Skip to main content

Advertisement

SpringerLink
Log in

Impact of semen-derived amyloid (SEVI) on sperm viability and motility: its implication in male reproductive fitness

  • Original Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

Human semen contains a large number of macromolecules, including proteins/enzymes and carbohydrates, regulating and protecting sperm cells. Proteomic analysis of human seminal fluid led to the discovery of semen amyloids derived from short peptide fragments of the proteins prostatic acid phosphatase (PAP) and semenogelin (SG) which are known to play a crucial role in enhancing HIV infection. However, the relevance of their existence in human semen and role in maintaining sperm behavior remains unclear. Distinct physiological, biochemical, and biophysical attributes might cause these amyloids to influence sperm behavior positively or negatively, affecting fertilization or other reproductive processes. We assessed the direct effect of amyloids derived from a PAP248–286 fragment, on sperm motility and viability, which are crucial parameters for assessment of sperm quality in semen. Co-incubation of human sperm with PAP248–286 amyloids at normal physiological concentrations formed in buffer led to significant reduction in sperm viability, though approximately a 10× higher concentration was needed to show a similar effect with amyloid formed in seminal fluid. Both forms of PAP248–286 amyloid also had a significant impact on sperm motility at physiological levels, in agreement with a previous report. Our study suggests that PAP248–286 amyloids can directly influence sperm motility and viability in a concentration-dependent manner. We hypothesise that the direct toxic effect of PAP248–286 amyloid is normally mitigated by other seminal fluid ingredients, but that in pathological conditions, where PAP248–286 concentrations are elevated and it plays a role in determining sperm health and viability, with relevance for male fertility as well as sterility.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

PAP:

Prostatic acid phosphatase

SG:

Semenogelin

SEVI:

Semen-derived enhancer of viral infection

SP:

Seminal plasma

DPBS:

Dulbecco’s phosphate buffer saline

RPM:

Revolutions per minutes

CR:

Congo red

TEM:

Transmission electron microscope

SMI:

Sperm motility index

PI:

Propidium iodide

FACS:

Fluorescence-activated cell sorting

References

  • Aagaard JE, Yi X, MacCoss MJ, Swanson WJ (2006) Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs. Proc Natl Acad Sci 103(46):17302–17307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Amaral A, Lourenço B, Marques M, Ramalho-Santos J (2013) Mitochondria functionality and sperm quality. Reproduction 146(5):R163–R174

    Article  CAS  PubMed  Google Scholar 

  • Barros C, Vigil P, Herrera E, Arguello B, Walker R (1984) Selection of morphologically abnormal sperm by human cervical mucus. Arch Androl 12:95–107

    PubMed  Google Scholar 

  • Berchowitz LE, Kabachinski G, Walker MR, Carlile TM, Gilbert WV, Schwartz TU, Amon A (2015) Regulated formation of an amyloid-like translational repressor governs gametogenesis. Cell 163(2):406–418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boke E, Ruer M, Wühr M, Coughlin M, Lemaitre R, Gygi SP, Alberti S, Drechsel D, Hyman AA, Mitchison TJ (2016) Amyloid-like self-assembly of a cellular compartment. Cell 166(3):637–650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Breydo L, Uversky VN (2015) Structural, morphological, and functional diversity of amyloid oligomers. FEBS Lett 589(19 Part A):2640–2648

    Article  CAS  PubMed  Google Scholar 

  • Cardullo RA, Baltz JM (1991) Metabolic regulation in mammalian sperm: mitochondrial volume determines sperm length and flagellar beat frequency. Cytoskeleton 19(3):180–188

    Article  CAS  Google Scholar 

  • Castellano LM, Shorter J (2012) The surprising role of amyloid fibrils in HIV infection. Biology 1(1):58–80

    Article  PubMed  PubMed Central  Google Scholar 

  • Castellano LM, Bart SM, Holmes VM, Weissman D, Shorter J (2015) Repurposing Hsp104 to antagonize seminal amyloid and counter HIV infection. Chem Biol 22(8):1074–1086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chau KM, Cornwall GA (2011) Reduced fertility in vitro in mice lacking the cystatin CRES (cystatin-related epididymal spermatogenic): rescue by exposure of spermatozoa to dibutyryl cAMP and isobutylmethylxanthine. Biol Reprod 84(1):140–152

