Concentrative nucleoside transporters (Cnts) are unidirectional carriers that mediate the energy-costly influx of nucleosides driven by the transmembrane sodium gradient. Cnts are transmembrane proteins that share a common structural organization and are found in all phyla. Although there have been studies on Cnts from a biochemical perspective, no deep research has examined their role at the organismal level. Here, we investigated the role of the Drosophila melanogaster cnt1 gene, which is specifically expressed in the testes. We used the CRISPR/Cas9 system to generate a mutation in the cnt1 gene. The cnt1 mutants exhibited defects in the duration of copulation and spermatid maturation, which significantly impaired male fertility. The most striking effect of the cnt1 mutation in spermatid maturation was an abnormal structure of the sperm tail, in which the formation of major and minor mitochondrial derivatives was disrupted. Our results demonstrate the importance of cnt1 in male fertility and suggest that the observed defects in mating behavior and spermatogenesis are due to alterations in nucleoside transport and associated metabolic pathways.

Biology Centre of the Czech Academy of Sciences Institute of Entomology Ceske Budejovice Czechia

Faculty of Science University of South Bohemia Ceske Budejovice Czechia

Institute of Molecular Genetics Czech Academy of Sciences Prague Czechia

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Acebes A., Grosjean Y., Everaerts C., Ferveur J. F. (2004). Cholinergic control of synchronized seminal emissions in Drosophila . Curr. Biol. 14, 704–710. 10.1016/j.cub.2004.04.003 PubMed DOI

Alzyoud E., Vedelek V., Réthi-Nagy Z., Lipinszki Z., Sinka R. (2021). Microtubule organizing centers contain testis-specific γ-turc proteins in spermatids of Drosophila . Front. Cell Dev. Biol. 9, 727264. 10.3389/fcell.2021.727264 PubMed DOI PMC

Augière C., Lapart J.-A., Duteyrat J.-L., Cortier E., Maire C., Thomas J., et al. (2019). salto/CG13164 is required for sperm head morphogenesis in Drosophila . Mol. Biol. Cell 30, 636–645. 10.1091/mbc.E18-07-0429 PubMed DOI PMC

Bader M., Arama E., Steller H. (2010). A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila . Development 137, 1679–1688. 10.1242/dev.050088 PubMed DOI PMC

Baker B. S., Taylor B. J., Hall J. C. (2001). Are complex behaviors specified by dedicated regulatory genes? Reasoning from Drosophila . Cell 105, 13–24. 10.1016/S0092-8674(01)00293-8 PubMed DOI

Baptissart M., Vega A., Martinot E., Volle D. H. (2013). Male fertility: Is spermiogenesis the critical step for answering biomedical issues? Spermatogenesis 3, e24114. 10.4161/spmg.24114 PubMed DOI PMC

Batut B., Hiltemann S., Bagnacani A., Baker D., Bhardwaj V., Blank C., et al. (2018). Community-driven data analysis training for biology. Cell Syst. 6, 752–758.e1. 10.1016/j.cels.2018.05.012 PubMed DOI PMC

Beaver L. M., Giebultowicz J. M. (2004). Regulation of copulation duration by period and timeless in Drosophila melanogaster . Curr. Biol. 14, 1492–1497. 10.1016/j.cub.2004.08.022 PubMed DOI

Cardullo R. A., Baltz J. M. (1991). Metabolic regulation in mammalian sperm: Mitochondrial volume determines sperm length and flagellar beat frequency. Cell Motil. Cytoskelet. 19, 180–188. 10.1002/cm.970190306 PubMed DOI

Chen X., Hiller M., Sancak Y., Fuller M. T. (2005). Tissue-specific TAFs counteract polycomb to turn on terminal differentiation. Science 310, 869–872. 10.1126/science.1118101 PubMed DOI

Chintapalli V. R., Wang J., Dow J. A. T. (2007). Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat. Genet. 39, 715–720. 10.1038/ng2049 PubMed DOI

Clark K., Karsch-Mizrachi I., Lipman D. J., Ostell J., Sayers E. W. (2016). GenBank. Nucleic Acids Res. 44, D67–D72. 10.1093/nar/gkv1276 PubMed DOI PMC

Crickmore M. A., Vosshall L. B. (2013). Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila . Cell 155, 881–893. 10.1016/j.cell.2013.09.055 PubMed DOI PMC

Demarco R. S., Eikenes Å. H., Haglund K., Jones D. L. (2014). Investigating spermatogenesis in Drosophila melanogaster . Methods 68, 218–227. 10.1016/j.ymeth.2014.04.020 PubMed DOI PMC

