Trehalose can effectively protect sheep epididymis epithelial cells from oxidative stress.

Zhaojin Luan,Yongchao Zhao,Jiaoxia Xu,Jiaxin Zhang,Xiaomei Fan,Wei Du,Zhaochen Wang,Wenguang Zhang,Huizi Song

doi:10.5194/aab-64-335-2021

Zhaojin Luan, Yongchao Zhao + Show 7 more

Open Access

https://doi.org/10.5194/aab-64-335-2021

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Abstract

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 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 () and decrease significantly the apoptotic rate of EECs () compared to the control. Moreover, the trehalose decreased ROS significantly () and increased CAT () and GSH-Px () activities significantly in EECs. GPX5 mRNA and protein expression were also significantly upregulated in trehalose-treated EECs ( and 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.

Highlights

The epididymis is an important male reproductive organ in mammals for sperm maturation (Qu et al, 2014)
The final concentrations of 0, 50, 100, and 200 mM trehalose were used for growth curve detection, and 0 and 100 mM trehalose were used for cell cycle, apoptosis, reactive oxygen species (ROS) content, enzyme activity, and glutathione peroxidase 5 (GPX5) expression detection, respectively
Immunofluorescence showed that isolated epithelial cell (EEC) could be reactive with cytokeratin-18-specific antibodies, whereas fibroblasts as a control did not express keratin 18 (Fig. 2)

Summary

Introduction

The epididymis is an important male reproductive organ in mammals for sperm maturation (Qu et al, 2014). The caput of the epididymis is crucial for the early and late processes of spermatozoa maturation (Leir et al, 2015). Sperm mature and acquire motility as they interact with proteins synthesized and secreted by epididymal epithelial cells (EECs) during passage through the epididymal lumen (Cornwall, 2009). Primary cells can help elucidate the molecular mechanisms of epididymis function in vitro (Leir et al, 2015). The expression patterns of many functional genes in epididymis are species-specific (Jalkanen et al, 2006; Thimon et al, 2007), which makes it necessary to establish different animal primary EEC lines

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