Guoqiu Shen1, Rongpei Wu2, Bo Liu1, Wenhan Dong1, Zhong Tu1, Jiarong Yang1, Zhe Xu3, Tiejun Pan4. 1. Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China. 2. Department of Urology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China. 3. Department of Pediatric Surgery, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China. 4. Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China. Electronic address: pantj-wgh@sohu.com.
Abstract
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.
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.
Authors: K O Skaftnesmo; R B Edvardsen; T Furmanek; D Crespo; E Andersson; L Kleppe; G L Taranger; J Bogerd; R W Schulz; A Wargelius Journal: BMC Genomics Date: 2017-10-18 Impact factor: 3.969
Authors: Kristina B V Døssing; Christina Kjær; Jonas Vikeså; Tina Binderup; Ulrich Knigge; Michael D Culler; Andreas Kjær; Birgitte Federspiel; Lennart Friis-Hansen Journal: Genes (Basel) Date: 2018-07-04 Impact factor: 4.096