High fat diet-induced obesity and gestational DMBA exposure alter folliculogenesis and the proteome of the maternal ovary

Abstract Obesity and ovotoxicant exposures impair female reproductive health with greater ovotoxicity reported in obese relative to lean females. The mother and developing fetus are vulnerable to both during gestation. 7,12-dimethylbenz[a]anthracene (DMBA) is released during carbon combustion including from cigarettes, coal, fossil fuels, and forest fires. This study investigated the hypothesis that diet-induced obesity would increase sensitivity of the ovaries to DMBA-induced ovotoxicity and determined impacts of both obesity and DMBA exposure during gestation on the maternal ovary. Female C57BL/6 J mice were fed a control or a High Sugar High Fat (45% kcal from fat; 20% kcal from sucrose) diet until ~30% weight gain was attained before mating with unexposed males. From gestation Day 7, mice were exposed intraperitoneally to either vehicle control (corn oil) or DMBA (1 mg/kg diluted in corn oil) for 7 d. Thus, there were four groups: lean control (LC); lean DMBA exposed; obese control; obese DMBA exposed. Gestational obesity and DMBA exposure decreased (P < 0.05) ovarian and increased liver weights relative to LC dams, but there was no treatment impact (P > 0.05) on spleen weight or progesterone. Also, obesity exacerbated the DMBA reduction (P < 0.05) in the number of primordial, secondary follicles, and corpora lutea. In lean mice, DMBA exposure altered abundance of 21 proteins; in obese dams, DMBA exposure affected 134 proteins while obesity alone altered 81 proteins in the maternal ovary. Thus, the maternal ovary is impacted by DMBA exposure and metabolic status influences the outcome.


Introduction
Ovarian function is vital for female reproductive health.Oocytes are the female gametes which develop within a follicular structure surrounded by somatic cells.The number of germ cells within primordial follicles that will grow into the follicular pool is limited [1] and folliculogenesis describes activation of oocyte and somatic cell growth and differentiation orchestrated both locally [2] and centrally [1].
During pregnancy, maternal exposure to ovotoxicants and/or endocrine disrupting chemicals could negatively affect fetal development leading to a broad spectrum of health complications or increased susceptibility to metabolic disorders for in utero exposed offspring [11][12][13][14][15][16][17][18].Less is understood regarding the global issue of maternal health, however, pregnant females subjected to environmental chemical exposure have increased risk of hypertension/preeclampsia [19] and breast cancer [20].Maternal morbidity rates are high in the United States suggesting increased anthropogenic chemical pollution [21] and obesity [22][23][24][25][26] as potential culpable factors.Women from socioeconomically disadvantaged backgrounds are considered to be at a higher risk for both of these contributing factors [27,28].
The majority of studies have been performed in adult nonpregnant mice or have examined the impacts of gestational exposure on offspring ovarian outcomes rather than investigating the maternal impacts.To determine an impact of DMBA exposure on the maternal ovary during gestation and an influence of obesity thereon, this study combined DMBA exposure with the physical stressor of gestational obesity to explore the effects of obesity and DMBA exposure on the maternal ovary.Female lean and obese mice were exposed to DMBA and ovaries collected at post-natal day 2. The impacts on ovarian, liver and spleen weight, ovarian follicle number, circulating progesterone (P 4 ) and the ovarian proteome were determined.

Animal handling and tissue collection
All animal procedures were approved by the Institutional Animal Care and Use Committee at Iowa State University.Female C57BL/6 J mice aged 5 weeks were obtained from Jackson Laboratories and fed a control (CT; Prolab RMH 1000, Lab-Diet, St. Louis, MO; n = 20) or a High Sugar High Fat (HSHF) diet representative of a human western diet (#D12451; 45% kcal from fat, 20% kcal from sucrose; Research Diets, NJ; n = 20).Females are referred to as lean or obese hereon.Four mice on the HSHF diet did not obtain 30% gain and were excluded from the continuation of the study.After ∼30% weight gain in the HFHS relative to the CT mice, females from both groups were mated to unexposed C57BL/6 J male mice for one week or until appearance of a vaginal plug.The day of an observed vaginal plug was designated as gestation day (GD) 0.5 and the male was removed from the cage.Body weights were recorded weekly before pregnancy and every 2nd day during the gestation to adjust the DMBA dose.Both lean and obese mice were intraperitoneally injected with vehicle control (corn oil) or DMBA (1 mg/kg diluted in corn oil) for 7 d starting from GD7.This dose of DMBA has been demonstrated previously to be ovotoxic [41].This time of gestation (GD) coincides with developing germ cells reaching the fetal ovary [66].Thus, there were four treatment groups: lean control (LC); lean DMBA (LD); obese control (OC); obese DMBA (OD); n = 10 per group; two technical replicates.Dams were euthanized at PND2 by CO 2 asphyxiation followed by cervical dislocation.Blood for serum collection was obtained via cardiac puncture.Liver and spleen were immediately weighed and snap-frozen in liquid nitrogen.Ovaries were either flash frozen in liquid nitrogen or fixed in 4% paraformaldehyde overnight followed by preservation in 70% ethanol for histological analysis.

