Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide

in Journal of Endocrinology
Correspondence should be addressed to S-Y Kim: soyoun.kim@unmc.edu
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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.

Downloadable materials

  • 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).

 

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    CPA does not increase the growing pool of follicles. (A) Three different strains of 8-week-old mice were injected with 150 mg/kg CPA for 24 h and the ovaries were fixed for H&E staining and follicle counting. Representative images and surviving primordial, primary and secondary follicles are shown for CD-1 (B), C57BL/6J (C) and BALB/cJ (D) mice.

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    Gonadotoxicity of CPA in mice is dose and time dependent. (A) 7-week-old CD-1 mice were injected with CPA 200 mg/kg (n = 3) (i) or 75 mg/kg (n = 3) (ii) and ovaries were harvested 1 week post injection. (B) 4-week-old CD-1 mice were injected with CPA 75 mg/kg (n = 3) (i) or 150 mg/kg (n = 3) (ii) weekly for 4 weeks and ovaries were harvested 1 week after the last injection. Surviving primordial follicles are marked with a blue arrow. A follicle-like structure without a primordial oocyte following CPA treatment is marked with a green arrow. All experiments were performed in triplicate. Scale bar, 50 µm.

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    CPA destroys primordial follicles and primary follicles. (A) Three different strains of mice (CD-1, C57BL/6J and BALB/cJ) were injected at P5 with 150 mg/kg CPA and the ovaries were harvested at days 1, 3 and 7 post injection (n = 3 animals for each timepoint). (B) Representative images of H&E staining of ovaries from each mouse strain at each time point. Scale bar, 50 µm.

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    CPA induces loss of primordial follicles and primary follicles. (A) Total number of surviving primordial, primary and secondary follicles per ovary at 1, 3 and 7 days after CPA or PBS injection. Change in the number of each class of follicles over time after treatment of three different strains of PD5 mice with CPA or PBS. (B) Ratio of growing follicles to dormant follicles at 1, 3 and 7 days after treatment of three different strains of PD5 mice with CPA or PBS. (C) Level in serum AMH over time after treatment of three different strains of PD5 mice with CPA or PBS (all n = 4 each). PF, primordial follicles; PM, primary follicles; SF, secondary follicles.

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    CPA induces cell death, not cell proliferation. (A) Representative images (n = 3) of Ki67 staining in PD5 (72 h) or 8-week-old mouse ovary 1 day (24 h) after injection of PBS or 150 mg/kg CPA. Granulosa (orange) and somatic cells (red) were marked with arrows. Green = Ki67, blue = DAPI. Scale bar, 50 µm. (B) Representative images of TUNEL staining of PD5 CD-1 mouse ovary 1, 3 and 7 days after injection of 150 mg/kg CPA or PBS. Green = apoptotic cells, blue = DAPI. Scale bar, 50 µm. (C) High magnification representative images of TUNEL staining 1 day after injection of 150 mg/kg CPA, showing positive TUNEL signals (green) in a primordial follicle (pr, a), primary follicle (pm, b), secondary follicle (sec, c) and somatic cell (sc, d). Blue=DAPI. Granulosa cells of secondary (sec) follicles and somatic cells (sc) are indicated with orange arrows. Scale bar, 50 µm. (D) Representative H&E images of PD5 CD-1 mouse ovary 1 and 3 days after injection of CPA. Dead granulosa cells (red arrow, i), dead primordial oocyte (yellow arrow, ii), healthy pregranulosa cells (blue arrow, ii), healthy stromal cells (blue arrow, iii), proliferating granulosa cells (orange arrow, iv), follicle-like structure without oocyte (green arrow, v) and healthy somatic cells (blue arrow, vi) are marked in each panel. Scale bar, 20 µm. (E) Representative images of p-H3 staining in PD5 mouse ovaries 3 and 7 days after injection of 150 mg/kg CPA or PBS. Green = p-H3, blue = DAPI. Scale bar, 200 µm.

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    4-HC destroys primordial follicles through activation of apoptosis. (A) Representative images of H&E and IHC of MSY2 (n = 3) (DAB) indicating damaged primordial oocytes (yellow arrows) and granulosa cells and somatic cells (red arrows). Scale bar, 100 μm. (B) The number of surviving primordial follicles significantly decreased after culturing ovaries in vitro with 4-HC compared with PBS (n = 5 each). ***, P < 0.001; n.s, non-significant. (C) Representative images of the time-dependent expression of BAX and cPARP in ovarian tissue cultured with 1 μM 4-HC. Green = BAX or cPARP, Blue = DAPI. Scale bar, 50 μm. (D) Representative images showing time-dependent expression of p-AKT in ovarian tissues cultured with 1 μM 4-HC. Green = p-AKT, Blue = DAPI. Scale bar, 50 μm. (E) Representative image showing co-expression of cPARP and p-AKT in PD5 ovaries 1 day after injection of 150 mg/kg CPA. Green = cPARP, Red = p-AKT, Blue = DAPI.

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    Apoptosis inhibitors protect the ovarian reserve from 4-HC‒induced follicle damage in vitro. (A) Representative images of γH2AX and p-p63 immunofluorescence (green) in ovaries of a PD5 CD-1 mouse 3 days after injection of 150 mg/kg CPA (n = 4). Each inset shows a surviving primordial oocyte positive for each signal in the panel. Blue = DAPI. Scale bar, 50 μm. (B) Representative images of p-ATR, p-ATM, p-CHK1 and p-CHK2 immunofluorescence (green) in PD5 CD-1 mouse ovaries after treatment with 3 µM 4-HC for 24 h in vitro. Each inset shows primordial oocytes positive for each signal in the panel. Blue = DAPI. Scale bar, 100 µm or 50 μm. (C) Representative images of H&E and DAB staining for MSY2 in PD5 CD-1 mouse ovaries cultured in vitro with the indicated doses of each inhibitor (CK2II or ETP46464) and PBS (control) or 3 µM 4-HC. Scale bar, 50 μm. (D) Number of surviving primordial follicles after culturing ovaries in vitro with each inhibitor and 2 µM 4-HC. Experiments were performed in triplicate. (E) Representative images of p-p63 immunofluorescence (green) in PD5 CD-1 mouse ovaries treated with each inhibitor and 3 µM 4-HC for 24 h in vitro. Blue=DAPI. Scale bar, 50 μm.

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    Schematic of the oocyte apoptotic pathway induced by CPA. The previously proposed hypothesis and newly tested hypothesis are shown. Based on our findings, CPA exposure induces apoptosis of primordial follicles, leading to oocyte death and depletion of the ovarian reserve. We did not observe activation of primordial follicles leading to an increase in the number of primary or secondary follicles.

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