Dos Santos-Rodrigues A., Pereira M. R., Brito R., de Oliveira N. A., Paes-de-Carvalho R. (2015). Adenosine transporters and receptors: Key elements for retinal function and neuroprotection. Vitam. Horm. 98, 487–523. 10.1016/bs.vh.2014.12.014 PubMed DOI

Edgar R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. 10.1093/nar/gkh340 PubMed DOI PMC

Ejsmont R. K., Ahlfeld P., Pozniakovsky A., Stewart A. F., Tomancak P., Sarov M. (2011). Recombination-mediated genetic engineering of large genomic DNA transgenes. Methods Mol. Biol. 772, 445–458. 10.1007/978-1-61779-228-1_26 PubMed DOI

Ejsmont R. K., Sarov M., Winkler S., Lipinski K. A., Tomancak P. (2009). A toolkit for high-throughput, cross-species gene engineering in Drosophila . Nat. Methods 6, 435–437. 10.1038/nmeth.1334 PubMed DOI

Fabian L., Brill J. A. (2012). Drosophila spermiogenesis: Big things come from little packages. Spermatogenesis 2, 197–212. 10.4161/spmg.21798 PubMed DOI PMC

Fatima R. (2011). Drosophila Dynein intermediate chain gene, Dic61B, is required for spermatogenesis. PLoS One 6, e27822. 10.1371/journal.pone.0027822 PubMed DOI PMC

Fleischmannova J., Kucerova L., Sandova K., Steinbauerova V., Broz V., Simek P., et al. (2012). Differential response of Drosophila cell lines to extracellular adenosine. Insect biochem. Mol. Biol. 42, 321–331. 10.1016/j.ibmb.2012.01.002 PubMed DOI

Gilchrist A. S., Partridge L. (2000). Why it is difficult to model sperm displacement in Drosophila melanogaster: The relation between sperm transfer and copulation duration. Evolution 54, 534–542. 10.1111/j.0014-3820.2000.tb00056.x PubMed DOI

Gray J. H., Owen R. P., Giacomini K. M. (2004). The concentrative nucleoside transporter family, SLC28. Pflugers Arch. 447, 728–734. 10.1007/s00424-003-1107-y PubMed DOI

Groth A. C., Fish M., Nusse R., Calos M. P. (2004). Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166, 1775–1782. 10.1534/genetics.166.4.1775 PubMed DOI PMC

Guindon S., Dufayard J.-F., Lefort V., Anisimova M., Hordijk W., Gascuel O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. 10.1093/sysbio/syq010 PubMed DOI

Hau R. K., Miller S. R., Wright S. H., Cherrington N. J. (2020). Generation of a hTERT-immortalized human Sertoli cell model to study transporter dynamics at the blood-testis barrier. Pharmaceutics 12, E1005. 10.3390/pharmaceutics12111005 PubMed DOI PMC

Huh J. R., Vernooy S. Y., Yu H., Yan N., Shi Y., Guo M., et al. (2004). Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization. PLoS Biol. 2, E15. 10.1371/journal.pbio.0020015 PubMed DOI PMC

Jayaramaiah Raja S., Renkawitz-Pohl R. (2005). Replacement by Drosophila melanogaster protamines and Mst77F of histones during chromatin condensation in late spermatids and role of sesame in the removal of these proteins from the male pronucleus. Mol. Cell. Biol. 25, 6165–6177. 10.1128/MCB.25.14.6165-6177.2005 PubMed DOI PMC

Jois S., Chan Y. B., Fernandez M. P., Leung A. K.-W. (2018). Characterization of the sexually dimorphic fruitless neurons that regulate copulation duration. Front. Physiol. 9, 780. 10.3389/fphys.2018.00780 PubMed DOI PMC

Kato R., Maeda T., Akaike T., Tamai I. (2005). Nucleoside transport at the blood-testis barrier studied with primary-cultured Sertoli cells. J. Pharmacol. Exp. Ther. 312, 601–608. 10.1124/jpet.104.073387 PubMed DOI

Klein D. M., Evans K. K., Hardwick R. N., Dantzler W. H., Wright S. H., Cherrington N. J. (2013). Basolateral uptake of nucleosides by Sertoli cells is mediated primarily by equilibrative nucleoside transporter 1. J. Pharmacol. Exp. Ther. 346, 121–129. 10.1124/jpet.113.203265 PubMed DOI PMC

