The Mechanism Of IGF2BP1 In Male Germ Cell Development

Spermatogenesis refers to the process by which primordial germ cells develop into mature sperm through a series of mitosis and meiosis.Studies have shown that abnormal spermatogenesis is a key factor in male infertility such as asthenospermia and oligozoospermia.As the core component of the m6 A methylation modification system,the m6 A methylation reader plays an important role in the regulation of gene expression in germ cell development.Insulin-like growth factor-2 mRNA-binding protein 1(IGF2BP1)is a member of the IGF2 BPs family of m6 A methylation readers.Studies have shown that IGF2BP1-deficient mice exhibit systemic malformations of tissue and organ development,suggesting that it play an important role in organ development.At present,the function of IGF2BP1 in male germ cell development and spermatogenesis has not been reported.This paper explored the effect of IGF2BP1 on male germ cell development,and identified its downstream target genes and analyzed its regulatory mechanism.Firstly,through the analysis of human HPA website and mouse germ cell database(GSE4193)combining with RT-PCR detection of multiple tissues and organs of normal mice,it was confirmed that IGF2BP1 is specifically highly expressed in testis tissue.Using immunohistochemical staining and tissue immunofluorescence techniques,IGF2BP1 was confirmed to be highly expressed in normal adult mouse seminiferous tubules,especially in spermatogonia,and was mainly located in the cytoplasm.In mouse C18-4 and GC-1spermatogonial cell lines with IGF2BP1 knock down,and the changes of cell proliferation and stemness ability were detected by CCK8,EDU,clone formation,and cell spheroidization experiments.The results indicated knock down of IGF2BP1 inhibits the proliferation and stemness of spermatogonia in vitro cell model.Western blot analysis further confirmed that after interfering with IGF2BP1,the expression of proliferation and stemness marker in C18-4and GC-1 cells decreased,indicating that IGF2BP1 promoted the proliferation and stemness of spermatogonia.Subsequently,using RIP-seq database information and GO database analysis,seven possible target molecules of IGF2BP1 in regulating cell proliferation and stemness were preliminarily screened: CDC20,CDK1,CDK2,CDK6,P16,CUL3,FXBW7.Among them,RT-q PCR and WB detection showed that the expression of cell-division cycle protein 20(CDC20)was most significantly down-regulated after interfering with IGF2BP1.RIP-q PCR experiments using mouse testis tissue showed that IGF2BP1 specifically binds to CDC20 mRNA.Then,C18-4 and GC-1 cells with RNA interfernce of IGF2BP1 were treated with actinomycin D,and RT-q PCR detection showed that knockdown of IGF2BP1 promoted the degradation of CDC20 mRNA.Functional rescue experiments by CCK8,EDU and cell spheroidization were performed after IGF2BP1 interference,CDC20 interference,and IGF2BP1/CDC20 double interference in spermatogonial cell lines were performed,and the results confirmed that IGF2BP1 promoted spermatogonia proliferation and stemness by targeting up-regulation of CDC20 expression.Finally,clinical samples of asthenozoospermia were collected and RT-q PCR technology was used to detect the expression of IGF2BP1 and CDC20.It was found that both IGF2BP1 and CDC20 were down-regulated in asthenospermia.In addition,the interacting proteins of IGF2BP1 in spermatogonia were screened by co-immunoprecipitation technology combined with mass spectrometry analysis in this study.It was found that DEAD-Box Helicase 5(DDX5)and IGF2BP1 can bind to each other,suggesting DDX5 may be involved in the regulation of CDC20 by IGF2BP1,which needs to be further verified in the future.In conclusion,IGF2BP1 promotes the CDC20 gene expression by increasing the mRNA stability of CDC20,thereby enhancing the proliferation and stemness ability of spermatogonial cells,and involving spermatogenesis.