Obesity partially potentiates dimethylbenz[a]anthracene-exposed ovotoxicity by altering the DNA damage repair response in mice†

Abstract

Obesity adversely affects reproduction, impairing oocyte quality, fecundity, conception, and implantation. The ovotoxicant, dimethylbenz[a]anthracene, is biotransformed into a genotoxic metabolite to which the ovary responds by activating the ataxia telangiectasia mutated DNA repair pathway. Basal ovarian DNA damage coupled with a blunted response to genotoxicant exposure occurs in obese females, leading to the hypothesis that obesity potentiates ovotoxicity through ineffective DNA damage repair. Female KK.Cg-a/a (lean) and KK.Cg-Ay/J (obese) mice received corn oil or dimethylbenz[a]anthracene (1 mg/kg) at 9 weeks of age for 7 days via intraperitoneal injection (n = 10/treatment). Obesity increased liver weight (P < 0.001) and reduced (P < 0.05) primary, preantral, and corpora lutea number. In lean mice, dimethylbenz[a]anthracene exposure tended (P < 0.1) to increase proestrus duration and reduced (P = 0.07) primordial follicle number. Dimethylbenz[a]anthracene exposure decreased (P < 0.05) uterine weight and increased (P < 0.05) primary follicle number in obese mice. Total ovarian abundance of BRCA1, γH2AX, H3K4me, H4K5ac, H4K12ac, and H4K16ac (P > 0.05) was unchanged by obesity or dimethylbenz[a]anthracene exposure. Immunofluorescence staining demonstrated decreased (P < 0.05) abundance of γH2AX foci in antral follicles of obese mice. In primary follicle oocytes, BRCA1 protein was reduced (P < 0.05) by dimethylbenz[a]anthracene exposure in lean mice. Obesity also decreased (P < 0.05) BRCA1 protein in primary follicle oocytes. These findings support both a follicle stage-specific ovarian response to dimethylbenz[a]anthracene exposure and an impact of obesity on this ovarian response.

Keywords: BRCA1; DMBA; DNA repair; obesity; ovary.

Figure 1

Impact of DMBA exposure on total body and organ weight in lean and hyperphagia obese mice. Following 7 days of exposure to corn oil (CT) or DMBA in lean (HPL) or obese (HPO) mice, weights of (A) body, (B) spleen, (C) liver, (D) ovary, and (E) uterus were recorded. (F) Amount of food consumed by each mouse during the dosing period was also recorded. Different letters indicate differences between treatments; P < 0.05; n = 10.

Figure 2

Impact of DMBA exposure on estrous cyclicity of lean and obese mice. The total percentage of days spent in (A) proestrus, (B) estrus, and (C) diestrus + metestrus were recorded during the dosing period (7 days) and until day 2 of diestrus. Data are presented as the percentage of days spent in each phase. # = P < 0.10; n = 10.

Figure 3

Impact of DMBA exposure on serum 17β-estradiol levels of lean and obese mice. The amount of circulating serum 17β-estradiol was measured via ELISA. Concentrations of estrogen in pg/mL are reported here. # = P < 0.1; n = 5–10 as some samples were below detection levels.

Figure 4

Impact of DMBA exposure on ovarian follicle number in lean and obese mice. Follicles were classified and counted after 7 days of exposure to corn oil (CT) or DMBA in lean or obese mice. Number of (A) primordial follicles (treatment effect P = 0.70; obesity effect P = 0.80; interaction effect P = 0.0180), (B) primary follicles (treatment effect P = 0.09; obesity effect P = 0.25; interaction effect P = 0.09), (C) secondary follicles (treatment effect P = 0.27; obesity effect P = 0.55; interaction effect P = 0.83), (D) pre-antral follicles (treatment effect P = 0.69; obesity effect P = 0.16; interaction effect P = 0.35), (E) antral follicles (treatment effect P = 0.11; obesity effect P > 0.99; interaction effect P = 0.68), and (F) corpora lutea were recorded (treatment effect P = 0.65; obesity effect P > 0.0027; interaction effect P = 0.99). Representative ovary sections stained by hematoxylin and eosin for each treatment group (G) HPL-CT, (H) HPL-DMBA, (I) HPO-CT, and (J) HPO-DMBA are shown. Scale bar = 0.5 mm. Different letters indicate differences between treatments; P < 0.05; n = 5.

