Pre-pubertal obesity compromises ovarian oxidative stress, DNA repair and chemical biotransformation.

Obesity in adult females impairs fertility by altering oxidative stress, DNA repair and chemical biotransformation. Whether prepubertal obesity results in similar ovarian impacts is under-explored. The objective of this study was to induce obesity in prepubertal female mice and assess puberty onset, follicle number, and abundance of oxidative stress, DNA repair and chemical biotransformation proteins basally and in response to 7,12-dimethylbenz(a)anthracene (DMBA) exposure. DMBA is a polycyclic aromatic hydrocarbon that has been shown to be ovotoxic. Lactating dams (C57BL6J) were fed either a normal rodent containing 3.5% kCal from fat (lean), or a high fat diet comprised of 60% kCal from fat, and 9% kCal from sucrose. The offspring were weaned onto the diet of their dam and exposed at postnatal day 35 to either corn oil or DMBA (1 mg/kg) for 7 d via intraperitoneal injection. Mice on the HFD had reduced (P < 0.05) age at puberty onset as measured by vaginal opening but DMBA did not impact puberty onset. Heart, spleen, kidney, uterus and ovary weight were increased (P < 0.05) by obesity and liver weight was increased (P < 0.05) by DMBA exposure in obese mice. Follicle number was largely unaffected by obesity or DMBA exposure, with the exception of primary follicle number, which were higher (P < 0.05) in lean DMBA exposed and obese control relative to lean control mice. There were also greater numbers (P < 0.05) of corpora lutea in obese relative to lean mice. In lean mice, DMBA exposure reduced (P < 0.05) the level of CYP2E1, EPHX1, GSTP1, BRCA1, and CAT but this DMBA-induced reduction was absent in obese mice. Basally, obesity reduced (P < 0.05) the abundance of CYP2E1, EPHX1, GSTP1, BRCA1, SOD1 and CAT. There was greater (P < 0.05) fibrotic staining in obese DMBA-exposed ovaries and PPP2CA was decreased (P < 0.05) in growing follicles by both obesity and DMBA exposure. Thus, prepubertal obesity alters the capacity of the ovary to respond to DNA damage, ovotoxicant exposure and oxidative stress.

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

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 (CT) or a High Sugar High Fat (HSHF; 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 (LD); obese control (OC); obese DMBA exposed (OD). 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.

Trajectory of primordial follicle depletion is accelerated in obese mice in response to 7,12-dimethylbenz[a]anthracene exposure.

Both obesity and exposure to environmental genotoxicants, such as 7,12-dimethylbenz[a]anthracene (DMBA), negatively impair female reproductive health. Hyperphagic lean KK.Cg-a/a (n = 8) and obese KK.Cg-Ay/J (n = 10) mice were exposed to corn oil as vehicle control (CT) or DMBA (1 mg/kg/day) for 7d intraperitoneally, followed by a recovery period. Obesity increased liver and spleen weight (P < 0.05), and DMBA exposure decreased uterine weight (P < 0.05) in obese mice. Primordial follicle loss (P < 0.05) caused by DMBA exposure was observed in obese mice only. Primary (lean P < 0.1; obese P < 0.05) and secondary (lean P < 0.05, obese P < 0.1) follicle loss initiated by DMBA exposure continued across recovery. Reduced pre-antral follicle number in lean mice (P < 0.05), regardless of DMBA exposure, was evident with no effect on antral follicles or corpora lutea number. Immunofluorescence staining of DNA damage marker, γH2AX, did not indicate ongoing DNA damage but TRP53 abundance was decreased in follicles (P < 0.05) of DMBA-exposed obese mice. In contrast, increased (P < 0.05) superoxide dismutase was observed in the corpora lutea of DMBA-exposed obese mice and reduced (P < 0.05) TRP53 abundance was noted in preantral and antral follicles of DMBA-exposed obese mice. This study indicates that obesity influences ovotoxicity caused by a genotoxicant, potentially involving accelerated primordial follicle activation and hampering normal follicular dynamics.

Biological sex differences in hepatic response to in utero dimethylbenz(a)anthracene exposure.

