RESUMEN
Trehalose, a naturally nontoxic disaccharide that does not exist in mammals, stabilizes cell membrane integrity under oxidative stress conditions, the mechanism of which is still unclear. Here, we analyzed the effects of trehalose on sheep epididymis epithelial cell (EEC) proliferation and its possible mechanisms. To study the effect of trehalose on EECs, EECs were isolated from testes of 12-month-old sheep; cell counting kit-8 (CCK-8) was used to measure the growth of the cells. Cell proliferation was evaluated by assaying cell cycle and apoptosis, and RT-PCR was utilized to identify the epididymal molecular markers glutathione peroxidase 5 (GPX5) and androgen receptor (AR). Next, reactive oxygen species (ROS) content was evaluated by a dichloro-dihydro-fluorescein diacetate (DCFH-DA) probe. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were evaluated by enzyme chemistry methods, and GPX5 expression was evaluated by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). The results showed that 100⯠mM trehalose significantly improved the proliferation potential of EECs, in which the cells could be serially passaged 14 times with continued normal GPX5 and AR marker gene expression in vitro. The trehalose can increase significantly a proportion of EECs in S phase ( P < 0.01 ) and decrease significantly the apoptotic rate of EECs ( P < 0.01 ) compared to the control. Moreover, the trehalose decreased ROS significantly ( P < 0.01 ) and increased CAT ( P < 0.01 ) and GSH-Px ( P < 0.05 ) activities significantly in EECs. GPX5 mRNA and protein expression were also significantly upregulated in trehalose-treated EECs ( P < 0.05 and P < 0.01 respectively). Our study suggested that exogenous trehalose exhibited antioxidant activity through increasing the activities of CAT, GSH-Px, and the expression level of GPX5 and could be employed to maintain vitality of sheep EECs during long-term in vitro culture.
RESUMEN
Androgens are involved in maintaining epididymal structure and function. In the present study, primary culture of goat EECs and effect of testosterone on expression of glutathione peroxidase-5 (GPX5) in goat epididymal epithelial cells (EECs) were investigated. The EECs isolated from 12-mo-old goat caput epididymis were cultured with testosterone in vitro, and expression of glutathione peroxidase-5 (GPX5) and androgen receptors (ARs) was analyzed. Our results showed that testosterone effectively increased EEC proliferation activity, and EECs cultured with testosterone could maintain molecular markers for up to 12 passages. Compared with the control group, 100 nM testosterone significantly increased the mRNA and protein expression of GPX5 (P < 0.05) and ARs (P < 0.01 and P < 0.05, respectively) in EECs, and this effect was blocked by the AR blocker enzalutamide. In conclusion, testosterone can promote the expression of GPX5 in EECs by up-regulating AR expression. We established an effective culture system for goat EECs which can be for further investigation on the regulation of epithelial function.
