The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs

Abstract

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time1. Recently, CD9 on the egg membrane was found to be essential for fusion2,3,4, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody5 and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

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Figure 1: Identification and characterization of Izumo.
Figure 2: Targeted disruption of Izumo gene.
Figure 3: Male infertility caused by Izumo disruption.
Figure 4: Involvement of Izumo in a xeno-species fusion system.

References

  1. 1

    Stein, K. K., Primakoff, P. & Myles, D. Sperm–egg fusion: events at the plasma membrane. J. Cell Sci. 117, 6269–6274 (2004)

    CAS  Article  Google Scholar 

  2. 2

    Miyado, K. et al. Requirement of CD9 on the egg plasma membrane for fertilization. Science 287, 321–324 (2000)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Le Naour, F., Rubinstein, E., Jasmin, C., Prenant, M. & Boucheix, C. Severely reduced female fertility in CD9-deficient mice. Science 287, 319–321 (2000)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Kaji, K. et al. The gamete fusion process is defective in eggs of CD9-deficient mice. Nature Genet. 24, 279–282 (2000)

    CAS  Article  Google Scholar 

  5. 5

    Okabe, M. et al. Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro . J. Reprod. Immunol. 11, 91–100 (1987)

    CAS  Article  Google Scholar 

  6. 6

    Inoue, N. et al. Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm. Mol. Cell. Biol. 23, 2614–2622 (2003)

    CAS  Article  Google Scholar 

  7. 7

    Nishimura, H., Kim, E., Nakanishi, T. & Baba, T. Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface. J. Biol. Chem. 279, 34957–34962 (2004)

    CAS  Article  Google Scholar 

  8. 8

    Cho, C. et al. Fertilization defects in sperm from mice lacking fertilin beta. Science 281, 1857–1859 (1998)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Saxena, D. K., Oh-Oka, T., Kadomatsu, K., Muramatsu, T. & Toshimori, K. Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice. Reproduction 123, 435–444 (2002)

    CAS  Article  Google Scholar 

  10. 10

    Bookbinder, L. H., Cheng, A. & Bleil, J. D. Tissue- and species-specific expression of sp56, a mouse sperm fertilization protein. Science 269, 86–89 (1995)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Hagaman, J. R. et al. Angiotensin-converting enzyme and male fertility. Proc. Natl Acad. Sci. USA 95, 2552–2557 (1998)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Ikawa, M. et al. Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility. Dev. Biol. 240, 254–261 (2001)

    CAS  Article  Google Scholar 

  13. 13

    Yamagata, K. et al. Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane. Dev. Biol. 250, 348–357 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Yanagimachi, R. Mammalian Fertilization (eds Knobil, E & Neill, J. D.) (Raven, New York, 1994)

    Google Scholar 

  15. 15

    Manandhar, G. & Toshimori, K. Exposure of sperm head equatorin after acrosome reaction and its fate after fertilization in mice. Biol. Reprod. 65, 1425–1436 (2001)

    CAS  Article  Google Scholar 

  16. 16

    Kimura, Y. & Yanagimachi, R. Intracytoplasmic sperm injection in the mouse. Biol. Reprod. 52, 709–720 (1995)

    CAS  Article  Google Scholar 

  17. 17

    Yanagimachi, R., Yanagimachi, H. & Rogers, B. J. The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod. 15, 471–476 (1976)

    CAS  Article  Google Scholar 

  18. 18

    Olson, E. N., Arnold, H. H., Rigby, P. W. & Wold, B. J. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85, 1–4 (1996)

    CAS  Article  Google Scholar 

  19. 19

    Rochwerger, L., Cohen, D. J. & Cuasnicu, P. S. Mammalian sperm–egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion. Dev. Biol. 153, 83–90 (1992)

    CAS  Article  Google Scholar 

  20. 20

    Anderson, D. J., Abbott, A. F. & Jack, R. M. The role of complement component C3b and its receptors in sperm–oocyte interaction. Proc. Natl Acad. Sci. USA 90, 10051–10055 (1993)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Ilayperuma, I. Identification of the 48-kDa G11 protein from guinea pig testes as sperad. J. Exp. Zool. 293, 617–623 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Hao, Z. et al. SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein. Biol. Reprod. 66, 735–744 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Blobel, C. P. et al. A potential fusion peptide and an integrin ligand domain in a protein active in sperm–egg fusion. Nature 356, 248–252 (1992)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Nishimura, H., Cho, C., Branciforte, D. R., Myles, D. G. & Primakoff, P. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta. Dev. Biol. 233, 204–213 (2001)

