Effect Of Shrb Gene On The Male Fertility Of Drosophila Melanogaster

shrub(shrb,CG8055)encodes the vps32/snf7 protein,a filament-forming subunit of the ESCRT(endosomal sorting complexes required for transport)-III complex involved in inward membrane budding.It regulates multiple cellular processes involving ESCRTs activity including multivesicular body biogenesis,cytokinesis,membrane repair and regulation of membrane associated signaling.Wolbachia is a class of intracellular symbiotic bacteria that widely exist in terrestrial arthropods.It can cause a variety of changes in host reproductive patterns,such as cytoplasmic incompatibility(CI),that is,the male host infected with Wolbachia cannot produce or produce very few offspring after mating with the uninfected wild type female host.This suggests that Wolbachia may affect the fertility of male hosts.By comparative proteomics in the spermatheca and seminal receptacle of uninfected female Drosophila melanogaster mated with Wolbachia-infected or uninfected males,our group has previously identified that shrb was downregulated(0.54-fold relative to control)due to Wolbachia infection.This implies that shrub protein could be involved in male fertility of D.melanogaster.In order to study the role of shrb gene in the male fertility of D.melanogaster,we first analyzed the spatiotemporal expression of shrb gene in D.melanogaster by quantitative reverse transcription PCR(qRT-PCR).The results showed that the expression levels of shrb gene were low in the early developmental stages,including embryonic stages and early larval stages,and increased from the 3rd instar larval stage.The highest expression level was found in the 1-day-old male fly.It was also found that the expression level of shrb gene in the testes of 1-day-old male flies was significantly higher than that in the ovary of 1-day-old females(p<0.05),suggesting that shrb gene may have a role in male fertility.Then we used nosGal4 driver to knock down shrb specifically in the fly testis to study the effect of shrb gene knockdown on male fertility of D.melanogaster.The results showed that knockdown of shrb in fly testes resulted in a reduction in male fertility.The hatch rate of eggs from the crosses of shrb knockdown males with wild type females was only 19.97 ± 2.73%,which was significantly lower than that in the control group(88.95± 3.94%)(p<0.001).However,the shrb knockdown male flies did not affect the egg laying of their mates.The spermatogenesis of D.melanogaster is a complex process.The primary spermatogonial cell produce 16 spermatogonial cells after four synchronous mitotic divisions,and then generate 64 spermatids clustered in one cyst following two consecutive meiotic divisions.Subsequently,the 64 haploid spermatids undergo dramatic changes in morphology.During the process of sperm morphogenesis,the 64 spermatids exhibit a tight spermatid bundle,and actin cones form around each of the spermatid nuclei.The individualization complex(IC)composed of the 64 actin cones moves down,separating the 64 connected spermatids into individualized sperm.In order to further study the underlying cause of the decreased egg hatch rate induced by shrb knockdown in fly testes,we dissected the testes of D.melanogaster and stained with DAPI and Vasa antibodies.Immunofluorescence staining showed that in the early stage of spermato-genesis(the first half of the testis),no obvious abnormality was found in the shrb knockdown testes comparing with the control.This indicates that knockdown of shrb did not affect the early spermatogenesis.However,contrary to the tightly clustered spermatid nuclei in the control testis,in the shrb knockdown testis we observed fewer spermatid nuclei bundles that were not tightly clusted(with a few spermatid muclei dragging behind).Furthermore,phalloidin staining exhibited that in late stage of spermatogenesis(spermiogenesis),control testes contained many ICs consisted of actin cones,while there were almost no any ICs in the shrb knockdown testis.Finally,in the control seminal vesicle,there was full of mature sperms with needle-like heads,while there were much less sperms(12 vs.96)in the seminal vesicle of shrb knockdown testis relative to the control.This may be the cause of the decreased male fertility induced by the knockdown of shrb gene.Since shrb encodes the subunit of the ESCRT-III complex,and many vesicle trafficking factors are necessary for conveying various materials such as RNAs and organelles to the specified location during spermiogenesis,we speculate that the abnormal spermiogenesis caused by knockdown of shrb could be involved in transport ability in cells.To test this hypothesis,we examined the expressions of four cytoskeleton-associated genes:CG9313,?Tub85D,Arp53D,and Klc,and some other genes that have been shown to cause the similar phenotypes,such as Hanabi,Yuri,and ATPsys-b.The results showed that all of these genes were significantly upregulated in shrb knockdown fly testes.These results suggest that knockdown of shrb in fly testes might cause disrupted spermiogenesis through affecting the transport ability during sperm differentiation.In conclusion,this study found that shrb gene plays an important role in the spermatogenesis of D.melanogaster.Knockdown of shrb in fly testes resulted in a significant decline in fertility of the male flies.Given that D.melanogaster is an important model organism,around over 2/3 genes that involved in human disease appear in Drosophila.The process of spermatogenesis in D.melanogaster is similar to that of mammals.Therefore,the study on the spermatogenesis of D.melanogaster can provide insights into the study of mammalian(including human)spermatogenesis.