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Maternal glyphosate (GLY) impacts remain unclear despite associations between urinary GLY and birth outcomes. Whether maternal pre-conceptional GLY exposure would have phenotypic and molecular impacts in the dam and offspring was tested. Female C57BL6 mice (6 wk) were exposed to saline (CT; n = 20) or GLY (2 mg/kg; n = 20) per os five d per week for 20 wk. Females were housed with males and on gestation day (GD) 14, divided into: CT non-pregnant (CNP), CT pregnant (CP), GLY non-pregnant (GNP), GLY pregnant (GP). Another cohort (CT; n = 10 or GLY; n = 10) completed three pregnancy rounds and pregnancy index (PI), number of pups per litter and pups surviving to postnatal day (PND) 5 calculated. The PI in GLY mice was higher in breeding rounds 1 and 2, but lower in round 3. Pregnancy increased (P ≤ 0.1) GD14 liver and ovary weight. Spleen weight was increased (P < 0.05) in GP relative to GNP mice. No offspring phenotypic impacts were observed. Approximately six months after cessation of exposure, secondary follicle number was reduced (P < 0.05) by pre-conceptional GLY exposure. The ovarian proteome analyzed by LC-MS/MS was altered (P < 0.05) by pregnancy (49 increased, 43 decreased) and GLY exposure (non-pregnant: 75 increased, 22 decreased, pregnant: 27 increased, 29 decreased; aged dams: 60 increased, 98 decreased) with several histone proteins being altered. These findings support ovarian transient and persistent impacts of GLY exposure and identify pathways as potential modes of action. • Pre-conceptional GLY exposure altered maternal ovarian proteome • No phenotypic offspring impacts of maternal GLY exposure observed • GLY reduced secondary follicle number [ABSTRACT FROM AUTHOR]

Obesity and overweight cause poor oocyte quality, miscarriage, infertility, polycystic ovarian syndrome, and offspring birth defects and affects 40% and 20% of US women and girls, respectively. Perfluorooctanoic acid (PFOA), a per - and poly-fluoroalkyl substance (PFAS), is environmentally persistent and has negative female reproductive effects including endocrine disruption, oxidative stress, altered menstrual cyclicity, and decreased fertility in humans and animal models. PFAS exposure is associated with non-alcoholic fatty liver disease which affects ∼24–26% of the US population. This study investigated the hypothesis that PFOA exposure impacts hepatic and ovarian chemical biotransformation and alters the serum metabolome. At 7 weeks of age, female lean, wild type (KK.Cg-a/a) or obese (KK.Cg-Ay/J) mice received saline (C) or PFOA (2.5 mg/Kg) per os for 15 d. Hepatic weight was increased by PFOA exposure in both lean and obese mice (P < 0.05) and obesity also increased liver weight (P < 0.05) compared to lean mice. The serum metabolome was also altered (P < 0.05) by PFOA exposure and differed between lean and obese mice. Exposure to PFOA altered (P < 0.05) the abundance of ovarian proteins with roles in xenobiotic biotransformation (lean - 6; obese - 17), metabolism of fatty acids (lean – 3; obese - 9), cholesterol (lean – 8; obese – 11), amino acids (lean – 18; obese – 19), glucose (lean – 7; obese – 10), apoptosis (lean – 18; obese – 13), and oxidative stress (lean – 3; obese – 2). Use of qRT-PCR determined that exposure to PFOA increased (P < 0.05) hepatic Ces1 and Chst1 in lean but Ephx1 and Gstm3 in obese mice. Also, obesity basally increased (P < 0.05) Nat2 , Gpi and Hsd17b2 mRNA levels. These data identify molecular changes resultant from PFOA exposure that may cause liver injury and ovotoxicity in females. In addition, differences in toxicity induced by PFOA exposure occurs in lean and obese mice. • Exposure to PFOA liver weight in lean and obese females. • Serum metabolites affected by PFOA exposure differed due to obesity. • Obesity impacted PFOA-induced hepatic chemical biotransformation gene mRNA changes. • PFOA exposure altered the ovarian proteome differently in lean and obese females. [ABSTRACT FROM AUTHOR]