    Article  CAS  PubMed  Google Scholar 

  • Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75:333–366

    Article  CAS  PubMed  Google Scholar 

  • Di Scala C, Yahi N, Boutemeur S, Flores A, Rodriguez L, Chahinian H, Fantini J (2016) Common molecular mechanism of amyloid pore formation by Alzheimer’s β-amyloid peptide and α-synuclein. Sci Rep 6:28781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dosch R, Wagner DS, Mintzer KA, Runke G, Wiemelt AP, Mullins MC (2004) Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev. Cell 6(6):771–780

    Article  CAS  PubMed  Google Scholar 

  • Easterhoff D (2013) Characterization of amyloid fibrils in seminal fluid and their interaction with pathogens. University of Rochester, New York

    Google Scholar 

  • Eisenberg D, Jucker M (2012) The amyloid state of proteins in human diseases. Cell 148(6):1188–1203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elia J, Imbrogno N, Delfino M, Mazzilli R, Rossi T, Mazzilli F (2010) The importance of the sperm motility classes-future directions. Open Androl J 2:42–43

    Google Scholar 

  • Elias AK (2015) The functional studies of amyloid fibrils and their toxicity. Doctoral dissertation

  • Eliasson R (1982) Biochemical analysis of human semen. Int J Androl 5(s5):109–119

    Article  CAS  Google Scholar 

  • Fändrich M, Meinhardt J, Grigorieff N (2009) Structural polymorphism of Alzheimer Aβ and other amyloid fibrils. Prion 3(2):89–93

    Article  PubMed  PubMed Central  Google Scholar 

  • Fowler DM, Koulov AV, Balch WE, Kelly JW (2007) Functional amyloid—from bacteria to humans. Trends Biochem Sci 32(5):217–224

    Article  CAS  PubMed  Google Scholar 

  • French KC, Makhatadze GI (2012) Core sequence of PAPf39 amyloid fibrils and mechanism of pH-dependent fibril formation: the role of monomer conformation. Biochemistry 51(51):10127–10136

    Article  CAS  PubMed  Google Scholar 

  • Gaharwar B, Gour S, Kaushik V, Gupta N, Kumar V, Hause G, Yadav JK (2015) Assessment of the effect of macromolecular crowding on aggregation behaviour of a model amyloidogenic peptide. Protein Pept Lett 22(1):87–93

    Article  CAS  PubMed  Google Scholar 

  • Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182(2):319–326

    Article  CAS  PubMed  Google Scholar 

  • Glabe CG (2008) Structural classification of toxic amyloid oligomers. J Biol Chem 283(44):29639–29643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Greve JM, Wassarman PM (1985) Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. J Mol Biol 181(2):253–264

    Article  CAS  PubMed  Google Scholar 

  • Gunia S, Koch S, May M, Dietel M, Erbersdobler A (2009) Expression of prostatic acid phosphatase (PSAP) in transurethral resection specimens of the prostate is predictive of histopathologic tumor stage in subsequent radical prostatectomies. Virchows Arch 454(5):573–579

    Article  CAS  PubMed  Google Scholar 

  • Guyonnet B, Egge N, Cornwall GA (2014) Functional amyloids in the mouse sperm acrosome. Mol Cell Biol 34(14):2624–2634

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hammer ND, Wang X, McGuffie BA, Chapman MR (2008) Amyloids: friend or foe? J Alzheimers Dis 13(4):407–419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hartjen P, Frerk S, Hauber I, Matzat V, Thomssen A, Holstermann B, Hohenberg H, Schulze W, Schulze Zur Wiesch J, van Lunzen J (2012a) Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG. AIDS Res Ther 9(1):2

    Article  PubMed  PubMed Central  Google Scholar 

  • Hewetson A, Do HQ, Myers C, Muthusubramanian A, Sutton RB, Wylie BJ, Cornwall GA (2017) Functional amyloids in reproduction. Biomolecules 7(3):46

    Article  CAS  PubMed Central  Google Scholar 

  • Iconomidou VA, Vriend G, Hamodrakas SJ (2000) Amyloids protect the silkmoth oocyte and embryo. FEBS Lett 479(3):141–145

