| Drosophila melanogaster is an ideal model organism for studying human diseases,which has been widely used to study various diseases such as cancer,immune response,and neurodegenerative diseases by researchers.The spermatogenesis process of Drosophila is similar to that of humans,including the renewal of reproductive stem cells,meiosis of primary spermatocytes,and morphological transformation of spermatocytes to sperms.Therefore,studying the fertility of male Drosophila can provide ideas and clues for the study of male reproductive mechanisms in animals.Previous exploration by the research team found that specific knockdown of the mitochondrial Val RS-m in the testis of Drosophila can lead to complete infertility in male Drosophila.And transcriptomic results showed that knockdown of Val RS-m in the testis could result in the significant downregulation of VhaM9.7-d.Since Val RS-m is also a highly expressed gene in the testis associated with mitochondria,and VhaM9.7-d is associated with ATP conversion that is highly expressed in the testis.We speculate that VhaM9.7-d may be involved in regulating male Drosophila fertility.The results of this study are summarized as follows:1.In order to explore the concrete function of VhaM9.7-d in male Drosophila fertility,we first used q RT-PCR to detect its spatiotemporal expression.The results showed that the expression level of VhaM9.7-d in the testes of one-day-old male Drosophila was significantly higher than that in the ovaries of one-day-old female Drosophila.Next,we used the driving strain of Drosophila to knock down the VhaM9.7-d in the testes of Drosophila by mating to produce offspring.Then we detected its expression level in the testes of the knockdown group and found it was significantly reduced,indicating that the knockdown effect was good and could be used for subsequent experiments.Then we found that the male fly that was knocked down the VhaM9.7-d can mate with the female fly normally,and the hatchability of embryos produced by the female fly is zero,which means that the specific knockdown of the VhaM9.7-d could result in male Drosophila infertility.2.In order to explore the causes of male infertility in Drosophila that was knocked down the VhaM9.7-d,we dissected the testes and seminal vesicles of Drosophila from the knockdown group and the control group for immunofluorescence staining of DAPI.The results showed that the morphology of the testes and seminal vesicles of male flies in the knockdown group was normal,and there were sperms in the seminal vesicles.So we further observed sperms in the seminal vesicles.The results showed that sperms in the seminal vesicles of male flies almost lose their motility and the sperm tails intertwine into clusters in the knockdown group compared to the active sperm motility in the control group,indicating that specific knockdown of the VhaM9.7-d inhibited the sperm motility in seminal vesicles.Since we observed the mating behavior between female and male flies in the knockdown group,we dissected the reproductive tract of female flies and performed immunofluorescence staining observations.The results showed that the sperm of male flies could be transferred to the uterus of female flies in the knockdown group after mating,but could not be stored in the seminal receptacles normally and failed to fertilize eggs.3.In order to further explore the causes of sperm motility inhibition,we used Vasa,Anti-β-Tubulin and Phalloidin antibody to mark germ cells,sperm tails,and individualized complexes respectively.The results showed that specific knockdown of VhaM9.7-d had no significant impact in early stages of spermatogenesis.Next we used transmission electron microscopy and mitochondrial membrane potential fluorescence probes to detect the structure and function of mitochondria respectively.Compared with the control group,the structure of mitochondrial derivative of the sperm tail in the knockdown group was normal and matched with the flagellar axoneme.In the knockdown group,the mitochondrial membrane potential of the sperm tail decreased significantly,indicating that specific knockdown of VhaM9.7-d would inhibit the production of mitochondrial membrane potential in sperm tails.4.Due to VhaM9.7-d is involved in the production of V-ATPase,and the role of V-ATPase is consuming ATP to transport protons(H~+)across the membrane to maintain an acidic environment in the local chamber.Therefore,we speculate that there is an acid environment in seminal vesicles to ensure sperm motility at normal level.We also speculate that VhaM9.7-d knockdown may lead to damage to the signaling pathway on sperm plasma membranes,inability to recognize the acid environment,or damage to the signaling pathway in seminal vesicles,inability to maintain the local acid environment.So we dissected the seminal vesicles of male flies in the knockdown and control group and performed transcriptome sequencing.The sequencing results showed that a total of528 differentially expressed genes were identified,including 270 upregulated differentially expressed genes and 258 downregulated differentially expressed genes.These differentially expressed genes participate in a variety of biological processes and are mainly enriched in the lysosomal pathway.We screened some differentially expressed genes that may be related to sperm motility for q RT-PCR detection,including upregulated differentially expressed genes(CG43672,ORY,CG13999,CG1271,ODA-Dnal1)and downregulated differentially expressed genes(Snapin,Cadps,Tpn C25D,Naxd,CG34423,CG11498).The detection results were consistent with transcriptome sequencing results.In summary,we found that specific knockdown of the VhaM9.7-d in Drosophila will cause a significant decrease of the mitochondrial membrane potential in sperms and the inhibition of sperm motility,leading to the failed storage in female seminal receptacles.The failure of sperms to fertilize eggs leads to male Drosophila infertility ultimately.Therefore,this study can provide new ideas and clues for the study of spermatogenesis mechanism and male infertility. |
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