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. [ABSTRACT FROM AUTHOR]

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced ( P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes ( Atm , Parp1 , Prkdc , Xrcc6 , and Brca1 ) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. [ABSTRACT FROM AUTHOR]

Abstract: 7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and cultured for 4 or 8days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P <0.05) at 4days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P <0.05) but CX43 protein increased (P <0.05) at 4days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P <0.05) but no changes in Bax expression were observed after 4days of DMBA exposures. In contrast, after 8days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. [Copyright &y& Elsevier]

Abstract: The mammalian ovary is a heterogeneous organ and contains oocyte-containing follicles at varying stages of development. The most immature follicular stage, the primordial follicle, comprises the ovarian reserve and is a finite number, defined at the time of birth. Depletion of all follicles within the ovary leads to reproductive senescence, known as menopause. A number of chemical classes can destroy follicles, thus hastening entry into the menopausal state. The ovarian response to chemical exposure can determine the extent of ovotoxicity that occurs. Enzymes capable of bioactivating as well as detoxifying xenobiotics are expressed in the ovary and their impact on ovotoxicity has been partially characterized for trichloroethylene, 7,12-dimethylbenz[a]anthracene, and 4-vinylcyclohexene. This review will discuss those studies, as well as illustrate where knowledge gaps remain for chemicals that have also been established as ovotoxicants. [Copyright &y& Elsevier]

Abstract: 7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO)±DMBA (1μM) or vehicle control (1% DMSO)±LY294002 (PI3K inhibitor; 20μM) for 1, 2, 4, or 6days. Total RNA or protein was isolated, followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2days. Ahr mRNA and protein increased after 2 and 4days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2days) and increased (4days) by PI3K inhibition, while Gstm mRNA increased (P <0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. [Copyright &y& Elsevier]

Abstract: The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80mg/kg/day; 15days, i.p.). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium±VCD (30μM) for 2–8days. VCD increased ovarian GSTp mRNA (P < 0.05) relative to control on d4-d8; whereas GSTp protein was increased (P <0.05) on d6–d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P =0.09), while unbound JNK was decreased (P< 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: 4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2–12 days in vehicle control, VCD (30 μM), or DMBA (1 μM), ±PI3 kinase inhibitor LY294002 (20 μM) or its inactive analog LY303511 (20 μM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P <0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P <0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P >0.05) at any time, but did cause loss (P <0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P <0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P <0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment. [Copyright &y& Elsevier]

Abstract: The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), targets and destroys all follicle types in rat and mouse ovaries. DMBA requires bioactivation to DMBA-3,4-diol-1,2-epoxide for ovotoxicity via formation of the intermediate, DMBA-3,4-diol (catalyzed by microsomal epoxide hydrolase; mEH). mEH was shown to be involved in DMBA bioactivation for ovotoxicity induction in B6C3F1 mouse ovaries. The current study compared DMBA and DMBA-3,4-diol mediated ovotoxicity, and investigated mEH involvement in DMBA-3,4-diol bioactivation in Fischer 344 (F344) rat ovary. F344 postnatal day (PND) 4 rat ovaries were cultured in vehicle control or media containing 1) DMBA or DMBA-3,4-diol (12.5 nM – 1 μM; 15 days); 2) DMBA (1 μM; 6 h – 15 days); and 3) DMBA (1 μM) or DMBA-3,4-diol (75 nM)±the mEH activity inhibitor cyclohexene oxide (CHO; 2 mM; 4 days). Ovaries were histologically evaluated and mEH mRNA and protein were measured by reverse transcriptase PCR or Western blotting, respectively. Ovotoxicity following 15 days of culture occurred (P <0.05) at lower concentrations of DMBA-3,4-diol (12.5 nM — primordial; 75 nM — primary) than DMBA (75 nM — primordial; 375 nM — primary). The temporal pattern of mEH expression following DMBA exposure showed mRNA up-regulation (P <0.05) on day 2, with increased protein (P <0.05) on day 4, the earliest time of observed follicle loss (P <0.05). mEH inhibition prevented DMBA-induced, but not DMBA-3,4-diol-induced ovotoxicity. These results demonstrate a conserved response in mice and rats for ovarian mEH involvement in DMBA bioactivation to its ovotoxic, 3,4-diol-1,2-epoxide form. [Copyright &y& Elsevier]

