Dynamics of PI3K and Hippo signaling pathways during in vitro human follicle activation

Abstract STUDY QUESTION How does biochemical stimulation or inhibition of the PI3K/Akt/mTOR pathway affect the activation and the growth of human primordial follicles in vitro? SUMMARY ANSWER The PI3K/Akt activators act synergistically with the Hippo signaling pathway, disrupted by tissue fragmentation, to accelerate primordial follicles recruitment while mTORC1 inhibitor partially prevents the massive spontaneous activation. WHAT IS KNOWN ALREADY Hippo disruption caused by ovarian fragmentation and exposure to PI3K/Akt activators have been successfully used to activate resting follicles prior to grafting in patients with premature ovarian insufficiency. Outside this indication, massive and premature activation is considered deleterious as it leads to the depletion of the follicular pool and developmental defects in vitro. STUDY DESIGN, SIZE, DURATION One hundred and twelve frozen-thawed ovarian cortex fragments from four patients were exposed to dimethylsulfoxide (DMSO-control group), everolimus (inhibitor group) or bpV (HOpic) and 740Y-P (activators group) during the first 24 and 48 h, respectively, and cultured for additional 5 days. PARTICIPANTS/MATERIALS, SETTING, METHODS The ovarian fragments (4×2×1 mm3) were analyzed at Days 0, 1, 3 and 5 of culture. Cortex were either directly processed for immunohistological or western blot analysis or subjected to follicular isolation for assessment of gene expression. The follicle number and the developmental stage were evaluated in sections of ovarian fragments to assess the early follicular development. Survival and developmental potential were evaluated using TUNEL labeling, GDF9 and Ki67 immunostainings, Kit ligand mRNA relative expression and measurement of 17β-estradiol (E2) levels in the culture medium. qPCR and western blotting were used to explore the expression of PI3K/Akt and Hippo pathways (TSC1, mTOR, LATS1, BIRC1 and CCN2 genes and PTEN/p-PTEN, Akt/p-Akt and rpS6/p-rps6 proteins), and localization of YAP in human ovarian tissue was performed by immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Around 80% of the follicles spontaneously activated during the culture period, triggered by the activation of the PI3K/Akt/mTOR signaling. Moreover, ovarian fragmentation immediately promoted translocation of the Hippo effector YAP into the nucleus of granulosa cells, leading to upregulation of BIRC1 and CCN2 downstream targets at Day 0 and an increase of follicular growth near the cutting site. Short t