Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

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 \u3c 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

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Cellular and molecular mechanisms of chemical-induced ovarian toxicity

The ovary is the ovum-producing female reproductive organ composed of follicles at different stages of development. Chemicals which selectively damage large growing or antral follicles only temporarily interrupt reproductive function because these follicles can be replaced by recruitment from the greater pool of primordial follicles. However, chemicals that destroy oocytes contained in primordial follicles can cause permanent infertility and premature ovarian failure (early menopause in women), since once a primordial follicle is destroyed, it cannot be replaced. We hypothesized that ovotoxic chemicals can bind to DNA to induce DNA damage and that the ovarian cells activate DNA repair, cell cycle arrest and apoptosis to cause chemical-induced toxicity. Additionally, we investigated cell to cell communication as targets of ovotoxicants. Gap junction proteins are involved in folliculogenesis and we questioned whether an external factor such as ovotoxicant exposure and obesity alters gap junction proteins to cause follicle loss. To test this hypothesis, ovarian mRNA and protein expression profiles involved in DNA repair, cell cycle arrest, apoptosis and gap junction formation were quantified using qRT-PCR, western blotting or immunohistochemistry techniques after exposure to two different chemicals: Phosphoramide mustard (PM) and 7,12-dimethylbenz[a]anthracene (DMBA). In addition, we investigated the impact of PM and/or DMBA in three physiological paradigms: 1) spontaneously immortalized rat granulosa cells (SIGC); 2) In vitro neonatal ovarian culture system; 3) In vivo obese mouse model. Overall, our data demonstrates that the ovarian DNA damage responses are activated and up-regulated in both a dose- and time-dependent manner. Also, chemical exposure alters the gap junction protein expression in ovaries, interfering with inter-cellular communication, leading to follicular demise. Taken together, our discoveries detailed herein shed new mechanistic light on the events that precipitate ovarian follicle depletion leading to female infertility, and potential targets for prevention of chemical-induced infertility are described

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

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 (1mg/kg; intraperitoneal injection) at 18 wks 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 \u3c 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P \u3c 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P \u3c 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P \u3c 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

Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

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 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. 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 \u3c 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P \u3c 0.05) but CX43 protein increased (P \u3c 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P \u3c 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, 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

Developmental Origins of Ovarian Disorder: Impact of Maternal Lean Gestational Diabetes on the Offspring Ovarian Proteome in Mice

Gestational diabetes mellitus (GDM) is an obstetric disorder affecting approximately 10% of pregnancies. The 4HFHS (High Fat High Sucrose) mouse model emulates GDM in lean women. Dams are fed a HFHS diet one week prior to mating and throughout gestation resulting in inadequate insulin response to glucose in mid-late pregnancy. The offspring of HFHS dams have increased adiposity, thus, we hypothesized that maternal metabolic alterations during lean GDM would compromise ovarian function in offspring both basally and in response to a control or HFHS diet in adulthood. Briefly, DLPL were lean dams and control diet pups; DLPH were lean dams and HFHS pups; DHPL were HFHS dams and control diet pups and DHPH were HFHS dams and HFHS pups. A HFHS challenge in the absence of maternal GDM (DLPL vs. DLPH) increased 3 and decreased 30 ovarian proteins. Maternal GDM in the absence of a dietary stress (DLPL vs. DHPL) increased abundance of 4 proteins and decreased abundance of 85 proteins in the offspring ovary. Finally, 87 proteins increased, and 4 proteins decreased in offspring ovaries due to dietary challenge and exposure to maternal GDM in utero (DLPL vs. DHPH). Canopy FGF signaling regulator 2 (CNPY2), Deleted in azoospermia-associated protein 1 (DAZAP1), Septin 7 (SEPT7), and Serine/arginine rich splicing factor 2 (SRSF2) were altered across multiple offspring groups. Together, these findings suggest a possible impact on fertility and oocyte quality in relation to GDM exposure in utero as well as in response to a western diet in later life