Fertilization 1: Sperm–Egg Interaction

Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1001)


In birds in the reproductive season, an egg is ovulated without cumulus cells from the largest follicle with the highest hierarchy in the ovary. The outermost part of the ovulated eggs is the perivitelline layer, a glycoprotein matrix consisting of a few ZP-glycoproteins. The fertilization starts from sperm penetration of the perivitelline layer predominantly in the germinal disc region, followed by uptake of the sperm into the egg, and goes through by the fusion of sperm male pronucleus with the female pronucleus in the egg. A series of these fertilization steps occurs in the infundibulum of the oviduct within a short period after ovulation. Some pioneering microstructural studies using electron microscopy and supporting biochemical data from later studies indicate that, in avian fertilization, sperm interacts with the perivitelline layer covering the germinal disc, locally degrade and dissolve the matrix of the perivitelline layer, and penetrate it through the hole made proteolytically at the sperm-binding site on the perivitelline layer. Several molecules and structures presumably involved in the sperm–perivitelline interaction have been characterized, especially sperm proteases and their targets in the egg perivitelline layer. On the other hand, no molecules involved in the sperm–egg membrane fusion for the male pronucleus uptake into the egg have yet been identified or characterized and, moreover, no orthologue but one have been annotated so far in the chicken genome for the mouse genes involved in the sperm–egg membrane fusion.


Stigma Perivitelline layer Granulosa cell Infundibulum Acrosome reaction Acrosin Germinal disc Membrane fusion 


  1. Bakst MR, Howarth B Jr. Hydrolysis of the hen’s perivitelline layer by cock sperm in vitro. Biol Reprod. 1977;17:370–9.CrossRefPubMedGoogle Scholar
  2. Bansal P, Chakrabarti K, Gupta SK. Functional activity of human ZP3 primary sperm receptor resides toward its C-terminus. Biol Reprod. 2009;81:7–15.CrossRefPubMedGoogle Scholar
  3. Bausek N, Waclawek M, Schneider WJ, Wohlrab F. The major chicken egg envelope protein ZP1 is different from ZPB and is synthesized in the liver. J Biol Chem. 2000;275:28866–72.CrossRefPubMedGoogle Scholar
  4. Bausek N, Ruckenbauer HH, Pfeifer S, Schneider WJ, Wohlrab F. Interaction of sperm with purified native chicken ZP1 and ZPC proteins. Biol Reprod. 2004;71:684–90.CrossRefPubMedGoogle Scholar
  5. Beebe SJ, Leyton L, Burks D, Ishikawa M, Fuerst T, Dean J, Saling P. Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction. Dev Biol. 1992;151:48–54.CrossRefPubMedGoogle Scholar
  6. Bellairs R, Harkness ML, Harkness RD. The vitelline membrane of the hen’s egg: a chemical and ultrastructural study. J Ultrastruct Res. 1963;8:339–59.CrossRefGoogle Scholar
  7. Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature. 2014;508:483–7.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Birkhead TR, Sheldon BC, Fletcher F. A comparative study of sperm–egg interactions in birds. J Reprod Fertil. 1994;101:353–61.CrossRefPubMedGoogle Scholar
  9. Bleil JD, Wassarman PM. Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte’s zona pellucida. Dev Biol. 1980;76:185–202.CrossRefPubMedGoogle Scholar
  10. Bleil JD, Wassarman PM. Sperm–egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol. 1983;95:317–24.CrossRefPubMedGoogle Scholar
