Synergistic Activation of Akt by IGF-1 and cAMP Is Correlated with Altered Expression of Paracrine Factors That Regulate Follicle Progression and Ovulation in Murine Granulosa Cells.

Abstract

Folliculogenesis and granulosa cell function are regulated, in part, by gonadotropin stimulation of cAMP-dependent signal transduction. For example, follicle stimulating hormone (FSH) regulates granulosa cell steroidogenesis, oocyte maturation, and the expression of paracrine factors which coordinate oocyte-granulosa cell growth and development. IGF-1 also contributes to follicular development via regulation of granulosa cell proliferation, apoptosis, and steroid production. These actions of IGF-1 are mediated through its interaction with the IGF-1 receptor, and in some cases, requires simultaneous gonadotropin-dependent signaling. Recent epidemiological studies demonstrate that IGF-1 levels decrease with increasing age, body mass index, and waist circumference. Given that advanced maternal age and obesity are the most common cause of anovulatory infertility, the objective of this study was to identify mechanisms by which IGF-1 regulates the expression of genes in granulosa cells which are crucial for follicle growth, follicle maturation, and ovulation. To achieve this objective, granulosa cells were isolated from the ovary of 23-day-old CF-1 mice and maintained in short-term culture. After 5 days, cells were exposed to no treatment, 1mM cAMP, 100ng/mL IGF-1, or a combination of cAMP and IGF-1 for 2 or 8 hours. The mRNA abundance of several paracrine factors which are produced by the granulosa cells and contribute to the coordination of oocyte and follicular growth and regulate the process of ovulation were examined using quantitative real-time PCR (QPCR). Genes associated with transzonal projections, which facilitate bi-directional communication between the granulosa cells and oocyte, were also examined. IGF-1 increased cAMP-dependent stimulation of kit ligand (Kitl), growth differentiation 9 (Gdf9), amphiregulin (Areg), and interleukin-6 (Il6) expression in both the 2 and 8 hour treatment groups. IGF-1 also increased cAMP-dependent stimulation of epiregulin (Ereg) expression 8 hours post-treatment. Conversely, anti-mullerian hormone (Amh) was downregulated upon combined treatment of granulosa cells with IGF-1 and cAMP for 2 or 8 hours. IGF-1 and cAMP alter gene expression through activation of the downstream signaling proteins Akt and Erk1/2. To assess the activity of these proteins, granulosa cells were maintained in short-term culture and exposed to no treatment, 1 mM cAMP, 100 ng/mL IGF-1, or a combination of cAMP and IGF-1 for 30 or 60 minutes. Western blot analyses demonstrated that cAMP alone or in combination with IGF-1 increased Erk1/2 phosphorylation compared to untreated cells 30 and 60 minutes post-treatment. IGF-1 or cAMP treatment alone also increased Akt phosphorylation in the granulosa cells. Interestingly, the combined treatment of IGF-1 and cAMP resulted in a synergistic increase in Akt phosphorylation indicating cross-talk between the cAMP and IGF-1 signaling pathways. Taken together, these data suggest that IGF-1 and cAMP may have an additive effect on paracrine factor gene expression due to the synergistic activation of Akt. Given that these genes are associated with follicle development and ovulation, these data also provide a plausible mechanism for age and obesity-dependent anovulatory infertility. (platform)