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Optimization of microelectrophoresis to select highly negatively charged sperm

  • Luke Simon
  • Kristin Murphy
  • Kenneth I. Aston
  • Benjamin R. Emery
  • James M. Hotaling
  • Douglas T. CarrellEmail author
Technological Innovations

Abstract

Purpose

The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge.

Methods

Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage.

Results

When raw semen was analyzed using microelectrophoresis, 94 % were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current.

Conclusion

The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.

Keywords

Membrane glycocalyx Micro-electrophoresis Sperm membrane charge Sperm DNA damage Sperm selection 

Notes

Acknowledgments

This project was supported by research grants from EMD Serono, Rockland, Massachusetts, and the Howard and Georgeanna Jones Foundation for Reproductive Medicine, Norfolk, Virginia. The authors wish to thank the UCRM IVF unit and laboratory staff for their commitment and support to this project, for preparing tissue samples, and for helping to collect data on ART outcomes.

References

  1. 1.
    Veres I. Negative electrical charge of the surface of bull sperm. Mikroskopie. 1968;23:166–9.PubMedGoogle Scholar
  2. 2.
    Nevo AC, Michaeli I, Schindler H. Electrophoretic properties of bull and of rabbit spermatozoa. Exp Cell Res. 1961;23:69–83.CrossRefPubMedGoogle Scholar
  3. 3.
    Yanagimachi R, Noda YD, Fujimoto M, Nicolson GL. The distribution of negative surface charges on mammalian spermatozoa. Am J Anat. 1972;135:497–520.CrossRefPubMedGoogle Scholar
  4. 4.
    Bedford JM. Changes in the electrophoretic properties of rabbit spermatozoa during passage through the epididymis. Nature. 1963;200:1178–80.CrossRefPubMedGoogle Scholar
  5. 5.
    Bostwick EF, Bentley MD, Hunter AG, Hammer R. Identification of surface glycoprotein on porcine spermatozoa and its alteration during epididymal maturation. Biol Reprod. 1980;23:161–5.CrossRefPubMedGoogle Scholar
  6. 6.
    Olson GE, Danzo BJ. Surface changes in the rat spermatozoa during epididymal transit. Biol Reprod. 1981;24:431–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Lassalle B, Testart J. Human zona pellucida recognition associated with removal of sialic acid from human sperm surface. J Reprod Fertil. 1994;101:703–11.CrossRefPubMedGoogle Scholar
  8. 8.
    Deng X, Czymmek K, Deleon M. Biochemical maturation of spam1 (PH-20) during epididymal transit of mouse sperm involves modifications of N-linked oligosaccharides. Mol Reprod Dev. 1999;52:196–206.CrossRefPubMedGoogle Scholar
  9. 9.
    Ainsworth CJ, Nixon B, Aitken RJ. The electrophoretic separation of spermatozoa: an analysis of genotype, surface carbohydrate composition and potential for capacitation. Int J Androl. 2011;34:422–34.CrossRefGoogle Scholar
  10. 10.
    Bedford JM. Sperm capacitation and fertilization in mammals. Biol Reprod. 1970;2:128–58.CrossRefPubMedGoogle Scholar
  11. 11.
    Bedford JM, Nicander L. Ultrastructural changes in the acrosome and sperm membranes during maturation of spermatozoa in the testis and epididymis of the rabbit and monkey. J Anat. 1971;108:527–43.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Kirchhoff C, Schroter S. New insights into the origin, structure and role of CD52: a major component of the mammalian sperm glycocalyx. Cells Tissues Organs. 2001;168:93–104.CrossRefPubMedGoogle Scholar
  13. 13.
    Oliva R. Protamines and male infertility. Hum Reprod Update. 2006;12:417–35.CrossRefPubMedGoogle Scholar
  14. 14.
    Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using Acridine Orange staining. Mol Hum Reprod. 1977;3:47–54.CrossRefGoogle Scholar
  15. 15.
    Turner TT. On the epididymis and its role in the development of the fertile ejaculate. J Androl. 1995;16:292–8.PubMedGoogle Scholar
  16. 16.
