Derivation of Mature Human Oocytes from Primordial Follicles
PI: A. Hsueh
The majority of ovarian primordial follicles remain dormant for decades in women before initiating growth. Based on recent reports showing premature activation of ALL dormant primordial follicles in both neonatal and adult mice with oocyte-specific deletion of the PTEN (Tumor-suppressor phosphatase with TENsin homology) gene, we demonstrated that treatment of ovaries with PTEN enzyme inhibitors and a phosphatidylinositol (Pl)- 3-kinase activating peptide activates dormant primordial follicles to initiate growth. After short term exposure of neonatal mouse ovaries and human cortical strips containing primordial follicles with PTEN inhibitor and a PI3 kinase activator in vitro, tissues were transplanted into kidney capsules of ovariectomized adult mice. After 2 (for mouse tissue) and 24 (for human tissue) weeks, activated follicles developed to the preovulatory stage and mature oocytes capable of undergoing germinal vesicle breakdown were obtained. After IVF, mouse oocytes developed into blastocysts and viable progeny was obtained. We will perform transcriptome profiling of mouse oocytes in activated follicles using DNA microarrays to elucidate molecular mechanisms underlying primordial follicle activation. We also propose to evaluate the safety of this approach before future human trials to activate residual human primordial follicles for auto-transplantation in patients with premature ovarian failure and in peri-menopausal women with diminishing ovarian reserve. We will evaluate chromosomal integrity, epigenetic modification, and mitochondrial DNA mutation using comparative genome hybridization, DNA methylation analyses, and mitochondrial DNA sequencing for individual mouse and human oocytes. Albeit with extremely low success rate, earlier studies have demonstrated the possibility to grow murine follicles from primary to preovulatory stages in culture, followed by the retrieval of mature oocytes for successful IVF, pregnancy, and delivery. In addition to the in vivo transplantation approach, we propose to establish a two-step culture system in which mouse and human primordial follicles in culture are activated, followed by the promotion of their growth into primary and secondary stages using ovarian growth factors. Secondary follicles will be isolated for further culture into the early antral stage with stage-specific growth factors. Competent oocytes will then be retrieved for in vitro maturation and evaluation of chromosomal integrity, epigenetic modification, and mitochondrial DNA mutation. We will also collaborate with the Reijo Pera group to activate primordial follicles derived from human ES or IPS cells using our optimized in vivo transplantation and in vitro culture approaches. Aims include: 1. Activation of dormant human primordial follicles in vitro and transplantation to obtain mature human oocytes; and 2. In vitro studies to activate dormant follicles and generate mature oocytes.