  11. Bleil JD, Greve JM, Wassarman PM. Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. Dev Biol. 1988;128:376–85.CrossRefPubMedGoogle Scholar
  12. Bramwell RK, Howarth B Jr. Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen’s egg. Biol Reprod. 1992;47:1113–7.CrossRefPubMedGoogle Scholar
  13. Bramwell RK, Marks HL, Howarth B. Quantitative determination of spermatozoa penetration of the perivitelline layer of the hen’s egg as assessed on oviposited eggs. Poult Sci. 1995;74:1875–83.CrossRefPubMedGoogle Scholar
  14. Brown CR, Cheng WT. Limited proteolysis of the porcine zona pellucida by homologous sperm acrosin. J Reprod Fertil. 1985;74:257–60.CrossRefPubMedGoogle Scholar
  15. Buruiana LM. Sur l’activité hyaluronidasique et trypsinique du sperme. Naturwissenschaften. 1956;43:523.CrossRefGoogle Scholar
  16. Chakravarty S, Suraj K, Gupta SK. Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C. Mol Hum Reprod. 2005;11:365–72.CrossRefPubMedGoogle Scholar
  17. Chakravarty S, Kadunganattil S, Bansal P, Sharma RK, Gupta SK. Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis. Mol Reprod Dev. 2008;75:75–88.CrossRefPubMedGoogle Scholar
  18. Dunbar BS, Dudkiewicz AB, Bundman DS. Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin. Biol Reprod. 1985;32:619–30.CrossRefPubMedGoogle Scholar
  19. Fronda FM. Studies on the fertility of the hen’s egg. Philippine Agr. 1926;15:349–60.Google Scholar
  20. Ganguly A, Bukovsky A, Sharma RK, Bansal P, Bhandari B, Gupta SK. In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis. Hum Reprod. 2010;25:1643–56.CrossRefPubMedGoogle Scholar
  21. Gilbert AB. Female genital organs. In: King AS, McLelland J, editors. Form and function in birds, vol. 1. London: Academic Press; 1979.Google Scholar
  22. Grayson P. Izumo1 and Juno: the evolutionary origins and coevolution of essential sperm–egg binding partners. R Soc Open Sci. 2015;2:150296.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Han L, Monné M, Okumura H, Schwend T, Cherry AL, Flot D, Matsuda T, Jovine L. Insights into egg coat assembly and egg–sperm interaction from the X-ray structure of full-length ZP3. Cell. 2010;143:404–15.CrossRefPubMedGoogle Scholar
  24. Ho JJ, Meizel S. Electrophoretic detection of multiple forms of trypsin-like activity in spermatozoa of the domestic fowl. J Reprod Fertil. 1970;23:177–9.CrossRefPubMedGoogle Scholar
  25. Ho JJ, Meizel S. Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes. J Exp Zool. 1975;194:429–37.CrossRefPubMedGoogle Scholar
  26. Horrocks AJ, Stewart S, Jackson L, Wishart GJ. Induction of acrosomal exocytosis in chicken spermatozoa by inner perivitelline-derived N-linked glycans. Biochem Biophys Res Commun. 2000;278:84–9.CrossRefPubMedGoogle Scholar
  27. Howarth B. Avian sperm–egg interaction: perivitelline layer possesses receptor activity for spermatozoa. Poult Sci. 1990;69:1012–5.CrossRefPubMedGoogle Scholar
  28. Howarth B. Carbohydrate involvement in sperm–egg interaction in the chicken. J Recept Res. 1992;12:255–65.CrossRefPubMedGoogle Scholar
  29. Howarth B Jr, Digby ST. Evidence for the penetration of the vitelline membrane of the hen’s egg by a trypsin-like acrosomal enzyme. J Reprod Fertil. 1973;33:123–5.CrossRefPubMedGoogle Scholar
  30. Inoue N, Ikawa M, Isotani A, Okabe M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature. 2005;434:234–8.CrossRefPubMedGoogle Scholar
  31. Ivanoff EJ. Compt Rend Soc Biol. 1913;75:371–4.Google Scholar
