Doxorubicin Has Dose-Dependent Toxicity on Mouse Ovarian Follicle Development, Hormone Secretion, and Oocyte Maturation

Abstract

Doxorubicin (DOX), one of the most commonly used anticancer medications, has been reported to affect fertility by damaging ovarian follicles; however, the dose-dependent toxicity of DOX on the dynamic follicle development and oocyte maturation has not been well-defined. Our objective is to determine the effects of human-relevant exposure levels of DOX on follicular functions across developmental time. In vitro cultured multilayered secondary mouse follicles were treated with DOX at 0, 2, 20, 100, and 200 nM for 24 h, and follicle development, hormone secretion, and oocyte maturation were analyzed. DOX caused dose-dependent toxicity on follicle growth, survival, and secretion of 17β-estradiol (E2). At 200 nM, DOX induced DNA damage and apoptosis in follicle somatic cells first and then in oocytes, which was correlated with the uptake of DOX first to the somatic cells followed by germ cells. Follicles treated with DOX at 0, 2, and 20 nM showed similar oocyte metaphase II (MII) percentages after in vitro oocyte maturation; however, 20 nM DOX significantly increased the number of MII oocytes with abnormal spindle morphology and chromosome misalignment. In an effort to harmonize the in vitro study to in vivo treatment, dose-dependent toxicity on oocyte meiotic maturation was found in 16-day-old CD-1 mice treated with DOX at 0, 0.4, 2, and 10 mg/kg, consistent with the in vitro oocyte maturation outcomes. Our study demonstrates that DOX has dose-dependent toxicity on ovarian follicle development, hormone secretion, and oocyte maturation, which are three key factors to support the female reproductive and endocrine functions.

Keywords: doxorubicin; follicle development; oocyte maturation; ovarian toxicity.

FIG. 1

Effect of doxorubicin (DOX) on follicle growth and survival during encapsulated in vitro follicle growth (eIVFG). A, Representative images of follicles treated with different doses of DOX on days 0, 4, and 8. B, Follicle diameters and C, follicle survival rates from days 0 to 8 after DOX exposure at 0–200 nM. Scale bar: 100 μm; error bar: standard deviation; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. N = 20–40 follicles for three replicates.

FIG. 2

Effect of DOX on 17β-estradiol (E2) secretion of in vitro cultured follicles on days 2, 4, and 8. **P < 0.01 compared to control group; error bar: standard deviation. N = 20–40 follicles for three replicates.

FIG. 3

Representative images of doxorubicin (DOX) fluorescence distribution in follicles after DOX exposure at 200 nM for 0, 1, 4, and 24 h. Blue: DAPI; red: DOX fluorescence. Scale bar: 25 μm. N = 5–10 follicles for each time points and replicate, three replicates were performed, and follicles at each time points showed similar DOX fluorescence distribution patterns.

FIG. 4

Representative images of follicular cell DNA damage after doxorubicin (DOX) exposure at 200 nM for 0, 4, and 24 h. Blue: DAPI; green: γ-H2ax. Scale bar: 50 μm. N = 5–10 follicles for each time points and replicate, three replicates were performed, and follicles at each time point showed similar DNA damage patterns.

FIG. 5

Follicle apoptosis after DOX exposure at 200 nM for 0, 4, and 24 h. A, Expression levels of follicle apoptotic genes after DOX exposure at 200 nM. *P < 0.05, **P < 0.01; error bar: standard deviation. B, Representative images of follicles with TUNEL staining. Blue: DAPI; green: DNA fragmentation. Scale bar: 100 μm. N = 40–50 follicles for RNA extraction and three replicates in total in (A) and N = 5–10 follicle for TUNEL staining in (B).

FIG. 6

Effect of DOX exposure on oocyte maturation in vitro. A, MII percentages after oocyte maturation for in vitro cultured follicles treated with DOX at 0, 2, 20, 100, and 200 nM on day 8, and the representative images of MII, shrunken, and fragmented oocytes. B, Incidence of spindle abnormality and chromosome misalignment in MII oocytes from follicles treated with DOX at 0, 2, and 20 nM. C, Representative images of meiotic spindles organization (green) and chromosome distribution (blue) of MII oocytes treated with DOX at 0 or 2 and 20 nM. *P < 0.05 and ***P < 0.001; error bar: standard deviation. Blue: DAPI; green: α-tubulin; red: F-actin; scale bar in (C): 50 μm. N = 30–40 follicles in each DOX exposure group and three replicates in total.

FIG. 7

Effect of DOX exposure on oocyte maturation in vivo. A, MII percentage of oocytes from mice treated with DOX at 0, 0.4, 2, and 10 mg/kg. B, Incidence of spindle abnormality and chromosome misalignment of MII oocytes from mice treated with DOX at 0, 0.4, 2, and 10 mg/kg. *P < 0.05, **P < 0.01, and ***P < 0.001; NS: nonsignificant; error bar: standard deviation. N = 4–6 mice and 20–30 oocytes per mice in each DOX-treated dosage.