Existing literature supports the idea that activation of the PI3K/AKT/mTOR signaling pathway stimulates dormant PFs to enter the pool of growing follicles [222324]. Intriguingly chemotherapeutic agents such as cisplatin and cyclophosphamide are reported to induce PI3K/AKT/mTOR signals in oocytes without promoting the death of oocytes in PFs. Furthermore, Roness et al. [25] and Kalich-Philosoph et al. [26] demonstrated that cyclophosphamide induces PI3K/AKT/mTOR signals in the oocyte of the mouse ovary, causing the initiation of recruitment and growth of all dormant PFs (direct activation). This eventually results in the depletion of the ovarian reserve, and has been labeled as the follicle ‘burnout’ theory. Cyclophosphamide also induces death of growing follicles through apoptosis in granulosa cells, which results in a decrease of Anti-Müllerian hormone (AMH) and paradoxically results in the recruitment of dormant PFs into a cohort of growing follicles (indirect activation). Accordingly, AS101, an immunomodulator, decreased PI3K/AKT/mTOR signaling and eventually prevented the decline of follicles by follicle activation and loss from the treatment of cyclophosphamide. This study suggested that the dormant follicle activation through PI3K/AKT/mTOR signaling is a new mechanism of ovotoxicity underlying the loss of follicles induced by chemotherapeutic agents and environmental carcinogens. Thus, agents that suppress PI3K signaling, such as AS101, may be ideal adjuvants to preserve viability and numbers of dormant PFs when patients are treated with ovotoxins.

A study conducted by Goldman et al. [27] in 8-week-old C57BL/6J female mice supports the findings that cyclophosphamide promotes follicle activation through PI3K/AKT/mTOR signaling. In this report the ooplasm of dormant PFs highly expressed the downstream markers of mTOR signaling after the treatment with cyclophosphamide. Correspondingly, treatment with the mTORC1/2 inhibitors, everolimus (RAD001) or INK128, preserved the ovarian reserve and serum AMH levels. As a consequence, the mTOR inhibitors extended the fertility span in mice treated with cyclophosphamide.

A recent in vitro study by Lande et al. [28] that examined PFs in human ovarian tissues supports the activation theory. These investigators observed a statistically significant increase in the number of growing follicles in the human ovarian tissues following the treatment with high concentrations of 4-hydroperoxy cyclophosphamide (4-HC) or phosphamide mustard, which are cyclophosphamide metabolites, as compared with untreated ovarian tissues. Although there was no proposed mechanism in this report, the results correlate with others showing more developing follicles after the treatment with cyclophosphamide as compared to the untreated controls [26].

Jang et al. [29] supported the concept that cisplatin activates the PI3K/AKT/mTOR signaling pathway and stimulates dormant PFs. This study also provided evidence for melatonin and ghrelin being fertoprotective against cisplatin-induced ovarian damage by decreasing PI3K signaling. In the ovary, dormant PFs highly express Forkhead box O3a (FOXO3a) in the nuclei of oocytes [30]. A wave of follicle activation signals initiates PI3K/AKT signaling resulting in the phosphorylation of FOXO3a inside the oocyte nucleus. Phosphorylated FOXO3a shuttles out of the nucleus, stimulating activation of dormant PFs and converting the squamous pregranulosa cells into cuboidal granulosa cells (direct activation). In this report, the authors demonstrated that the treatment of mice with low doses of cisplatin (2.5 mg/kg) for 12 days activated dormant PFs through phosphorylation of FOXO3a. They also found that the treatment with melatonin and ghrelin synergistically suppressed the phosphorylation of FOXO3a by cisplatin and retained FOXO3a in the nucleus of oocytes of PFs. They further showed that the ovarian follicles express receptors for melatonin (MT1 and MT2) and ghrelin (GHSR1) in PFs [29]. These findings suggest that the dormant PFs may be protected from ovotoxins like cisplatin through high nuclear expression of FOXO3a. Furthermore, the combination of melatonin and ghrelin may serve as ovoprotectants/fertoprotectants to mitigate the adverse effects of cisplatin treatment.

The indirect activation concept for loss of the ovarian reserve was recently highlighted by Kano et al. [31] demonstrating that AMH/Müllerian inhibiting substance (MIS) produced by granulosa cells protects the ovarian reserve from chemotherapeutic agents like carboplatin, doxorubicin or cyclophosphamide [32]. AMH/MIS plays an important role in the development of male and female gonads and the differentiation of the urogenital ridge. In females, AMH/MIS is mainly produced by granulosa cells from primary to medium sized antral follicles [33] and thus it is considered by the fertility clinics to be a biomarker for the ovarian reserve [34353637]. It serves an important role as a negative regulator of PF activation [3839]. The study by Kano et al. [31] demonstrated that co-treatment of AMH/MIS with chemotherapeutic agents protects PFs through suppression of PFs. According to this concept, chemotherapeutic agents destroy the growing follicles and thus reduce the production of AMH/MIS from them. Consequently, the lack of AMH/MIS production removes the suppressive effects allowing the PFs to be recruited into the pool of growing follicles (indirect activation). They also demonstrated that the treatment of AMH/MIS is reversible, showing that the cessation of treatment allows dormant PFs to resume ovarian folliculogenesis. Thus, co-treatment of AMH/MIS with chemotherapeutic agents is a therapeutic option for protecting the ovarian reserve.