    Article  CAS  PubMed  Google Scholar 

  • Johansson J, Olson L, Andersson J, Johansson G, Winblad B (2016) Amyloid: a multifaceted player in human health and disease. J Intern Med 280(2):136–138

    Article  CAS  PubMed  Google Scholar 

  • Juyena NS, Stelletta C (2012) Seminal plasma: an essential attribute to spermatozoa. J Androl 33(4):536–551

    Article  PubMed  Google Scholar 

  • Kim KA, Yolamanova M, Zirafi O, Roan NR, Staendker L, Forssmann WG, Burgener A, Dejucq-Rainsford N, Hahn BH, Shaw GM, Greene WC, Kirchhoff F, Munch J (2010) Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI. Retrovirology 7:55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klunk WE, Pettegrew J, Abraham DJ (1989) Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem 37(8):1273–1281

    Article  CAS  PubMed  Google Scholar 

  • Kordan W, Fraser L, Wysocki P, Strzeżek R, Lecewicz M, Mogielnicka-Brzozowska M, Dziekońska A, Soliwoda D, Koziorowska-Gilun M (2013) Semen quality assessments and their significance in reproductive technology. Pol J Vet Sci 16(4):823–833

    Article  CAS  PubMed  Google Scholar 

  • Last NB, Rhoades E, Miranker AD (2011) Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity. Proc Natl Acad Sci 108(23):9460–9465

    Article  PubMed  PubMed Central  Google Scholar 

  • Litscher ES, Janssen WG, Darie CC, Wassarman PM (2008) Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions. J Cell Physiol 214(1):153–157

    Article  CAS  PubMed  Google Scholar 

  • Louros NN, Petronikolou N, Karamanos T, Cordopatis P, Iconomidou VA, Hamodrakas SJ (2014) Structural studies of “aggregation-prone” peptide-analogues of teleostean egg chorion ZPB proteins. J Pept Sci 102(6):427–436

    Article  CAS  Google Scholar 

  • Macleod J, Wang Y (1979) Male fertility potential in terms of semen quality: a review of the past a study of the present. Fertil Steril 31(2):103–116

    Article  CAS  PubMed  Google Scholar 

  • Malm J, Sonesson A, Hellman J, Bjartell A, Frohm B, Hillarp A (2008) The pentraxin serum amyloid P component is found in the male genital tract and attached to spermatozoa. Int J Androl 31(5):508–517

    Article  CAS  PubMed  Google Scholar 

  • Martellini JA, Cole AL, Svoboda P, Stuchlik O, Chen L-M, Chai KX, Gangrade BK, Sørensen OE, Pohl J, Cole AM (2011) HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma. PLoS ONE 6(1):e16285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Munch J, Rucker E, Standker L, Adermann K, Goffinet C, Schindler M, Wildum S, Chinnadurai R, Rajan D, Specht A, Gimenez-Gallego G, Sanchez PC, Fowler DM, Koulov A, Kelly JW, Mothes W, Grivel JC, Margolis L, Keppler OT, Forssmann WG, Kirchhoff F (2007) Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131(6):1059–1071

    Article  CAS  PubMed  Google Scholar 

  • Nanga RP, Brender JR, Vivekanandan S, Popovych N, Ramamoorthy A (2009) NMR structure in a membrane environment reveals putative amyloidogenic regions of the SEVI precursor peptide PAP(248–286). J Am Chem Soc 131(49):17972–17979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Navarro S, Ventura S (2014) Fluorescent dye ProteoStat to detect and discriminate intracellular amyloid-like aggregates in Escherichia coli. Biotechnol J 9(10):1259–1266

    Article  CAS  PubMed  Google Scholar 

  • Owen DH, Katz DF (2005) A review of the physical and chemical properties of human semen and the formulation of a semen simulant. J Androl 26(4):459–469

    Article  CAS  PubMed  Google Scholar 

  • Podrabsky JE, Carpenter JF, Hand SC (2001) Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers. Am J Physiol Regul Integr Comp Physiol 280(1):R123–R131

    Article  CAS  PubMed  Google Scholar 

  • Poiani A (2006) Complexity of seminal fluid: a review. Behav Ecol Sociobiol 60(3):289–310