Abstract: 4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F1 mice were incubated with VCD (15 μM) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p >0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p <0.05) both primordial and primary follicle numbers. Increased (p <0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased (p <0.05) after 10 days of exposure. mEH and GST pi proteins were elevated (p <0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented. [Copyright &y& Elsevier]

Abstract: The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid. [Copyright &y& Elsevier]

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. • Pre-pubertal obesity altered ovarian and uterine weight. • Chemical biotransformation was reduced by pre-pubertal obesity. • Ovarian DNA repair was compromised in pre-pubertal obese females. [ABSTRACT FROM AUTHOR]

Glyphosate (GLY) usage for weed control is extensive. To investigate ovarian impacts of chronic GLY exposure, female C57BL6 mice were orally administered saline as vehicle control (CT) or GLY at 0.25 (G0.25), 0.5 (G0.5), 1.0 (G1.0), 1.5 (G1.5), or 2 (G2.0) mg/kg for five days per wk. for 20 wks. Feed intake increased (P <.05) in G1.5 and G2.0 mice and body weight increased (P <.05) in G1.0 mice. There was no impact of GLY on estrous cyclicity, nor did GLY affect circulating levels of 17β-estradiol or progesterone. Exposure to GLY did not impact heart, liver, spleen, kidney or uterus weight. Both ovarian weight and follicle number were increased (P <.05) by G2.0 but not affected at lower GLY concentrations. There were no detectable effects of GLY on ovarian protein abundance of pAKT, AKT, pAKT:AKT, γH2AX, STAR, CYP11A1, HSD3B, CYP19A, ERA or ERB. Increased (P <.05) abundance of ATM protein was observed at G0.25 but not higher GLY doses. A dose-dependent effect (P <.10) of GLY exposure on ovarian protein abundance as quantified by LC-MS/MS was observed (G0.25–4 increased, 19 decreased; G0.5–5 increased, 25 decreased; G1.0–65 increased, 7 decreased; G1.5–145 increased, 2 decreased; G2.0–159 increased, 4 decreased). Pathway analysis was performed using DAVID and identified glutathione metabolism, metabolic and proteasome pathways as GLY exposure targets. These data indicate that chronic low-level exposure to GLY alters the ovarian proteome and may ultimately impact ovarian function. • A dose-dependent altered ovarian proteome resulted from glyphosate exposure. • The highest glyphosate dose increased ovarian weight and follicle number. • Glyphosate did not impact ovarian steroid hormone production. [ABSTRACT FROM AUTHOR]

Obesity impairs oocyte quality, fertility, pregnancy maintenance, and is associated with offspring birth defects. The model ovotoxicant, 7,12-dimethylbenz[a]anthracene (DMBA), causes ovarian DNA damage and follicle loss. Both DMBA-induced chemical biotransformation and the DNA damage response are partially attenuated in obese relative to lean female mice but whether weight loss could improve the DNA damage response to DMBA exposure has not been explored. Thus, at six weeks of age, C57BL/6 J female mice were divided in three groups: 1) Lean (L; n = 20) fed a chow diet for 12 weeks, 2) obese (O; n = 20) fed a high fat high sugar (HFHS) diet for 12 weeks and, 3) slim-down (S; n = 20). The S group was fed with HFHS diet for 7 weeks until attaining a higher body relative to L mice on week 7.5 and switched to a chow diet for 5 weeks to achieve weight loss. Mice then received either corn oil (CT) or DMBA (D; 1 mg/kg) for 7 d via intraperitoneal injection (n = 10/treatment). Obesity increased (P < 0.05) kidney and spleen weight, and DMBA decreased uterine weight (P < 0.05). Ovarian weight was reduced (P < 0.05) in S mice, but DMBA exposure increased ovary weight in the S mice. LC-MS/MS identified 18, 64, and 7 ovarian proteins as altered (P < 0.05) by DMBA in the L, S and O groups, respectively. In S and O mice, 24 and 8 proteins differed, respectively, from L mice. These findings support weight loss as a strategy to modulate the ovarian genotoxicant response. • Body weight was restored to lean level by diet intervention. • Ovary weight reduced by slim-down. • Ovarian proteome altered by obesity and slim-down. [ABSTRACT FROM AUTHOR]

