Optimization of microelectrophoresis to select highly negatively charged sperm
- 325 Downloads
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.
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.
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.
The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.
KeywordsMembrane glycocalyx Micro-electrophoresis Sperm membrane charge Sperm DNA damage Sperm selection
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.
- 16.Cooper TG. Interactions between epididymal secretions and spermatozoa. J Reprod Fertil. 1998;53:119–36.Google Scholar
- 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.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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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.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