    Article  Google Scholar 

  • Roan NR, Greene WC (2007) A seminal finding for understanding HIV transmission. Cell 131(6):1044–1046

    Article  CAS  PubMed  Google Scholar 

  • Roan NR, Münch J, Arhel N, Mothes W, Neidleman J, Kobayashi A, Smith-McCune K, Kirchhoff F, Greene WC (2009) The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection. J Virol 83(1):73–80

    Article  CAS  PubMed  Google Scholar 

  • Roan NR, Sowinski S, Münch J, Kirchhoff F, Greene WC (2010) Aminoquinoline surfen inhibits the action of SEVI (semen-derived enhancer of viral infection). J Biol Chem 285(3):1861–1869

    Article  CAS  PubMed  Google Scholar 

  • Roan NR, Muller JA, Liu H, Chu S, Arnold F, Sturzel CM, Walther P, Dong M, Witkowska HE, Kirchhoff F, Munch J, Greene WC (2011) Peptides released by physiological cleavage of semen coagulum proteins form amyloids that enhance HIV infection. Cell Host Microbe 10(6):541–550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roan NR, Liu H, Usmani SM, Neidleman J, Muller JA, Avila-Herrera A, Gawanbacht A, Zirafi O, Chu S, Dong M, Kumar ST, Smith JF, Pollard KS, Fandrich M, Kirchhoff F, Munch J, Witkowska HE, Greene WC (2014) Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection. J Virol 88(13):7221–7234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roan NR, Sandi-Monroy N, Kohgadai N, Usmani SM, Hamil KG, Neidleman J, Montano M, Standker L, Rocker A, Cavrois M, Rosen J, Marson K, Smith JF, Pilcher CD, Gagsteiger F, Sakk O, O'Rand M, Lishko PV, Kirchhoff F, Munch J, Greene WC (2017) Semen amyloids participate in spermatozoa selection and clearance. eLife 6:e24888

    Article  PubMed  PubMed Central  Google Scholar 

  • Roberts RG (2016) Good amyloid, bad amyloid—what’s the difference? PLoS Biol 14(1):e1002362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Röcker A, Roan NR, Yadav JK, Fändrich M, Münch J (2018) Structure, function and antagonism of semen amyloids. Chem Commun 54(55):7557–7569

    Article  Google Scholar 

  • Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, Dorin J, Cooke HJ (1997) The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389(6646):73

    Article  CAS  PubMed  Google Scholar 

  • Sciacca MF, Kotler SA, Brender JR, Chen J, Lee D-K, Ramamoorthy A (2012) Two-step mechanism of membrane disruption by Aβ through membrane fragmentation and pore formation. Biophys J 103(4):702–710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sheftic SR, Snell JM, Jha S, Alexandrescu AT (2012) Inhibition of semen-derived enhancer of virus infection (SEVI) fibrillogenesis by zinc and copper. Eur Biophys J 41(9):695–704

    Article  CAS  PubMed  Google Scholar 

  • Shen D, Coleman J, Chan E, Nicholson TP, Dai L, Sheppard PW, Patton WF (2011) Novel cell-and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies. Cell Biochem Biophys 60(3):173–185

    Article  CAS  PubMed  Google Scholar 

  • Southern PJ (2013) Missing out on the biology of heterosexual HIV-1 transmission. Trends Microbiol 21(5):245–252

    Article  CAS  PubMed  Google Scholar 

  • Suarez S, Pacey A (2006) Sperm transport in the female reproductive tract. Hum Reprod Update 12(1):23–37

    Article  CAS  PubMed  Google Scholar 

  • Ta HP, Berthelot K, Coulary-Salin B, Castano S, Desbat B, Bonnafous P, Lambert O, Alves I, Cullin C, Lecomte S (2012) A yeast toxic mutant of HET-s amyloid disrupts membrane integrity. BBA Biomembranes 1818(9):2325–2334

    Article  CAS  PubMed  Google Scholar 

  • Tan S, Lu L, Li L, Liu J, Oksov Y, Lu H, Jiang S, Liu S (2013) Polyanionic candidate microbicides accelerate the formation of semen-derived amyloid fibrils to enhance HIV-1 infection. PLoS ONE 8(3):e59777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanaka M, Komi Y (2015) Layers of structure and function in protein aggregation. Nat Chem Biol 11(6):373