Histological analysis
Four ovaries per treatment were randomly selected for hematoxylin and eosin staining.Followed by fixation in 4% paraformaldehyde for 24 h, ovaries were first preserved in 70% ethanol, and then paraffin embedded to be sectioned in 5-micron thickness using a microtome.Every 6th section was mounted on the microscopy slides, and every 12th ovarian section was counted to establish follicular composition.Healthy follicles were counted as previously described [67].Follicles undergoing apoptosis or necrosis containing the pyknotic bodies and intense eosinophilic staining were designated and counted as atretic.

Serum P 4 hormone level quantification
The obtained serum from PND2 dams from the second replicate trial was used to quantify P 4 at the Ligand Assay & Analysis Core of the Center for Research in Reproduction, University of Virginia with two technical replicates per sample (reportable range = 0.15-40.0ng/mL).

LC-MS/MS analysis
Dams' ovarian tissue homogenates were analyzed by Protein Facility of the Iowa State University Office of Biotechnology using Q Exactive Tandem Mass Spectrometry.After homogenate samples were reduced with dithiothreitol, proteins were treated with iodoacetamide to modify the cysteine groups followed by overnight digestion with trypsin/Lys-C.Afterwards, samples were treated with formic acid and dried in a SpeedVac chamber.C18 MicroSpin Columns (Nest Group SEM SS18V) were used to desalt the samples.The internal control spiked into the peptide samples used was Peptide Retention Time Calibration standard (Pierce part #88320).The peptides were separated using EASY nLC-1200 system with pulled glass emitter 75 μm X 20 cm column (Agilent capillary, part #160-2644-5) packed with a NextAdvance Presssure Injection Cell and UChrom 3-micron material from nanoLCMS Solutions (part #80002) coupled to a Nanospray FlexIon source (Thermofisher Scientific).Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with an HCD fragmentation cell (Thermofisher Scientific) was utilized for peptide separation and MS/MS analysis.Proteins were identified by MS/MS database as previously described [13,68].
Protein abundance and fold changes (FC) between treatment groups were analyzed by the Genome Informatics Facility at Iowa State University.Pathways in which altered proteins have functional roles were identified using DAVID v 6.8 software.The false discovery rate (FDR) for pathways was calculated using DAVID v 6.8 software.

Statistical analysis
Unpaired t-test using Prism 9.0.1 software (GraphPad Prism) were used for data sets that met normal distribution parameters.For the data sets with skewed distributions, Wilcoxon Mann-Whitney comparison was utilized.Statistical significance was defined as P < 0.05 with a tendency for biological meaning if P < 0.1.Pathway enrichment analyses were generated using proteins identified by LC-MS/MS results for which P < 0.1.The results in bar charts represent mean ± standard error of mean (SEM).

Effect of obesity on pregnancy outcome
Female mice were fed either a CT or HSHF diet for 10 weeks before mating to males who were fed a standard chow diet.Pregnancy success was achieved in 52.5% of females from CT group and 37.5% of females from the HFHS group (data not shown).The mean body weight for females at the beginning and the end of 10 wk of assigned dietary treatment (CT n = 19; HSHF n = 16) determined that the females on the HFHS diet were heavier than their CT diet counterparts (P < 0.0001; Figure 1) prior to mating with males.
Impact of gestational DMBA exposure on maternal body and organ weight in lean and obese mice Exposure to DMBA did not impact maternal body weight at the cessation of dosing relative to the respective controls; however the lean mice treated with DMBA had lower (P < 0.05) body weight compared to both of the obese groups (Figure 2A).There was an additive effect of gestational obesity and DMBA exposure on ovary weight with a decrease (P < 0.05) in ovarian weight in OD relative to LC dams, which experienced ∼25% loss of ovarian mass (Figure 2B).There was no effect of gestational obesity nor DMBA exposure on maternal spleen weight at PND2 (Figure 2C).Gestational DMBA exposure also did not impact hepatic weight in lean dams, but liver weight was increased in obese DMBA treated dams compared with both lean groups (P < 0.05) (Figure 2D).
Impact of gestational DMBA exposure on circulating serum P 4 level in postpartum lean and diet-induced obese mice The level of P 4 was measured in the serum obtained from PND2 dams.There was no treatment effect on serum P 4 (P > 0.05; Figure 3) in either lean or obese dams.

