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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54HD068158-02
Application #
8377044
Study Section
Special Emphasis Panel (ZHD1-DSR-L)
Project Start
Project End
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$324,623
Indirect Cost
$127,702
Name
Stanford University
Department
Type
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Woglar, Alexander; Villeneuve, Anne M (2018) Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination. Cell 173:1678-1691.e16
Gustin, Stephanie L F; Wang, Guangwen; Baker, Valerie M et al. (2018) Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers. J Assist Reprod Genet :
Karakikes, Ioannis; Termglinchan, Vittavat; Cepeda, Diana A et al. (2017) A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circ Res 120:1561-1571
Feng, Yi; Zhu, Shoujun; Antaris, Alexander L et al. (2017) Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore. Chem Sci 8:3703-3711
Fan, Qianlan; Cheng, Yuan; Chang, Hsun-Ming et al. (2017) Sphingosine-1-phosphate promotes ovarian cancer cell proliferation by disrupting Hippo signaling. Oncotarget 8:27166-27176
Feng, Yi; Cui, Peng; Lu, Xiaowei et al. (2017) CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions. Sci Rep 7:44810
Panula, Sarita; Reda, Ahmed; Stukenborg, Jan-Bernd et al. (2016) Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells. PLoS One 11:e0165268
Kawamura, Kazuhiro; Kawamura, Nanami; Hsueh, Aaron J W (2016) Activation of dormant follicles: a new treatment for premature ovarian failure? Curr Opin Obstet Gynecol 28:217-22
Durruthy-Durruthy, Jens; Sebastiano, Vittorio; Wossidlo, Mark et al. (2016) The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming. Nat Genet 48:44-52
Zhai, Jun; Yao, Guidong; Dong, Fangli et al. (2016) In Vitro Activation of Follicles and Fresh Tissue Auto-transplantation in Primary Ovarian Insufficiency Patients. J Clin Endocrinol Metab 101:4405-4412

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