The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal cells surrounding the ovary that exhibits both epithelial and mesenchymal characteristics . During monthly ovulations, the primary function of the OSE is to remodel the ovarian surface and underlying extracellular matrix to allow for rupture of a mature follicle. Following oocyte extrusion, the OSE proliferates to heal the wound in the surface of the ovary . OSE have receptors for steroid hormones and growth factors, both of which are found in abundance in follicular fluid released during ovulation . In particular, the OSE has been shown to express insulin receptor (IR) and insulin-like growth factor receptors (IGF1Rs); additionally, at high concentrations insulin can signal through IGF1R or through hybrid receptors of IR and IGF1R [4, 5]. Activation of IR or IGF1R by ligand binding activates downstream signaling pathways including the phosphatidylinositiol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. In turn, proliferative and anti-apoptotic pathways are activated, including Akt, glycogen synthase kinase 3 β (GSK3β), Bcl2, and Bad .
In immortalized OSE cell lines and many primary cell cultures, insulin is a critical component of the culture medium required for propagation of the cells; however, the ovary is not a classically insulin-responsive tissue [7, 8]. Crosstalk can occur between IR and IGF1R signaling when high concentrations of insulin initiate signaling through IGF1R . Interestingly, IGF-I is secreted into follicular fluid by granulosa cells, providing a local source for this cytokine . While it is known that insulin and IGF are proliferative in immortalized OSE cell lines , it is unknown whether these growth factors may exhibit additional changes in cell growth when the ovary is cultured in three dimensions (3D). By growing normal OSE as a component of ovarian organoids cultured within alginate hydrogels [11, 12], the effects of insulin and IGF on tissue architecture can be determined. The use of alginate hydrogels for organ culture permits growth of tissues in their normal three-dimensional architecture without disruption of signaling pathways downstream of extracellular matrix, as can be observed with other culture materials such as Matrigel .
In America, 64% of adult women are considered obese, and this negatively influences reproductive health and fertility . High circulating levels of insulin and IGFs are associated with obesity and diabetes; in the female reproductive system, increased levels of these growth factors are associated with polycystic ovary syndrome (PCOS) and ovarian cancer . For example, PCOS is a leading cause of infertility that affects 5-10% of reproductive-aged women and is diagnosed when patients exhibit at least two of the three following symptoms: anovulation, hyperinsulinemia, and hyperandrogenism . Ovarian cancer is the deadliest gynecological malignancy affecting American women, and obesity and diabetes are associated with a worse prognosis due in part to the effects of elevated levels of insulin and IGF on cancer cells [17, 18]. Primary cell lines established from ovarian carcinomas demonstrate that the components of the IGF pathway are present in ovarian cancer, including secreted IGF-I and IGF-II, IGFR-I and IGFR-II, and IGFBPs [19, 20]. IGF-II is overexpressed in ovarian cancer cells compared to normal OSE . IGF signaling exerts a pro-proliferative, anti-apoptotic effect on ovarian cancer cells and has also been shown to play a role in mediating cisplatin resistance [22, 23].
The current study examines the effects of high insulin and IGF levels on the OSE and ovarian follicles using an alginate hydrogel culture system. High proliferation rates in the OSE following culture with insulin or IGF were observed as described in previous studies [3, 24, 25]; however, by utilizing a 3D organ culture system, the present study demonstrates that high levels of insulin and IGF induce hyperplasia and formation of multiple cell layers in the OSE. Treatment of organ cultures with the IR/IGF1R inhibitor tyrphostin AG1024 restored the OSE to a single layer of epithelium and reduced proliferation to basal rates. Both the MAPK and PI3K pathways were involved in OSE hyperplasia, as small molecule inhibitors for these pathways inhibited insulin or IGF-induced hyperplasia and proliferation. Upon further examination of ovarian organ cultures, insulin and IGF reduced proliferation of granulosa cells, decreased Müllerian inhibiting substance (MIS) expression, and altered collagen deposition, which were restored upon blockage of IR/IGF1R function with tyrphostin AG1024. In summary, this study highlights the use of a 3D tissue culture system in demonstrating the differential effects that insulin and IGF signaling have on the ovarian surface and follicles.