Impact of both obesity and DMBA on the number of follicles in the ovaries from PND2 dams
There was no effect (P > 0.05) of DMBA exposure on primordial follicle number in lean dams.The number of primordial follicles was decreased (P < 0.05) in obese dams exposed to DMBA relative to lean DMBA exposed dams (Figure 4A).In lean mice, DMBA exposure increased the number of primary follicles (P < 0.05; Figure 4B) but this was not observed in the obese mice.Exposure to DMBA increased the number of secondary follicles in lean mice, but in obese dams secondary follicle number was decreased (P < 0.05; Figure 4C).There was no effect of body composition or DMBA exposure on pre-antral follicle number (Figure 4D).The number of atretic follicles was increased (P < 0.05) by both obesity and DMBA exposure (Figure 4E).Ovaries from DMBA exposed obese dams had decreased (P < 0.05) numbers of corpora lutea (CLs) compared with ovaries from lean dams (Figure 4F).

Additive effects of obesity and DMBA exposure on the ovarian proteome in postpartum dams
In postpartum dams, the proteomic analyses of ovarian tissue were compared between four groups: LC vs. LD, LC vs. OC, LD vs. OD, and OC vs. OD (Table 7).
Considering the impact of obesity alone (LC vs. OC), HFHS diet-induced obesity changed (P < 0.05) the levels of 81 proteins (Table 1).Obesity increased the abundance of 39 proteins and decreased 42 proteins.Out of multiple proteins altered by obesity with FC > 2, RNA-binding motif protein 3 (RBM3) was increased almost 14-fold (Figure 5).Two proteins with roles in detoxification of xenobiotics, Glutathione S-transferase P1 (GSTP) and Quinone oxidoreductase (NADPH:quinone reductase), were decreased by 0.7-fold and 0.4-fold, respectively.There was a total of 13 pathways as possible targets of obesity in postpartum dams recognized by DAVID statistical analysis with FDR ≤ 0.08 including "Glutathione" and "Metabolic" pathways, "Carbon metabolism", "Thermogenesis", and "Oxidative phosphorylation" (Table 2).
DMBA exposure in lean mice (LC vs. LD) altered (P < 0.05) the abundance of 21 ovarian proteins with 5 increased and 16 decreased (Table 3).Of these, only four had a FC >2 in expression: Keratin, Type II cytoskeletal 79 (KRT79) and EFhand domain-containing protein (S100A9) were increased and (Phospholipase A-2-activating protein [PLA2P] and Omega-amidase [NIT2]) were decreased (Figure 6).In the table for identified pathways, "Gene Found" indicates the number of genes identified from proteomic analyses, and "Gene Pathway" represents the total number of genes in the particular pathway according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database.Pathway analysis of the ovarian altered proteins with P < 0.1 identified only "Metabolic pathway" as a possible target with FDR = 0.05.
DMBA-induced alterations in the ovaries of obese postpartum dams (OC vs. OD) identified the greatest number of altered proteins (total n = 134; P < 0.05; Table 4).The abundance of 72 proteins were increased, and 62 proteins were decreased.Within these 18 proteins were increased and 34 were decreased with FC > 2 (Figure 7).The same proteins altered in obese control relative to lean control mice that participate in xenobiotic defense were identified with altered levels in the ovaries from DMBA-exposed obese mice: GSTP1 (1.4-fold increase) and CRYZ (2.2-fold increase).In addition, Catalase, an important antioxidant protein had an ∼2-fold increase in abundance due to DMBA exposure in the ovaries of obese mice.Caspase 3, an apoptotic cascade protein, was decreased 0.4-fold by DMBA exposure in obese dams.Thirty pathway targets with FDR ≤ 0.08, were identified and 29 of them had an FDR ≤ 0.05 including "Metabolic pathway", "Carbon metabolism", "Tight junction", and "Oxidative phosphorylation" (Table 5).
Finally, the differential impacts of DMBA exposure in lean and obese mice were determined (LD vs. OD) and 21 proteins had differential abundance with 11 increased and 10 decreased (P < 0.05; Table 6).Peroxisomal-CoA oxidase 3 and coronin proteins were increased ∼2-fold, and 5 proteins levels were decreased with FC > 2 (Figure 8)."Metabolic pathway" was identified as an altered pathway (FDR < 0.5) in the ovaries of obese relative to lean DMBA exposed dams.

