eam of IR IGF1R is the PI3K pathway, which plays a role in cell proliferation, regulation of apoptosis, and directional cell growth. Activation of the PI3K pathway alters orientation of the cytoskeleton through the Rho Rac Cdc42 GTPases, as well as affecting other components required for cell polarity and migration. Targets of the PI3K pathway were altered in response to insulin and IGF and the OSE exhibited altered morphology, hyperplasia, and multilayering in response to insulin and IGF, indicating that activation of the PI3K pathway may be involved in this phenotype. Organoids cultured with 10 uM LY294002, a PI3K inhibitor, exhibited a single layer of OSE with only 1% of OSE proliferating. To determine if LY294002 could effect ively block insulin or IGF induced hyperplasia and prolif eration, organoids were cultured with LY294002 and insulin or IGF.

Culture of organoids with insulin plus LY294002 or IGF I plus LY294002 resulted in selleck growth of a single layer of OSE, unlike organoids cultured with UO126, which only completely blocked insulin induced OSE hyperplasia. LY294002 reduced insulin induced OSE proliferation from 41% to 10%, and reduced IGF induced OSE proliferation from 41% to 4%. High levels of insulin and IGF I decrease secondary follicle MIS expression In the mouse ovary, immature primordial and primary fol licles are located in the cortex close to the surface of the ovary, with maturing follicles found in the medulla and perimedullary zone. As follicles become activated and begin to mature into secondary and preantral follicles, granulosa cells proliferate to form multiple cell layers around the oocyte and begin to secrete Müllerian Inhibit ing Substance.

IGF secreted by granulosa cells is required for follicle maturation beyond the antral stage, however, SH-4-54 ic50 high levels of insulin or IGF can be detri mental to follicle development, resulting in polyovular fol licles, ovarian cysts, and poor oocyte quality. To determine if insulin or IGF affected the follicles as well as the OSE, the expression of MIS by the secondary follicles was analyzed. All organoids exhibited localization of MIS to the ovarian surface as expected, with organoids cultured with insulin or IGF exhibiting several cell layers of OSE expressing MIS, providing a second marker indicating ex pansion of this cell type in response to insulin and IGF sig naling.

Secondary follicles were classified morphologically based on the appearance of at least 2 layers of granulosa cells surrounding the oocyte. In basal cultured organoids, most secondary follicles exhibited MIS expression, however, addition of insulin or IGF to the culture media resulted in reduced expression of MIS in secondary follicles, which could be rescued by addition of tyrphostin AG1024 to the media to block IR and IGF