Jumping the gun: Smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress

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Abstract

Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5 mg/kg/daily) and high (3 mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility.

Highlights

► BaP exposure up-regulates canonical pathways linked with follicular growth/atresia. ► BaP causes primordial follicle activation and developing follicle atresia. ► BaP causes oocyte mitochondrial ROS and lipid peroxidation, impairing fertilisation. ► Short term neonatal BaP exposure compromises adult oocyte quality.

Introduction

Cigarette smoke is a known reproductive hazard associated with delayed conception, premature ovarian failure (POF), and lower fertilisation rates in women undergoing assisted conception (El-Nemr et al., 1998, Freour et al., 2008, Gruber et al., 2008, Howe et al., 1985, Sun et al., 2011, Windham et al., 2005). In addition to direct inhalation, side-stream cigarette exposure also adversely affects female fertility, resulting in reduced embryo implantation and pregnancy rates in IVF patients (Neal et al., 2005). Although we are aware of the destructive nature of cigarette smoke on female fertility, limited data exists on the chemical constituents which cause its reported ovotoxicity.

Cigarette smoke is composed of over 4,000 chemicals, including nicotine, nitroso compounds, aromatic amines, protein pyrolysates and polycyclic aromatic hydrocarbons (PAH) (Rustemeier et al., 2002). Of these chemicals, of particular interest is the polycyclic aromatic hydrocarbon Benzo(a)pyrene (BaP). BaP is a mutagenic toxicant produced via the incomplete combustion of carbon, and is present at relatively high levels in cigarette smoke (Lodovici et al., 2004). Studies of the effects of BaP on ovarian function have identified this chemical as a potent ovotoxicant capable of inducing POF through the rapid depletion of the primordial follicle pool (Mattison and Thorgeirsson, 1979). In the ovary, BaP is converted into a 7,8-dihydrodiol-9,10-epoxide bioactive metabolite by Aryl hydrocarbon receptor (Ahr) induced members of the cytochrome P450 family of oxidases (Bengtsson et al., 1983, Mattison et al., 1983). This bioactive metabolite then binds to the 2-amino group of DNA guanosine to form BaP-DNA adducts, resulting in mutagenesis and follicular atresia (Zhou et al., 2009). Interestingly, BaP-DNA adducts have been detected in the granulosa cells of women who smoke, indicating BaP may be a contributing factor in cigarette induced POF (Zenzes, 2000). Although the overall effect of BaP on follicular development has been well documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. Recent studies conducted by ourselves and others have identified a novel mechanism of premature ovarian failure involving primordial follicle activation and developing follicle atresia for a number of ovotoxic xenobiotics, including the PAH 7,12-Dimethylbenz-[a]anthracene (DMBA) (Keating et al., 2010, Sobinoff et al., 2010, Sobinoff et al., 2011). Given the similarities between DMBA and BaP induced ovotoxicity, we sought to confirm that BaP also induces POF via a similar mechanism (Bengtsson et al., 1983, Mattison et al., 1983).

In addition to POF, evidence also suggests BaP may induce oocyte dysfunction. BaP is detected at high concentrations in the follicular fluid of female IVF patients who smoke, and was associated with a decreased rate of conception (Neal et al., 2008). One potential mechanism by which BaP may adversely affect oocyte quality is through the production of reactive oxygen species (ROS). Xenobiotic-enhanced ROS formation can occur via several mechanisms, including the unnatural “uncoupling” of phase I cytochrome P450 detoxification, quinone formation, and glutathione peroxidise depletion (Sobinoff et al., in press). Indeed, BaP is converted into 3,6-dione and 6,12-dion redox cycling quinones by cyp1A1, both of which have been detected in rodent ovaries after a single dose exposure of BaP (Ramesh et al., 2010). We have previously demonstrated that neonatal xenobiotic exposure severely reduces the ability of oocytes obtained in adulthood to undergo fertilisation, and that exposure to the same xenobiotics in vitro causes dose dependant lipid peroxidation in cultured MII oocytes (Sobinoff et al., 2010). Therefore, we hypothesised that BaP induces oocyte dysfunction through xenobiotic induced lipid peroxidation, resulting in impaired membrane fluidity and therefore sperm-oocyte fusion.

In order to gain a better understanding of the mechanisms behind BaP induced ovotoxicity, we examined the effects of BaP exposure on the ovarian transcriptome of cultured neonatal mouse ovaries, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis revealed a complex mechanism of BaP induced ovotoxicity targeting a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Mitochondrial ROS, lipid peroxidation, and sperm-oocyte fusion assays supported our hypothesis of BaP induced oocyte dysfunction, with short-term BaP exposure causing long term mitochondrial membrane damage, altered oolemma membrane fluidity, and impaired sperm/oocyte fusion and fertilisation.

Section snippets

Reagents

BaP (> 95% purity), Menadione (MEN; > 95% purity) and custom designed primers were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse monoclonal anti-Proliferating Cell Nuclear Antigen antibody (anti-PCNA, NA03T) was obtained from Merck KGaA (Darmstadt, Germany). Rabbit polyclonal anti-active Caspase 3 antibody (anti-Casp3, ab13847), Rabbit polyclonal anti-active Caspase 2 antibody (anti-Casp2, ab2251) were obtained from Abcam (Cambridge, MA). Mouse monoclonal anti-human Anti-Müllerian

Effects of BaP exposure on the neonatal ovarian transcriptome

BaP exposure significantly altered the expression of a small cohort of genes representing 0.7% of the total number present on the array, suggesting a specific gene-regulatory response in the neonatal ovary (Fig. 1A). Significantly altered genes were then analysed for networks and molecular functions through the use of Ingenuity Pathway Analysis software. BaP altered genes were identified as components of molecular networks for gene expression, the cell cycle, cellular growth and proliferation,

Discussion

In this study we characterised the effects of the ovotoxic smoking constituent BaP on the ovarian transcriptome of neonatal mice in vitro. Microarray analysis revealed an elaborate mechanism of BaP induced ovotoxicity involving a small cohort of genes implicated in follicular growth and development, cell cycle regulation, cell to cell signalling, cell death, cancer, and genetic disorder (Fig. 1; Table 1). These results support previous studies which have identified BaP as a potent ovotoxicant

Role of the funding source

Funding sources approved the overall study design as a condition of their support (Project grants).

Disclosure statement

The authors declare no conflict of interest.

Acknowledgments

The authors gratefully acknowledge the financial assistance to EAM by the Australian Research Council, Hunter Medical Research Institute and the Newcastle Permanent Building Society Charitable Trust. APS is the recipient of an Australian Postgraduate Award PhD scholarship. This work was supported by National Health and Medical Research Council (Project grant #510735) to EAM, SDR and BN.

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