Discussion
Ovarian function is critical for both reproductive and general female health.Follicles at different stages of growth can be targeted by ovotoxicants which can results in premature ovarian aging and, subsequently, negatively impact female health [6].Exposure to environmental chemicals may also contribute to maternal morbidity [69,70] prompting research to address risks from environmental pollutants on gestational and postpartum health of exposed mothers.The PAH family impair female reproduction [16,32,33] and are released by incomplete combustion of organic matter.DMBA is biotransformed to an active, ovotoxic metabolite [36,37] in the   ovary that result in follicle loss [36,41,47,48,71].Female offspring exposed in utero to DMBA have reduced numbers of oocytes and poor oocyte quality in adult life [72].However, the maternal ovarian effects of DMBA exposure are not yet fully elucidated.
Obesity is a metabolic disorder that impedes female reproductive function through impaired oocyte quality [49], endocrine dysregulation [55][56][57] and concomitant infertility [50][51][52][53][54].In addition, obesity has been shown to associate with maternal mortality [26].The ovaries of non-pregnant adult obese mice have molecular alterations that suggest enhanced sensitivity to environmental ovotoxicants, including alterations to proteins with roles in DNA repair [43][44][45]73], follicular viability [62,63], steroidogenesis [74], and gap junction communication [64].This current study queried if obesity during gestation would impact the maternal response to ovotoxicant exposure.Thus, the hypothesis investigated was that diet-induced obesity would impact the ovarian response to gestational DMBA exposure.In addition, the experimental design permitted comparison of the basal obesity impacts on the ovarian proteome during pregnancy.
A negative impact of high-caloric diet on pregnancy outcome was observed with the number of pregnant mice on HSHF diet being reduced by 15% compared with the control diet fed mice.Similar to a previous study in non-pregnant mice [45], there was lack of a DMBA effect on female body weight post-gestation.Though spleen weight did not differ across treatment groups, there was an additive impact of obesity and DMBA exposure on hepatic weight which was increased in DMBA exposed obese mice.Metabolic dysregulation including non-alcoholic fatty liver disease have been reported due to obesity [75][76][77] and alterations to hepatic xenobiotic biotransformation could further potentiate ovarian dysfunction.In obese DMBA exposed mice, there was also a reduction in ovary weight, potentially demonstrating a higher susceptibility of ovaries from obese dams to DMBA toxicity in pregnant females.
Progesterone is vital for pregnancy maintenance, and its regulation by PGF2α pathway at the end of gestation is important for the initiation of parturition.Therefore, circulating P 4 was measured in the postpartum dam serum to examine whether DMBA administration and/or metabolic dysregulation during the gestation could adversely affect this hormone's level but no treatment effect on circulating P 4 was observed, discounting an endocrine disrupting effect of obesity and DMBA exposure on this hormone parameter.
Follicular composition in the ovaries was affected by both obesity and DMBA exposure.In lean mice, DMBA exposure increased both primary and secondary follicle number, potentially indicating that normal ovarian function, that should be reduced during pregnancy, is being impacted through alterations to follicular growth dynamics.Obesity alone in the absence of DMBA exposure, increased the number of atretic follicles observed-potentially with negative implications for long-term fertility post-partum.In the obese dams that were exposed to DMBA gestationally, decreased secondary follicle number, decreased CL number and increased atretic follicles were noted.All three of these could be negative in terms of pregnancy outcomes, since endocrine disruption could ensue.A disconnect between circulating P 4 and the reduced number of CL due to DMBA exposure in obese dams could indicate that placental P 4 production was not affected but this cannot be concluded from this study alone.Overall, follicular composition in the maternal ovary was altered by obesity and DMBA exposure.Measuring local ovarian regulators of folliculogenesis such as anti-müllerian hormone would be a logical consideration for future work.
Ovarian proteomic profiling was examined to further investigate the adverse effects of both DMBA and obesity during the pregnancy.In lean mice, DMBA exposure altered the abundance of 21 proteins and the metabolic pathway was the sole identified target of DMBA.Contrarily, in obese compared to lean mice, 81 proteins were identified as targets of obesity representing 16 pathways including metabolic, glutathione and oxidative phosphorylation pathways.Interestingly, the glutathione (GSH) pathway is documented to be involved in biotransformation of DMBA in the ovary [41,47,[78][79][80].The GST family of enzymes conjugate GSH to toxicants generally facilitating excretion and cell protection [81][82][83][84][85][86].In nonpregnant lean mice exposed to DMBA, GSTP was increased in the ovary, while in obese mice, basal levels of GSTP were higher than in lean mice, but the DMBA-induced increase in GSTP noted in lean mice was absent in obese females [41].