    Cooper TG. Interactions between epididymal secretions and spermatozoa. J Reprod Fertil. 1998;53:119–36.Google Scholar
  17. 17.
    Schroter S, Osterhoff C, McArdle W, Ivell R. The glycocalyx of the sperm surface. Hum Reprod Update. 1999;5:302–13.CrossRefPubMedGoogle Scholar
  18. 18.
    Giuliani V, Pandolfi C, Santucci R, Pelliccione F, Macerola B, Focarelli R, et al. Expression of gp20, a human sperm antigen of epididymal origin, is reduced in spermatozoa from subfertile men. Mol Reprod Dev. 2004;69:235–40.CrossRefPubMedGoogle Scholar
  19. 19.
    Schroter S, Derr P, Conradt HS, Nimtz M, Hale G, Kirchhoff C. Male-specific modification of human CD52. J Biol Chem. 1999;274:29862–73.CrossRefPubMedGoogle Scholar
  20. 20.
    Ishijima SA, Okuno M, Mohri H. Zeta potential of human X- and Y-bearing sperm. Int J Androl. 1991;14:340–7.CrossRefPubMedGoogle Scholar
  21. 21.
    Yudin AI, Generao SE, Tollner TL, Treece CA, Overstreet JW, Cherr GN. Beta-defensin 126 on the cell surface protects sperm from immunorecognition and binding of anti-sperm antibodies. Bio Reprod. 2005;73:1243–52.CrossRefGoogle Scholar
  22. 22.
    Chan PJ, Jacobson JD, Corselli JU, Patton WC. A simple zeta method for sperm selection based on membrane charge. Fertil Steril. 2006;85:481–6.CrossRefPubMedGoogle Scholar
  23. 23.
    Kam TL, Jacobson JD, Patton WC, Corselli JU, Chan PJ. Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection. J Assist Reprod Genet. 2007;24:429–34.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ainsworth C, Nixon B, Aitken RJ. Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum Reprod. 2005;20:2261–70.CrossRefPubMedGoogle Scholar
  25. 25.
    Ainsworth C, Nixon B, Jansen RP, Aitken RJ. First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa. Hum Reprod. 2007;22:197–200.CrossRefPubMedGoogle Scholar
  26. 26.
    Razavi SH, Nasr-Esfahani MH, Deemeh MR, Shayesteh M, Tavalaee M. Evaluation of zeta and HA-binding methods for selection of spermatozoa with normal morphology, protamine content and DNA integrity. Andrologia. 2010;42:13–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Deemeh MR, Nasr-Esfahani MH, Razavi S, Nazem H, Moghadam MS, Tavalaee M. The comparison of HA binding and Zeta methods efficiency in selection of sperm with normal morphology and intact chromatin. J Isfahan Med School. 2009;27:46–56.Google Scholar
  28. 28.
    Khajavi NA, Razavi S, Mardani M, Tavalaee M, Deemeh MR, Nasr-Esfahani MH. Can Zeta sperm selection method, recover sperm with higher DNA integrity compare to density gradient centrifugation? Iranian J Reprod Med. 2009;7:73–7.Google Scholar
  29. 29.
    Aitken RJ, Hanson AR, Kuczera L. Electrophoretic sperm isolation: optimization of electrophoresis conditions and impact on oxidative stress. Hum Reprod. 2011;26:1955–64.CrossRefPubMedGoogle Scholar
  30. 30.
    Fleming SD, Ilad RS, Griffin AM, Wu Y, Ong KJ, Smith HC, et al. Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation. Hum Reprod. 2008;23:2646–51.CrossRefPubMedGoogle Scholar
  31. 31.
    Deemeh MR, Tavalaee M, Ahmadi SM, Kalantari SA, Nasab SVA, Najafi MH, et al. The first report of successfully pregnancy after ICSI with combined DGC/Zeta sperm selection procedure in a couple with eleven repeated fail IVF/ICSI cycles. Int J Fertil Steril. 2010;4:41–3.Google Scholar
  32. 32.
    Yetunde I, Vasiliki M. Effects of advanced selection methods on sperm quality and ART outcome. Minerva Ginecol. 2013;65:487–96.PubMedGoogle Scholar
  33. 33.
    Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, et al. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril. 2003;80:1413–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M. “Physiologic ICSI”: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertil Steril. 2010;93:598–604.CrossRefPubMedGoogle Scholar
  35. 35.
