FSH Regulates IGF-2 Expression in Human Granulosa Cells in an AKT-Dependent Manner

Context: IGF-2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF-2 gene or the interaction of IGF-2 and FSH during follicle development. Objective: To examine the mechanisms involved in the regulation of the IGF-2 gene by FSH and the interplay between FSH and IGF-2 during granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus granulosa cells were separated from cumulus-oocyte complexes isolated from the follicular aspirates of in vitro fertilization patients and cultured for in vitro studies. Main Outcome: Protein and mRNA levels of IGF-2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF-2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF-2. Results: FSH significantly enhanced IGF-2 expression after 8 hours of treatment and at low doses (1 ng/mL). Reciprocally, IGF-2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF-2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF-2 promoter usage in human cumulus cells showed that the IGF-2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter-derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF-2 transcripts was blocked by cotreatment with inhibitors of AKT or IGF-1 receptor (IGF-1R). The inhibitory effect of the IGF-1R inhibitor on FSH-induced IGF-2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct. Conclusions: FSH is a potent enhancer of IGF-2 expression in human granulosa cells. In return, IGF-2 activation of the IGF-1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF-2 that is critical for human granulosa cell proliferation and differentiation.