b. Significance: The majority of ovarian follicles stay dormant at the primordial stage for decades in women and the depletion of this dormant pool leads to menopause. Due to genetic and environmental influences, some women suffer from premature ovarian failure/insufficiency associated with a diminishing follicle reserve. Under physiological conditions, uncharacterized intraovarian mechanisms activate -1,000 dormant primordial follicles to initiate growth per month, whereas the remaining follicles stay quiescent for years or decades. Once recruited into the growing pool, primordial follicles continue growth to the early antral stage with minimal loss. At the early antral stage, select follicles respond to gonadotropins and one preovulatory follicle releases the mature oocyte for fertilization. Studies using mutant mice indicated that oocyte-specific deletion of the PTEN gene or the downstream transcriptional factor Foxo3 promoted the growth of all primordial follicles [1,2]. The PTEN gene encodes a phosphatase enzyme that negatively regulates the PI3K and Akt signaling pathway. Deletion of PTEN in the oocyte increases the phosphorylation of Akt and the nuclear exclusion of the downstream Foxo3 proteins, leading to the activation of all dormant primordial follicles. We used PTEN inhibitors and PI3K activators to activate primordial follicles in mice. Neonatal mouse ovaries were exposed transiently to PTEN inhibitors and an PI3K activating peptide in vitro. After transplantation into the kidney capsule of FSH-treated adult recipients, treated ovaries showed marked increases in follicle growth. Mature oocytes could be retrieved from these ovaries for IVF and blastocyst formation. Our recent data also demonstrated the efficacy of the PTEN inhibitor in activating dormant HUMAN primordial follicles in cortical strips donated by a cancer patient to yield mature oocytes. The Fertility and Cancer program at Stanford have stored frozen human ovarian cortical strips containing mainly primordial follicles from cancer patients. Here, we propose to incubate human follicles in vitro with PTEN inhibitors and PISK activators, followed by transplantation into immune-deficient mice to optimize the follicle activation approach for the derivation of mature human oocytes. Chromosomal integrity, epigenetic programming, and mitochondrial DNA sequences will be investigated for individual mature oocytes generated after transplantation to determine the safety of the present protocol. In addition, we will amplify RNA from individual mature oocytes for DNA microarray analyses to elucidate follicle activation mechanisms. Furthermore, we will use a two-step in vitro culture approach to treat mouse neonatal ovaries and human cortical strips with PTEN inhibitor and PI3K activator for the activation of dormant follicles, followed by the in vitro promotion of their growth to secondary and preantral follicles using stage-specific paracrine factors. We will investigate the chromosomal, epigenetic, and mitochondrial DNA changes of individual mature oocytes derived from in vitro cultures. Although fertility is compromised in patients with premature ovarian failure and peri-menopausal women, their ovaries still contain many primordial follicles. The present in vitro follicle activation approach, followed by auto-transplantation, could allow retrieval of functional mature oocytes for infertile women with diminishing ovarian reserve and for cancer patients requiring fertility preservation. Future development of a two-step culture approach to allow the development of primordial follicles to the antral stage, followed by the generation of mature oocytes, could have important clinical implications for the treatment of infertility in diverse populations of women.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZHD1-DSR-L)
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Stanford University
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Durruthy-Durruthy, Jens; Briggs, Sharon F; Awe, Jason et al. (2014) Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions. PLoS One 9:e94231
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