To assess FOXO3 gene expression levels and members of the KL-PI3K-AKT-FOXO3 pathway in pre- and postmenopausal human ovaries. To evaluate in a temporal manner protein localization of FOXO3 in pre- and postmenopausal human ovaries by immunohistochemistry (IHC).METHODS:
Total RNA was isolated from human ovary collected from pre- and postmenopausal patients undergoing oophorectomy and used for real-time qPCR for members of the KL-PTEN-PI3K-FOXO3 pathway, as well as other genes. IHC was performed on archived paraffin embedded ovaries to visualize the presence and localization of FOXO3 in human ovaries ranging from fetal to menopausal.RESULTS:
FOXO3 is expressed in premenopausal human ovary and to a significantly reduced level in postmenopausal ovary. Nine additional genes in the KL-PTEN-PI3K-FOXO3 pathway were also expressed in premenopausal ovaries but like FOXO3 displayed significantly decreased levels in the postmenopausal cohort. IHC analysis revealed strong nuclear FOXO3 staining in primordial follicles from pre-pubertal ovaries through menarche/post-pubertal. A dominant population of primordial follicles were identified in older post-pubertal ovaries and in the reproductive/premenopausal groups that displayed light to negative nuclear staining for FOXO3, with a second much less abundant population of primordial follicles retaining strong positive nuclear FOXO3 staining. Postmenopausal ovaries were devoid of follicles but had strong stromal FOXO3 staining.CONCLUSION:
As predicted from mouse genetic models, FOXO3 mRNA and protein is present in premenopausal human ovary as are nine members of the KL- PI3K-AKT-FOXO3 pathway. FOXO3 localization in the human ovary undergoes an age-dependent transition from strongly positive nuclei in young ovaries to predominantly light to negative staining in reproductive/premenopausal. Given FOXO3's importance to genome stability, primordial follicles with high nuclear FOXO3 levels may define high quality oocytes lacking detrimental levels of DNA damage, so that the ratio of FOXO3 positive to negative oocytes may explain the consequences of decreasing ovarian reserve.