There were additive impacts of the combined insults of obesity and DMBA exposure on the ovarian proteome of obese DMBA exposed relative to obese control treated dams resulting in 134 total number of altered proteins and 30 pathways.The levels of two apoptotic proteins, NIT1 and HSP70, and two antioxidative stress enzymes, GSTP and CRYZ (quinone oxidoreductase) were changed and of interest.HSP70 functions along with NF-E2-related factor 2 (NRF2) transcription factor in a protective cellular signaling initiated by oxidative stress [87].Both GSTP and CRYZ are downstream targets of the NRF2 pathway [88].Exposure to DMBA increased both GSTP and NRF2 protein in ex vivo cultured ovaries [80,89], indicating a role for NRF2 signaling in the ovarian DMBA response.Interestingly, there was lower basal GSTP in the ovary of obese mice in the absence of DMBA exposure, but during DMBA exposure in the obese ovary, GSTP was increased.Thus, there are deficits in the ovary of an obese female for GSTP-regulated processes.In terms of HSP70, in the obese ovary, HSP70-2 was reduced basally but increased  due to DMBA exposure.This protein is identified in sperm [90,91] and is proposed to have a role in ovarian cancer [92].The function of a routine ovarian role for HSP70-2 is currently unclear.
The level of zona pellucida sperm-binding protein 3 (ZP3) protein which is expressed in extracellular matrix surrounding an ovulated ovum was increased by almost 6-fold in obese mice.Highly conserved in many mammalian species, including  humans and mice, this protein is essential for the formation of the zona pellucida and successful sperm binding [93].Human females with missense mutations in the ZP3 gene are infertile [94,95] thus altered ZP3 due to obesity might compromise female fertility.
Proteins involved in apoptosis were perhaps unsurprisingly altered by DMBA exposure.Proteins related to cytochrome c function including UGCRH, COX5A, COX2 were decreased in ovaries of obese mice exposed to DMBA.Additionally, proapoptotic caspase 3 which was reduced in abundance in obese DMBA-exposed mice, which was unexpected considering the higher levels of atretic follicles observed in both obese and DMBA-exposed mouse ovaries but it is important to consider that the proteomic response was captured at a single timepoint, and protein changes prior to this timepoint could have initiated atresia but may not have been captured herein.
In obese mice exposed to DMBA, three coronin (CORO) proteins were increased >2-fold in the ovary-CORO1A, CORO1B, and CORO1C.Changes in these proteins were not detected in the lean mice exposed to DMBA nor were they evident due to obesity alone.CORO proteins are actinrelated proteins [96] and immunodeficiency-related disease in humans have been linked to dysfunction of CORO proteins [97,98].Since PAH chemicals are documented as immunotoxicants [99,100], there is a potential interaction between obesity and DMBA that alters ovarian function related to immune cell function and actin organization.Additionally, CORO1A is implicated in autophagy [101] and cigarette smoke exposure induces autophagy in the ovary as a means of ovotoxicity [35,102].The literature on an ovarian-specific CORO protein role is scant, however, use of serum CORO1A as a biomarker of neural tube defects in offspring is proposed [103].Interestingly, there is increased risk of neural tube defects in offspring from both obese [104,105] and PAH exposed [106][107][108] females.
In conclusion, our findings are impactful considering environmental exposure of pregnant women to ovotoxicants and high incidences of obesity in society.The maternal ovary follicle composition findings of both obesity and DMBA exposure are concerning and could result in long-term ovarian function impairments.Moreover, identification of the ovarian proteomic effects of basal obesity and of DMBA exposure in the absence and presence of obesity identified ovarian adverse outcome pathways, and DMBA exposure for one third of gestation caused differential alterations in lean and obese dams.The long-term maternal reproductive consequences cannot be deduced from this study alone but there is potential for impaired subsequent reproduction in ovotoxicant exposed, both lean and obese, dams similar to that previously demonstrated in offspring of obese dams who received a second hit exposure to an obesogenic diet in adulthood [109].Future work to examine local ovarian folliculogenesis regulators and cellular location in which proteome changes occur are warranted.

