The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with α6 integrin subunit, which forms a dimer with β4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.

Department of Biochemistry Faculty of Science Charles University Hlavova 8 128 40 Prague 2 Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources University of Life Sciences Prague Kamycka 129 165 00 Prague 6 Czech Republic

Department of Zoology Faculty of Science Charles University Vinicna 7 128 44 Prague 2 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Laboratory of Reproductive Physiology Institute of Animal Biochemistry and Genetics Centre of Biosciences Slovak Academy of Sciences Dubravska cesta 9 845 05 Bratislava Slovak Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Zobrazit více v PubMed

Hemler ME. Integrin associated proteins. Curr. Opin. Cell Biol. 1998;10:578–585. doi: 10.1016/S0955-0674(98)80032-X. PubMed DOI

Spinardi L, Ren YL, Sanders R, Giancotti FG. The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes. Molecular biology of the cell. 1993;4:871–884. doi: 10.1091/mbc.4.9.871. PubMed DOI PMC

Ramírez‐Ramírez, D. et al. Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa. J. Cell. Biochem. (2019). PubMed

Mainiero F, et al. Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. The EMBO journal. 1995;14:4470–4481. doi: 10.1002/j.1460-2075.1995.tb00126.x. PubMed DOI PMC

Boucheix C, Rubinstein E. Tetraspanins. Cellular and Molecular Life Sciences CMLS. 2001;58:1189–1205. doi: 10.1007/pl00000933. PubMed DOI PMC

Colburn ZT, Jones JC. α6β4 integrin regulates the collective migration of epithelial cells. American journal of respiratory cell and molecular biology. 2017;56:443–452. doi: 10.1165/rcmb.2016-0313OC. PubMed DOI PMC

Kaji K, et al. The gamete fusion process is defective in eggs of Cd9-deficient mice. Nat. Genet. 2000;24:279. doi: 10.1038/73502. PubMed DOI

Miyado K, et al. Requirement of CD9 on the egg plasma membrane for fertilization. Science. 2000;287:321–324. doi: 10.1126/science.287.5451.321. PubMed DOI

Le Naour F, Rubinstein E, Jasmin C, Prenant M, Boucheix C. Severely reduced female fertility in CD9-deficient mice. Science. 2000;287:319–321. doi: 10.1126/science.287.5451.319. PubMed DOI

Ohnami N, et al. CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte. Biology open. 2012;1:640–647. doi: 10.1242/bio.20121420. PubMed DOI PMC

Jankovicova J, et al. Detection of CD9 and CD81 tetraspanins in bovine and porcine oocytes and embryos. International journal of biological macromolecules. 2019;123:931–938. doi: 10.1016/j.ijbiomac.2018.11.161. PubMed DOI

Antalíková J, et al. Localization of CD 9 Molecule on Bull Spermatozoa: Its Involvement in the Sperm–Egg Interaction. Reproduction in domestic animals. 2015;50:423–430. doi: 10.1111/rda.12508. PubMed DOI

Frolikova M, et al. CD9 and CD81 Interactions and Their Structural Modelling in Sperm Prior to Fertilization. International journal of molecular sciences. 2018;19:1236. doi: 10.3390/ijms19041236. PubMed DOI PMC

Jankovicova, J. et al. Characterization of tetraspanin protein CD81 in mouse spermatozoa and bovine gametes. Reproduction, REP-16-0304 (2016). PubMed

Parthasarathy V, et al. Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells. Immunology. 2009;127:237–248. doi: 10.1111/j.1365-2567.2008.02945.x. PubMed DOI PMC

Hemler ME. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu. Rev. Cell. Dev. Biol. 2003;19:397–422. doi: 10.1146/annurev.cellbio.19.111301.153609. PubMed DOI

Glander HJ, Schaller J. Beta 1‐integrins of spermatozoa: a flow cytophotometric analysis. International journal of andrology. 1993;16:105–111. doi: 10.1111/j.1365-2605.1993.tb01162.x. PubMed DOI

Reddy VRK, Rajeev SK, Gupta V. α6β1 Integrin is a potential clinical marker for evaluating sperm quality in men. Fertility and Sterility. 2003;79:1590–1596. doi: 10.1016/S0015-0282(03)00368-6. PubMed DOI

Barraud-Lange V, et al. Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization. BMC Dev. Biol. 2007;7:102. doi: 10.1186/1471-213X-7-102. PubMed DOI PMC

Frolikova M, et al. Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm. International journal of molecular sciences. 2019;20:1004. doi: 10.3390/ijms20051004. PubMed DOI PMC

Lammerding J, Kazarov AR, Huang H, Lee RT, Hemler ME. Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening. Proceedings of the National Academy of Sciences. 2003;100:7616–7621. doi: 10.1073/pnas.1337546100. PubMed DOI PMC

