llagen expression in the OSE and theca cells as expected, with low levels observed in the granulosa cells. However, insulin dramatically increased colla gen IV expression in the granulosa cells, which may cor relate with reduced expression of MIS in secondary follicles. Inhibition of IR IGF1R function with tyrphostin AG1024 resulted in collagen IV expression restricted to the OSE and theca as well as increased MIS expression in granulosa cells. Studies from the Woodruff lab have demonstrated that altered cortical rigidity can disrupt folliculogenesis, as a more rigid environment favors androgen secretion and reduced follicle growth. As high levels of insulin cause hyperplastic OSE and increased collagen deposition in the OSE and granulosa cells, this may possibly increase cor tical tension on the ovarian follicles to restrict their growth and reduce MIS expression.

The detrimental effects of high levels of insulin or IGF on follicle growth may be also be mediated directly by increased MAPK and PI3K signaling. The MAPK and PI3K pathways are canonical signaling pathways downstream of IR and IGF1R activation. Ovarian organoids cultured extra resources with inhibitors of the insu lin IGF pathway appeared to have more MIS expression in the granulosa cells indicating that the ovary has en dogenous production of IGF that in ex vivo 3D culture is detrimental to the tissue. In the current study, inhib ition of the MAPK pathway more effectively blocked insulin induced OSE hyperplasia and follicular degener ation and was less effective at attenuating the effects of IGF I.

When the MAPK inhibitor UO126 was included along with selleck insulin in the culture medium, the OSE grew as a single layer of cells and the secondary follicles pro duced MIS. However, collagen IV expression was still detected in the granulosa cells, indicating that additional signaling pathways may be involved in the process of altered ECM deposition in response to insulin. The PI3K inhibitor LY294002 effectively reduced OSE multilayering and proliferation induced by either insulin or IGF I as well as restoring MIS expression. This cor related with expression of collagen IV being restricted to the OSE and theca cells similar to when organoids were cultured with the IR IGF1R inhibitor AG1024, indicating that PI3K signaling may control collagen IV synthesis or deposition in the ovary, although future work is necessary to delineate the role of each of these pathways in the OSE.

Use of an alginate hydrogel 3D culture system facilitates observation of how different cell types in the ovary interact with one another when stimulated with insulin or IGF I. As an example, IGF I is produced locally from the granulosa cells and may be responsible for the low levels of collagen IV observed in basal cultured organoids while inhib