    CAS  Article  Google Scholar 

  25. 25

    He, Z. Y. et al. None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm–egg binding and fusion. Dev. Biol. 254, 226–237 (2003)

    CAS  Article  Google Scholar 

  26. 26

    Ellerman, D. A., Ha, C., Primakoff, P., Myles, D. G. & Dveksler, G. S. Direct binding of the ligand PSG17 to CD9 requires a CD9 site essential for sperm–egg fusion. Mol. Biol. Cell 14, 5098–5103 (2003)

    CAS  Article  Google Scholar 

  27. 27

    Inoue, N. et al. A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization. J. Immunol. 166, 424–431 (2001)

    CAS  Article  Google Scholar 

  28. 28

    Toyoda, Y., Yokoyama, M. & Hoshi, T. Studies on the fertilization of mouse egg in vitro . Jpn. J. Anim. Reprod. 16, 147–151 (1971)

    Article  Google Scholar 

  29. 29

    Overstreet, J. W., Yanagimachi, R., Katz, D. F., Hayashi, K. & Hanson, F. W. Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil. Steril. 33, 534–542 (1980)

    CAS  Article  Google Scholar 

  30. 30

    Nakanishi, T. et al. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett. 449, 277–283 (1999)

    CAS  Article  Google Scholar 

  31. 31

    Okabe, M. et al. A human sperm antigen possibly involved in binding and/or fusion with zona-free hamster eggs. Fertil. Steril. 54, 1121–1126 (1990)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank K. Toshimori for providing anti-MN9 antibody; K. Yamagata for discussions; G. L. Gerton and S. Moss for critically reviewing the draft; and Y. Maruyama, A. Kawai and Y. Koreeda for technical assistance with gene disruption. This work was supported by grant-in-aid for Scientific Research and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Correspondence to Masaru Okabe.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

a. Transgene to express mouse Izumo under the control of calmegin promoter. The locations of primers A to E were indicated in this figure. b. lane 1; Izumo +/- mouse with intrinsic Izumo, lane 2 and 3; Izumo -/- mouse with transgenically expressed Izumo and Izumo His-tag, respectively. c. Litter size obtained by mating male mice with C57BL/6 wild-type mice. The group numbers are equal to those shown in b. The numbers in parentheses indicate the numbers of matings. (PPT 11631 kb)

41586_2005_BFnature03362_MOESM2_ESM.mov

First half. Mouse eggs 6 hours after insemination of Izumo +/- sperm. The eggs were fertilized and at the beginning of pronuclear stage after extruded their second polar body. Note that excess sperm bound to zona pellucida were already detached from probably due to the zona reaction. Second half. Mouse eggs 6 hours after insemination of Izumo -/- sperm. Unlike Izumo +/-, the eggs inseminated with Izumo -/- sperm remained unfertilized without extrusion of their second polar body and had many sperm on their zona pellucida, probably due to the absence of zona-reaction. Note that the Izumo -/- sperm has normal zona-binding ability and normal motility as wild-type sperm. (MOV 3356 kb)

Supplementary Movie

First half. Mouse eggs 6 hours after insemination of Izumo +/- sperm. The eggs were fertilized and at the beginning of pronuclear stage after extruded their second polar body. Note that excess sperm bound to zona pellucida were already detached from probably due to the zona reaction. Second half. Mouse eggs 6 hours after insemination of Izumo -/- sperm. Unlike Izumo +/-, the eggs inseminated with Izumo -/- sperm remained unfertilized without extrusion of their second polar body and had many sperm on their zona pellucida, probably due to the absence of zona-reaction. Note that the Izumo -/- sperm has normal zona-binding ability and normal motility as wild-type sperm. (MOV 3356 kb)

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Inoue, N., Ikawa, M., Isotani, A. et al. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434, 234–238 (2005). https://doi.org/10.1038/nature03362

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