Quantitative Proteomics Suggests That STIM1 Plays An Important Role In Mouse Testicular Cord Formation

Expression of sex-determining gene Sry begins after primitive germ cells migration alongside the intestinal mesangial,which triggered cascade reactions involving a series of downstream genes,inducing Sertoli cell differentiation,and then caused other cells in testis,such as primitive germ cells,Leydig cells,interstitial cells,and endothelial cells to differentiate.Testis eventually formed with the interaction between germ cells and somatic cells.Male genital ridge dysplasia congenital can lead to male reproductive organs disease such as much testosterone,Muller pipe resistance syndrome,and epididymis and vas deferens absent..Male gonad development is a complex process involving multiple cells to form a functional tissue,however,little is known about its precise molecular mechanism.With the high-throughput quantitative proteomics technology,a comparative proteomic profile of male mouse embryonic male gonads were analyzed at three time points(11.5,12.5 and 13.5 days post coitum),corresponding to critical stages of testicular cord formation in gonadal development.4070 proteins were identified,and 338 were differentially expressed.Identification and function analysis of the differences in protein will help us further understand the male gonads forming mechanism of testicular cord.Bioinformatics methods analysis of differential protein in fetalmouse gonad testicular function,of which the Sertoli cell specific genes were significant enrichment,with mainly increased expression across testis cord development.Additionally,we found overrepresentation of proteins related to oxidative stress in these Sertoli cell specific genes.Of these differentially expressed oxidative stress-associated Sertoli cell specific protein,stromal interaction molecule 1(STIM1),was found to have discrepant mRNA and protein regulations,with increased protein expression but decreased mRNA levels during testicular cord development.The mRNA and protein expression patterns of the same gene can be on the contrary,which hinting post-transcriptional regulation,and thus STIM1 may play an important role in testicular cord formation.We found that STIM1 was located in Sertoli cell cytoplasm.On the basis of results above,we studied the role of STIM1 in male testicular cord development by using Translation-Blocking-Vivo-Morpholino interference Knockdown technology,combining with gonads invitro culture platform.We cultured 12.0 dpc male genital ridge with morpholino for 30 hours in vitro,Western Blot results showed that morpholino-added group had less protein expression,causing severe defects in male gonad development including testicular cord disruption,germ cell and somatic cell morphological and number abNormal,followed with angiogenesis failure.Further study showed that these phenotypes were caused by increased level of reactive oxygen species(ROS)in STIM1 Knockdown tissues,which activate the MAPK and Wnt/?-catenin signaling pathway.Suppressing the aberrant elevation of ROS by NAC could partly rescue the defects of testicular cord development:The numbers of Sertoli cells,Primitive germ cells and Leydig cells were higher in the NAC-treatedKnockdown group than in the Knockdown group.Our results suggested that STIM1 was very important during testicular cord development.Knocking down of STIM1 in Sertoli cells cause reactive oxygen species level increase in genital ridge,which activate MAPK and Wnt/p-catenin signaling pathway,and eventually cause male gonad development defects.Removing reactive oxygen species with NAC treatment can partly rescue the phenotype.Taken together,STIM1 played an important regulating role in testicular cord development.At the same time,quantitative proteomic data could be helpful for us to know the mechanism of testicular cord formation.This work provides a number of potential molecular targets for clinical treatment of early embryonic gonad dysplasia,embryonic death and birth defects and other reproductive disorders.