Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

Abstract

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

References

  1. Abeydeera LR, Wang WH, Prather RS, Day BN (1998) Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro. Biol Reprod 58(5):1316–1320

    Article  CAS  PubMed  Google Scholar 

  2. Baarends WM, Roest HP, Grootegoed JA (1999) The ubiquitin system in gametogenesis. Mol Cell Endocrinol 151(1–2):5–16

    Article  CAS  PubMed  Google Scholar 

  3. Baker MA, Hetherington L, Reeves G, Muller J, Aitken RJ (2008a) The rat sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification. Proteomics 8(11):2312–2321

    Article  CAS  PubMed  Google Scholar 

  4. Baker MA, Hetherington L, Reeves GM, Aitken RJ (2008b) The mouse sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification. Proteomics 8(8):1720–1730

    Article  CAS  PubMed  Google Scholar 

  5. Baska KM, Sutovsky P (2005) Protein modification by ubiquitination and is consequences for spermatogenesis, sperm maturation, fertilization and pre-implantation embryonic development. In: Tokumoto T (ed) New impact on protein modifications in the regulation of reproductive system. Research Signpost, Kerala, pp 83–114

    Google Scholar 

  6. Bebington C, Doherty FJ, Fleming SD (2001) The possible biological and reproductive functions of ubiquitin. Hum Reprod Update 7(1):102–111

    Article  CAS  PubMed  Google Scholar 

  7. Belote JM, Miller M, Smyth KA (1998) Evolutionary conservation of a testes-specific proteasome subunit gene in Drosophila. Gene 215(1):93–100

    Article  CAS  PubMed  Google Scholar 

  8. Berruti G, Martegani E (2002) mUBPy and MSJ-1, a deubiquitinating enzyme and a molecular chaperone specifically expressed in testis, associate with the acrosome and centrosome in mouse germ cells. Ann N Y Acad Sci 973:5–7

    Article  CAS  PubMed  Google Scholar 

  9. Berruti G, Martegani E (2005) The deubiquitinating enzyme mUBPy interacts with the sperm-specific molecular chaperone MSJ-1: the relation with the proteasome, acrosome, and centrosome in mouse male germ cells. Biol Reprod 72(1):14–21

    Article  CAS  PubMed  Google Scholar 

  10. Bialy LP, Ziemba HT, Marianowski P, Fracki S, Bury M, Wojcik C (2001) Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study. Folia Histochem Cytobiol 39(2):129–130

    CAS  PubMed  Google Scholar 

  11. Brooks DE (1978) Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat. Biochem J 174(3):741–752

    CAS  PubMed  Google Scholar 

  12. Chakravarty S, Bansal P, Sutovsky P, Gupta SK (2008) Role of proteasomal activity in the induction of acrosomal exocytosis in human spermatozoa. Reprod Biomed Online 16(3):391–400

    Article  CAS  PubMed  Google Scholar 

  13. Davidová N, Jonáková V, Manásková-Postlerová P (2009) Expression and localization of acrosin inhibitor in boar reproductive tract. Cell Tissue Res 338(2):303–311

    Google Scholar 

  14. Fischer DF, De Vos RA, Van Dijk R, De Vrij FM, Proper EA, Sonnemans MA, Verhage MC, Sluijs JA, Hobo B, Zouambia M, Steur EN, Kamphorst W, Hol EM, Van Leeuwen FW (2003) Disease-specific accumulation of mutant ubiquitin as a marker for proteasomal dysfunction in the brain. FASEB J 17(14):2014–2024

    Article  CAS  PubMed  Google Scholar 

  15. Fischer KA, Van Leyen K, Lovercamp KW, Manandhar G, Sutovsky M, Feng D, Safranski T, Sutovsky P (2005) 15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet. Reproduction 130(2):213–222

    Article  CAS  PubMed  Google Scholar 

  16. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2):373–428

    CAS  PubMed  Google Scholar 

  17. Guterman A, Glickman MH (2004) Deubiquitinating enzymes are IN/(trinsic to proteasome function). Curr Protein Pept Sci 5(3):201–211

