1 Division of Reproductive Science in Medicine, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
2 Olson Center for Women’s Health, Department of Obstetrics and Gynecology, and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985860 Nebraska Medical Center, Omaha, Nebraska, USA
Correspondence should be addressed to S-Y Kim: email@example.com
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.
Supplemental Figure 1. Effect of CPA on granulosa cells and cumulus cells in growing follicles. (A) Representative H&E images of granulosa cells and cumulus cells in ovaries from an 8-week-old CD-1 mouse (n=3). Scale bar, 50 µm. Red arrows indicate pyknotic cells. (B) Representative images of TUNEL staining of ovaries from an 8-week-old CD-1 mouse. Green=apoptotic cells, Blue-DAPI. Scale bar, 100 µm. (C) Ovary and (D) kidney weights of CD-1 mice (n=3) treated with PBS or CPA. n.s., non-significant.
Supplemental Figure 2. Effect of CPA on morphological phenotypes. (A) Comparison of morphological phenotypes in PD5 CD-1 mice 3 to 7 days after injection with 150 mg/kg CPA. Body size and hair density were compared. Comparison of morphological phenotypes in (B) PD5 C57BL/6J and (C) BALB/cJ mice 7 days after injection with 150 mg/kg CPA. Body size and hair density were compared. (D) Change in body weight in C57BL/6J mice 1 day, 3 days, and 7 days after injection with 150 mg/kg CPA. The numbers of animals assessed at each time point are shown.
Supplemental Figure 3. Effect of CPA on proliferating ovarian cells. The number of Ki67 positive cells per section was counted in ovarian sections of PD5 CD-1 mice 3 days post injection with either PBS or 150 mg/kg CPA (n=4 each) (A), and the ovaries of 8-week-old mice 24 hours post injection with the same dosages (n=6 for PBS or n=5 for CPA) (B).