhibited follicle growth and reduced follicular fluid oestradiol concentrations. We suggest that the successful functioning of healthy follicles requires the activation of the Akt and Erk signal transduction pathways, and that these pathways are necessary for ovarian follicle growth and development.