196 GLUTATHIONE S-TRANSFERASE THETA1 (GSTT1) IS DIFFERENTIALLY REGULATED FROM OTHER GLUTATHIONE S-TRANSFERASES IN GRANULOSA CELLS

Abstract

Oxidative stress is a major source of aging that damages genomic and mitochondrial DNA, causing female reproductive disorders. Glutathione S-transferases (GST) are known to detoxify the metabolites of genotoxic molecules to more water-soluble and readily excretable forms. Therefore, most GST have been considered to be down-regulated by aging. We recently demonstrated that only GSTT1 is up-regulated in granulosa cells from aged, infertile patients and aged mice, although other kinds of GST are down-regulated in these cells (Ito et al. 2008 Fertil. Steril. 90, 1026–1035). Measurement of the GSTT1 level in granulosa cells binding to the patient’s oocytes could be used in the selection of oocytes of high quality, meaning that the GSTT1 level in granulosa cells could be a good indicator of age-related infertility. To do that, an understanding of the concrete relationship between aging and GSTT1 up-regulation is necessary. However, the regulation mechanism of GSTT1 in granulosa cells remains unclear. In the present study, we attempted to examine the difference in expression mechanisms between GSTT1 and other GST. First, the expression patterns of GSTT1 and GSTP (a major member of the GST) in aged granulosa cells from patients were observed by immunostaining. The GSTT1 was up-regulated (young: 25 to 34 years old, N = 9; aged: 38 to 43 years old, N = 12; P < 0.05) but the GSTP was down-regulated (young: 28 to 30 years, N = 5; aged: 38 to 42 years, N = 7; P < 0.05) in the aged group. Nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor that regulates the expression of most GST, so in the next experiment, we examined the expression patterns of GSTT1 and GSTP in granulosa cells from Nrf2-disrupted (Nrf2–/–) mice by immunostaining. The GSTP was down-regulated in granulosa cells of Nrf2–/– mice compared with those of Nrf2+/– mice, although a significant difference was not observed in GSTT1. In addition, we confirmed these results by using mouse embryonic fibroblasts (MEF) from Nrf2–/– and Nrf2+/– mice, and the same results were obtained. Our previous experiment showed that GSTT1 was induced by FSH in human granulosa-like tumour cells (KGN cells). We analysed the expression level of other genes in FSH-stimulated KGN cells. The transcription levels of GSTP and Nrf2 were not induced by FSH. These results suggest that the expression of GSTT1 is not regulated by Nrf2 and is differentially regulated from other GST. The difference in regulation mechanisms between GSTT1 and other GST may result in the up-regulation of GSTT1 in the aging granulosa cells, and it may contribute to the constitution of the diagnosis of oocyte quality using the GSTT1 measurement.