    Article  CAS  PubMed  Google Scholar 

  • Tasken K, Angelsen A, Svindland A, Eide T, Berge V, Wahlquist R, Karlsen S (2005) Markers for diagnosis, prediction and prognosis of prostate cancer. Tidsskrift for den Norske laegeforening: tidsskrift for praktisk medicin, ny raekke 125(23):3279–3282

    Google Scholar 

  • Usmani SM, Zirafi O, Muller JA, Sandi-Monroy NL, Yadav JK, Meier C, Weil T, Roan NR, Greene WC, Walther P, Nilsson KP, Hammarstrom P, Wetzel R, Pilcher CD, Gagsteiger F, Fandrich M, Kirchhoff F, Munch J (2014) Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen. Nat Commun 5:3508

    Article  CAS  PubMed  Google Scholar 

  • Vaubourdolle M, Clavel J, Gonzales J, Galli A (1985) Evaluation of acid phosphatase isoenzymes in seminal fluid from normozoospermic, oligozoospermic, azoospermic and asthenoteratozoospermic men. Andrologia 17(6):598–604

    Article  CAS  PubMed  Google Scholar 

  • Walsh P, Vanderlee G, Yau J, Campeau J, Sim VL, Yip CM, Sharpe S (2014) The mechanism of membrane disruption by cytotoxic amyloid oligomers formed by prion protein (106–126) is dependent on bilayer composition. J Biol Chem 289(15):10419–10430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang C, Swerdloff RS (2014) Limitations of semen analysis as a test of male fertility and anticipated needs from newer tests. Fertil Steril 102(6):1502–1507

    Article  PubMed  PubMed Central  Google Scholar 

  • Whelly S, Johnson S, Powell J, Borchardt C, Hastert MC, Cornwall GA (2012) Nonpathological extracellular amyloid is present during normal epididymal sperm maturation. PLoS ONE 7(5):e36394

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Whelly S, Serobian G, Borchardt C, Powell J, Johnson S, Hakansson K, Lindstrom V, Abrahamson M, Grubb A, Cornwall GA (2014) Fertility defects in mice expressing the L68Q variant of human cystatin CA role for amyloid in male infertility. J Biol Chem 289(11):7718–7729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Whelly S, Muthusubramanian A, Powell J, Johnson S, Hastert MC, Cornwall GA (2016) Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen. MHR Basic Sci Reprod Med 22(11):729–744

    Article  CAS  Google Scholar 

  • Ye Z, French KC, Popova LA, Lednev IK, Lopez MM, Makhatadze GI (2009) Mechanism of fibril formation by a 39-residue peptide (PAPf39) from human prostatic acidic phosphatase. Biochemistry 48(48):11582–11591

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are thankful to the technical staff of central instrument facility at RGCB, and KJK hospital Thiruvananthapuram, Kerala, India for their unconditional support. Mr. Vijay Kumar is thankful to the lab members of molecular reproduction at RGCB for their help during this project. Mr. Vijay Kumar acknowledges University Grants Commission, India and Social Justice and Empowerment Department, Govt. of India for RGN fellowship.

Funding

This study was partially supported by Department of Science and Technology, Government of India (Grant number SB/YS/LS-130/2013).

Author information

Authors and Affiliations

  1. Department of Biotechnology, Central University of Rajasthan, NH-8 Jaipur-Ajmer Highway, Bandarsindri, Kishangarh, Ajmer, Rajasthan, 305817, India

    Vijay Kumar & Jay Kant Yadav

  2. Molecular Reproduction Division, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Poojappura, Thiruvananthapuram, Kerala, 695014, India

    Pradeep G. Kumar

Authors
  1. Vijay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pradeep G. Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jay Kant Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jay Kant Yadav.

Ethics declarations

Conflict of interest

The authors do not have any conflict of interest to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumar, V., Kumar, P.G. & Yadav, J.K. Impact of semen-derived amyloid (SEVI) on sperm viability and motility: its implication in male reproductive fitness. Eur Biophys J 48, 659–671 (2019). https://doi.org/10.1007/s00249-019-01391-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-019-01391-2

Keywords

Search

Navigation