Obesity alters the ovarian proteomic response to zearalenone exposure†

Abstract

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, is detrimental to female reproduction. Altered chemical biotransformation, depleted primordial follicles and a blunted genotoxicant response have been discovered in obese female ovaries, thus, this study investigated the hypothesis that obesity would enhance ovarian sensitivity to ZEN exposure. Seven-week-old female wild-type nonagouti KK.Cg-a/a mice (lean) and agouti lethal yellow KK.Cg-Ay/J mice (obese) received food and water ad libitum, and either saline or ZEN (40 μg/kg) per os for 15 days. Body and organ weights, and estrous cyclicity were recorded, and ovaries collected posteuthanasia for protein analysis. Body and liver weights were increased (P < 0.05) in the obese mice, but obesity did not affect (P > 0.05) heart, kidney, spleen, uterus, or ovary weight and there was no impact (P > 0.05) of ZEN exposure on body or organ weight in lean or obese mice. Obese mice had shorter proestrus (P < 0.05) and a tendency (P = 0.055) for longer metestrus/diestrus. ZEN exposure in obese mice increased estrus but shortened metestrus/diestrus length. Neither obesity nor ZEN exposure impacted (P > 0.05) circulating progesterone, or ovarian abundance of EPHX1, GSTP1, CYP2E1, ATM, BRCA1, DNMT1, HDAC1, H4K16ac, or H3K9me3. Lean mice exposed to ZEN had a minor increase in γH2AX abundance (P < 0.05). In lean and obese mice, LC-MS/MS identified alterations to proteins involved in chemical metabolism, DNA repair and reproduction. These data identify ZEN-induced adverse ovarian modes of action and suggest that obesity is additive to ZEN-induced ovotoxicity.

Keywords: DNA damage repair; chemical metabolism; obesity; ovarian proteome; ovary; zearalenone.

Figure 1

Impact of obesity and ZEN exposure on body weight. Mice were weighed prior to euthanasia. Bars represent body weight (g) ± SEM. Superscript letters indicate significant differences; P < 0.05; n = 5/treatment. Lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ.

Figure 2

Effect of obesity or ZEN exposure on organ weight. After euthanasia, (A) heart, (B) liver, (C) kidney, (D) spleen, (E) uterus, and (F) ovary were collected and weighed (g). Bars represent mean weight ± SEM. Superscript letters indicate statistical difference; P < 0.05; n = 5/treatment. Lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ.

Figure 3

Estrous cyclicity impacts of obesity and ZEN exposure. The number of days at each stage of the estrous cycle were calculated over a 14-day period and presented as a percentage. Bars represent percentage of d at proestrus (black bars), estrus (dark gray bars), and metestrus + diestrus (light gray bars) ± SEM. Asterix indicates differences between treatments; P < 0.05; n = 5/treatment. Lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ.

Figure 4

Ovarian steroid hormone effects of obesity and ZEN exposure. Circulating progesterone was measured by ELISA in lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ. Bars represent mean concentration ± SEM.

Figure 5

Impact of obesity and ZEN exposure on ovarian proteins involved in chemical biotransformation. Total ovarian protein homogenates were analyzed for (A) EPHX1, (B) GSTP, or (C) CYP2E1 protein abundance. Bars represent mean values relative to total protein staining ± SEM; n = 3/treatment. Lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ.

Figure 6

Impact of obesity and ZEN exposure on ovarian proteins involved in DNA repair. Total ovarian protein homogenates were analyzed for (A) ATM, (B) γH2AX (C) BRCA1, (D) DNMT1, (E) HDAC1, (F) H4K16ac, and (G) H3K9me3. Bars represent mean values relative to total protein staining ± SEM. Asterix indicates differences between treatment; P < 0.05; n = 3/treatment. Lean control-treated mice = LC; lean zearalenone-exposed mice = LZ; obese control-treated mice = OC; obese zearalenone-exposed mice = OZ.

Figure 7

Ovarian protein identification and quantification via LC–MS/MS. Total ovarian protein homogenates were analyzed by LC–MS/MS and bioinformatic comparison performed between peptides identified in (A) LC vs. LZ, (B) OC vs. OZ, and (C) LC vs. OC. Pink dots above the solid horizontal line indicate increased (upper right corner) or decreased (upper left corner) proteins; n = 5/treatment; P < 0.05. The top-five increased and decreased proteins per comparison are illustrated as fold-change in (D) LC vs, LZ, (E) OC vs. OZ, and (F) LC vs. OC treated mice.