The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased ( P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased ( P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced ( P < 0.05) by obesity while total CX37 protein was reduced ( P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased ( P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced ( P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased ( P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. [ABSTRACT FROM AUTHOR]

7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated ( Atm ), X-ray repair complementing defective repair in Chinese hamster cells 6 ( Xrcc6 ), breast cancer type 1 ( Brca1 ), Rad 51 homolog ( Rad51 ), poly [ADP-ribose] polymerase 1 ( Parp1 ) and protein kinase, DNA-activated, catalytic polypeptide ( Prkdc ) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased ( P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased ( P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased ( P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase ( P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: 7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and maintained in culture for 4 or 8days. Alternately, PND4 F344 rat ovaries were exposed to 1μM DMBA at the start of culture for 2days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. γH2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and γH2AX protein. Additionally, DMBA (12.5nM and 1μM) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75nM d4; 12.5nM d8), while pATM was localized in large primary and secondary follicles and increased after 8days of 75nM DMBA exposure compared to both control and 12.5nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss. [Copyright &y& Elsevier]

Abstract: Anti-Müllerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30μM, 2–8days) decreased (P <0.05) AMH mRNA (d4–8) and protein (d6–8). Recombinant AMH (100–400mg/ml) in PND4 ovaries cultured 8days±VCD (30μM) caused an increase (P <0.05) in primordial, and a decrease (P <0.05) in small primary follicles, supporting that AMH retarded primordial follicle recruitment. However, no concentration of AMH had an effect on VCD-induced ovotoxicity. Whereas, VCD caused a reduction in expression of AMH (d4–d8), it followed previously reported initial disruptions in Kit signaling induced by VCD (d2). Thus, collectively, these results do not support a mechanism whereby VCD causes ovotoxicity via generalized activation of primordial follicle recruitment, but instead provide further support for the specificity of other intracellular mechanisms involved in VCD-induced ovotoxicity. [ABSTRACT FROM AUTHOR]

Assessing health outcomes associated with exposure to polychlorinated biphenyls (PCBs) is important given their persistent and ubiquitous nature. PCBs are classified as a Group 1 carcinogen, but the full range of potential noncancer health effects from exposure to PCBs has not been systematically summarized and evaluated. We used systematic review methods to identify and screen the literature using combined manual review and machine learning approaches. A protocol was developed that describes the literature search strategy and Populations, Exposures, Comparators, and Outcomes (PECO) criteria used to facilitate subsequent screening and categorization of literature into a systematic evidence map of PCB exposure and noncancer health endpoints across 15 organs/systems. A comprehensive literature search yielded 62,599 records. After electronic prioritization steps, 17,037 studies were manually screened at the title and abstract level. An additional 900 studies identified by experts or supplemental searches were also included. After full-text screening of 3889 references, 1586 studies met the PECO criteria. Relevant study details such as the endpoints assessed, exposure duration, and species were extracted into literature summary tables. This review compiles and organizes the human and mammalian studies from these tables into an evidence map for noncancer health endpoints and PCB mixture exposure to identify areas of robust research as well as areas of uncertainty that would benefit from future investigation. Summary data are available online as interactive visuals with downloadable metadata. Sufficient research is available to inform PCB hazard assessments for most organs/systems, but the amount of data to inform associations with specific endpoints differs. Furthermore, despite many years of research, sparse data exist for inhalation and dermal exposures, which are highly relevant human exposure routes. This evidence map provides a foundation for future systematic reviews and noncancer hazard assessments of PCB mixtures and for strategic planning of research to inform areas of greater uncertainty. • A wide variety of endpoints have been assessed with PCB exposure. • For each organ/system examined, at least 45 studies were identified. • Reproductive, hepatobiliary, and nervous system effects were the most studied. • Sparse data exist for PCB inhalation and dermal exposures. [ABSTRACT FROM AUTHOR]

