title: Upstream and downstream mechanisms for the promoting effects of IGF-1 on differentiation of spermatogonia to primary spermatocytes.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
authors.forename: Guoqiu
authors.initials: G
authors.lastname: Shen
authors.affiliation: Department of Urology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Rongpei
authors.initials: R
authors.lastname: Wu
authors.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
authors.forename: Wenhan
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Tu
authors.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China.
authors.forename: Jiarong
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Pediatric Surgery, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Urology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei 430070, China. Electronic address: pantj-wgh@sohu.com.
authors.forename: Tiejun
authors.initials: T
authors.lastname: Pan
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D055785:Gene Knockdown Techniques; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis / Q000235:genetics; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis; D015398:Signal Transduction / Q000502:physiology; D013091:Spermatogenesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
chemical_list: D002070:Butadienes; D002867:Chromones; D007334:Insulin-Like Growth Factor I; D035683:MicroRNAs; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017526:Receptor, IGF Type 1; C113580:U 0126; C542687:mirnlet7 microRNA, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
keywords: Differentiation; ERK1/2; IGF1 receptor (IGF1R); Insulin growth factor I (IGF1); Let-7; PI3K; Primary spermatocytes; Spermatogenesis; Spermatogonia