Figure 3 .
Figure 3. Impact of gestational obesity and DMBA exposure on circulating serum progesterone in postpartum dams.Lean (n = 19) and obese (n = 16) pregnant dams were intraperitoneally (i.p) dosed with corn oil or DMBA (1 mg/kg) for 7 d starting from GD7. Mice were euthanized at PND2 and progesterone (ng/ml) was measured in serum (n = 4 per group).Data points represent mean ± SEM.

Figure 5 .
Figure 5. Gestational DMBA exposure alters the ovarian proteome of lean postpartum dams.Lean pregnant dams were intraperitoneally (i.p) dosed with corn oil or DMBA (1 mg/kg) for 7 d starting from GD7. Mice were euthanized at PND2 and protein homogenates analyzed by LC-MS/MS.Bioinformatic analyses were performed to determine differences in protein abundance in lean mice exposed to DMBA relative to vehicle control mice.The dots in the volcano plots above horizontal line indicate proteins that differed in abundance at P < 0.05 with dots to the right and left of the vertical lines indicating increased and decreased in abundance, respectively.

Figure 6 .
Figure 6.Gestational obesity affects the basal ovarian proteome of postpartum dams.Lean and obese pregnant dams were intraperitoneally (i.p) dosed with corn oil or DMBA (1 mg/kg) for 7 d starting from GD7. Mice were euthanized at PND2 and protein homogenates analyzed by LC-MS/MS.Bioinformatic analyses were performed to determine differences in protein abundance between lean and obese vehicle control treated mice.The dots in the volcano plots above the horizontal line indicate proteins that differed in abundance at P < 0.05 with dots to the right and left of the vertical lines indicating increased and decreased in abundance, respectively.

Figure 7 .
Figure 7. Impact of gestational DMBA maternal exposure on the ovarian proteome of obese dams.Obese pregnant dams were intraperitoneally (i.p) dosed with corn oil or DMBA (1 mg/kg) for 7 d starting from GD7. Mice were euthanized at PND2 and protein homogenates analyzed by LC-MS/MS.Bioinformatic analyses were performed to determine differences in protein abundance between obese mice exposed to vehicle control or DMBA.The dots in the volcano plots above horizontal line indicate proteins that differed in abundance at P < 0.05 with dots to the right and left of the vertical lines indicating increased and decreased in abundance, respectively.

Figure 8 .
Figure 8. Gestational obesity alters the maternal ovarian proteomic response to DMBA exposure.Lean and obese pregnant dams were intraperitoneally (i.p) dosed with corn oil or DMBA (1 mg/kg) for 7 d starting from GD7. Mice were euthanized at PND2 and protein homogenates analyzed by LC-MS/MS.Bioinformatic analyses were performed to determine differences in protein abundance in the ovaries due to DMBA exposure in lean and obese dams at PND2.The dots in the volcano plots above horizontal line indicate proteins that differed in abundance at P < 0.05, with dots to the right and left of the vertical lines indicating increased and decreased in abundance, respectively.

Table 1 .
Ovarian proteins affected by obesity in postpartum dams (LC vs. OC)

Table 2 .
Ovarian KEGG pathway targets changed by diet-induced obesity in postpartum dams (LC vs. OC)

Table 3 .
Ovarian proteins altered by DMBA exposure in lean postpartum dams (LC vs. LD)

Table 4 .
Ovarian proteins altered by DMBA exposure in postpartum obese dams (OC vs. OD)

Table 5 .
Ovarian KEGG pathway targets affected by gestational DMBA exposure in postpartum obese dams (OC vs. OD)

Table 6 .
Differential ovarian protein abundance due to gestational DMBA exposure in postpartum lean and obese dams (LD vs. OD)

Table 7 .
Number of proteins altered by obesity basally and in the presence and absence of DMBA exposure