    Article  CAS  PubMed  Google Scholar 

  18. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479

    Article  CAS  PubMed  Google Scholar 

  19. Jelinkova P, Manaskova P, Ticha M, Jonakova V (2003) Proteinase inhibitors in aggregated forms of boar seminal plasma proteins. Int J Biol Macromol 32(3–5):99–107

    Article  CAS  PubMed  Google Scholar 

  20. Jonakova V, Sanz L, Calvete JJ, Henschen A, Cechova D, Topfer-Petersen E (1991) Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa. FEBS Lett 280(1):183–186

    Article  CAS  PubMed  Google Scholar 

  21. Jonakova V, Calvete JJ, Mann K, Schafer W, Schmid ER, Topfer-Petersen E (1992) The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor. FEBS Lett 297(1–2):147–150

    Article  CAS  PubMed  Google Scholar 

  22. Jonakova V, Kraus M, Veselsky L, Cechova D, Bezouska K, Ticha M (1998) Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma. J Reprod Fertil 114(1):25–34

    Article  CAS  PubMed  Google Scholar 

  23. Kessler BM, Tortorella D, Altun M, Kisselev AF, Fiebiger E, Hekking BG, Ploegh HL, Overkleeft HS (2001) Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits. Chem Biol 8(9):913–929

    Article  CAS  PubMed  Google Scholar 

  24. Lam YA, Pickart CM, Alban A, Landon M, Jamieson C, Ramage R, Mayer RJ, Layfield R (2000) Inhibition of the ubiquitin-proteasome system in Alzheimer’s disease. Proc Natl Acad Sci USA 97(18):9902–9906

    Article  CAS  PubMed  Google Scholar 

  25. Lindsten K, de Vrij FM, Verhoef LG, Fischer DF, van Leeuwen FW, Hol EM, Masucci MG, Dantuma NP (2002) Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J Cell Biol 157(3):417–427

    Article  CAS  PubMed  Google Scholar 

  26. Morales P, Kong M, Pizarro E, Pasten C (2003) Participation of the sperm proteasome in human fertilization. Hum Reprod 18(5):1010–1017

    Article  CAS  PubMed  Google Scholar 

  27. Morales P, Pizarro E, Kong M, Jara M (2004) Extracellular localization of proteasomes in human sperm. Mol Reprod Dev 68(1):115–124

    Article  CAS  PubMed  Google Scholar 

  28. Oko R, Maravei D (1995) Distribution and possible role of perinuclear theca proteins during bovine spermiogenesis. Microsc Res Tech 32(6):520–532

    Article  CAS  PubMed  Google Scholar 

  29. Pasten C, Morales P, Kong M (2005) Role of the sperm proteasome during fertilization and gamete interaction in the mouse. Mol Reprod Dev 71(2):209–219

    Article  CAS  PubMed  Google Scholar 

  30. Rivkin E, Kierszenbaum AL, Gil M, Tres LL (2009) Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, tether to the acrosome membranes and the head-tail coupling apparatus during rat spermatid development. Dev Dyn 238(7):1851–1861

    Article  CAS  PubMed  Google Scholar 

  31. Saeki Y, Tanaka K (2008) Cell biology: two hands for degradation. Nature 453(7194):460–461

    Article  CAS  PubMed  Google Scholar 

  32. Saitoh Y, Sawada H, Yokosawa H (1993) High-molecular-weight protease complexes (proteasomes) of sperm of the ascidian, Halocynthia roretzi: isolation, characterization, and physiological roles in fertilization. Dev Biol 158(1):238–244

    Article  CAS  PubMed  Google Scholar 

  33. Sakai N, Sawada MT, Sawada H (2004) Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis. Int J Biochem Cell Biol 36(5):776–784

    Article  CAS  PubMed  Google Scholar 

  34. Sanz L, Calvete JJ, Jonakova V, Topfer-Petersen E (1992) Boar spermadhesins AQN-1 and AWN are sperm-associated acrosin inhibitor acceptor proteins. FEBS Lett 300(1):63–66

    Article  CAS  PubMed  Google Scholar 

  35. Sawada H, Yokosawa H, Hoshi M, Ishii S (1983) Ascidian sperm chymotrypsin-like enzyme; participation in fertilization. Experientia 39(4):377–378