  32. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K, Hirohashi N. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci U S A. 2011;108:4892–6.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Johnson AL. Reproduction in the female. In: Sturkie PD, editor. Avian physiology. 4th ed. New York: Springer-Verlag; 1986. p. 403–31.CrossRefGoogle Scholar
  34. Koyanagi F, Masuda S, Nishiyama H. Acrosome reaction of cock spermatozoa incubated with the perivitelline layer of the hen’s egg. Poult Sci. 1988;67:1770–4.CrossRefPubMedGoogle Scholar
  35. Kuroki M, Mori M. Binding of spermatozoa to the perivitelline layer in the presence of a protease inhibitor. Poult Sci. 1997;76:748–52.CrossRefPubMedGoogle Scholar
  36. La Spina FA, Puga Molina LC, Romarowski A, Vitale AM, Falzone TL, Krapf D, Hirohashi N, Buffone MG. Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct. Dev Biol. 2016;411:172–82.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Martin JH, Anderson WB. J Am Assoc Instr Invest Poultry Husbandry. 1918;5:22–3.Google Scholar
  38. Mimura H. On the mechanism of travel of spermatozoa through the oviduct in the domestic fowl, with special reference to the artificial insemination. Okajimas Folia Anat Jpn. 1939;17:459–76.CrossRefGoogle Scholar
  39. Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F, Suzuki K, Kosai K, Inoue K, Ogura A, Okabe M, Mekada E. Requirement of CD9 on the egg plasma membrane for fertilization. Science. 2000;287:321–4.CrossRefPubMedGoogle Scholar
  40. Nishio S, Kohno Y, Iwata Y, Arai M, Okumura H, Oshima K, Nadano D, Matsuda T. Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs. Biol Reprod. 2014;91:107.CrossRefPubMedGoogle Scholar
  41. Okamura F, Nishiyama H. The passage of spermatozoa through the vitelline membrane in the domestic fowl, Gallus gallus. Cell Tissue Res. 1978a;188:497–508.CrossRefPubMedGoogle Scholar
  42. Okamura F, Nishiyama H. Penetration of spermatozoon into the egg and transformation of the sperm nucleus into the male pronucleus in the domestic fowl, Gallus gallus. Cell Tissue Res. 1978b;190:89–98.CrossRefPubMedGoogle Scholar
  43. Okumura H, Kohno Y, Iwata Y, Mori H, Aoki N, Sato C, Kitajima K, Nadano D, Matsuda T. A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm–egg interaction. Biochem J. 2004;384:191–9.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Olsen MW. Maturation, fertilization, and early cleavage in the hen’s egg. J Morphol. 1942;70:513–33.CrossRefGoogle Scholar
  45. Olsen MW. Intra-ovarian insemination in the domestic fowl. J Exp Zool. 1952;119:461–81.CrossRefGoogle Scholar
  46. Olsen MW, Fraps RM. Maturation, fertilization, and early cleavage of the egg of the domestic turkey. J Morphol. 1944;74:297–309.CrossRefGoogle Scholar
  47. Pan J, Sasanami T, Kono Y, Matsuda T, Mori M. Effects of testosterone on production of perivitelline membrane glycoprotein ZPC by granulosa cells of Japanese quail (Coturnix japonica). Biol Reprod. 2001;64:310–6.CrossRefPubMedGoogle Scholar
  48. Robertson L, Wishart GJ, Horrocks AJ. Identification of perivitelline N-linked glycans as mediators of sperm–egg interaction in chickens. J Reprod Fertil. 2000;120:397–403.PubMedGoogle Scholar
  49. Romanoff AL. Fertilization and fertility. In:The avian embryo: structural and functional development. New York: Macmillan; 1960.Google Scholar
  50. Romanoff AL, Romanoff AJ. The avian egg. New York: John Wiley & Sons; 1949.Google Scholar
  51. Sasanami T, Pan J, Mori M. Expression of perivitelline membrane glycoprotein ZP1 in the liver of Japanese quail (Coturnix japonica) after in vivo treatment with diethylstilbestrol. J Steroid Biochem Mol Biol. 2003;84:109–16.CrossRefPubMedGoogle Scholar
