LAUSR

Thiazolidinedione (TZD) is an oral anti-diabetic drug that exhibits some side effects on the male reproductive system by interfering with the steroidogenesis and androgenic activity and also shows anti-proliferative effect on several cell types. This study investigated the effect of TZD on immature chicken Sertoli cell (SC) proliferation and the potential mechanism by which 17β-estradiol regulated this process. Chicken SC viability was investigated under different treatment concentration and time of TZD. 17β-estradiol (0.001 μM, 24 h) was added to analyze its effects on TZD-mediated cell viability, cell metabolic activity, cell growth, cell cycle progression, reactive oxygen species (ROS) level, antioxidant enzyme activity, mitochondria activity, oxygen consumption rate, adenosine triphosphate (ATP) level, and mitochondrial respiratory chain enzyme activity, adiponectin expression and several cell proliferation-related genes mRNA and protein levels. We performed the microRNA (miRNA) array to find TZD-induced differentially expressed miRNAs and validated whether miR-1577 can target on adiponectin via the dual luciferase reporter assay, as well as verified the effect of adiponectin addition with different concentrations on the SC viability. Further, SCs were transfected with miR-1577 agomir (a double-stranded synthetic miRNA mimic) in the presence or absence of TZD and antagomir (a single-stranded synthetic miRNA inhibitor) in the presence or absence of 17β-estradiol to analyze whether miR-1577 was involved in TZD-mediated SC proliferation and whether 17β-estradiol regulated this process. Results showed that TZD significantly inhibited SC viability, cell metabolic activity, cell growth, and cell cycle progression, while increased adiponectin level and ROS generation. TZD-treated SCs presented decreases of antioxidant enzyme activity, mitochondria activity, basal and maximal respiration, ATP production and level, mitochondrial respiratory chain enzyme activity, and mRNA and protein expressions of several cell proliferation-related genes, as well as the significant alteration of miRNA expressions (a total number of 55 miRNAs were up-regulated whereas 53 miRNAs down-regulated). Whereas, 17β-estradiol played a positive role in chicken SC proliferation and rescued the damage of TZD on SC proliferation by up-regulating miR-1577 expression whose target gene was validated to be the adiponectin. In addition, exogenous adiponectin (more than 1 μg/ml) treatment exhibited a significant inhibition on the SC viability. Transfection of miR-1577 agomir promoted the SC proliferation via down-expressed adiponectin, and increased the mitochondrial function and cell proliferation-related gene expression, while TZD weakened the positive effect of miR-1577 agomir on SCs. On the other hand, transfection of miR-1577 antagomir inhibited SC proliferation by producing the opposite effects on above parameters, while 17β-estradiol attenuated the negative effect of miR-1577 antagomir on SCs. These findings suggest down-expressed miR-1577 is involved in the regulation of TZD-inhibited SC proliferation through increasing adiponectin level, and this damage of TZD on the immature chicken SC proliferation can be ameliorated by appropriate dose of exogenous 17β-estradiol treatment. This study provides an insight into the cytoprotective effect of 17β-estradiol on TZD-damaged SC proliferation and may suggest a potential strategy for reducing the risk of SC dysfunction caused by the abuse of TZD.