The Function And Mechanism Of CG8005 In Mediating Oxidative Stress In The Microenvironment Of Drosophila Testes Germ Stem Cells

Objective:Drosophila testis germline stem cells(GSCs)are the source of sperm production.Their self-renewal and differentiation processes are strictly regulated by many factors in the microenvironment of stem cells,which is essential for the occurrence of male infertility and germ cell tumors.Reactive oxygen free radicals(ROS)are widely present in the body’s metabolic reactions,which can participate in the proliferation,differentiation and apoptosis of stem cells and affect stem cell behavior.The Kelch-like epichlorohydrin-related protein-1(Keap1)-nuclear factor E2-related factor 2(Nrf2)-antioxidant response element(ARE)signaling pathway is a key pathway in the cellular oxidative stress response,and is a research hotspot in the field of anti-oxidative stress in recent years.CG8005 is one of the regulatory factors screened from the microenvironmental regulatory network of Drosophila testicular reproductive stem cells.Its regulatory mechanism is still unclear.This topic mainly focuses on the research and discussion of the function and mechanism of CG8005 in the microenvironment of Drosophila testis germ stem cells.Methods:(1)The RNAi pathway mediated by UAS/Gal4 allows Nos-gal4,Tj-gal4 and Bam-gal4 to be expressed in the microenvironment of stem cells,thereby mediating the detection of CG8005 staining markers and analyzing the function of CG8005 gene in Drosophila testis.(2)Using the principle of small interfering RNA(si RNA)in Drosophila S2 cells to silence CG8005 gene expression,the growth of Drosophila S2 cells was detected by CCK-8 assay,cell proliferation was detected by Phospho-Histone H3(PH3)immunofluorescence staining.In order to preliminary explore the function of CG8005 in vitro.(3)The interference principle of si RNA was used to reduce the expression of CG8005 in Drosophila S2 cells.The survival of Drosophila S2 cells were observed by flow cytometry apoptosis detection technology.The cell apoptosis was analyzed by TUNEL immunofluorescence.(4)CG8005 was konckdown in Drosophila testis by UAS / Gal4 system,and the superoxide anion fluorescent probe DHE was used to detect the production of ROS in the testis of Drosophila.(5)According to the detection principle of the peroxide-sensitive fluorescent probe DCFH-DA and the superoxide anion fluorescent probe DHE,the immunofluorescence staining method was used to compare the production of ROS in Drosophila S2 cells between the control group and the si CG8005 group..(6)Detect the pro-oxidation factors and antioxidant factors that are highly related to CG8005 by real-time fluorescent quantitative PCR.(7)Using the Drosophila UAS/Gal4 system,the cell-specific knockdown of the antioxidant factor(cnc)identified in q RT-PCR was performed.In order to further explore the regulation mechanism of CG8005 in the microenvironment of Drosophila testicular germ cells.The immunofluorescence staining and ROS detection were used to analyze whether the cnc has a similar phenotype to CG8005.(8)Detect the m RNA level of a batch of spliceosome subunits with real-time fluorescent quantitative PCR.Results:(1)The results of laser confocal microscopy showed that the microenvironment of the germline stem cells at the top of the testis of Drosophila has changed.Using Nos-gal4 to knock down CG8005 in early germ cells,a small testicular phenotype appeared and germ cells were completely absent.The use of Tj-gal4 to knock down CG8005 in encapsulated cells resulted in undifferentiated germ cell clumps and eventually developed into germ cell tumors.And Bam-gal4 knocks down CG8005 in spermatogonia,causing germ cells to accumulate,and the differentiation process is hindered to affect spermatogenesis.(2)Knockdown of CG8005 in Drosophila S2 cells resulted in weakened cell proliferation and inhibited growth.(3)Knockdown of CG8005 in Drosophila S2 cells leaded to increased levels of apoptosis and cell survival disorded.(4)Tj-gal4 and Bam-gal4 knock down CG8005 in cyst cells and spermatogonia,respectively,resulted in ROS levels increased.(5)CG8005 knockdown significantly increased the level of ROS in Drosophila S2 cells.(6)In the absence of CG8005 in Drosophila S2 cells,the level of antioxidant factor m RNA was up-regulated,and the level of pro-oxidant factor m RNA was down-regulated.(7)The self-renewal and differentiation caused by cnc RNAi are blocked,just like the phenotype of CG8005 gene missing in the microenvironment of Drosophila testicular germ stem cells,and knocking down cnc also inhibited the proliferation of Drosophila S2 cells and promoted the apoptosis of Drosophila S2 cells.(8)Knock down the CG8005 gene in Drosophila S2 cells,and the m RNA levels of a batch of spliceosome subunits are up-regulated.Conclusion:The in vivo experiments of Drosophila show that the CG8005 for the self-renewal and differentiation of germ cells in Drosophila testes.Drosophila in vitro experiments show that CG8005 participates in the steady-state regulation of Drosophila S2 cell proliferation and apoptosis,causing a significant down-regulation of the antioxidant factor cnc,and it can also competitively regulate the m RNA level of spliceosome subunits.Similarly,cnc completely mimics the self-renewal and differentiation of germ cells,proliferation and apoptosis of Drosophila S2 cells in vivo and in vitro.It is suggested that CG8005 may play an important role in the self-renewal and differentiation process of Drosophila testis germ cells.And regulate spermatogenesis,and its expression is mediated by oxidative stress regulated by cnc activity.The study proved the role of CG8005 to provide new insights into the pathogenesis of male non-obstructive azoospermia and germ cell tumors.Down-regulation of CG8005 and cnc may be involved in the pathogenesis of male infertility and germ cell tumors.Therefore,these two factors may be effective for treatment goals.