Protein ubiquitination is a stable, reversible post-translational modification, targeting proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies have revealed the role of UPS in the regulation of fertilization, including sperm-zona pellucida interactions and the early event of sperm capacitation. The present study investigates the changes in proteasome compartmentalization, subunit composition and post-translational modifications during in vitro capacitation of fresh boar spermatozoa. We observed capacitation-dependent shedding of both 20S core and 19S regulatory particles from the acrosome that was associated with decreased plasma membrane integrity, independent of proteasomal inhibition. Subunits PSMA1-7 of the 20S core did not appear to undergo post-translational modifications during capacitation, based on invariant molecular masses before and after capacitation; however, we observed multiple PSMD4 forms of 19S regulatory particles (50, 53, 70, 115-140, 160 and >176 kDa) sequentially released from spermatozoa. PSMD4 subunit was found to be post-translationally modified during the course of capacitation, resulting in changes of apparent molecular mass, some of which were dependent on proteasomal inhibition. These results show that the sperm proteasomes are being modified during sperm capacitation. Additional studies of individual 26S proteasome subunits will be required to elucidate these modifications and to understand how UPS modulates sperm capacitation.

Department of Obstetrics Gynecology and Women's Health University of Missouri Columbia MO 65211 USA

Division of Animal Sciences University of Missouri Columbia MO 65211 5300 USA

Laboratory of Reproductive Biology Institute of Biotechnology CAS v v i 25242 Vestec Czech Republic

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Austin CR. The capacitation of the mammalian sperm. Nature. 1952;170:326. PubMed

Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ. Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. Biochem J. 2004;378:177–184. PubMed PMC

Crosas B, Hanna J, Kirkpatrick DS, Zhang DP, Tone Y, Hathaway NA, Buecker C, Leggett DS, Schmidt M, King RW, Gygi SP, Finley D. Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell. 2006;127:1401–1413. PubMed

Deveraux Q, Ustrell V, Pickart C, Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem. 1994;269:7059–7061. PubMed

Diaz ES, Kong M, Morales P. Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm. Hum Reprod. 2007;22:1420–1430. PubMed

Feng Y, Longo DL, Ferris DK. Polo-like kinase interacts with proteasomes and regulates their activity. Cell Growth Differ. 2001;12:29–37. PubMed

Fernandez Murray P, Pardo PS, Zelada AM, Passeron S. In vivo and in vitro phosphorylation of Candida albicans 20S proteasome. Arch Biochem Biophys. 2002;404:116–125. PubMed

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

Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature. 1951;168:697–698. PubMed

Isasa M, Katz EJ, Kim W, Yugo V, Gonzalez S, Kirkpatrick DS, Thomson TM, Finley D, Gygi SP, Crosas B. Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome. Mol Cell. 2010;38:733–745. PubMed PMC

Jacobson AD, Macfadden A, Wu Z, Peng J, Liu CW. Autoregulation of the 26S proteasome by in situ ubiquitination. Mol Biol Cell. 2014;25:1824–1835. PubMed PMC

Kerns K, Morales P, Sutovsky P. Regulation of Sperm Capacitation by the 26S Proteasome: An Emerging New Paradigm in Spermatology. Biol Reprod. 2016;94:117. PubMed

Kong M, Diaz ES, Morales P. Participation of the human sperm proteasome in the capacitation process and its regulation by protein kinase A and tyrosine kinase. Biol Reprod. 2009;80:1026–1035. PubMed

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. PubMed

Lipinszki Z, Kovacs L, Deak P, Udvardy A. Ubiquitylation of Drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors. Biochemistry. 2012;51:2461–2470. PubMed

Miles EL, O'gorman C, Zhao J, Samuel M, Walters E, Yi YJ, Sutovsky M, Prather RS, Wells KD, Sutovsky P. Transgenic pig carrying green fluorescent proteasomes. Proc Natl Acad Sci U S A. 2013;110:6334–6339. PubMed PMC

Mochida K, Tres LL, Kierszenbaum AL. Structural features of the 26S proteasome complex isolated from rat testis and sperm tail. Mol Reprod Dev. 2000;57:176–184. PubMed

Morales P, Diaz ES, Kong M. Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa. Soc Reprod Fertil Suppl. 2007;65:269–273. PubMed

Sanchez R, Deppe M, Schulz M, Bravo P, Villegas J, Morales P, Risopatron J. Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle. Andrologia. 2011;43:114–120. PubMed

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;144:423–431. PubMed

Satoh K, Sasajima H, Nyoumura KI, Yokosawa H, Sawada H. Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. Biochemistry. 2001;40:314–319. PubMed

Sawada H, Pinto MR, De Santis R. Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis. Mol Reprod Dev. 1998;50:493–498. PubMed

Sutovsky P. Sperm proteasome and fertilization. Reproduction. 2011;142:1–14. PubMed

Sutovsky P, Manandhar G, Mccauley TC, Caamano JN, Sutovsky M, Thompson WE, Day BN. Proteasomal interference prevents zona pellucida penetration and fertilization in mammals. Biol Reprod. 2004;71:1625–1637. PubMed

Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350–4354. PubMed PMC

Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, editors. The physiology of reproduction. Vol. 1. Raven Press; New York: 1994. pp. 189–317.

Yi YJ, Manandhar G, Oko RJ, Breed WG, Sutovsky P. Mechanism of sperm-zona pellucida penetration during mammalian fertilization: 26S proteasome as a candidate egg coat lysin. Soc Reprod Fertil Suppl. 2007;63:385–408. PubMed

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

Yi YJ, Manandhar G, Sutovsky M, Zimmerman SW, Jonakova V, Van Leeuwen FW, Oko R, Park CS, Sutovsky P. Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization. Cell Tissue Res. 2010b;341:325–340. PubMed

Yi YJ, Park CS, Kim ES, Song ES, Jeong JH, Sutovsky P. Sperm-surface ATP in boar spermatozoa is required for fertilization: relevance to sperm proteasomal function. Syst Biol Reprod Med. 2009;55:85–96. PubMed

Yi YJ, Zimmerman SW, Manandhar G, Odhiambo JF, Kennedy C, Jonakova V, Manaskova-Postlerova P, Sutovsky M, Park CS, Sutovsky P. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization. Int J Androl. 2012;35:196–210. PubMed

Yokota N, Kataoka Y, Hashii N, Kawasaki N, Sawada H. Sperm-specific C-terminal processing of the proteasome PSMA1/alpha6 subunit. Biochem Biophys Res Commun. 2011;410:809–815. PubMed

Yokota N, Sawada H. 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. 2007;308:222–231. PubMed

Zimmerman S, Sutovsky P. The sperm proteasome during sperm capacitation and fertilization. J Reprod Immunol. 2009;83:19–25. PubMed

Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, Powell MD, Miller DJ, Sutovsky P. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization. PLoS One. 2011;6:e17256. PubMed PMC

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Compartmentalization of the proteasome-interacting proteins during sperm capacitation