Roles of reactive oxygen species and antioxidants in ovarian toxicity

Abstract

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.

FIG. 1.

Reactive oxygen species (ROS) generation and detoxification. ROS are formed by the sequential addition of electrons to molecular oxygen, forming superoxide anion radical, H₂O₂, and hydroxyl radical. Peroxynitrite (ONOO⁻) is formed when superoxide anion radical reacts with nitric oxide (NO). Key antioxidant enzymes (in bold blue) and the reactions they catalyze are shown. CAT, catalase; GPX, glutathione peroxidase; GSR, glutathione reductase; GST, glutathione-S-transferase; PRDX, peroxiredoxin; SOD, superoxide dismutase. GPXs, PRDXs, and GSTs require glutathione (GSH) as a cofactor, and GSH can also scavenge free radicals through direct chemical reactions. GSR reduces the oxidized form of GSH (GSSG, glutathione disulfide).

FIG. 2.

VCD-induced reductions in hepatic (A) but not ovarian (B) GSH levels. GSH levels were measured by HPLC in tissue homogenates of female Fischer 344 rats 2, 6, or 26 h after a single i.p. dose or 2 or 26 h following 15 days of dosing of 80 mg/kg VCD in sesame oil, 450 mg/kg (2 mmol/kg) BSO in saline, or vehicle control (sesame oil or saline). Values represent means ± SEM; n = 8–12 animals per group. *Significantly different from control, P < 0.05. Modified from Devine et al. [66] with permission of the Society of Toxicology and Oxford University Press.

FIG. 3.

Cyclophosphamide (CP)-induced apoptosis in granulosa cells of ovarian follicles involves activation of caspases 9 and 3. Proestrous female rats received sequential i.p. injections 2 h apart: normal saline followed by normal saline with 10% DMSO (sal/sal), saline followed by 50 mg/kg CP in DMSO (sal/50), saline followed by 300 mg/kg CP (sal/300), or 5 mmol/kg buthionine sulfoximine followed by 50 mg/kg CP (BSO/50) [41]. The animals were euthanized 24 h later and ovaries (n = 3 per group) were processed for immunostaining with antibodies to activated (cleaved) caspase 9 or activated caspase 3 (both from Cell Signaling Technologies, Beverly, MA) using the Vectastain ABC Kit. Secondary and antral follicles with more than three positively stained granulosa cells per largest cross section were counted in 16 sections per ovary blind to treatment group. A) Representative images of antral follicles with positively stained (brown) granulosa cells in ovaries from rats treated with 300 mg/kg CP. The graphs show the mean ± SEM percentages of secondary and antral follicles that stained positively for activated caspase 9 (B) and activated caspase 3 (C). *Significantly different from respective sal/sal control, P < 0.05. (U. Luderer, previously unpublished data obtained on same ovaries as TUNEL data published in Lopez and Luderer [41]).