Ziyyat A, et al. CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion. J. Cell Sci. 2006;119:416–424. doi: 10.1242/jcs.02730. PubMed DOI

Kajiji S, Tamura RN, Quaranta V. A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. The EMBO journal. 1989;8:673–680. doi: 10.1002/j.1460-2075.1989.tb03425.x. PubMed DOI PMC

Serru V, et al. Selective tetraspan–integrin complexes (CD81/α4β1, CD151/α3β1, CD151/α6β1) under conditions disrupting tetraspan interactions. Biochem. J. 1999;340:103–111. PubMed PMC

Lotus MT, et al. The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin α6β4 and may regulate the spatial organization of hemidesmosomes. The Journal of cell biology. 2000;149:969–982. doi: 10.1083/jcb.149.4.969. PubMed DOI PMC

Evinger AJ, III, Levin ER. Requirements for estrogen receptor α membrane localization and function. Steroids. 2005;70:361–363. doi: 10.1016/j.steroids.2005.02.015. PubMed DOI

Berditchevski F, Odintsova E, Sawada S, Gilbert E. Expression of the palmitoylation-deficient CD151 weakens the association of α3β1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling. J. Biol. Chem. 2002;277:36991–37000. doi: 10.1074/jbc.M205265200. PubMed DOI

Nielsen M, Bøgh IB, Schmidt M, Greve T. Immunohistochemical localization of estrogen receptor-α in sex ducts and gonads of newborn piglets. Histochemistry and Cell Biology. 2001;115:521–526. doi: 10.1007/s004180100269. PubMed DOI

Kitiyanant Y, Chaisalee B, Pavasuthipaisit K. Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187. International Journal of Andrology. 2002;25:215–222. doi: 10.1046/j.1365-2605.2002.00350.x. PubMed DOI

Satouh Y, Inoue N, Ikawa M, Okabe M. Visualization of the moment of mouse sperm–egg fusion and dynamic localization of IZUMO1. J. Cell Sci. 2012;125:4985–4990. doi: 10.1242/jcs.100867. PubMed DOI

Ito, C., Yamatoya, K. & Toshimori, K. In Sexual Reproduction in Animals and Plants 85–95 (Springer, Tokyo, 2014).

Frolikova M, Sebkova N, Ded L, Dvorakova-Hortova K. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction. Scientific Reports. 2016;6:33714. doi: 10.1038/srep33714. PubMed DOI PMC

Frolíková M, et al. Role of complement regulatory proteins CD46, CD55 and CD59 in reproduction. Folia Zoologica. 2012;61:84–95. doi: 10.25225/fozo.v61.i1.a12.2012. DOI

Sebkova N, Ded L, Vesela K, Dvorakova-Hortova K. Progress of sperm IZUMO1 relocation during spontaneous acrosome reaction. Reproduction. 2014;147:231–240. doi: 10.1530/REP-13-0193. PubMed DOI

Jankovicova J, et al. Characterization of tetraspanin protein CD81 in mouse spermatozoa and bovine gametes. Reproduction. 2016;152:785–793. doi: 10.1530/REP-16-0304. PubMed DOI

Nakanishi T, et al. Real‐time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett. 1999;449:277–283. doi: 10.1016/S0014-5793(99)00433-0. PubMed DOI

Fitter S, Tetaz TJ, Berndt MC, Ashman LK. Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3. Blood. 1995;86:1348–1355. doi: 10.1182/blood.V86.4.1348.bloodjournal8641348. PubMed DOI

Zola, H., Swart, B., Nicholson, I. & Woss, E. Leukocyte and Stromal Cell Molecules: The CDMarkers. (John Wiley & Sons, Inc., Hoboken, New Jersey, 2007).

Wang Z, et al. CD151-mediated adhesion is crucial to osteosarcoma pulmonary metastasis. Oncotarget. 2016;7:60623. PubMed PMC

Spiro RG. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology. 2002;12:43R–56R. doi: 10.1093/glycob/12.4.43R. PubMed DOI

Inoue N, Ikawa M, Okabe M. The mechanism of sperm–egg interaction and the involvement of IZUMO1 in fusion. Asian journal of andrology. 2011;13:81. doi: 10.1038/aja.2010.70. PubMed DOI PMC

Cohen DJ, et al. Participation of epididymal cysteine‐rich secretory proteins in sperm‐egg fusion and their potential use for male fertility regulation. Asian journal of andrology. 2007;9:528–532. doi: 10.1111/j.1745-7262.2007.00283.x. PubMed DOI

Yoshida K, et al. A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin. Reproduction. 2010;139:533–544. doi: 10.1530/REP-09-0434. PubMed DOI

Ito C, et al. Tetraspanin family protein CD9 in the mouse sperm: unique localization, appearance, behavior and fate during fertilization. Cell Tissue Res. 2010;340:583–594. doi: 10.1007/s00441-010-0967-7. PubMed DOI

