Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide

Abstract

Cancer therapy can cause off-target effects including ovarian damage, which may result in primary ovarian insufficiency in girls and premenopausal women. Loss of ovarian follicles within the ovarian reserve leads to ovarian endocrine dysfunction and impaired fertility. Cyclophosphamide (CPA), a commonly used chemotherapeutic and immunosuppressant agent, is a gonadotoxic agent that destroys ovarian cells by crosslinking DNA. To protect the ovary against CPA damage, we sought to precisely map the mechanism by which the ovarian reserve is depleted by CPA. We found that CPA specifically depletes primordial follicles without affecting primary and secondary follicles in three independent murine strains (CD-1, C57BL/6J and BALB/cJ) in vivo. We directly tested the effect of the active metabolite of CPA, 1 μM 4-hydroxyperoxycyclophophamide (4-HC), in vitro and confirmed the loss of primordial oocytes but no change in the number of primary and secondary follicles. We demonstrated that phospho-AKT (p-AKT) and cleaved PARP (cPARP) are present in primordial oocytes 3 days after CPA injection, consistent with the role of these markers as part of the apoptotic cascade. Interestingly, p-AKT positive primordial oocytes co-expressed cPARP. Treatment of animals with specific inhibitors of apoptotic pathway components, ETP46464 and CHK2, blocked 4-HC‒induced DNA damage in vitro. These data suggest that CPA targets primordial germ cells in the ovarian reserve by stimulating apoptosis pathways. Adjuvant therapies to protect primordial germ cells from the off-target effects of CPA may reduce the risk of POI.

Ataxia–telangiectasia mutated coordinates the ovarian DNA repair and atresia-initiating response to phosphoramide mustard

“…Despite a dramatic increase in CASP3 positive cells in the WT mice post-PM exposure, fewer cells positively stained for CASP3 in Atm +/female mice after PM exposure. This finding in the WT mice is consistent with previous studies [21,25,43,67]. DNA damage can also activate the mitochondrial apoptosis pathway and CYCS release [68].…”

“…Direct toxicity to the follicle or oocyte may occur [40][41][42][43] or accelerated hyperactivation of primordial follicles [44] in response to targeted death of larger growing follicles, resulting in follicular "burnout" [45][46][47]. PM induces ovarian DNA damage in the oocyte and granulosa cells [22,24,25] and our group discovered that ATM inhibition prevents PM-induced follicle depletion [31].…”

Early prepubertal cyclophosphamide exposure in mice results in long-term loss of ovarian reserve, and impaired embryonic development and blastocyst quality

“…Oocytes within primordial follicles exist in a relatively quiescent state, and hence may be relatively resistant to antimitotic agents [8], but the effects of alkylating agents are independent of the cell cycle. On the other hand, recent studies have shown that CY is also capable of damaging the primordial follicles in murine ovaries [7,9]. Rodents exposed to CY in pregnancy (i.e.…”

Extended Adverse Effects of Cyclophosphamide on Mouse Ovarian Function

“…Of them, Zbtb1 is a member of the Krüppel C2H2-type zinc-nger protein family and regulated by the PI3K/PTEN/AKT pathway, which has a critical role in regulating dormancy and initial primordial follicle activation [28]. However, exposure to Cy disturbs this balance by destroying growing follicles or activating the PI3K/PTEN/Akt pathway, causing reservoir "burnout" [29,30].…”