    Nasr-Esfahani MH, Razavi S, Vahdati AA, Fathi F, Tavalaee M. Evaluation of sperm selection procedure based on hyaluronic acid binding ability on ICSI outcome. J Assist Reprod Genet. 2008;25:197–203.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Kheirollahi-Kouhestani M, Razavi S, Tavalaee M, Deemeh MR, Mardani M, Moshtaghian J, et al. Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome. Hum Reprod. 2009;24:2409–16.CrossRefPubMedGoogle Scholar
  37. 37.
    Polak de Fried E, Denaday F. Single and twin ongoing pregnancies in two cases of previous ART failure after ICSI performed with sperm sorted using annexin V microbeads. Fertil Steril. 2010;94(351):e15–8.PubMedGoogle Scholar
  38. 38.
    Wilding M, Coppola G, di Matteo L, Palagiano A, Fusco E, Dale B. Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa. J Assist Reprod Genet. 2011;28:253–62.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Simon L, Murphy K, Aston KI, Emery BR, Hotaling JM, Carrell DT. Micro-electrophoresis: a non-invasive method of sperm selection based on membrane charge. Fertil Steril. 2015;103:361–6.CrossRefPubMedGoogle Scholar
  40. 40.
    World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press; 1999.Google Scholar
  41. 41.
    Boitrelle F, Ferfouri F, Petit JM, Segretain D, Tourain C, Bergere M, et al. Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod. 2011;26:1650–8.CrossRefPubMedGoogle Scholar
  42. 42.
    Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl. 2006;27:53–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Cornwall GA, Lareyre JJ, Matusik RJ, Hinton BT, Orgebin-Crist MC. Gene expression and epididymal function. In: The epididymis: from molecules to clinical practice—a comprehensive survey of the efferent ducts, the epididymis and the vas deferens. Roubaire, B., Hinton, B.T., (Eds.) New York, 2002, pp. 575.Google Scholar
  44. 44.
    Syntin P, Dacheux X, Gatti JL, Okamura N, Dacheux JL. Characterization and identification of proteins secreted in the various regions of the adult boar epididymis. Bio Reprod. 1996;55:956–74.CrossRefGoogle Scholar
  45. 45.
    Yeung CH, Cooper TG, Wagenfeld A, Kirchhoff C, Kliesch S, Poser D, et al. Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkey. Mol Reprod Develop. 1997;48:267–75.CrossRefGoogle Scholar
  46. 46.
    Howes EA, Hurst S, Laslop A, Jones R. Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa. Mol Hum Reprod. 1998;4:673–81.CrossRefPubMedGoogle Scholar
  47. 47.
    Kirchhoff C, Osterhoff C, Pera I, Schroter S. Function of human epididymal proteins in sperm maturation. Andrologia. 1998;30:225–32.CrossRefPubMedGoogle Scholar
  48. 48.
    Leahy T, Gadella BM. Sperm surface changes and physiological consequences induced by sperm handling and storage. Reprod. 2011;142:759–78.CrossRefGoogle Scholar
  49. 49.
    Gadella BM, Lopescardozo M, Vangolde LMG, Colenbrander B, Gadella TWJ. Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction—evidence for a primary capacitation event in boar spermatozoa. J Cell Sci. 1995;108:935–46.PubMedGoogle Scholar
  50. 50.
    Bedford JM, Chang MC. Removal of decapacitation factor from seminal plasma by high-speed centrifugation. Am J Physiol. 1962;202:179–87.PubMedGoogle Scholar
  51. 51.
    Yanagimachi R. Mammalian fertilization. Knobil E, Neill JD. (Eds.) In: The Physiology of Reproduction, 2nd edition, New York, 1994, pp. 189–317.Google Scholar
  52. 52.
    Norbury CJ, Hickson ID. Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol. 2001;41:367–401.CrossRefPubMedGoogle Scholar
  53. 53.
    Mourdjeva M, Kyurkchiev D, Mandinova A, Altankova I, Kehayov I, Kyurkchiev S. Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody. Apoptosis. 2005;10:209–17.CrossRefPubMedGoogle Scholar
  54. 54.
    Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182:1545–56.CrossRefPubMedGoogle Scholar
  55. 55.
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods. 1995;184:39–51.CrossRefPubMedGoogle Scholar
