Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources University of Life Sciences Prague Prague Czechia

Department of Zoology Faculty of Science Charles University Prague Czechia

Imaging Methods Core Facility at BIOCEV Faculty of Science Charles University Vestec Czechia

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czechia

Light Microscopy Core Facility Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

Zobrazit více v PubMed

Alam M. S. (2018). Proximity ligation assay (PLA). Curr. Protoc. Immunol. 123 (1), e58. 10.1002/cpim.58 PubMed DOI PMC

Andreu Z., Yáñez-Mó M. (2014). Tetraspanins in extracellular vesicle formation and function. Front. Immunol. 5, 442. 10.3389/fimmu.2014.00442 PubMed DOI PMC

Benammar A., Ziyyat A., Lefèvre B., Wolf J. P. (2017). Tetraspanins and mouse oocyte microvilli related to fertilizing ability. Reprod. Sci. 24 (7), 1062–1069. 10.1177/1933719116678688 PubMed DOI

Bianchi E., Wright G. J. (2016). Sperm meets egg: the Genetics of mammalian fertilization. Annu. Rev. Genet. 50, 93–111. 10.1146/annurev-genet-121415-121834 PubMed DOI

Bianchi E., Wright G. J. (2020). Find and fuse: unsolved mysteries in sperm–egg recognition. PLOS Biol. 18 (11), e3000953. 10.1371/journal.pbio.3000953 PubMed DOI PMC

Bianchi E., Doe B., Goulding D., Wright G. J. (2014). Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508 (7497), 483–487. 10.1038/nature13203 PubMed DOI PMC

Bogan A. A., Thorn K. S. (1998). Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280 (1), 1–9. 10.1006/jmbi.1998.1843 PubMed DOI

Brock K., Talley K., Coley K., Kundrotas P., Alexov E. (2007). Optimization of electrostatic interactions in protein-protein complexes. Biophys. J. 93 (10), 3340–3352. 10.1529/biophysj.107.112367 PubMed DOI PMC

Chalbi M., Barraud-Lange V., Ravaux B., Howan K., Rodriguez N., Soule P., et al. (2014). Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization. Development 141 (19), 3732–3739. 10.1242/dev.111534 PubMed DOI

de Vries S. J., van Dijk M., Bonvin A. M. J. J. (2010). The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5 (5), 883–897. 10.1038/nprot.2010.32 PubMed DOI

Demirel M. C., Keskin O. (2005). Protein interactions and fluctuations in a proteomic network using an elastic network model. J. Biomol. Struct. Dyn. 22 (4), 381–386. 10.1080/07391102.2005.10507010 PubMed DOI

Erijman M., Rosenthal E., Shifman J. M. (2014). How structure defines affinity in protein-protein interactions. PLoS ONE 9 (10), e110085. 10.1371/journal.pone.0110085 PubMed DOI PMC

Frolikova M., Manaskova-Postlerova P., Cerny J., Jankovicova J., Simonik O., Pohlova A., et al. (2018). CD9 and CD81 interactions and their structural modelling in sperm prior to fertilization. Int. J. Mol. Sci. 19 (4), 1236. 10.3390/ijms19041236 PubMed DOI PMC

Frolikova M., Blazikova M., Capek M., Chmelova H., Valecka J., Kolackova V., et al. (2023). A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte. bioRxiv, 531472. 10.1101/2023.03.07.531472 DOI

Grigoryan G., Keating A. E. (2008). Structural specificity in coiled-coil interactions. Curr. Opin. Struct. Biol. 18 (4), 477–483. 10.1016/j.sbi.2008.04.008 PubMed DOI PMC

Grünberg R., Nilges M., Leckner J. (2006). Flexibility and conformational entropy in protein-protein binding. Structure 14 (4), 683–693. 10.1016/j.str.2006.01.014 PubMed DOI

