Cellular Nucleic Acid-binding Protein Is Vital To Testis Development And Spermatogenesis In Mice

The cellular nucleic acid-binding protein(CNBP),also known as zinc finger protein 9(ZNF9),is a highly conserved zinc finger protein that is strikingly conserved among vertebrates.It binds DNA or RNA in a sequence-specific manner.It contains seven tandem zinc finger repeats that are composed of 14 same amino acids of Cys-X2-Cys-X4-His-X4-Cys.So far,CNBP cDNAs have been cloned from human,mouse,rat,chicken,Xenopus,Bufo arenarum,Danio rerio and gibel carp.Data collected from low vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis.However,the location and function of CNBP in mammalian testes is not well known.In order to clarify the location and functional mechanisms of CNBP in mammalian testis and spermatogenesis,we used the mature SSCs in vitro culture platform and the in vitro culture platform of spermatogenic tubules to study the effect of CNBP knockdown on neonatal testicular development and spermatogenesis.Firstly,combined with Qrt-PCR and Western Blot techniques,it was found that CNBP expression was higher in the testes of day 0 and day 7 and decreased significantly from day 14 and the CNBP expression was very low in testes of adult mice.Immunofluorescence staining revealed that CNBP was located in germ cells and sertoli cells in mouse testis.This result suggests that CNBP is required for neonatal testicular development in mice.Further we use morpholino to knockdown CNBP in neonatal mouse testis and SSCs cultured in vitro.The results of H&E staining and immunofluorescence staining showed that the disruption of seminiferous tubules,mislocation of Sertoli cells,loss of germ cells,and expansion of Leydig cells.In addition,it was observed in this study that the knockdown of CNBP caused abnormal activation of the WNT/β-catenin pathway in the testis,and that intervention with the inhibitor XAV939 of this pathway could partly rescue these phenotypes.In addition,SSCs grew normally when morpholino was used to inhibit the expression of CNBP in cultured SSCs.In conclusion,our study suggests that CNBP could maintain testicular development by inhibiting the WNT/β-catenin pathway of sertoli cells.