Abstract: The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4days before being exposed once to PM (10 or 30μM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P <0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60μM) exposure destroyed all stage follicles after 4days (P <0.05). VC from nearby wells depleted primordial follicles after 4days (P <0.05), temporarily reduced secondary follicle number after 2days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors. [Copyright &y& Elsevier]

Abstract: Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75nM) and has not (12.5nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12nM; low-concentration or 75nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4days of exposure, but induced large primary follicle loss at both concentrations after 8days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4days after exposure. With the exception of Atg7 and cKit, DMBA increased (P <0.05) expression of all genes investigated. Also, BECN1 and pAKTThr308 protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures. [Copyright &y& Elsevier]

Abstract: 4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30μM) for 2–8days; 2) VCD (30μM) for 2days, followed by incubation in control media for 4days (acute VCD exposure); or 3) LY294002 (20μM) for 6days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P <0.05) after 6days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P <0.05) relative to control after 6days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P <0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P <0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. [Copyright &y& Elsevier]

Abstract: 4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P<0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P <0.05) on day 4 of VCD (30μM) exposure, followed by increased (P <0.05) mEH protein after 6days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. [Copyright &y& Elsevier]

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals with documented, though mechanistically ill-defined, reproductive toxicity. The toxicity of dioxin-like PCBs, such as PCB126, is mediated via the aryl hydrocarbon receptor (AHR) in non-ovarian tissues. The goal of this study was to examine the uterine and ovarian effects of PCB126 and test the hypothesis that the AHR is required for PCB126-induced reproductive toxicity. Female Holzman-Sprague Dawley wild type (n = 14; WT) and Ahr knock out (n = 11; AHR−/−) rats received a single intraperitoneal injection of either corn oil vehicle (5 ml/kg: WT_O and AHR−/−_O) or PCB126 (1.63 mg/kg in corn oil: WT_PCB and AHR−/−_PCB) at four weeks of age. The estrous cycle was synchronized and ovary and uterus were collected 28 days after exposure. In WT rats, PCB126 exposure reduced (P < 0.05) body and ovary weight, uterine gland number, uterine area, progesterone, 17β-estradiol and anti-Müllerian hormone level, secondary and antral follicle and corpora lutea number but follicle stimulating hormone level increased (P < 0.05). In AHR−/− rats, PCB126 exposure increased (P ≤ 0.05) circulating luteinizing hormone level. Ovarian or uterine mRNA abundance of biotransformation, and inflammation genes were altered (P < 0.05) in WT rats due to PCB126 exposure. In AHR−/− rats, the transcriptional effects of PCB126 were restricted to reductions (P < 0.05) in three inflammatory genes. These findings support a functional role for AHR in the female reproductive tract, illustrate AHR's requirement in PCB126-induced reprotoxicity, and highlight the potential risk of dioxin-like compounds on female reproduction. • PCB126 reduced body and ovarian weight and depleted follicles in wildtype rats. • PCB126 exposure disrupted the endocrine environment in wildtype rats. • PCB126 exposure altered chemical metabolism and inflammation mRNA level. • Lack of AHR in rats was protective against many PCB126 alterations. [ABSTRACT FROM AUTHOR]