Function And Mechanism Of CG6015 In Germline Stem Cell Differentiation Of Drosophila Testes

Objective: This study aims to explore the function of somatic CG6015 in Germline Stem Cells(GSCs)microenvironment and clarify the regulatory mechanism of CG6015 mediated GSCs differentiation in somatic cyst cells,providing new clues for elucidating the mechanism of male non-obstructive azoospermia(NOA).Methods: 1.The UAS-GAL4 system was used to achieve cell-specific knock-down in Drosophila testes.And the morphological analysis of testes was analyzed by immunostaining.2.Small interferingRNA(siRNA)was utilized to downregulate the expression level of CG6015,Downstream of Raf1(Dsor1)and rolled(rl)in Drosophila S2 cells.The relative mRNA expression level of CG6015,Dsor1 and rl was detected by quantitative Real-time Polymerase Chain Reaction(qRT-PCR)separately.Cell Counting Kit 8(CCK-8)test was conducted to analyze cell growth curve.Cell proliferation was determined by Phospho-Histone H3(PH3)staining.TUNEL staining was performed to detect cell apoptosis,and flow cytometry was carried out to evaluate the proportion of viable cells and apoptotic cells.Results:1.Immunofluorescence staining results showed that after knockdown of somatic CG6015,the normal morphology of the testes disappeared completely,instead showed an abnormal phenotype of smaller testes in comparison with the control testes.Further analysis displayed that Somatic Cyst Stem Cells(CySCs)and mature somatic cyst cells were undetected in Tj>CG6015RNAi testes while a large number of undifferentiated germ cells accumulated into cell masses,suggesting that CG6015 affects the maintenance of Cy SCs and somatic cyst and the differentiation of germ cells.Moreover,knockdown of CG6015 led to dysregulated proliferation and apoptosis of germ cells as well as the abnormal expression of double phospho-extracellular regulated protein kinase(dpERK)in germ cell masses,indicating the role of CG6015 in regulating GSCs differentiation was related to Epidermal Growth Factor Receptor(EGFR)signaling pathway.2.After knockdown of CG6015 in Drosophila S2 cells,q RT-PCR revealed that the relative mRNA level of Prp submit complex and Sm submit complex were upregulated.CCK-8 test suggested that the cell growth of si CG6015-1331 group was lower than that of NC group.PH3 staining displayed that the proportion of PH3 positive cells in the si CG6015-1331 group was reduced compared with NC group.While TUNEL staining showed that,the percentage of TUNEL positive cells in the si CG6015-1331 group was increased in comparison with NC group.The results of flow cytometry revealed that the proportion of apoptotic cells in the si CG6015-1331 group was higher than that in the NC group.It is indicated that CG6015 regulates the transcriptional level ofRNA spliceosome subunits in S2 cells,as well as the function of proliferation and apoptosis of S2 cells.3.It is uncovered that Dsor1 mimics the role of CG6015 in somatic cyst cells by immunofluorescence staining.Downregulation of Dsor1 leads to complete loss of Cy SCs and somatic cyst cells with disrupted germline differentiation,mediating smaller testes.Knockdown of somaticrl also resulted in differentiation defects of germ cells in Drosophila testes.Moreover,the normal expression pattern of dpERK was also destroyed by down-regulating Dosr1 and rl in somatic cyst cells.These results suggested that Dsor1 and rl may be involved in the regulation of GSCs differentiation mediated by CG6015.4.Knockdown of Dsor1 and rl in Drosophila S2 cells led to decreased cell growth and the proportion of PH3 positive cells obviously.While the percentage of TUNEL positive cells was significantly increased,as well as the proportion of apoptotic cells detected by flow cytometry.Conclusion: CG6015 regulates CySCs maintenance and GSCs differentiation through EGFR signaling pathway,and inhibits the abnormal activation of dpERK signals through somatic cyst cells.This study provides a new idea for investigating the regulatory mechanism of the stem cell niche homeostasis of Drosophila testis.