Fetal hepatic dimethylbenz(a)anthracene (DMBA) biotransformation is not defined, thus, this study investigated whether the fetal liver metabolizes DMBA and differs with biological sex. KK.Cg-a/a (lean; n = 20) or KK.Cg-Ay/J (obese; n = 20) pregnant mice were exposed to corn oil (CT) or DMBA (1 mg/kg bw/day) by intraperitoneal injection (n = 10/treatment) from gestation day 7-14. Postnatal day 2 male or female offspring livers were collected. Total RNA (n = 6) and protein (n = 6) were analyzed via a PCR-based array or LC-MS/MS, respectively. The level of Mgst3 was lower (P < 0.05) in livers of female compared to male offspring. Furthermore, in utero DMBA exposure increased (P < 0.1) Cyp2c29 and Gpx3 levels (P < 0.05) in female offspring. In male offspring, the abundance of Ahr, Comt (P < 0.1), Alox5, and Asna1 (P < 0.05) decreased due to DMBA exposure. Female and male offspring had 34 and 21 hepatic proteins altered (P < 0.05) by in utero DMBA exposure, respectively. Opposing patterns for hepatic CD81 and KRT78 occurred, being decreased in females but increased in males, while YWHAG was decreased by DMBA exposure in both. Functional KEGG pathway analysis identified enrichment of 26 and 13 hepatic metabolic proteins in male and female offspring, respectively, due to in utero DMBA exposure. In silico transcription factor analysis of differentially expressed proteins predicted involvement of female NRF1 but male AHR. Thus, hepatic biological sex differences and capacity to respond to toxicants in utero are supported.

Altered histone abundance as a mode of ovotoxicity during 7,12-dimethylbenz[a]anthracene exposure with additive influence of obesity†.

Histones are slowly evolving chromatin components and chromatin remodeling can incorporate histone variants differing from canonical histones as an epigenetic modification. Several identified histone variants are involved with the environmental stress-induced DNA damage response (DDR). Mechanisms of DDR in transcriptionally inactive, prophase-arrested oocytes and epigenetic regulation are under-explored in ovarian toxicology. The study objective was to identify ovarian proteomic and histone modifications induced by DMBA exposure and an influence of obesity. Post-pubertal wildtype (KK.Cg-a/a; lean) and agouti (KK.Cg-Ay/J; obese) female mice, were exposed to either corn oil (control; CT) or DMBA (1 mg/kg) for 7d via intraperitoneal injection (n = 10/treatment). Ovarian proteome analysis (LC-MS/MS) determined that obesity altered 225 proteins (P < 0.05) with histone 3 being the second least abundant (FC = -5.98, P < 0.05). Histone 4 decreased by 3.33-fold, histone variant H3.3 decreased by 3.05-fold, and H1.2, H1.4 and H1.1(alpha) variants increased by 1.59, 1.90 and 2.01-fold, respectively (P < 0.05). DMBA exposure altered 48 proteins in lean mice with no observed alterations in histones or histone variants. In obese mice, DMBA exposure altered 120 proteins and histone 2B abundance increased by 0.30-fold (P < 0.05). In DMBA-exposed mice, obesity altered the abundance of 634 proteins. Histones 4, 3 and 2A type 1-F decreased by 4.03, 3.71, 0.43-fold, respectively, whereas histone variant H1.2 and linker histone, H15 increased by 2.72- and 3.07-fold, respectively (P < 0.05). Thus, DMBA exposure alters histones and histone variants, and responsivity is more pronounced during obesity, potentially altering ovarian transcriptional regulation.

Maternal pre-conceptional glyphosate exposure impacts the offspring hepatic and ovarian proteome.

