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. 2023 Oct 20;9(42):eadg0898.
doi: 10.1126/sciadv.adg0898. Epub 2023 Oct 20.

CHEK2 signaling is the key regulator of oocyte survival after chemotherapy

Chihiro Emori  1   2 , Zachary Boucher  1 , Ewelina Bolcun-Filas  1
Affiliations

CHEK2 signaling is the key regulator of oocyte survival after chemotherapy

Chihiro Emori et al. Sci Adv. .

Abstract

Cancer treatments can damage the ovarian follicle reserve, leading to primary ovarian insufficiency and infertility among survivors. Checkpoint kinase 2 (CHEK2) deficiency prevents elimination of oocytes in primordial follicles in female mice exposed to radiation and preserves their ovarian function and fertility. Here, we demonstrate that CHEK2 also coordinates the elimination of oocytes after exposure to standard-of-care chemotherapy drugs. CHEK2 activates two downstream targets-TAp63 and p53-which direct oocyte elimination. CHEK2 knockout or pharmacological inhibition preserved ovarian follicle reserve after radiation and chemotherapy. However, the lack of specificity for CHEK2 among available inhibitors limits their potential for clinical development. These findings demonstrate that CHEK2 is a master regulator of the ovarian cellular response to damage caused by radiation and chemotherapy and warrant the development of selective inhibitors specific to CHEK2 as a potential avenue for ovario-protective treatments.

