The Roles of Oxidative Stress, Mitochondria, and Lipid Droplets in Glutathione Deficiency and Benzo(a)pyrene-induced Germ Cell Death and Reduced Oocyte Developmental Competence
Polycyclic aromatic hydrocarbons (PAHs) are ovotoxicants. Benzo[a]pyrene (BaP) is a PAH that depletes the ovarian reserve when females are exposed directly or in utero. Oocyte quality is developmental competence acquired through folliculogenesis. Among the attributes of the oocyte that determine its competence include mitochondria, lipid droplets (LDs), and small antioxidant molecules such as glutathione (GSH). Whether the developing germ cells in the embryo are more sensitive to BaP exposure during proliferation or meiosis has been unexplored. Further, how gestational BaP exposure and persistent oxidative stress impact the developmental competence of the oocyte remains unclear.
C57BL/6J female mice were mated with males and orally dosed with 0 or 2 mg/kg/day of BaP from either embryonic day (E)6.5-11.5 (proliferative phase, BaP6.5 and Oil6.5) or E12.5-17.5 (meiotic phase, BaP12.5 and Oil 12.5). F1 pubertal ovaries were examined for follicle counts and persistent oxidative stress. BaP6.5 and BaP12.5 had statistically significant depletion in primordial, primary, and secondary follicles as well as increased lipid peroxidation compared with controls. Oocytes from F1 BaP6.5 and Oil6.5 females were examined and BaP6.5 oocytes were observed to have increased levels of superoxide while significantly decreased mitochondrial membrane potential and LD volume.
We exposed female mice to concentrated air particulates at 2.5 µm or less (PM2.5) in aerodynamic diameter using a Versatile Air Concentration Enrichment System (VACES) for 6 h/ day, 5 days/ week, for 12 weeks. With our collaborators, I developed and validated an extraction and HPLC-MS/MS method to measure ovarian 17β-estradiol concentrations. The concentration of 17β-estradiol did not differ among exposure groups. However, the 17β-estradiol content of ovaries from wild-type, unexposed mice were significantly higher than either exposure group of the transgenic mouse model (Apoe -/-) that were used in this study.
GSH deficiency reduction mature oocyte developmental competence was explored. Using a transgenic mouse model, deleting the modifier subunit of the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase (Gclm), we demonstrated that Gclm -/- oocytes have increased levels of ROS, superoxide and decreased mitochondrial membrane potential. Additionally, Gclm -/- oocytes had decreased LD content compared with Gclm +/+ oocytes. The data in this work demonstrate that the developing ovary is sensitive to BaP exposure across the entirety of its development resulting in F1 ovarian follicular depletion, and persistent F1 ovarian oxidative stress which reduces oocyte developmental competence through disrupted redox homeostasis and lipid dysregulation, and that BaP has similar effects on the ovary as PM2.5.