Yang XH, et al. CD151 restricts the α6 integrin diffusion mode. J. Cell Sci. 2012;125:1478–1487. doi: 10.1242/jcs.093963. PubMed DOI PMC

Singethan K, Schneider-Schaulies J. Tetraspanins: Small transmembrane proteins with big impact on membrane microdomain structures. Communicative & integrative biology. 2008;1:11–13. doi: 10.4161/cib.1.1.6406. PubMed DOI PMC

Stipp CS, Kolesnikova TV, Hemler ME. Functional domains in tetraspanin proteins. Trends Biochem. Sci. 2003;28:106–112. doi: 10.1016/S0968-0004(02)00014-2. PubMed DOI

Goschnick MW, et al. Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice. Blood. 2006;108:1911–1918. doi: 10.1182/blood-2006-02-004267. PubMed DOI

Charrin S, et al. Lateral organization of membrane proteins: tetraspanins spin their web. Biochem. J. 2009;420:133–154. doi: 10.1042/BJ20082422. PubMed DOI

Sabetian S, Shamsir MS, Naser MA. Functional features and protein network of human sperm-egg interaction. Systems biology in reproductive medicine. 2014;60:329–337. doi: 10.3109/19396368.2014.955896. PubMed DOI

Hemler M, Crouse C, Sonnenberg A. Association of the VLA alpha 6 subunit with a novel protein. A possible alternative to the common VLA beta 1 subunit on certain cell lines. J. Biol. Chem. 1989;264:6529–6535. PubMed

Simon‐Assmann P, et al. Differential expression of laminin isoforms and α6‐β4 integrin subunits in the developing human and mouse intestine. Dev. Dyn. 1994;201:71–85. doi: 10.1002/aja.1002010108. PubMed DOI

Sadej R, Grudowska A, Turczyk L, Kordek R, Romanska HM. CD151 in cancer progression and metastasis: a complex scenario. Lab. Invest. 2014;94:41. doi: 10.1038/labinvest.2013.136. PubMed DOI

Liu L, et al. Tetraspanin CD151 promotes cell migration by regulating integrin trafficking. J. Biol. Chem. 2007;282:31631–31642. doi: 10.1074/jbc.M701165200. PubMed DOI

Winterwood NE, Varzavand A, Meland MN, Ashman LK, Stipp CS. A critical role for tetraspanin CD151 in α3β1 and α6β4 integrin–dependent tumor cell functions on laminin-5. Molecular biology of the cell. 2006;17:2707–2721. doi: 10.1091/mbc.e05-11-1042. PubMed DOI PMC

te Molder L, et al. Tetraspanin CD151 and integrin α3β1 contribute to the stabilization of integrin α6β4-containing cell-matrix adhesions. J. Cell Sci. 2019;132:jcs235366. doi: 10.1242/jcs.235366. PubMed DOI

Kierszenbaum AL, Rivkin E, Tres LL. Molecular biology of sperm head shaping. Society of Reproduction and Fertility supplement. 2007;65:33. PubMed

Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002;110:673–687. doi: 10.1016/S0092-8674(02)00971-6. PubMed DOI

Lozahic S, et al. CD46 (membrane cofactor protein) associates with multiple β1 integrins and tetraspans. Eur. J. Immunol. 2000;30:900–907. doi: 10.1002/1521-4141(200003)30:3<900::AID-IMMU900>3.0.CO;2-X. PubMed DOI

Kazarov AR, Yang X, Stipp CS, Sehgal B, Hemler ME. An extracellular site on tetraspanin CD151 determines α3 and α6 integrin–dependent cellular morphology. The Journal of cell biology. 2002;158:1299–1309. doi: 10.1083/jcb.200204056. PubMed DOI PMC

Chang Y-F, Lee-Chang JS, Panneerdoss S, MacLean JA, Rao MK. Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis. BioTechniques. 2011;51:341–344. doi: 10.2144/000113764. PubMed DOI PMC

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Yang J, et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods. 2015;12:7–8. doi: 10.1038/nmeth.3213. PubMed DOI PMC

The UniProt, C. UniProt: the universal protein knowledgebase. Nucleic. Acids. Res. 2017;45:D158–D169. doi: 10.1093/nar/gkw1099. PubMed DOI PMC

Kozakov D, et al. How good is automated protein docking? Proteins. 2013;81:2159–2166. doi: 10.1002/prot.24403. PubMed DOI PMC

Kozakov D, Brenke R, Comeau SR, Vajda S. PIPER: an FFT-based protein docking program with pairwise potentials. Proteins. 2006;65:392–406. doi: 10.1002/prot.21117. PubMed DOI

Schrodinger, L. L. C. The PyMOL Molecular Graphics System, Version 2.1 (2015).

Role of Integrins in Sperm Activation and Fertilization

αV Integrin Expression and Localization in Male Germ Cells