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Figures

Fig. 1.
Fig. 1.. CHEK2 deficiency prevents primordial oocyte depletion after ex vivo treatment with MAFO, CDDP, DOX, and ETO.
(A) Schematic representation of the ovary explant organ culture system. Explants were exposed to drugs for 48 hours, then monitored in drug-free culture for 5 days (7 days total, circles = days). Media changes are indicated by arrows. Whole ovaries from wild-type (B) and Chek2−/− (C) females after 7-day ex vivo culture with MAFO (1 μg/ml), CDDP (0.5 μg/ml), DOX (0.1 μg/ml), or ETO (0.5 μg/ml) were immunostained for oocyte markers DDX4 (cytoplasmic green) and p63 (nuclear magenta). Representative whole ovaries are shown in the top row (i) and white boxes mark regions magnified below (ii). White bars indicate regions where primordial follicles (PMFs) are typically found in cultured ovaries. Small cells marked by arrowheads in magnified regions indicate primordial oocytes in PMF (sensitive to treatments) and large cells marked by arrows indicate oocytes in growing follicles (resistant to treatments). Asterisk: unspecific staining. Scale bars, 200 μm for whole ovary images and 50 μm for magnified regions. (D) Primordial and (E) growing oocytes were counted in ovaries harvested after 7-day organ culture. (N) = number of ovaries per group. Data are expressed as mean ± SEM; *P < 0.05, ****P < 0.0001 (Mann-Whitney nonparametric test). (A) was created with BioRender.
Fig. 2.
Fig. 2.. CHEK2-deficient females maintain PMF reserve after in vivo treatment with CTX and CDDP.
Ovarian histology from Chek2+/− (A) and Chek2−/− (B) females 2 weeks after in vivo treatment with CTX (150 mg/kg) and CDDP (5 mg/kg). Representative sections are shown in the top row (i) and white boxes mark regions magnified below (ii). Arrowheads indicate PMFs and asterisks indicate follicle remnants devoid of oocytes. Arrows indicate growing follicles with abnormal granulosa cell layers. Abnormal growing follicles are occasionally found in untreated 3-week-old ovaries. Scale bars, 200 μm for whole ovary sections and 50 μm for magnified regions. (C and D) PMF numbers counted in Chek2+/− and Chek2−/− ovaries 2 weeks after injection with CTX and CDDP, respectively. Data are expressed as mean ± SEM; ****P < 0.0001, ns, nonsignificant (Mann-Whitney nonparametric test). (E) Body weight changes in Chek2+/− and Chek2−/− females injected with CTX (7 to 21 dpp). (N) = number of females per group. Data are expressed as mean ± SEM; ***P < 0.001, ns, nonsignificant (Mann-Whitney nonparametric test).
Fig. 3.
Fig. 3.. MAFO and CDDP treatment of oocytes causes DNA DSBs that activate HR, but not NHEJ, repair.
Ovaries were exposed to vehicle and chemotherapy drugs ex vivo for 24 hours (MAFO: 1 μg/ml, CDDP: 0.5 μg/ml, DOX: 0.1 μg/ml, and ETO 0.5 μg/ml). Oocytes in PMF were analyzed by immunostaining for general DNA damage marker γ-H2AX (A), DNA repair markers RAD51 for HR (B), and 53BP1 for NHEJ (C) in green as indicated on the left. Background levels of γ-H2AX and RAD51 are detected in untreated primordial oocytes (white arrowheads). Oocytes were labeled with DDX4 (magenta) and DNA counterstained with Hoechst (blue). Top panels show merged representative immunofluorescence images and bottom panels show corresponding grayscale image of DNA damage and repair markers. Arrowheads and arrows indicate primordial oocytes and somatic cells, respectively. Yellow arrowheads and arrows show cells with DNA DSBs and white arrowheads and arrows show cells without damage. Scale bars, 10 μm. (D and E) Quantification of the number of DSBs per oocyte using γ-H2AX (D) and RAD51 (E) markers. Sample number (N); number of cells per group. Data are expressed as mean ± SD; ****P < 0.0001 (one-way ANOVA, Bonferroni multiple-comparison test).
Fig. 4.
Fig. 4.. Radiation and chemotherapy treatments lead to activation of CHEK2 in oocytes.
Ovaries were exposed to radiation and chemotherapy drugs ex vivo for 24 hours (IR: 0.5 Gy, MAFO: 1 μg/ml, CDDP: 0.5 μg/ml, DOX: 0.1 μg/ml, and ETO: 0.5 μg/ml). Ovarian sections were immunostained for phospho-CHEK2 T68 (green) and DDX4 (magenta) as indicated on the left. pCHEK2 T68 is readily detected in all primordial oocytes exposed to radiation. Few pCHEK2-positive oocytes are also detected in MAFO-, CDDP-, and DOX-treated ovaries, while rare pCHEK2-positive granulosa cells are detected on ETO-treated ovaries (yellow arrowheads and yellow arrows). Scale bars, 50 μm. Top panels show merged image and bottom panels show corresponding grayscale image of pCHEK2.
Fig. 5.
Fig. 5.. Inhibition of CHEK2-dependent TAp63 phosphorylation is not sufficient to prevent complete primordial oocyte elimination in response to MAFO (1 μg/ml) and CDDP (0.5 μg/ml) treatments indicating a role for p53.
(A) CDDP and MAFO fail to induce TAp63 hyperphosphorylation within 24 hours after treatment. TAp63 hyperphosphorylation leads to mobility shift (asterisk) observed after radiation. Compared to untreated controls, increased levels of total p53, indicative of its phosphorylation and stabilization, are observed after drug treatments. DDX4 (oocyte marker); γ-H2AX (DNA damage marker). (B) Primordial oocytes expressing nonphosphorylatable mutant TAp63 are not fully resistant to MAFO and CDDP toxicity while double mutant oocytes lacking active TAp63 and p53 are highly resistant and display almost normal survival. Ovaries from wild-type, Trp63A/A (mutation at S621A), and Trp63A/A Trp53−/− double mutant females after 7-day ex vivo culture after treatments with MAFO and CDDP were immunostained for oocyte markers DDX4 (green) and p63 (magenta). Representative immunofluorescence images of ovaries are shown. Scale bars, 50 μm. Arrowheads indicate oocytes in PMF and arrows indicate oocytes in growing follicles. Asterisk: oocytes with abnormal localization of p63 staining. (C) phospho-p53 detection in oocytes and granulosa cells after treatment with MAFO and CDDP by IHC. (D) Primordial oocyte (PMF) counts in ovaries treated with MAFO and CDDP. (E) Survival rates (%) were normalized to the average PMF number of the control group in each genotype (wild type, Chek2−/−, Trp63A/A, and Trp63A/A Trp53−/−). Sample number (N); number of ovaries per group. Data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 (Mann-Whitney nonparametric test). (F) Primordial oocyte survival in mouse mutants used in this study reveals the contribution of p53 and TAp63 to oocyte apoptosis.