Figure 5

Impact of DMBA on ovarian DNA damage repair and histone modification proteins in lean and obese mice. Following 7 days of exposure to vehicle control (CT) or DMBA, (A) BRCA1 (treatment effect P = 0.70; obesity effect P = 0.45; interaction effect P = 0.95), (B) γH2AX (treatment effect P = 0.39; obesity effect P = 0.55; interaction effect P = 0.52), (C) H3K4me (treatment effect P = 0.87; obesity effect P = 0.68; interaction effect P = 0.87), (D) H4K5ac (treatment effect P = 0.03; obesity effect P = 0.62; interaction effect P = 0.87), (E) H4K12ac (treatment effect P = 0.23; obesity effect P = 0.75; interaction effect P = 0.50), and (F) H4K16ac (treatment effect P = 0.18; obesity effect P = 0.80; interaction effect P = 0.74) abundances were measured through western blot. P > 0.05; n = 5 ovaries from individual mice per treatment.

Figure 6

Impact of DMBA on ovarian γH2AX localization in lean and obese mice. Following 7 days of exposure to vehicle control (CT) or DMBA, healthy follicles immunostained to detect γH2AX protein (red) in (A), (E), (I) HPL-CT; (B), (F), (J) HPL-DMBA; (C), (G), (K) HPO-CT; and (D), (H), (L) HPO-DMBA were analyzed. Cellular DNA is stained in green. γH2AX abundance was quantified by counting γH2AX foci (indicated by arrows in F-H) in (M) secondary follicles (treatment effect P = 0.48; obesity effect P = 0.08; interaction effect P = 0.41), (N) antral follicles (treatment effect P = 0.18; obesity effect P = 0.06; interaction effect P = 0.28, (O) corpora lutea (treatment effect P = 0.0017; obesity effect P = 0.82; interaction effect P = 0.82), and (P) all growing follicles (treatment effect P = 0.90; obesity effect P = 0.08; interaction effect P = 0.73). Images were captured at 20× magnification. Different letters indicate differences between treatments; P < 0.05; n = 4 ovaries from individual mice per treatment.

Figure 7

Impact of DMBA on oocyte BRCA1 localization in lean and obese mice. Following 7 days of exposure to vehicle control (CT) or DMBA, oocyte membranes of healthy follicles immunostained by BRCA1 (red) in (A) HPL-CT, (B) HPL-DMBA, (C) HPO-CT, and (D) HPO-DMBA were observed. Positive BRCA1 staining was also observed in (E) thecal cells and antral fluid and (F) atretic follicles (indicated by arrows). Cellular DNA is stained in green. Intensities of the BRCA1 stain were quantified in oocytes of (G) primary follicles (treatment effect P = 0.02; obesity effect P = 0.06; interaction effect P = 0.04), (H) secondary follicles (treatment effect P = 0.50; obesity effect P = 0.37; interaction effect P = 0.54), and (I) all follicles combined (treatment effect P = 0.22; obesity effect P = 0.005; interaction effect P = 0.0009), and the averages from each treatment are reported here. Images were captured at 20× magnification. Different letters indicate differences between treatments; P < 0.05; n = 4 ovaries from individual mice per treatment.

Figure 8

Impact of DMBA on oocyte pBRCA1 localization in lean and obese mice. Following 7 days of exposure to vehicle control (CT) or DMBA, immunostaining of pBRCA1 (red) was observed in oocyte nuclei of healthy follicles (indicated by arrows) in (A) HPL-CT, (B) HPL-DMBA, (C) HPO-CT, and (D) HPO-DMBA. Staining of pBRCA1 was also observed in atretic follicles (indicated by arrows) in (E) HPL-CT, (F) HPL-DMBA, (G) HPO-CT, and (H) HPO-DMBA and quantified in (I) (treatment effect P = 0.003; obesity effect P = 0.85; interaction effect P = 0.37). Green stain represents cellular DNA. Images were captured at 20× magnification. n = 4 ovaries from individual mice per treatment.