Role Of Rpt4R In Male Fertility Of Drosophila Melanogaster

The ubiquitin-proteasome pathway(UPP)is involved in the removal of abnormal proteins,which is the most important protein removal system in organisms.Ubiquitination refers to the process of ubiquitin-specific modification and target classification of proteins by the action of specific enzymes(ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2,ubiquitin ligase E3,etc.)in cells.The 26s proteasome pathway is responsible for recognizing and degrading ubiquitinated proteins,and comprises two multisubunit subcomplexes as follows:20s proteasome and 19s regulatory particle.Therefore,UPP plays an important role in regulating the localization and function of protein in vivo.It is investigated that UPP plays an important role in the degradation of proteins and organelles during spermatogenesis.In Drosophila,about a third of the 32 proteasome subunits are found to have testis-specific isoforms,encoded by paralogous genes.In every case,one form of each subunit is widely expressed at all developmental stages and in all tissues(these will be referred to as conventional proteasome subunits),whereas all of the additional isoforms are testis specific.Here,we characterize in detail the spermatogenic expression of the regulatory particle subunit Rpt4R.Rpt4,representing the conventional counterpart of Rpt4R,belongs to one of the AAA+superfamily ATPase subunits in the 19s regulatory subunit.While there might be a testis-specific proteasome subunit,it is dynamically assembled during spermatogenesis.What’s more,It is reported that the testis-specific proteasomes might not be qualitatively that much different in their function from that of the conventional somatic proteasomes,although it is possible that they have a subtly distinct function,or that they are more efficient at mediating rapid protein degradation during spermiogenesis.Wolbachia is an endosymbiotic bacterium that induces a wide range of effects in its insect hosts including cytoplasmic incompatibility(CI),which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females,or with different strains Wolbachia-infected females.The previous results in our lab shows Wolbachia can affect the expression of certain genes in the host,resulting in defects in the process of spermatogenesis in Drosophila.Studies have reported that protein ubiquitination is increased in Wolbachia-infected cells.Therefore,we first performed western blot experiments and found that compared with uninfected Drosophila melanogaster(Dmel T)testes,the protein ubiquitination of Wolbachia infected(Dmel wMel)testis increased.We analyzed this increased protein ubiquitination through mass spectrometry,which revealed peptides including Rpt4R of the 19S regulatory particle.Through QRT-PCR found that Rpt4R expression was significantly increased(p<0.05)in 1-day-old Dmel wMel testis compared to non-infected Drosophila melanogaste.Since Wolbachia can modify male sperm and disrupt male fertility,we speculated that Rpt4R may be associated with male fertilization.In order to verify whether the upregulation of Rpt4R by infection with Wolbachia was related to Wolbachia-induced CI,knock down of Rpt4R in the Wolbachia-infected Drosophila testis[bamGal4;Rpt4R-hp(Ⅰ)],then which mating with normal female flies and estimate the hatching rate of the female embryos.The results showed that the hatching rate from 44.42±3.30 recovered to 61.47%,indicating that Wolbachia infection resulted in up-regulation of Rpt4R and ubiquitination of Rpt4R may be one of the reasons that Wolbachia infection causes insect host to produce CI.In order to study the role of Rpt4R in the reproductive process of Drosophila,Specifically knock down of Rpt4R in Wolbachia-free fly testis and detecet the hatching rate of the embryos of the offspring in knock down the male fly.After knock down of Rpt4R,the hatching rate of the offspring and egg production were extremely significant decreased(p<0.01).In order to further study the mechanism of Rpt4R affecting the fertility of male flies,we used immunofluorescence staining technique to analyze the spermatogenesis in Rpt4R knock-down testes.We stained nuclei with DAPI,in the Rpt4R knock down testis,the tip of the testes produces 1-several unusually large gonadal tumor-like cysts and the abnormal cell nuclei prolonged,the sperm bundles at the base of testis are scattered.The seminal vesicle had large number of mature sperm in the control group,however the sperm in seminal vesicle of knock down group is only the 25%of control group.Due to the testis-specific subunit Rpt4R was none expressed somatically or during the early gonial stages of spermatogenesis,was prominently expressed in the cytoplasm and nuclei of 64-cell spermatids and elongating stage cysts.Therefore,Vasa and FasⅢ antibodies were used for immunofluorescence staining.The results showed that the expression of Hub cells was normal and the number of germ cells was lower than that of the control group,especially there was no signal of Vasa antibody at the location of tumor-like cyst,indicating that it was not a germ cell.Because the phenotype of Rpt4R knock down was similar to that in these genes prosa6T,DuBa and poe mutants,we used qRT-PCR to test the expression of these genes in the Rpt4R knock down flies,the results showed that Rpt4R knock down in the testis of flies caused the expression of DuBa and poe significantly decreased(p<0.05)and prosa6T extremely significant increased(p<0.01)in the testis.A preliminary judgment of the knockdown of Rpt4R affects the expression of these genes,which may further affect the spermatogenesis of Drosophila.In summary,Rpt4R plays an important regulatory role in the spermatogenesis process,and affects the fertility of male flies.