Upstream and downstream mechanisms for the promoting effects of IGF-1 on differentiation of spermatogonia to primary spermatocytes.

AIM: 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.

MAIN METHODS: 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.

KEY FINDINGS: 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.

SIGNIFICANCE: 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.