| Dynamitin(Dmn),also known as DCTN2-p50, which encodes the core subunit of dynactin, is a key factor in the polymerization of dynactin complex and the attachment with Arp1 filament and the shoulder structure, as well as an important structure in the connection between dynactin complex and dynein, tubulin. Many motor events in cells are regulated by the dynein-dynactin complex.The previous results in our lab shows: the symbiotic bacteria Wolbachia infection can induce cytoplasmic incompatibility (CI) on Drosophila, it means When male flies infection with Wolbachia mates with female uninfection or infection with different strains of Wolbachia, the hatching rate of embryo was significantly reduced or nothing progeny could survive. In order to further explore the molecular mechanism of CI on Drosophila caused by Wolbachia, our lab tested the gene expression of the third instar larvae of uninfected and infected Wolbachia through microarray, we identified 296 differentially expression genes (the fold difference was over 1.5 times), 167 genes were up-regulated,while 129 genes were down_regulated. Our group also studied the differential expression proteins in the female spermathetes and the seminal vesicles after 2h mating with Wolbachia infection or uninfection males by proteomics, and identified 83 differentially expression proteins. In the test of microarray and proteomics, the expression of Dmn was down-regulated in Wolbachia infection flies,suggesting that it may play an important role in the reproductive process of male Drosophila,therefore we selected RNAi strain, and used UAS/Ga14 system to knock down Dmn in Drosophila to study the function of Dmn in the reproductive process of male flies and the relationship with the CI.We found the expression level of Dmn in the male testis was dramatically down regulated in the Wolbachia flies (P<0.01). We also used nosGa14 to knock down Dmn in the gland to investigate the role of Dmn in the process of male reproduction. We used two independent Dmn RNAi lines Dmn-hp-1 and Dmn-hp-2 to cross with nosGal4/TM6B, and get the nosGal4/Dmn-hp-1,rnosGal4/Dmn-hp-2,then used these male flies to cross with wildtype females,the hatching rate respectively were 28.97 ± 4.14% and 42.34±4.40%, was significantly lower than those in the control group (90.6%±3.32%),this is a similar CI phenotype caused by Wolbachia. This result suggests that Wolbachia infection causes a significant reduction of Dmn in the testis of Drosophila, which may be one of the causes of CI caused by Wolbachia. To further investigate whether the expression of Dmn reduction in the Wolbachia infection flies testis is associated with CI, we over-expressed Dmn in Wolbachia infection flies to see if it could save fertility of males, the results showed that over-expression of Dmn in the testis of Wolbachia infection flies, could really save the male fertility,the hatching rate recovered to 77.84%, indicating that the reduction of Dmn in Wolbachia infection male flies, is the cause of CI.In order to study the mechanism of Dmn affecting the fertility of male flies, we used immunohistochemistry and transmission electron microscopy to analyze the spermatogenesis in Dmn knock down testis (We used the Dmn-hp-1 strain in the following study).We stained nuclei with DAPI,in the Dmn knock down testis, the front part is relatively larger, the sperm bundle at the base of testis are less, and the concentration of sperm nuclei is lower, and some canoe stage sperm cells are scattered around the spermatozoa. At the end of the testis, there are highly concentrated needle-shaped spermatozoa in the control group, while in the Dmn knock down group, the sperm get out of the cysts without highly concentration. The seminal vesicle is full of sperm in the control group, however the sperm in seminal vesicle of knock down group is only the 40% of control group. This suggests that Dmn knock down in the testis destroys the spermatogenesis process and reduced the amount of sperm,which could reduce the fertility of male flies.Normally, at the end of meiosis ? of Drosophila spermatogenesis, mitochondria should aggregate together to form nebenkern. Results of transmission electron microscopy showed,there were many mitochondria tended to coalesce together, some of them began to fuse. In the Dmn knockdown testes, however, therewere much less number of mitochondria and these mitochondria did not aggregate, but scattered around. After completion of meiosis, the formation of 64 interconnected spermatid cells begin to undergo the morphological change to complete the development process of sperm. Here we found that in the control testes, spermatids were tightly packed and highly oriented.Each spermatid had clear integral plasma membrane, which kept apart from each other. Dark-staining material accumulated in the major mitochondrial derivative next to the axoneme. While in the Dmn knockdown testes, we frequently observed that the spermatid cell membrane fused together, i.e. more than one axoneme-mitochondria sets were found within a single cell envelope. We also observed multiple mitochondrial derivatives associated with one axoneme. In the sperm elongation stage, in