Klein D. M., Harding M. C., Crowther M. K., Cherrington N. J. (2017). Localization of nucleoside transporters in rat epididymis. J. Biochem. Mol. Toxicol. 31, e21911. 10.1002/jbt.21911 PubMed DOI

Knight D., Harvey P. J., Iliadi K. G., Klose M. K., Iliadi N., Dolezelova E., et al. (2010). Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila . J. Neurosci. 30, 5047–5057. 10.1523/JNEUROSCI.6241-09.2010 PubMed DOI PMC

Kondo S., Ueda R. (2013). Highly improved gene targeting by germline-specific Cas9 expression in Drosophila . Genetics 195, 715–721. 10.1534/genetics.113.156737 PubMed DOI PMC

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. 10.1093/molbev/msy096 PubMed DOI PMC

Larkin A., Marygold S. J., Antonazzo G., Attrill H., dos Santos G., Garapati P. V., et al. (2021). FlyBase: Updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49, D899–D907. 10.1093/nar/gkaa1026 PubMed DOI PMC

Leatherman J. L., Dinardo S. (2008). Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal. Cell Stem Cell 3, 44–54. 10.1016/j.stem.2008.05.001 PubMed DOI PMC

Lee G., Hall J. C. (2001). Abnormalities of male-specific FRU protein and serotonin expression in the CNS of fruitless mutants in Drosophila . J. Neurosci. 21, 513–526. 10.1523/JNEUROSCI.21-02-00513.2001 PubMed DOI PMC

Lee G., Villella A., Taylor B. J., Hall J. C. (2001). New reproductive anomalies in fruitless-mutant Drosophila males: Extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs. J. Neurobiol. 47, 121–149. 10.1002/neu.1021 PubMed DOI

Lefort V., Longueville J.-E., Gascuel O. (2017). SMS: Smart model selection in PhyML. Mol. Biol. Evol. 34, 2422–2424. 10.1093/molbev/msx149 PubMed DOI PMC

Leung G. P., Ward J. L., Wong P. Y., Tse C. M. (2001). Characterization of nucleoside transport systems in cultured rat epididymal epithelium. Am. J. Physiol. Cell Physiol. 280, C1076–C1082. 10.1152/ajpcell.2001.280.5.C1076 PubMed DOI

Lin Y.-H., Maaroufi H. O., Ibrahim E., Kucerova L., Zurovec M. (2019). Expression of human mutant huntingtin protein in Drosophila hemocytes impairs immune responses. Front. Immunol. 10, 2405. 10.3389/fimmu.2019.02405 PubMed DOI PMC

Lin Y.-H., Maaroufi H. O., Kucerova L., Rouhova L., Filip T., Zurovec M. (2021). Adenosine receptor and its downstream targets, mod(mdg4) and Hsp70, work as a signaling pathway modulating cytotoxic damage in Drosophila . Front. Cell Dev. Biol. 9, 651367. 10.3389/fcell.2021.651367 PubMed DOI PMC

Lindsley D. I., Tokuyasu K. T. (1980). “Spermatogenesis,” in Genetics and Biology of Drosophila (New York: Academic Press; ), 225–294.

Lotti F., Maggi M. (2018). Sexual dysfunction and male infertility. Nat. Rev. Urol. 15, 287–307. 10.1038/nrurol.2018.20 PubMed DOI

MacBean I. T., Parsons P. A. (1967). Directional selection for duration of copulation in Drosophila melanogaster . Genetics 56, 233–239. 10.1093/genetics/56.2.233 PubMed DOI PMC

Machado J., Abdulla P., Hanna W. J. B., Hilliker A. J., Coe I. R. (2007). Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter. Physiol. Genomics 28, 337–347. 10.1152/physiolgenomics.00087.2006 PubMed DOI

Masino S., Boison D. (2013). Adenosine: A key link between metabolism and brain activity. New York, NY: Springer. 10.1007/978-1-4614-3903-5 DOI

Molina-Arcas M., Pastor-Anglada M. (2013). Nucleoside transporters (SLC28 and SLC29) family. Pharmacogenomics of Human Drug Transporters. 11, 243–270. 10.1002/9781118353240.ch11 DOI

Morton D. B., Clemens-Grisham R., Hazelett D. J., Vermehren-Schmaedick A. (2010). Infertility and male mating behavior deficits associated with Pde1c in Drosophila melanogaster . Genetics 186, 159–165. 10.1534/genetics.110.118018 PubMed DOI PMC