  56. 56.
    Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Gibbons E, Pickett KR, Streeter MC, Warcup AO, Nelson J, Judd AM, et al. Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A2 activity. Biochim Biophys Acta. 1828;2013:887–95.Google Scholar
  58. 58.
    Roux C, Tripogney C, Joanne C, Bresson JL. Sperm chromatin packaging as an indicator of in-vitro fertilization rates. Gynecol Obstet Fertil. 2004;32:792–8.CrossRefPubMedGoogle Scholar
  59. 59.
    Hou JW, Chen D, Jeyendran RS. Sperm nuclear maturity in spinal cord-injured men: evaluation by acidic aniline blue stain. Arch Phys Med Rehab. 1995;76:444–5.CrossRefGoogle Scholar
  60. 60.
    Dadoune JP. Expression of mammalian spermatozoal nucleoproteins. Microscopy Res Tech. 2003;61:56–75.CrossRefGoogle Scholar
  61. 61.
    Dadoune JP, Mayaux MJ, Guihard-Moscato ML. Correlation between defects in chromatin condensation of human spermatozoa stained by aniline blue and semen characteristics. Andrologia. 1988;20:211–7.CrossRefPubMedGoogle Scholar
  62. 62.
    Hofmann N, Hilscher B. Use of aniline blue to assess chromatin condensation inmorphologically normal spermatozoa in normal and infertile men. Hum Reprod. 1991;6:979–82.PubMedGoogle Scholar
  63. 63.
    Kim HS, Kang MJ, Kim SA, Oh SK, Kim H, Ku SY, et al. The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis. Clin Exp Reprod Med. 2013;40:23–8.CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Sellami A, Chakroun N, Zarrouk SB, Sellami H, Kebaili S, Rebai T, et al. Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility. Adv Urol. 2013;578631:8.Google Scholar
  65. 65.
    de Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, et al. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biol Reprod. 2009;81:517–24.CrossRefPubMedGoogle Scholar
  66. 66.
    Schulte RT, Ohl DA, Sigman M, Smith GD. Sperm DNA damage in male infertility: etiologies, assays, and outcomes. J Assist Reprod Genet. 2010;27:3–12.CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Morel F, Mercier S, Roux C, Elmrini T, Clavequin MC, Bresson JL. Interindividual variations in the disomy frequencies of human spermatozoa and their correlation with nuclear maturity as evaluated aniline blue staining. Fertil Steril. 1998;69:1122–7.CrossRefPubMedGoogle Scholar
  68. 68.
    Ovari L, Sati L, Stronk J, Borsos A, Ward DC, Huszar G. Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies. Fertil Steril. 2010;93:2255–61.CrossRefPubMedGoogle Scholar
  69. 69.
    Boue F, Blais J, Sullivan R. Surface localization of P34H, an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa. Biol Reprod. 1996;54:1009–17.CrossRefPubMedGoogle Scholar
  70. 70.
    Boue F, Duquenne C, Lassalle B, Lefèvre A, Finaz C. FLB1, a human protein of epididymal origin that is involved in the sperm-oocyte recognition process. Biol Reprod. 1995;52:267–8.CrossRefPubMedGoogle Scholar
  71. 71.
    Lefevre A, Ruis CM, Chokomian S, Duquenne C, Finaz C. Characterization and isolation of SOB2, a human sperm protein with a potential role in oocyte membrane binding. Mol Hum Reprod. 1997;3:507–16.CrossRefPubMedGoogle Scholar
  72. 72.
    Yeung CH, Cooper TG, Schroter S, Kirchhoff C, Nieschlag E. Epididymal secretion of CD52 as measured in human seminal plasma by a fluorescence immunosaasy. Mol Hum Reprod. 1998;4:447–51.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York (outside the USA) 2016

Authors and Affiliations

  • Luke Simon
    • 1
  • Kristin Murphy
    • 1
  • Kenneth I. Aston
    • 1
  • Benjamin R. Emery
    • 1
  • James M. Hotaling
    • 1
  • Douglas T. Carrell
    • 1
    • 2
    • 3
    Email author
  1. 1.Andrology and IVF Laboratory, Department of Surgery (Urology)Salt Lake CityUSA
  2. 2.Department of Obstetrics and GynecologySalt Lake CityUSA
  3. 3.Department of Human GeneticsUniversity of UtahSalt Lake CityUSA

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