The Mechanism Of RpS13 Regulating Germline Stem Cells Self-renewal And Differentiation In Drosophila Testes

Objective:Germ cell tumors can be accompanied by spermatogenesis disorder,which is an important cause of male infertility.Abnormal function of germline stem cells（GSCs）can lead to germ cell tumors.The potential self-renewal and differentiation of GSCs are regulated by their intrinsic signals and stem cell microenvironment.For researchers,Drosophila testis is an ideal model to study GSC.Ribosomal small subunit protein RpS13 has been identified as an important factor in regulating GSCs self-renewal and differentiation in Drosophila testes.However,its specific regulatory mechanism in germline stem cell niche is not well understood.The purpose of this study was to explore and discuss the mechanism of RpS13,which regulates the self-renewal and differentiation of germline stem cells in Drosophila testes.Methods:（1）Using RNAi interference silencing method mediated by UAS/Gal4 system,the specific loss of RpS13 function（Nos>RpS13 RNAi,Tj>RpS13 RNAi and Bam >RpS13 RNAi）was induced in early germ cells（Nos-Gal4）,cyst cells（Tj-Gal4）and spermatogonia（Bam-Gal4）of Drosophila testes.The fertility of male Drosophila melanogaster was tested to observe whether RpS13 affected male fertility.（2）Immunofluorescence staining was performed with antibodies Vasa,1B1,Eya,Zfh1 and Fas III,which labeled different cell types in testis,and PhosphoHistone H3（PH3）labeled proliferating cells in WT wild-type Drosophila testes（control group）and RpS13 RNAi Drosophila testes（experimental group）to observe the effect of RpS13 on GSCs self-renewal and differentiation in Drosophila testes.（3）Small interfering RNA（siRNA）was utilized to silence RpS13 gene expression in Drosophila embryonic stem cells（S2 cells）.Quantitative real-time PCR was used to detect the expression of RpS13 m RNA in negative control group（NC）and experimental group（siRpS13-16 and siRpS13-234）to verify the efficiency of interference.The cell proliferation was detected by PH3 immunofluorescence staining,and cell apoptosis was detected by TUNEL staining and flow cytometry.（4）The expression of Rho1 in germ cells,somatic cells and spermatogonia was detected by immunofluorescence staining of UAS/Gal4 system and marker protein in Drosophila melanogaster.The testicular phenotype was similar to that of RpS13 RNAi（changes of fertility and testicular function in male Drosophila）.（5）QRT-PCR was used to detect the expression of Rho1 m RNA in S2 cells of negative control group（NC）and experimental group（siRho1-457 and siRho1-374）to verify the interference efficiency.The proliferation and apoptosis of S2 cells were respectively detected by PH3 immunofluorescence staining,TUNEL staining and flow cytometry to observe whether Rho1 regulates the growth of S2 cells and whether its function in vitro is similar to that of RpS13.（6）The expression patterns of adhesion proteins Rho1,DE-cad and Arm were detected by immunofluorescence staining in germline cells and somatic cells of RpS13 RNAi Drosophila.At the same time,immunofluorescence staining was used to detect whether the expression patterns of DE-cad and Arm in Rho1 RNAi Drosophila mimicked those of RpS13 RNAi in testis.（7）QRT-PCR was used to detect the m RNA expression levels of large ribosomal subunits（Rp L6,Rp L14,Rp L19,Rp L22,Rp L27 and Rp L30）and small ribosomal subunits（RpS2,RpS8,RpS9,RpS14 a and RpS16）in RpS13 and Rho1 silenced S2 cells.Results:（1）The results of fertility test showed that the fertility of male Drosophila with Nos>RpS13 RNAi,Tj>RpS13 RNAi and Bam>RpS13 RNAi was lower than that of WT Drosophila.（2）Immunofluorescence staining and laser confocal microscopy showed that on the one hand,knockdown of RpS13 in early germ cells resulted in complete loss of germ cells and abnormal proliferation of somatic cells,along with tiny testicular morphology;On the other hand,RpS13 deficiency in somatic cells leads to an accumulation of undifferentiated germ cells and the differentiation of cyst cells were blocked,and the formation of germ cell like tumor;What’s more,RpS13 deficiency in spermatogonia caused spermatogonia differentiation defects.（3）The deletion of RpS13 increased the proliferation and apoptosis of Drosophila S2 cells;（4）The results of immunofluorescence staining and confocal laser microscopy showed that Rho1 RNAi mimicked the phenotype of RpS13 RNAi in Drosophila testes leading to the dysfunction of GSCs self-renewal and differentiation.（5）Knocking down of Rho1 in Drosophila S2 cells also led to the increase of cell proliferation and apoptosis,which fully simulated the function of RpS13 in regulating the growth of S2 cells.（6）Immunofluorescence staining results showed that the functional defect of RpS13 in Drosophila testes led the expression level of adhesion proteins Rho1,DE-cad and Arm increased;at the same time,the expression level of Rho1 in Drosophila testes was decreased in Nos>Rho1 RNAi and Tj>Rho1 RNAi,and the expression levels of DE-cad and Arm were ectopic accumulation.（7）QRT-PCR analysis showed that RpS13 and Rho1 could competitively regulate the m RNA expression of important ribosomal subunits.Conclusions:Herein,using a genetic manipulation approach,we investigated the role of ribosomal protein S13（RpS13）in testes and S2 cells.We reported that RpS13 was required for GSCs self-renewal and differentiation,and further affected the differentiation of spermatogonia.We also demonstrated that RpS13 participated in the regulation of proliferation and apoptosis of Drosophila S2 cells.Mechanistically,we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression patterns of the Rho1,DE-cad and Arm proteins.Notably,Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells.These results suggest that RpS13 and Rho1 compete with each other and damage cell adhesions,which was mediated by the DE-cad and Arm proteins,to disorder cell adhesion signal,thus leading to the defects of stem cell niche in Drosophila testes.These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis,which may provide new insights into the regulatory network of stem cell niche and the pathogenesis of testicular germ cell tumors.