Harris T. K., Mildvan A. S. (1999). High-precision measurement of hydrogen bond lengths in proteins by nuclear magnetic resonance methods. Proteins 35 (3), 275–282. 10.1002/(sici)1097-0134(19990515)35:3<275::aid-prot1>3.0.co;2-v PubMed DOI

Inoue N., Ikawa M., Isotani A., Okabe M. (2005). The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434 (7030), 234–238. 10.1038/nature03362 PubMed DOI

Inoue N., Saito T., Wada I. (2020). Unveiling a novel function of CD9 in surface compartmentalization of oocytes. Development 147 (15), dev189985. 10.1242/dev.189985 PubMed DOI

Jankovičová J., Neuerová Z., Sečová P., Bartóková M., Bubeníčková F., Komrsková K., et al. (2020). Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos. Med. Microbiol. Immunol. 209 (4), 407–425. 10.1007/s00430-020-00676-0 PubMed DOI

Jégou A., Ziyyat A., Barraud-Lange V., Perez E., Wolf J. P., Pincet F., et al. (2011). CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization. Proc. Natl. Acad. Sci. U. S. A. 108 (27), 10946–10951. 10.1073/pnas.1017400108 PubMed DOI PMC

Kaji K., Oda S., Shikano T., Ohnuki T., Uematsu Y., Sakagami J., et al. (2000). The gamete fusion process is defective in eggs of Cd9-deficient mice. Nat. Genet. 24 (3), 279–282. 10.1038/73502 PubMed DOI

Kozakov D., Hall D. R., Xia B., Porter K. A., Padhorny D., Yueh C., et al. (2017). The ClusPro web server for protein–protein docking. Nat. Protoc. 12 (2), 255–278. 10.1038/nprot.2016.169 PubMed DOI PMC

Laskowski R. A., Macarthur M. W., Moss D. S., Thornton J. M. (1993). Procheck - a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291. 10.1107/S0021889892009944 DOI

Lee J., Patel D. S., Ståhle J., Park S. J., Kern N. R., Kim S., et al. (2019). CHARMM-GUI membrane builder for complex biological membrane simulations with glycolipids and lipoglycans. J. Chem. Theory. Comput. 15 (1), 775–786. 10.1021/acs.jctc.8b01066 PubMed DOI

Le Naour F., Rubinstein E., Jasmin C., Prenant M., Boucheix C. (2000). Severely reduced female fertility in CD9-deficient mice. Science 287 (5451), 319–321. 10.1126/science.287.5451.319 PubMed DOI

Macindoe G., Mavridis L., Venkatraman V., Devignes M.-D., Ritchie D. W. (2010). HexServer: an FFT-based protein docking server powered by graphics processors. Nucleic Acids Res. 38 (2), W445–W449. 10.1093/nar/gkq311 PubMed DOI PMC

Miyado K., Yamada G., Yamada S., Hasuwa H., Nakamura Y., Ryu F., et al. (2000). Requirement of CD9 on the egg plasma membrane for fertilization. Science 287 (5451), 321–324. 10.1126/science.287.5451.321 PubMed DOI

Miyado K., Yoshida K., Yamagata K., Sakakibara K., Okabe M., Wang X., et al. (2008). The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice. Proc. Natl. Acad. Sci. U. S. A. 105 (35), 12921–12926. 10.1073/pnas.0710608105 PubMed DOI PMC

Mori M., Yao T., Mishina T., Endoh H., Tanaka M., Yonezawa N., et al. (2021). RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization. J. Cell. Biol. 220 (10), e202012001. 10.1083/jcb.202012001 PubMed DOI PMC

Pierce B. G., Wiehe K., Hwang H., Kim B.-H., Vreven T., Weng Z. (2014). ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 30 (12), 1771–1773. 10.1093/bioinformatics/btu097 PubMed DOI PMC

Runge K. E., Evans J. E., He Z. Y., Gupta S., McDonald K. L., Stahlberg H., et al. (2007). Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution. Dev. Biol. 304 (1), 317–325. 10.1016/j.ydbio.2006.12.041 PubMed DOI

