Although LH has been reported to activate the cAMP PKA pathway and the ERK MAPK pathway in theca cells, whether LH stim ulates the PI3K Akt cascade in theca cells remains unclear. Results of this study show for the first time that 1 LH stimulates Akt phosphorylation in cultured bovine theca cells, and that 2 activation of PI3K Akt is involved in CYP17A1 mRNA expression and androgen production in stimulated Akt phosphorylation in theca cells is responsi ble for these observations reported earlier. Both wortmannin and LY294002 are inhibitors of the lipid modifying enzymes known as PI3K, and many researchers perform a parallel study by using both inhibi tors to probe the roles of PI3K in biological processes. However, depending on the concentration examined, LY294002 are the minimal effective concentrations for blocking the LH induced androstenedione production in theca cells.

Nevertheless, only LY294002 suppressed LH induced CYP17A1 mRNA expression, whereas wortman nin did not affect this response. While the reason for this apparent discrepancy is not clear, it is worth noting that wortmannin has been reported to be unstable in aqueous solution and less specific than LY294002. Higher concentration of wortmannin induced inhibitor L-Mimosine theca cell detachment and apoptosis in our serum free culture system. Numerous reports have described that an activation of the intracellular signaling is a rapid reaction in most cells. However, in this study, it took 12 h for LH induced increase in phos theca cells. Reportedly, LH induced Akt phosphorylation in whole rat ovary, and the PI3K inhibitor, LY294002, suppressed androstenedione production by theca cells in rat and cattle.

It is possible that LH As described earlier, H89, a potent and selective inhibitor of PKA, did not affect LH mediated changes in phospho Akt, indicating that a pathway distinct from that of PKA is involved in LH induced Akt phosphorylation in theca cells. Until recently, the effects of cAMP were generally thought to be mediated by activation recommended site BMS-863233 of cAMP dependent PKA, a major cAMP target, followed by phosphorylation of many intracellular targets, such as cAMP responsive ele ment binding protein, resulting in changes in ovarian gene expression such as CYP17A1. Nevertheless, some effects of cAMP appear to be inexplicable by activa tion of PKA. For instance, TSH and cAMP regulate prolif eration of thyroid cells by mechanisms independent of PKA.

Actually, cAMP binds specific guanine nucle otide exchange factors, cAMP GEFs. Gonzalez Robayna et al. reported that cAMP GEFs are expressed in rat granulosa cells and that the cAMP GEFs play a role in FSH induced activation of the PI3K Akt pathway in gran ulosa cells by PKA independent manner. Whether theca cells also express these regulatory components and whether the cAMP GEFs mechanism is involved in LH induced Akt phosphorylation in theca cells remains to be elucidated.