Nagarkar-Jaiswal S., Lee P.-T., Campbell M. E., Chen K., Anguiano-Zarate S., Cantu Gutierrez M., et al. (2015). A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila . Elife 4, e05338. 10.7554/eLife.05338 PubMed DOI PMC

Ng C. L., Qian Y., Schulz C. (2019). Notch and Delta are required for survival of the germline stem cell lineage in testes of Drosophila melanogaster . PLoS One 14, e0222471. 10.1371/journal.pone.0222471 PubMed DOI PMC

Noguchi T., Koizumi M., Hayashi S. (2011). Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila . Curr. Biol. 21, 805–814. 10.1016/j.cub.2011.04.016 PubMed DOI

Noguchi T., Miller K. G. (2003). A role of actin dynamics in individualization during spermatogenesis in Drosophila melanogaster . Development 130, 1805–1816. 10.1242/dev.00406 PubMed DOI

Osborne D. M., Sandau U. S., Jones A. T., Vander Velden J. W., Weingarten A. M., Etesami N., et al. (2018). Developmental role of adenosine kinase for the expression of sex-dependent neuropsychiatric behavior. Neuropharmacology 141, 89–97. 10.1016/j.neuropharm.2018.08.025 PubMed DOI PMC

Pavlou H. J., Goodwin S. F. (2013). Courtship behavior in Drosophila melanogaster: Towards a ‘courtship connectome. Curr. Opin. Neurobiol. 23, 76–83. 10.1016/j.conb.2012.09.002 PubMed DOI PMC

Pérez-Torras S., Mata-Ventosa A., Drögemöller B., Tarailo-Graovac M., Meijer J., Meinsma R., et al. (2019). Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure. Biochim. Biophys. Acta. Mol. Basis Dis. 1865, 1182–1191. 10.1016/j.bbadis.2019.01.013 PubMed DOI

Phatarpekar P. V., Wen J., Xia Y. (2010). Role of adenosine signaling in penile erection and erectile disorders. J. Sex. Med. 7, 3553–3564. 10.1111/j.1743-6109.2009.01555.x PubMed DOI PMC

Porter M. E. (1996). Axonemal dyneins: Assembly, organization, and regulation. Curr. Opin. Cell Biol. 8, 10–17. 10.1016/S0955-0674(96)80042-1 PubMed DOI

Ranz J. M., Ponce A. R., Hartl D. L., Nurminsky D. (2003). Origin and evolution of a new gene expressed in the Drosophila sperm axoneme. Genetica 118, 233–244. 10.1023/A:1024186516554 PubMed DOI

Resende L. P. F., Boyle M., Tran D., Fellner T., Jones D. L. (2013). Headcase promotes cell survival and niche maintenance in the Drosophila testis. PLoS One 8, e68026. 10.1371/journal.pone.0068026 PubMed DOI PMC

Rideout E. J., Billeter J.-C., Goodwin S. F. (2007). The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song. Curr. Biol. 17, 1473–1478. 10.1016/j.cub.2007.07.047 PubMed DOI PMC

Ruiz-Pesini E., Diez C., Lapeña A. C., Pérez-Martos A., Montoya J., Alvarez E., et al. (1998). Correlation of sperm motility with mitochondrial enzymatic activities. Clin. Chem. 44, 1616–1620. 10.1093/clinchem/44.8.1616 PubMed DOI

Santel A., Blümer N., Kämpfer M., Renkawitz-Pohl R. (1998). Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa. J. Cell Sci. 111 (2), 3299–3309. 10.1242/jcs.111.22.3299 PubMed DOI

Santel A., Winhauer T., Blümer N., Renkawitz-Pohl R. (1997). The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif. Mech. Dev. 64, 19–30. 10.1016/s0925-4773(97)00031-2 PubMed DOI

Sarov M., Barz C., Jambor H., Hein M. Y., Schmied C., Suchold D., et al. (2016). A genome-wide resource for the analysis of protein localisation in Drosophila . Elife 5, e12068. 10.7554/eLife.12068 PubMed DOI PMC

Sawyer E. M., Brunner E. C., Hwang Y., Ivey L. E., Brown O., Bannon M., et al. (2017). Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila . BMC Cell Biol. 18, 16. 10.1186/s12860-017-0132-1 PubMed DOI PMC

Schneider C. A., Rasband W. S., Eliceiri K. W. (2012). NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. 10.1038/nmeth.2089 PubMed DOI PMC