Sala-Valdés M., Ursa A., Charrin S., Rubinstein E., Hemler M. E., Sánchez-Madrid F., et al. (2006). EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins. J. Biol. Chem. 281 (28), 19665–19675. 10.1074/jbc.M602116200 PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9 (7), 676–682. 10.1038/nmeth.2019 PubMed DOI PMC

Shalgi R., Phillips D. M. (1980). Mechanics of in vitro fertilization in the hamster. Biol. Reprod. 23 (2), 433–444. 10.1095/biolreprod23.2.433 PubMed DOI

Shashikala H. B. M., Chakravorty A., Alexov E. (2019). Modeling electrostatic force in protein-protein recognition. Front. Mol. Biosci. 6, 94. 10.3389/fmolb.2019.00094 PubMed DOI PMC

Siu K. K., Serrão V. H. B., Ziyyat A., Lee J. E. (2021). The cell biology of fertilization: gamete attachment and fusion. J. Cell. Biol. 220 (10), e202102146. 10.1083/jcb.202102146 PubMed DOI PMC

Sur V. P., Simonik O., Novotna M., Mazumdar A., Liska F., Vimberg V., et al. (2022). Dynamic study of small toxic hydrophobic proteins PepA1 and PepG1 of Staphylococcus aureus . Int. J. Biol. Macromol. 219, 1360–1371. 10.1016/j.ijbiomac.2022.07.192 PubMed DOI

Tobi D., Bahar I. (2005). Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state. Proc. Natl. Acad. Sci. 102 (52), 18908–18913. 10.1073/pnas.0507603102 PubMed DOI PMC

Umeda R., Satouh Y., Takemoto M., Nakada-Nakura Y., Liu K., Yokoyama T., et al. (2020). Structural insights into tetraspanin CD9 function. Nat. Commun. 11 (1), 1606. 10.1038/s41467-020-15459-7 PubMed DOI PMC

Vondrakova J., Frolikova M., Ded L., Cerny J., Postlerova P., Palenikova V., et al. (2022). MAIA, Fc receptor-like 3, supersedes JUNO as IZUMO1 receptor during human fertilization. Sci. Adv. 8 (36), eabn0047. 10.1126/sciadv.abn0047 PubMed DOI PMC

Vriend G. (1990). What if: a molecular modeling and drug design program. J. Mol. Graph. 8 (1), 52. 10.1016/0263-7855(90)80070-V PubMed DOI

Vyas V. K., Ukawala R. D., Ghate M., Chintha C. (2012). Homology modeling a fast tool for drug discovery: current perspectives. Indian J. Pharm. Sci. 74 (1), 1–17. 10.4103/0250-474x.102537 PubMed DOI PMC

Wang H., Kinsey W. H. (2022). Signaling proteins recruited to the sperm binding site: role of β-catenin and rho A. Front. Cell. Dev. Biol. 10, 886664. 10.3389/fcell.2022.886664 PubMed DOI PMC

Webb B., Sali A. (2016). Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinforma. 54 (1), 5.6.1–5.6.37. 10.1002/cpbi.3 PubMed DOI PMC

Yanagimachi R. (1994). “Mammalian fertilization,” in The physiology of reproduction. Editors Knobil E., Neill J. (New York: Raven Press; ).

Zhu G. Z., Miller B. J., Boucheix C., Rubinstein E., Liu C. C., Hynes R. O., et al. (2002). Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion. Development 129 (8), 1995–2002. 10.1242/dev.129.8.1995 PubMed DOI

Ziyyat A., Rubinstein E., Monier-Gavelle F., Barraud V., Kulski O., Prenant M., et al. (2006). CD9 controls the formation of clusters that contain tetraspanins and the integrin alpha 6 beta 1, which are involved in human and mouse gamete fusion. J. Cell. Sci. 119 (3), 416–424. 10.1242/jcs.02730 PubMed DOI

Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte