1R signaling. Inhibitors of the MAPK and PI3K pathway did not equivalently restore MIS expression fol lowing treatment with insulin or IGF I, as culture of orga noids with UO126 restored MIS expression when organoids were cultured with insulin, but LY294002 restored expression of MIS when organoids were cultured with IGF I. Culture of organoids with insulin or IGF I disorders collagen IV organization Inclusion of high levels of insulin or IGF I in ovarian orga noid culture medium resulted in hyperplastic OSE and reduced follicle MIS expression. Recent work suggests that the mechanical forces within the ovary may be involved in follicle maturation and ovu lation. Expression of extracellular matrix proteins in the ovary has been well characterized, with collagen IV expressed abundantly in the OSE and theca cells, with very low levels in the granulosa cells and stroma.

To determine if culture of organoids with insulin or IGF I resulted in altered ECM deposition or organization, organoids were analyzed for localization of FR 180204 FLT inhibitor collagen IV. Organoids cultured in basal medium exhibited strong ex pression of collagen IV in the OSE and theca, but collagen IV was also detected in the granulosa cells. Addition of insulin to the medium resulted in a dra matic increase in collagen IV expression in the granu losa cells, with little expression observed in the theca. Organoids cultured with IGF I exhibited a similar ex pression pattern as basal cultured organoids, with colla gen IV expressed primarily in the OSE and theca, with low expression in the granulosa cells.

Abrogation of IR and IGF1R signaling by AG1024 alone altered the de position of collagen such Etizolam structure that the follicles were sur rounded with collagen and very little expression was detected in the granulosa cells which was a phenotype that resembled uncultured ovaries and was different than basal organs. The resulting phenotype from AG1024 alone suggested antagonizing endogenous IGF resulted in collagen deposition more similar to uncul tured ovaries. AG1024 in combination with insulin also resulted in collagen IV expression restricted to the OSE and theca, resembling normal, uncultured ovaries. However, addition of AG1024 to organoids cultured with exogenous IGF did not alter the collagen IV distribution back to resembling uncultured ovaries, suggesting that 10 uM of the inhibitor could not effectively block all the en dogenous and exogenous IGF.

Although inhibition of MAPK by UO126 did not rescue collagen IV localization, inhibition of the PI3K pathway by LY294002 reduced granulosa cell expression of collagen IV to those of organoids cultured with AG1024 alone, in dicating that the PI3K pathway may play a central role in altered collagen synthesis and deposition downstream of insulin and IGF signaling. Discussion