  52. Sasanami T, Murata T, Ohtsuki M, Matsushima K, Hiyama G, Kansaku N, Mori M. Induction of sperm acrosome reaction by perivitelline membrane glycoprotein ZP1 in Japanese quail (Coturnix japonica). Reproduction. 2007;133:41–9.Google Scholar
  53. Sasanami T, Yoshizaki N, Dohra H, Kubo H. Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica). Reproduction. 2011;142:267–76.CrossRefPubMedGoogle Scholar
  54. Sasanami T, Sugiura K, Tokumoto T, Yoshizaki N, Dohra H, Nishio S, Mizushima S, Hiyama G, Matsuda T. Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica). Reproduction. 2012;144:423–31.CrossRefPubMedGoogle Scholar
  55. Słowińska M, Ciereszko A. Identification of the second form of acrosin in Turkey spermatozoa. Reprod Domest Anim. 2012;47:849–55.CrossRefPubMedGoogle Scholar
  56. Słowińska M, Olczak M, Liszewska E, Watorek W, Ciereszko A. Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa. Comp Biochem Physiol B Biochem Mol Biol. 2010;157:127–36.CrossRefPubMedGoogle Scholar
  57. Stambaugh R, Buckley J. Zona pellucida dissolution enzymes of the rabbit sperm head. Science. 1968;161:585–6.CrossRefPubMedGoogle Scholar
  58. Steele MG, Meldrum W, Brillard JP, Wishart GJ. The interaction of avian spermatozoa with the perivitelline layer in vitro and in vivo. J Reprod Fertil. 1994;101:599–603.CrossRefPubMedGoogle Scholar
  59. Stewart SG, Bausek N, Wohlrab F, Schneider WJ, Janet Horrocks A, Wishart GJ. Species specificity in avian sperm: perivitelline interaction. Comp Biochem Physiol A Mol Integr Physiol. 2004;137:657–63.CrossRefPubMedGoogle Scholar
  60. Takeuchi Y, Nishimura K, Aoki N, Adachi T, Sato C, Kitajima K, Matsuda T. A 42-kDa glycoprotein from chicken egg-envelope, an avian homolog of the ZPC family glycoproteins in mammalian Zona pellucida. Its first identification, cDNA cloning and granulosa cell-specific expression. Eur J Biochem. 1999;260:736–42.CrossRefPubMedGoogle Scholar
  61. Takeuchi Y, Cho R, Iwata Y, Nishimura K, Kato T, Aoki N, Kitajima K, Matsuda T. Morphological and biochemical changes of isolated chicken egg-envelope during sperm penetration: degradation of the 97-kilodalton glycoprotein is involved in sperm-driven hole formation on the egg-envelope. Biol Reprod. 2001;64:822–30.CrossRefPubMedGoogle Scholar
  62. Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A. Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev. 2002;61:414–24.CrossRefPubMedGoogle Scholar
  63. Toshimori K, Saxena DK, Tanii I, Yoshinaga K. An MN9 antigenic molecule, equatorin, is required for successful sperm–oocyte fusion in mice. Biol Reprod. 1998;59:22–9.CrossRefPubMedGoogle Scholar
  64. Urch UA, Wardrip NJ, Hedrick JL. Limited and specific proteolysis of the zona pellucida by acrosin. J Exp Zool. 1985a;233:479–83.CrossRefPubMedGoogle Scholar
  65. Urch UA, Wardrip NJ, Hedrick JL. Proteolysis of the zona pellucida by acrosin: the nature of the hydrolysis products. J Exp Zool. 1985b;236:239–43.CrossRefPubMedGoogle Scholar
  66. Waclawek M, Foisner R, Nimpf J, Schneider WJ. The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells. Biol Reprod. 1998;59:1230–9.CrossRefPubMedGoogle Scholar
  67. Wishart GJ. Quantitative aspects of sperm: egg interaction in chickens and turkeys. Anim Reprod Sci. 1997;48:81–92.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Department of Applied Molecular BiosciencesGraduate School of Bioagricultural Sciences, Nagoya UniversityNagoyaJapan

Personalised recommendations