the control group cysts we can observed each sperm contained a nebenkem with axoneme, arranged in the same direction, in the Dmn knock down group, sperm arranged disorder, and sometimes two pairs of axon-mitochondria connects together through a small mitochondrial derivatives,part of the nebenkern is not highly concentrated. In the individual stages of sperm, in the Dmn knock down group, the membrane of cyst is rapidly retracted, and some sperm were degenerated in the cysts. This may be the reason that immature sperm is released from the cyst too early and the reduction of sperm.The results Dmn monoclonal antibody staining showed that Dmn did not express in male reproductive stem cells. When the germ cell stem began to differentiate, after four rounds of synchronic division, 16 spermatogonial cells formed cysts, Dmn began to express. In the midterm of spermatocyte division of control group, Dmn signal is strong in the stellate microtubules and spindle poles, Dmn also expresses in the centromere, in this process dynein is almost co-located with Dmn.The signal intensity of Dmn was significantly lower in Dmn knock down group than that in the control group (P <0.05) in the midterm of spermatocyte division. As the sperm matured, the signals of Dmn and dynein gradually diminished and eventually disappeared, indicating that they may be discharged from the sperm with the excess cytoplasm. Dmn knock down also led to decreased expression of dynein and tubulin in sperm cells, which may also be the reason why sperm can not form normally.The last steps inDrosophila spermiogenesis are the individualization and coiling of mature sperm. Individualization proceeds via formation of the individualization complex (IC). IC is composed of 64 actin cones around the 64 needle shaped nuclei. F-actin of head cyst cell accumulated and projected into the interstitial spaces between the individualization spermatid heads. In the individual process, the IC moved from the head to the tail of sperm, removed the excess cytoplasm and organelles,each sperm formed new cell membrane,finished the sperm individual process, formed independent and dynamic mature sperm. In the Dmn knock down testis, the sperm bundle structure was dispersed, and most of the sperm bundle did not form a complete and orderly individualized complex, F-actin scattered in the testis. In addition, compared with the control group, in the Dmn knock down testis, the individualized complex moved slowly along the sperm bundle, the sperm is still in the initial state of individual, there are some of sperm bundle without formation individual complex. This result shows that Dmn can control the aggregation of F-actin and the formation of IC,thus affecting the individual sperm process.Because the phenotype of Dmn knock down was similar to that in these genes Lis-1, Spag4, Yuri,Ddlc1, DCTN1-p150 and ATPsys-b mutants, we used qRT-PCR to test the expression of these genes in the Dmn knock down flies, the results showed that Dmn knock down in the testis of flies also caused the expression of these genes significantly decreased in the testis. In order to investigate whether these genes interact with Dmn, we chose Ddlcl (encoding Dynein light chain) for further exploration. We over-expressed Ddlcl in Dmn knock down flies, to determine whether it can save the phenotype caused by the Dmn knock down, the results showed it did, the hatching rate returned to 89.95 ± 2.73%.Therefore, the Dmn does interact with these spermatogenic genes and regulate the spermatogenesis of Drosophila.We also used actGal4 to knock down Dmn in the whole body to explore the role of Dmn in development of Drosophila. The results showed that we knocked down Dmn in the whole body of flies, flies could only grow to the larvae stage, could not develop into pupa, and eventually died in the larvae. According to the related papers, the signal pathways affecting the development of Drosophila larvae are juvenile hormone and ecdysone signal pathways. In this study, we observed the molting of the larvae and found Dmn knock down group had no significantly difference with control group.Therefore, we inferred that Dmn knock down affected the juvenile hormone signaling pathway. Next we examined the expression level of Jh1-26, Met and Kr-hlin Dmn knock down flies, three genes expression were significantly up-regulated. Therefore, we conclude that Dmn plays a significant role in larval growth mediated by up-regulation of JhI-26, Met and Kr-h1, it induces excessive expression of juvenile hormones, blocking the pupa of larvae.In summary, Dmn not only plays an important regulatory role in the spermatogenesis process, but also has an important function in the control of larval growth and development. This study also found that Wolbachia infection caused down-regulation of Dmn in testis of Drosophila melanogaster, was one of the mechanisms that Wolbachia induced CI of insect. It creats a new idea for the study of spermatogenesis, at the same time, provides some basic information for the study of male infertility because of the similarities in spermatogenesis between Drosophila and human beings. |
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