Year 3

We continue to obtain ovarian cortical fragments from cancer patients for activating dormant primordial follicles using xenografting in immune-deficient mice. In addition to the activation of primordial follicles, we discovered that the growth of isolated secondary follicle from mice could also be promoted following treatment with PTEN inhibitor and PI3K (phosphatidyl inositol-3 kinase) stimulators to activate the Akt pathway. In addition, ovarian fragmentation could disrupt the ovarian Hippo signaling system and promote secondary follicle growth. Following Akt activation together with ovarian fragmentation to disrupt the Hippo signaling pathway, we found additive increases in ovarian graft weights and secondary follicle growth in mice. Of particular interest, human secondary follicles, following Akt stimulation and Hippo signaling disruption, could grow into the antral stage in four weeks in xeno-grafts. We are using this approach to obtain more human mature oocytes for re-programming and embryonic stem cell derivation. For Aim 2, we have collected immature and failed-to-be-fertilized oocytes from IVF patients in addition to mature oocytes obtained after xeno-transplantation. We found that it is easier to first perform ICSI (intra-cytoplasmic sperm injection) followed by removing female pronuclei from injected oocytes to derive early embryos. We, therefore, concentrated our studies to derive these early embryos using in vitro matured oocytes and mature oocytes from xenografts as recipients. For Aim 3, we have established mouse in vitro culture models to generate mature oocytes. We further demonstrated that mouse ovarian explants containing secondary follicles were stimulated by CCN growth factors downstream of Hippo signaling disruption, leading to the promotion of follicle growth. We are checking if combined Akt stimulation and CCN2 growth factor treatment can lead to cumulative promotion of human secondary follicle growth.