Gonadotropin-releasing hormone (GnRH) agonists/antagonists are clinically used as ovoprotectants/fertoprotectants [404142]. Several reports show that the loss of ovarian cyclicity is prevented by co-treatment of GnRH agonist/antagonists with chemotherapeutic agents as compared with the chemotherapeutic agents alone [43]. Although the use of GnRH agonist has been promising, its usefulness has been debated due to the inconsistency of results in protection of ovarian follicles [1244]. A proposed mechanism indicates an increase in cAMP levels by GnRH agonist in oocytes leading to the suppression of oocyte apoptosis, although this finding was not confirmed in studies using mice and human [45]. This is thought to occur as a result of the passage of cAMP between oocytes and granulosa cells through gap junctions, eventually resulting in the survival of the ovarian follicles. The critical role of cAMP in ovoprotection is also supported by studies showing that treatment with LH provides protection against cisplatin. Research from Dr. Klinger's group showed that LH protects oocytes against cisplatin through the activation of the LH/choriogonadotropin receptor in ovarian cells [46]. In postnatal day 8 and adult mouse ovary, the LHCGR is expressed in cells surrounding follicles. Accordingly, exogenous 8-Bromo-cAMP and forskolin, factors mimicking the signaling pathway activated by LH, protect the ovarian follicles against cisplatin and initiate DNA repair kinetics in oocytes. Furthermore, LH stimulated the expression of KIT-ligand (Stem cell factor, SCF, KL, steel factor) and KIT (receptor), and protected oocytes against cisplatin-induced death in, both, oocytes and granulosa cells of the ovarian tissues in vitro. They proposed that protein kinase A activity, downstream of KL/KIT, protects oocytes from ovotoxicity by cisplatin followed by the activation of PI3K/AKT pathway to promote oocyte survival. However, it is not known whether the activation of PI3K/AKT pathway induced by LH treatment is necessary for protecting oocytes in PFs; in the case that it is needed, it must be on the level of threshold to promote oocyte survival.

Recent studies from our laboratory indicate that the chemotherapeutic agents directly destroy oocytes of dormant PFs and supports the inactivation concept. In support of the notion, cyclophosphamide treatment in three different mouse strains (C57BL/6J, CD-1 and BALB/cJ) did not promote any increase in the total number of primary and secondary follicles [47]. Our findings showed that the oocytes within the PFs are the most sensitive cells to DNA damage induced by cyclophosphamide treatment; this is in accordance with the previous data with cisplatin and X-rays [16]. Accordingly, oocytes of PFs were destroyed prior to damage to other ovarian cells without the activation of oocytes of dormant PFs, preserving the quiescent pregranulosa cells surrounding the oocytes of PFs. However, granulosa cells in growing follicles were also damaged. In addition, our results showed that the damaged granulosa cells are replaced within 3 days, if the follicles keep a portion of healthy granulosa cells, supported by proliferation of Ki67 markers and a subsequent increase in AMH production [47]. This suggests that the elevation of AMH does not represent the recruitment of dormant follicles into the pool of growing follicles. Furthermore, we found that the oocytes within the PFs presented temporal expression of phosphorylated-AKT as well as considerably prolonged expression of cleaved poly (ADP-ribose) polymerase (PARP) inside of the same PFs upon the treatment with cyclophosphamide or 4-HC, the active metabolite of cyclophosphamide in vivo. Moreover, cyclophosphamide induced time-dependent increases in apoptosis of oocytes of PFs as was evident by elevated expression of BAX and cleaved PARP. The oocytes expressed additional markers of apoptosis including γH2AX and phosphorylated TAp63α. Additional studies have demonstrated that cyclophosphamide-induced damage in oocytes of dormant PFs occurs via the p-ataxia telangiectasia and Rad3-related (ATR)>p-CHEK1/p-CHEK2>p-p63 apoptotic pathway [47], while cisplatin induces the p-ATR>p-CHEK2>p63/p73>PUMA/NOXA apoptotic pathway [172148]. The apoptotic pathway induced by cyclophosphamide eventually leads to the formation of TAp63α tetramers in the nucleus of oocytes, resulting in the induction of an irreversible apoptotic pathway. As shown in another report, ataxia-telangiectasia mutated (ATM), CHEK2, and CK1 inhibitors inhibit the tetramerization of TAp63α induced by cisplatin and doxorubicin [18], suggesting that the apoptotic inhibitors can block oocyte death at the molecular level [214849]. This suggests that PFs are destined to die rather than activate upon cyclophosphamide treatment, even though oocytes in PFs temporally present phosphorylated-AKT signals in response to chemotherapeutic agents. We assume that those follicles may contain genomic mutations or damaged DNA if the follicles with both the signals manage to survive. Taken together, these findings suggest that the apoptotic inhibitors instead of inhibitors of PI3K pathway should be used to protect dormant PFs from ovotoxins such as cisplatin, cyclophosphamide and X-ray [162147].

Oxidative stress affects reproductive health in females through the production of reactive oxygen species (ROS). Although ROS play an important role in signal transduction during folliculogenesis, oocyte maturation, and embryogenesis [50], excessive ROS can induce damage to the ovary through the induction of granulosa cell death [51], follicle atresia or oocyte damage. In addition, it adversely affects the production of ovarian hormones and mature oocytes from healthy follicles. Of relevance to the preservation of fertility, reports suggest that the antioxidants such as S1P [51], ceramide-1-phosphate [52], zingerone [53], proanthocyanidin [54], gallic acid [55], mangafodipir [56], resveratrol [57], curcumin [58], and capsaicin [58] protect the ovary from ovotoxins including chemotherapeutic agents. Thus, oxidative stress-induced damage can be prevented by antioxidants through attenuating the free radical induced ovarian damages. However, there are only few reports that show direct evidences of ROS in oocytes of PFs. Thus, the effect of ROS on mitochondria in oocytes or granulosa cells should be further investigated [5960].