Sex-specific anti-oxidant capacities in astrocytes

Introduction: Neuronal cell death after exposure to various cytotoxins is sex-specific; male cells being more sensitive to nitrosative stress and excitotoxity than female neurons (1). We previously demonstrated that astrocytes cultured from female brain (XX) are less susceptible to cell death induced by oxygen-glucose deprivation (OGD) and hydrogen peroxide (H2O2) than male (XY) astrocytes (2). In the current study, we tested the hypothesis that male and female astrocytes respond differently to oxidative stress, in part due to differences in antioxidant capacity between male and female astrocytes. Methods: Primary cultured cortical astrocytes were prepared from 1-3-day old male and female rat pups separately and grown to confluency in steroid-free medium. Male and female rat pups were distinguished by a larger genital papilla and longer ano-genital distance in male vs. female pups. Sex was confirmed by inspecting internal organs and gonads after laporatomy, e.g. uterine horns in females and ductus deferens in males, and by multiplex polymerase chain reaction (PCR) using male-specific marker SRY (sex determination region on the Y chromosome, responsible for testes formation) and the universal marker myogenin (Myog), which is expressed in both males and females. At day 14 in vitro (div), cultures were homogenized for protein and mRNA isolation. The protein levels of three antioxidant enzymes: glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), were determined by Western blotting in male and female astrocytes. Furthermore, GPx mRNA level was determined by RT-PCR, and GPx activity was measured in male and female astrocytes using a glutathione peroxidase activity assay kit measures GPx activity indirectly by a coupled reaction with glutathione reductase. Results: The protein levels of SOD and CAT were not different between male and female astrocytes. However, the level of GPx protein was 3-fold higher in female compared to male astrocytes (3±0.6 fold, n=4, p<0.05). Similarly, GPx mRNA and activity were significantly higher in female compared to male astrocytes in female compared to male astrocytes (2±0.2 fold, n=3, p<0.05 for the mRNA and 2.4±0.1 fold, n=3, p<0.05 for the activity). Conclusions: We conclude that female astrocytes express higher levels of GPx and exhibit higher GPx activity than male astrocytes. The sex difference in GPx expression and activity may afford female astrocytes with higher anti-oxidant capacity, and may underlie the difference in ischemic cell death between male and female astrocytes.