    Article  CAS  PubMed  Google Scholar 

  36. Sawada H, Sakai N, Abe Y, Tanaka E, Takahashi Y, Fujino J, Kodama E, Takizawa S, Yokosawa H (2002a) Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor. Proc Natl Acad Sci USA 99(3):1223–1228

    Article  CAS  PubMed  Google Scholar 

  37. Sawada H, Takahashi Y, Fujino J, Flores SY, Yokosawa H (2002b) Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi. Mol Reprod Dev 62(2):271–276

    Article  CAS  PubMed  Google Scholar 

  38. Sixt SU, Dahlmann B (2008) Extracellular, circulating proteasomes and ubiquitin—incidence and relevance. Biochim Biophys Acta 1782(12):817–823

    CAS  PubMed  Google Scholar 

  39. Sixt SU, Beiderlinden M, Jennissen HP, Peters J (2007) Extracellular proteasome in the human alveolar space: a new housekeeping enzyme? Am J Physiol Lung Cell Mol Physiol 292(5):L1280–L1288

    Article  CAS  PubMed  Google Scholar 

  40. Sutovsky P (2003) Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 61(1):88–102

    Article  CAS  PubMed  Google Scholar 

  41. Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G (2000) Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol Reprod 63(2):582–590

    Article  CAS  PubMed  Google Scholar 

  42. Sutovsky P, McCauley TC, Sutovsky M, Day BN (2003) Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132. Biol Reprod 68(5):1793–1800

    Article  CAS  PubMed  Google Scholar 

  43. Sutovsky P, Manandhar G, McCauley TC, Caamano JN, Sutovsky M, Thompson WE, Day BN (2004a) Proteasomal interference prevents zona pellucida penetration and fertilization in mammals. Biol Reprod 71(5):1625–1637

    Article  CAS  PubMed  Google Scholar 

  44. Sutovsky P, Van Leyen K, McCauley T, Day BN, Sutovsky M (2004b) Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance. Reprod Biomed Online 8(1):24–33

    Article  CAS  PubMed  Google Scholar 

  45. van Leeuwen FW, de Kleijn DP, van den Hurk HH, Neubauer A, Sonnemans MA, Sluijs JA, Koycu S, Ramdjielal RD, Salehi A, Martens GJ, Grosveld FG, Peter J, Burbach H, Hol EM (1998) Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer’s and Down patients. Science 279(5348):242–247

    Article  PubMed  Google Scholar 

  46. Van Leeuwen FW, Hol EM, Hermanussen RW, Sonnemans MA, Moraal E, Fischer DF, Evans DA, Chooi KF, Burbach JP, Murphy D (2000) Molecular misreading in non-neuronal cells. FASEB J 14(11):1595–1602

    Article  PubMed  Google Scholar 

  47. van Leeuwen FW, Kros JM, Kamphorst W, van Schravendijk C, de Vos RA (2006a) Molecular misreading: the occurrence of frameshift proteins in different diseases. Biochem Soc Trans 34(Pt 5):738–742

    PubMed  Google Scholar 

  48. van Leeuwen FW, van Tijn P, Sonnemans MA, Hobo B, Mann DM, Van Broeckhoven C, Kumar-Singh S, Cras P, Leuba G, Savioz A, Maat-Schieman ML, Yamaguchi H, Kros JM, Kamphorst W, Hol EM, de Vos RA, Fischer DF (2006b) Frameshift proteins in autosomal dominant forms of Alzheimer disease and other tauopathies. Neurology 66(2 Suppl 1):S86–S92

    PubMed  Google Scholar 

  49. van Tijn P, de Vrij FM, Schuurman KG, Dantuma NP, Fischer DF, van Leeuwen FW, Hol EM (2007) Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease. J Cell Sci 120(Pt 9):1615–1623

    Article  PubMed  Google Scholar 

  50. Veselsky L, Jonakova V, Sanz ML, Topfer-Petersen E, Cechova D (1992) Binding of a 15 kDa glycoprotein from spermatozoa of boars to surface of zona pellucida and cumulus oophorus cells. J Reprod Fertil 96(2):593–602

    Article  CAS  PubMed  Google Scholar 

  51. Voges D, Zwickl P, Baumeister W (1999) The 26 S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068

    Article  CAS  PubMed  Google Scholar 

  52. Wilkinson CR, Ferrell K, Penney M, Wallace M, Dubiel W, Gordon C (2000) Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised. J Biol Chem 275(20):15182–15192