Glyphosate (GLY) is an herbicide used for rural and urban weed control. Urinary GLY in women is associated with shortened gestational length yet effects of GLY on offspring due to maternal exposure are unclear. This study tested the hypothesis that maternal chronic pre-conceptional GLY exposure would cause phenotypic and molecular changes in F1 offspring. Female C57BL/6 mice (7-week-old; n = 40) received saline vehicle control (CT; n = 20) or GLY (2 mg/kg; n = 20) daily per os for 10 weeks. At dosing completion, females were housed with unexposed males and divided into Cohort 1 who were euthanized at gestation day 14 (n = 10 per treatment) and Cohort 2 who completed gestation (n = 10 per treatment). F1 female ovarian and liver samples underwent LC-MS/MS and bioinformatic analysis. Maternal exposure did not affect litter (P > .05) sex ratio, or embryonic or neonatal gross phenotypes. In Cohort 2 offspring, no treatment effect on (P > .05) offspring anogenital distance, puberty onset, or ovarian follicular composition was noted. Body weight was increased (P < .05) in male GLY-exposed compared with CT dam offspring. F1 females from GLY-exposed dams had altered (P < .05) abundance of 54 ovarian and 110 hepatic proteins. Pathways altered in the ovary (false discovery rate [FDR] ≤ 0.07) included thermogenesis and phosphatidylinositol-3 kinase-AKT signaling and in liver (FDR ≤ 0.08) included metabolic, glutathione metabolism, oxidative phosphorylation, non-alcoholic fatty liver disease, and thermogenesis. Thus, pre-conceptional GLY exposure affected offspring phenotypic and molecular profiles potentially impacting reproductive health.

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

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.

Pre-Conceptional Exposure to Glyphosate Affects the Maternal Hepatic and Ovarian Proteome.

Exposure to glyphosate (GLY), a commonly used herbicide, is supported by urinary detection and associated with shortened gestation in women. This study tested the hypothesis that chronic low-dose pre-conceptional GLY exposure would affect maternal ovarian function mid- and post-gestation. Mice (C57BL/6; n = 40) were exposed per os to saline vehicle control (CT; n = 20) or GLY (2 mg/kg; n = 20) daily for 10 weeks starting at 7 weeks of age. Post-exposure, females were impregnated and euthanized at gestation day 14 (GD14) or post-weaning (PW). Pregnancy success was reduced from 75% to 55% by GLY exposure. No treatment effect (p > .05) on body weight, maternal serum 17ß-estradiol, or litter size was noted. Ovarian weight was unaffected or reduced (p < .05) by GLY in GD14 and PW dams, respectively. Exposure to GLY decreased (p < .05) PW ovarian secondary follicle number with no other follicle composition impacts. Protein abundance analysis by LC-MS/MS identified that GLY altered (p < .05) 26 ovarian and 41 hepatic proteins in GD14 dams and 39 hepatic proteins in PW dams. In GD14 dams, GLY increased ovarian protein abundance of SEC16A (p < .05; 29-fold) and hepatic RPS27L and GM4952 (p < .05; ∼4-fold). In both GD14 and PW dams, GLY exposure increased (p < .05) hepatic RPS4 and decreased (p < .05) ECHDC3. Pathway analysis using DAVID identified 10 GLY hepatic pathway targets with FDR ≤ 0.07 in GD14 dams.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos.

Changes in abundance of mRNAs during oocyte growth and maturation and during pre-implantation embryo development have been documented using quantitative real-time RT-PCR (qPCR), microarray analyses, and whole genome sequencing. However, these techniques require amplification of mRNAs, normalization using housekeeping genes, can be biased for abundant transcripts, and/or require large numbers of oocytes and embryos which can be difficult to acquire from mammalian species. We optimized a single molecule RNA fluorescence in situ hybridization (RNA-FISH) protocol, which amplifies fluorescence signal to detect candidate transcripts, for use with individual oocytes and embryos. Quantification using the software Localize showed patterns of Gdf9 and Pou5f1 mRNA expression in oocytes and embryos that were consistent with previously published data. Interestingly, low levels of Nanog mRNA were also accurately and reproducibly measured in oocytes and one- and two-cell embryos suggesting that RNA-FISH could be used to detect and quantify low abundance transcripts. Unlike other techniques, RNA-FISH is also able to detect changes in the localization patterns of mRNAs which may be used to monitor post-transcriptional regulation of a transcript. Thus, RNA-FISH represents an important technique to investigate potential mechanisms associated with the synthesis and stability of candidate mRNAs in mammalian oocytes and embryos.

Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.