The PTEN/PI3K/AKT/FOXO3 pathway has been implicated as a major signaling pathway involved in cancer; however, biochemical and genetic studies in mice have revealed that this pathway also plays an important role in the regulation of dormancy and initial follicular activation in ovary. FOXO subclasses are transcription factors that have important roles in cell cycle arrest, apoptosis, and stress responses in vitro [21]. While disruption of the FOXO1 gene results in embryonic lethality as a consequence of incomplete vascular development, deletion of the FOXO3a gene in a mouse model resulted in offspring that were viable and grossly indistinguishable from their littermate controls. FOXO3a-null females, however, showed age-dependent infertility and abnormal ovarian follicle development. These mice exhibited normal assembly of the primordial follicles followed by immediate global activation, resulting in ovarian hyperplasia, follicle depletion, premature ovarian failure, and infertility [22]. Within oocytes, FOXO3a is regulated by nucleocytoplasmic shuttling, essentially being imported into the nucleus during primordial follicle assembly and exported upon activation. Within the oocytes, FOXO3a may have a suppressive effect on initiation of follicular growth by affecting the mechanisms intrinsic to the ovary [21].

Oocyte-specific deletion of PTEN also caused global primordial follicle activation, similar to the phenotype induced by FOXO3a knock-out, and resulted in POI [23]. Interestingly, both the FOXO3- and the PTEN-knockout female mice are initially fertile, indicating that the deletion of either of the factors does not appear to disrupt subsequent steps of follicle maturation, ovulation, and fertilization. Another study utilizing pharmacological inhibition of PI3K showed that PVI3K suppressed the PTEN-knockout but not the FOXO3-knockout ovarian phenotype, suggesting that FOXO3 lies downstream of PTEN [24]. Furthermore, oocyte-specific ablation of PTEN resulted in PI3K-induced conversion of secondary messenger phosphatidylinositol-4, 5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3), which then activates phosphatidylinositol-dependent kinase 1 (PDK1), resulting in AKT activation [24]. Within the nucleus, AKT activation leads to FOXO3a hyperphosphorylation and nuclear export, thereby triggering primordial follicle activation. These findings suggest that hypoactivation or negative regulation of the PTEN/PI3K/AKT/FOXO3 pathway may cause retardation of follicle activation and excessive primordial follicle atresia. On the contrary, inhibition of PTEN and activation of PI3K may cause activation of dormant follicles, since PTEN is a negative regulator of the PI3K/AKT signaling pathway. In addition, accumulating evidence shows that the PTEN/PI3K/AKT/FOXO3 pathway is responsible for chemotherapy-induced POI [25] and that chronic stress and bisphenol A are related to pathway modulation [26,27]. Numerous inhibitors and activators directed against either individual or multiple components of this pathway have already been developed, based on the primary importance of modulating this pathway for cancer treatment (Figure 1) [28]. Currently, several PI3K/AKT/mammalian target of rapamycin (mTOR) modulating agents, primarily agents to induce pathway inhibition, have been identified. LY294002 and wortmannin, which are the best characterized inhibitors, prevent ATP binding and activation of PI3K. Both induce apoptosis of cancer cells and rescue drug sensitivity [29,30]. Perifosine is a water-soluble synthetic alkylphosphocholine with oral bioavailability, and this agent inhibits AKT, reduces cell proliferation, and induces apoptosis in a wide variety of cancers [31]. Rapamycin is an mTOR inhibitor that results in inhibition of proliferation and sensitization to chemotherapeutic agents, and a recent study suggested that rapamycin can inhibit the excessive activation of primordial follicles in PTEN-knockout rat ovaries, reducing follicle consumption and maintaining ovarian reserve [32]. It has also been suggested that administration of PI3K/AKT/mTOR inhibitors can give rise to potentially life-threatening adverse events, such as pneumonitis and others [33]. While these PI3K/AKT/mTOR inhibitors have been developed for therapeutic use, very few PI3k/AKT/mTOR activators have been identified, since activation of this signaling pathway has been demonstrated to induce malignancies, be associated with poor prognosis, and enhance drug resistance after cancer treatment [28]. Nonetheless, PTEN inhibitors, such as bisperoxovanadium (HOpic), bpV (HOpic), have been shown to effectively activate primordial follicles without any known side effects.

First discovered in genetic screens for tumor suppressors in Drosophila, the Hippo signaling pathway is a conserved regulator of organ size, halting organ growth at the appropriate time during development and regeneration [34]. Central to this pathway is a kinase cascade that leads from the tumor suppressor Hippo (MST1 and MST2 in mammals) to the oncoprotein Yorkie (yes-associated protein [YAP] transcriptional coactivator with PDZ-binding motif [TAZ] in mammals), which is a transcriptional coactivator of target genes involved in cell proliferation and survival (Table 1). Activation of YAP/TAZ signaling results in increased expression of intercellular signaling protein (CCN; CYR61, connective tissue growth factor [CTGF], and nephroblastoma overexpressed [NOV] proteins) [35]. The Hippo pathway, however, actively prevents aberrant tissue growth by deactivating YAP/TAZ and other transcription factors via phosphorylation.

Dysregulation of Hippo signaling leads to development of various human diseases, including cancer development. In the ovary, Hippo signaling specifically prevents activation of primordial follicles. Mechanical manipulation of ovarian tissue by cutting this tissue into cubes can disrupt this pathway (Figure 2), as ovarian fragmentation changes intercellular tension and facilitates conversion of globular actin (monomer) to fibrous actin (polymer) and this actin polymerization process disrupts Hippo pathway-related genes. Decreasing phospho-YAP (pYAP) levels in concert with increased nuclear localization of YAP leads to increased CCN growth factors and baculoviral inhibitors of apoptosis repeat containing apoptosis inhibitors, which promote follicle growth [35-37].