Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
Department of Urology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.
Life Sci. 2014 Apr 17;101(1-2):49-55. doi: 10.1016/j.lfs.2014.02.016. Epub 2014 Feb 26.
The aim of this study is to delineate the mechanisms for the promoting the effects of insulin growth factor I (IGF1) on the differentiation of spermatogonia into primary spermatocytes.
We used organ culture of testicular fragments from mice to observe the effects of varying agents and siRNAs. Real-time RT-PCR and immunoblotting analysis were employed to quantify expression of genes. Luciferase reporter gene assay was employed for verifying the targeting relationship between miRNA and protein-coding genes.
During spermatogenesis while spermatozoa pass through epididymis and vas deferens for maturation, expression of IGF1 and its receptor IGF1R, p44/ERK1 and p42/ERK2, and PI3K was all upregulated, whereas let-7 miRNA family members let-7a/b/d/e/g/ were downregulated. We established both IGF1 and IGF1R as cognate target genes for let-7; downregulation of let-7 resulted in upregulation of IGF1 and IGF1R during the early stage of differentiation from spermatogonia to primary spermatocytes. Transfection of let-7 inhibited, whereas transfection of anti-let-7 inhibitor enhanced the differentiation. The promoting effect of anti-let-7 was eliminated by PPP to block IGF1R phosphorylation. IGF1 activated ERK1/2 and PI3K and induced differentiation. PPP eliminated the activation of ERK1/2 and PI3K and inhibited the differentiation induced by IGF1. Specific inhibition of ERK1/2 by U0126 or PI3K by LY294002 reduced the IGF1-induced differentiation. Knockdown of ERK1/ERK2 or PI3K by siRNAs also blocked IGF1-induced spermatogenesis.
Our study therefore identified downregulation of let-7 as an upstream mechanism for IGF1/IGF1R upregulation and activation of ERK1/2 and PI3K as a downstream mechanism mediating the IGF1 signaling cascade promoting differentiation of spermatogonia to primary spermatocytes.
本研究旨在阐明胰岛素样生长因子 I（IGF1）促进精原细胞向初级精母细胞分化的作用机制。
我们使用从小鼠睾丸组织中分离出来的器官培养物来观察不同试剂和 siRNA 的作用。采用实时 RT-PCR 和免疫印迹分析来定量基因的表达。采用荧光素酶报告基因实验来验证 miRNA 与蛋白编码基因之间的靶向关系。
在精子发生过程中，当精子通过附睾和输精管进行成熟时，IGF1 及其受体 IGF1R、p44/ERK1 和 p42/ERK2 以及 PI3K 的表达均上调，而 let-7 miRNA 家族成员 let-7a/b/d/e/g/则下调。我们将 IGF1 和 IGF1R 确立为 let-7 的同源靶基因；let-7 的下调导致精原细胞向初级精母细胞分化的早期 IGF1 和 IGF1R 的上调。let-7 的转染抑制了分化，而抗 let-7 抑制剂的转染增强了分化。抗 let-7 抑制剂的促进作用被 PPP 阻断 IGF1R 磷酸化所消除。IGF1 激活 ERK1/2 和 PI3K 并诱导分化。PPP 消除了 IGF1 诱导的分化所激活的 ERK1/2 和 PI3K。U0126 特异性抑制 ERK1/2 或 LY294002 特异性抑制 PI3K 减少了 IGF1 诱导的分化。ERK1/ERK2 或 PI3K 的 siRNA 特异性敲低也阻断了 IGF1 诱导的精子发生。
因此，我们的研究确定了 let-7 的下调作为 IGF1/IGF1R 上调的上游机制，以及 ERK1/2 和 PI3K 的激活作为介导 IGF1 信号级联促进精原细胞向初级精母细胞分化的下游机制。