    Article  CAS  PubMed  Google Scholar 

  53. Wojcik C, Benchaib M, Lornage J, Czyba JC, Guerin JF (2000) Proteasomes in human spermatozoa. Int J Androl 23(3):169–177

    Article  CAS  PubMed  Google Scholar 

  54. Yi YJ, Manandhar G, Sutovsky M, Li R, Jonakova V, Oko R, Park CS, Prather RS, Sutovsky P (2007) Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization. Biol Reprod 77(5):780–793

    Article  CAS  PubMed  Google Scholar 

  55. Yi YJ, Manandhar G, Sutovsky M, Jonakova V, Park CS, Sutovsky P (2010) Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro. J Reprod Immunol 84(2):154–163

    Article  CAS  PubMed  Google Scholar 

  56. Yokota N, Sawada H (2007) Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus. Dev Biol 308(1):222–231

    Article  CAS  PubMed  Google Scholar 

  57. Young P, Deveraux Q, Beal RE, Pickart CM, Rechsteiner M (1998) Characterization of two polyubiquitin binding sites in the 26S protease subunit 5a. J Biol Chem 273(10):5461–5467

    Article  CAS  PubMed  Google Scholar 

  58. Yu Y, Xu W, Yi YJ, Sutovsky P, Oko R (2006) The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38). Dev Biol 290(1):32–43

    Article  CAS  PubMed  Google Scholar 

  59. Yu Y, Vanhorne J, Oko R (2009) The origin and assembly of a zona pellucida binding protein, IAM38, during spermiogenesis. Microsc Res Tech 72(8):558–565

    Article  CAS  PubMed  Google Scholar 

  60. Ziemba H, Bialy LP, Fracki S, Bablok L, Wojcik C (2002) Proteasome localization and ultrastructure of spermatozoa from patients with varicocele-immunoelectron microscopic study. Folia Histochem Cytobiol 40(2):169–170

    CAS  PubMed  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Young-Joo Yi.

Additional information

This work was supported by National Research Initiative Competitive Grant #2007–01319 from the USDA Cooperative State Research, Education and Extension Service, and the Food for the 21st Century Program of the University of Missouri-Columbia. Y.-J.Y. was in part supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (No. 2010–0001356). F.W.v.L. was supported by the Internationale Stichting Alzheimer Onderzoek (ISAO grant no.06502/09514), The International Parkinson Foundation and the Hersenstichting, projects, grants no.12F04.01 and 15F07.48). V.J. was supported by grants from GACR (grant no. 303/09/1285) and MSMT (grant no.1M06011) and IGA, Ministry of Health of the Czech Republic (grant no. NS 10009-4).

Electronic Supplementary Material

Supplemental Data Fig. 1 (for web publication)

Visualization of acrosomal exocytosis and binding of the antibodies against subunit PSMD4 to the zona-bound boar sperm acrosomes during porcine IVF. In order to examine whether subunit PSMD4 contributes to sperm-ZP binding and acrosomal exocytosis shortly after sperm-oocyte mixing, ova were fertilized in the presence of different concentrations of mouse anti-PSMD4 antibody or without antibody, and fixed at 30 min after fertilization. The fixed ova, fertilized in the presence of anti-PSMD4 antibody, were incubated with a rabbit polyclonal antibody against sperm acrosomal tyrosine kinase YES, which detects intact acrosomes in ZP-bound boar spermatozoa (Sutovsky et al. 2004a), followed by incubation with a mixture of GAM-IgG-FITC and DAPI (DNA stain; blue). Panel (a) shows double-positive labeling at high magnification, panel (b) is a negative control for PSMD4 (with positive signal for YES), and panel (c) is a double positive labeling at low magnification (JPEG 120 kb).

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yi, YJ., Manandhar, G., Sutovsky, M. et al. Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization. Cell Tissue Res 341, 325–340 (2010). https://doi.org/10.1007/s00441-010-0988-2

Download citation

Keywords

  • Sperm
  • Proteasome
  • Fertilization
  • Zona pellucida
  • Acrosome
  • Alzheimer’s
  • Porcine