Fig. 6.
Fig. 6.. CCT241533, LY2606368 and PF477736 fail to prevent primordial oocyte elimination after radiation treatment.
Ovaries were treated with inhibitors ex vivo for 2 hours before IR and for 24 hours after IR. After 24 hours, inhibitors were withdrawn, and ovaries were cultured for six additional days without inhibitors. Graphs show numbers of oocytes per ovary present after treatment with increasing doses of inhibitors (μM). Sample number (N); number of ovaries per group. Data are expressed as mean ± SEM; **P < 0.01, ****P < 0.0001 (one-way ANOVA, Kruskal-Wallis with Dunn’s multiple-comparison test for nonparametric data). Below, panels show examples of treated ovaries immunostained with oocyte markers DDX4 (green) and p63 (magenta). White bars indicate regions where PMFs are typically found in cultured ovaries. Arrowheads indicate primordial oocytes and arrows denote larger growing oocytes. Scale bar, 50 μm.
Fig. 7.
Fig. 7.. Cotreatment with AZD7762 reduces primordial oocyte loss in prepubertal ovaries after treatment with radiation and chemotherapy drugs.
(A) Cotreatment with AZD7762 exhibited protective effect and presence of primordial oocytes after treatments. Example ovaries cotreated with AZD7762 and radiation, MAFO, or CDDP in ex vivo organ culture. Ovaries were treated with AZD7762 ex vivo for 2 hours before treatments and for 48 hours after treatment start. Inhibitors and drugs were withdrawn, and ovaries were cultured for five additional days without inhibitors or drugs. Oocyte markers DDX4 (green) and p63 (magenta). Representative whole ovaries are shown in the top row (i) and dotted boxes mark regions magnified below (ii). White bars indicate regions where primordial oocytes are typically found in cultured ovaries. Arrowheads indicate primordial oocytes and arrows denote larger growing oocytes. Scale bars, 200 μm for whole ovary images and 50 μm for magnified regions. (B) Graphs show numbers of primordial oocytes (PMF oocytes) per ovary present after cotreatment with increasing doses of AZD7762 inhibitor. Sample number (N); number of ovaries per group. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA, Kruskal-Wallis with Dunn’s multiple-comparison test for nonparametric data). (C) Ovarian extracts with and without AZD7762 treatment were analyzed by Western blot for activation of CHEK2 targets TAp63 and p53. Ovarian protein extracts were collected 6 hours after IR with 0.5 Gy in vivo or ex vivo. In contrast to ovaries irradiated without AZD7762, TAp63 mobility shift (asterisk) indicative of phosphorylation and p53 expression were not detected in AZD7762-treated ovaries. Increased levels of pCHEK1(S317) were present only in AZD7762-treated ovaries indicating accumulation of CHEK1.
Fig. 8.
Fig. 8.. Inhibition of CHEK2 with AZD7762 facilitates DNA damage repair after treatment with chemotherapy drugs.
(A) Drug-treated oocytes in PMF were analyzed by immunostaining for general DNA damage marker γ-H2AX after 24 hours treatment with MAFO (1 μg/ml) or CDDP (0.5 μg/ml) and after 48 hours cotreatment with AZD7762 (1 μM) at the end of 7 days of culture. Oocytes were labeled with DDX4 (magenta) and DNA was counterstained with Hoechst (blue). Top panels show merged representative immunofluorescence images and bottom panels show corresponding grayscale image of DNA damage marker. Scale bars, 10 μm. (B) Quantification of the number of γ-H2AX foci at 24 hours and 7 days of culture. (N); number of cells per group. Data are expressed as mean ± SD; ***P < 0.001, ****P < 0.0001 (one-way ANOVA, Bonferroni multiple-comparison test).
Fig. 9.
Fig. 9.. Dual CHEK1/2 inhibitor AZD7762 exhibits cytotoxic effects in proliferating ovarian somatic cells.
Ovarian explants treated with vehicle or AZD7762 for a total of 8 hours without and with radiation (0.5 Gy) were immunostained for oocyte marker DDX4 (magenta), DNA damage marker γ-H2AX (green), apoptosis marker TUNEL (green), and granulosa cells marker FOXL2 (white). DAPI (blue). (A) AZD7762 treatment caused increased levels of γ-H2AX staining in somatic cells of growing follicles (arrows) but not oocytes even in the absence of IR. Background levels of γ-H2AX are detected in healthy oocytes (white arrowheads). Bright γ-H2AX foci indicate IR-induced DNA damage in primordial oocytes (yellow arrowheads) and granulosa cells in growing follicles (yellow arrows). Scale bars, 50 and 10 μm. Top panels show merged image and bottom panels show grayscale image. White boxes indicate magnified regions (i, ii). (B) TUNEL staining shows increased apoptosis in granulosa cells of growing follicles in ovaries treated with AZD7762. Yellow arrows show growing follicles with apoptotic granulosa cells. Scale bars, 10 and 50 μm. Growing follicles are outlined.
Fig. 10.
Fig. 10.. Schematic illustration of the conclusions of this study.
Many chemotherapy drugs, including CDDP, CTX, DOX, and ETO, induce DNA damage and oxidative stress, which kill cancer cells (on-target effect), but they can also damage healthy cells such as those in the primordial follicles in ovaries, leading to their death (off-target effect). CHEK2 kinase coordinates response to chemotherapy toxicity by activating p53 in somatic cells, and p53 and/or TAp63 in oocytes depending on the amount and type of damage (e.g., DNA damage versus oxidative stress). Low levels of cellular damage are sufficient to activate TAp63-dependent apoptosis while p53 is activated at higher levels of DNA damage and oxidative stress; thus, the combined action of both pro-apoptotic factors regulates oocyte survival and death. CHEK2 inhibitors prevent oocyte elimination by blocking CHEK2 and its downstream signaling (red lines). CHEK2 and p53 are expressed in all cell types, therefore blocking CHEK2 activity in somatic cells in the ovary (e.g., granulosa) may indirectly contribute to primordial follicle survival (dashed line). Figure created with BioRender.com
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