Siegel R. W., Hall J. C. (1979). Conditioned responses in courtship behavior of normal and mutant Drosophila . Proc. Natl. Acad. Sci. U. S. A. 76, 3430–3434. 10.1073/pnas.76.7.3430 PubMed DOI PMC

Soulavie F., Piepenbrock D., Thomas J., Vieillard J., Duteyrat J.-L., Cortier E., et al. (2014). Hemingway is required for sperm flagella assembly and ciliary motility in Drosophila . Mol. Biol. Cell 25, 1276–1286. 10.1091/mbc.e13-10-0616 PubMed DOI PMC

Steinhauer J. (2015). Separating from the pack: Molecular mechanisms of Drosophila spermatid individualization. Spermatogenesis 5, e1041345. 10.1080/21565562.2015.1041345 PubMed DOI PMC

Tokuyasu K. T. (1975). Dynamics of spermiogenesis in Drosophila melanogaster. VI. Significance of “onion” nebenkern formation. J. Ultrastruct. Res. 53, 93–112. 10.1016/S0022-5320(75)80089-X PubMed DOI

Vedelek V., Bodai L., Grézal G., Kovács B., Boros I. M., Laurinyecz B., et al. (2018). Analysis of Drosophila melanogaster testis transcriptome. BMC Genomics 19, 697. 10.1186/s12864-018-5085-z PubMed DOI PMC

Wakimoto B. T., Lindsley D. L., Herrera C. (2004). Toward a comprehensive genetic analysis of male fertility in Drosophila melanogaster . Genetics 167, 207–216. 10.1534/genetics.167.1.207 PubMed DOI PMC

White-Cooper H. (2004). Spermatogenesis: Analysis of meiosis and morphogenesis. Methods Mol. Biol. 247, 45–75. 10.1385/1-59259-665-7:45 PubMed DOI

Witt E., Benjamin S., Svetec N., Zhao L. (2019). Testis single-cell RNA-seq reveals the dynamics of de novo gene transcription and germline mutational bias in Drosophila . Elife 8, e47138. 10.7554/eLife.47138 PubMed DOI PMC

Xie P., Dou S.-X., Wang P.-Y. (2006). Model for unidirectional movement of axonemal and cytoplasmic dynein molecules. Acta Biochim. Biophys. Sin. 38, 711–724. 10.1111/j.1745-7270.2006.00223.x PubMed DOI

Yeh S.-D., Do T., Abbassi M., Ranz J. M. (2012). Functional relevance of the newly evolved sperm dynein intermediate chain multigene family in Drosophila melanogaster males. Commun. Integr. Biol. 5, 462–465. 10.4161/cib.21136 PubMed DOI PMC

Young J. D. (2016). The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: A 30-year collaborative odyssey. Biochem. Soc. Trans. 44, 869–876. 10.1042/BST20160038 PubMed DOI

Young J. D., Yao S. Y. M., Baldwin J. M., Cass C. E., Baldwin S. A. (2013). The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol. Asp. Med. 34, 529–547. 10.1016/j.mam.2012.05.007 PubMed DOI

Yu J., Chen B., Zheng B., Qiao C., Chen X., Yan Y., et al. (2019). ATP synthase is required for male fertility and germ cell maturation in Drosophila testes. Mol. Med. Rep. 19, 1561–1570. 10.3892/mmr.2019.9834 PubMed DOI PMC

Yuan X., Zheng H., Su Y., Guo P., Zhang X., Zhao Q., et al. (2019). Drosophila Pif1A is essential for spermatogenesis and is the homolog of human CCDC157, a gene associated with idiopathic NOA. Cell Death Dis. 10, 125. 10.1038/s41419-019-1398-3 PubMed DOI PMC

Zhang S. D., Odenwald W. F. (1995). Misexpression of the white (w) gene triggers male-male courtship in Drosophila . Proc. Natl. Acad. Sci. U. S. A. 92, 5525–5529. 10.1073/pnas.92.12.5525 PubMed DOI PMC

Zhao J., Klyne G., Benson E., Gudmannsdottir E., White-Cooper H., Shotton D. (2010). FlyTED: The Drosophila testis gene expression database. Nucleic Acids Res. 38, D710–D715. 10.1093/nar/gkp1006 PubMed DOI PMC

Ziegler A. B., Berthelot-Grosjean M., Grosjean Y. (2013). The smell of love in Drosophila . Front. Physiol. 4, 72. 10.3389/fphys.2013.00072 PubMed DOI PMC