17ß-estradiol rescues the damage of thiazolidinedione on chicken Sertoli cell proliferation via adiponectin.

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 17ß-estradiol regulated this process. Chicken SC viability was investigated under different treatment concentration and time of TZD. 17ß-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 17ß-estradiol to analyze whether miR-1577 was involved in TZD-mediated SC proliferation and whether 17ß-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, 17ß-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 17ß-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 17ß-estradiol treatment. This study provides an insight into the cytoprotective effect of 17ß-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.

Estradiol ameliorates metformin-inhibited Sertoli cell proliferation via AMPK/TSC2/mTOR signaling pathway.

Metformin is a commonly used for treating type 2 diabetes and it acts on a variety of organs including the male reproductive system. 17ß-estradiol plays an important role in Sertoli cell (SC) proliferation which determines the germ cell development and spermatogenesis. The aim of this study is to investigate the effect of metformin on immature chicken SC proliferation and the potential mechanisms by which 17ß-estradiol regulate this process. Results showed that metformin significantly inhibited SC proliferation, whereas 17ß-estradiol weakened the inhibitory effects of metformin on SC viability, cell growth, and cell cycle progression. SC proliferation-inhibiting effect of metformin exposure was regulated by decreasing adenosine triphosphate level and respiratory enzyme activity in the mitochondria; this process was possibly mediated by the adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) signaling pathway, which was regulated by the down-expressed miR-1764 and by the decreased antioxidant enzyme activity and excessive reactive oxygen species generation. In addition, SCs transfected with the miR-1764 agomir led to an improvement of proliferation capacity through down-regulating AMPKα2 level, which further decreased TSC2 expression and induced mTOR activation. However, the anti-proliferative effect of miR-1764 antagomir can be alleviated by 17ß-estradiol treatment via the up-expression of miR-1764 in transfected SCs. Our findings suggest appropriate dose of exogenous 17ß-estradiol treatment can ameliorate the inhibitory effect of metformin on SC proliferation via the regulation of AMPK/TSC2/mTOR signaling pathway, this might reduce the risk of poor male fertility caused by the abuse of anti-diabetic agents.

Identification and function prediction of novel microRNAs in adenosine monophosphate activated protein kinase-activated Sertoli cells of immature boar.

This study was carried out with the objective to identify function prediction of novel microRNAs (miRNAs) in immature boar Sertoli cells (SCs) treated with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), which is an agonist of adenosine monophosphate-activated protein kinase (AMPK) for regulating cellular energy homeostasis. Two small RNA libraries (control and AICAR treatment) prepared from immature boar SCs were constructed and sequenced by the Illumina small RNA deep sequencing. We identified 77 novel miRNAs and predicted 177 potential target genes for 26 differential novel miRNAs (four miRNAs up-regulation and 22 miRNAs down-regulation) in AICAR-treated SCs. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway suggested that target genes of differential novel miRNAs were implicated in many biological processes and metabolic pathways. Our findings provided useful information for the functional regulation of novel miRNAs and target mRNAs on AMPK-activated immature boar SCs.