Abstract
Purpose: To develop a method that could accommodatemicrovolumes of solubilized human zona pellucida (ZP) andsperm for assessing the induction of the acrosome reaction. Methods: A microassay using 1 μl of 2.5, 1.25, 0.6, 0.3,and 0.125 ZP/μl incubated with 1 μl of a highly motilesperm suspension for 60 min. As a control and parallelto the microassay a standard acrosome reaction techniquewas performed. Results: No significant differences were observed betweenthe percentage acrosome reacted sperm reported by the twoassays under basal conditions (spontaneous) or after inductionwith a Ca2+ ionophore or solubilized ZP. At a ZPconcentration of 0.6 ZP/μl, the percentages of acrosome-reactedspermatozoa in both techniques were significantlyhigher compared to the spontaneous acrosome reactionresults, namely, 18% and 17%, compared to 10% and 10%,respectively. An approximately 30% level of acrosomal exocytosiswas induced with 2.5 ZP/μl in both methods. Conclusions: This newly devised microtechnique is easy andrapid to perform, is repeatable and facilitates the use ofminimal volumes of solubilized human ZP (even a single ZP)for assessment of the inducibility of the acrosome reaction ofa homologous sperm population.
Keywords: Human sperm, acrosome reaction, zona pellucida, microassay
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REFERENCES
- 1.Oehninger S, Acosta A, Veeck L, Brzyski R, Kruger TF, Muasher SJ, Hodgen GD. Recurrent failure of in vitro fertilization: Role of the hemizona assay in the sequential diagnosis of specific sperm-oocyte defects. Am J Obstet Gynecol. 1991;164:1210–1215. doi: 10.1016/0002-9378(91)90684-j. [DOI] [PubMed] [Google Scholar]
- 2.Amman RP, Hammerstedt RH. In vitro evaluation of sperm quality: An opinion. J Androl. 1993;14:397–406. [PubMed] [Google Scholar]
- 3.Oehninger SC, Franken DR, Kruger TF. Approaching the next millenium: How should we manage andrology diagnosis in the intracytoplasmic sperm injection era? Fertil Steril. 1997;67:434–436. doi: 10.1016/s0015-0282(97)80065-9. [DOI] [PubMed] [Google Scholar]
- 4.WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 3rd ed. Cambridge, England: Cambridge University Press; 1992. [Google Scholar]
- 5.Oehninger SC. An update on the laboratory assessment of male fertility. Hum Reprod. 1995;10(Suppl1):38–45. doi: 10.1093/humrep/10.suppl_1.38. [DOI] [PubMed] [Google Scholar]
- 6.Consensus Workshop on Advanced Diagnostic Andrology Techniques. ESHRE Andrology Special Interest Group. Hum Reprod. 1996;11:1463–1479. [PubMed] [Google Scholar]
- 7.Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartman PE. A test of the human sperm acrosome reaction following ionophore challenge: Relationship to fertility and other seminal parameters. J Androl. 1991;12:98–103. [PubMed] [Google Scholar]
- 8.Tesarik J. Acrosome reaction testing. Consensus Workshop on Advanced Diagnostic Andrology Techniques. 1996;11:5–8. [Google Scholar]
- 9.Henkel R, Müller C, Miska W, Gips H, Schill W.B. Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro. Hum Reprod. 1993;12:2128–2132. doi: 10.1093/oxfordjournals.humrep.a137994. [DOI] [PubMed] [Google Scholar]
- 10.Henkel R, Franken D.R., Habenicht UF. Zona pellucida physiological trigger for the induction of the acrosome reaction. Andrologia. 1998;30:275–280. doi: 10.1111/j.1439-0272.1998.tb01171.x. [DOI] [PubMed] [Google Scholar]
- 11.Cross NL, Morales P, Overstreet JP, Hanson FW. Induction of the acrosome reaction by the human zona pellucida. Biol Reprod. 1988;38:235–244. doi: 10.1095/biolreprod38.1.235. [DOI] [PubMed] [Google Scholar]
- 12.Mahony M, Oehninger S, Clark G, Acosta A, Hodgen GD. Inhibition of zona pellucida-induction of the acrosome reaction in human spermatozoa by the complex polysaccharide, fucoidin. Contraception. 1991;44:657–665. doi: 10.1016/0010-7824(91)90085-t. [DOI] [PubMed] [Google Scholar]
- 13.Bielfeld PP, Faridi A, Zaneveld LJD, de Jonge CJ. The zona pellucida induced acrosome reaction of human spermatozoa is mediated by protein kinases. Fertil Steril. 1994;61:536–541. [PubMed] [Google Scholar]
- 14.Liu DY, Baker HWG. Disordered acrosome reaction of spermatozoa bound to the zona pellucida: A newly discovered sperm defect causing infertility with reduced sperm-zona penetration and reduced fertilization in vitro. Hum Reprod. 1994;9:1694–1700. doi: 10.1093/oxfordjournals.humrep.a138776. [DOI] [PubMed] [Google Scholar]
- 15.Franken DR, Morales P, Habenicht UF. Inhibition of G protein in human sperm and its influence on acrosome reaction and zona pellucida binding. Fertil Steril. 1996;66:1009–1011. [PubMed] [Google Scholar]
- 16.Franken DR, Bastiaan HS, Kidson A, Wranz P, Habenicht U-F. Zona pellucida mediated acrosome reaction and sperm morphology. Andrologia. 1997;29:311–317. doi: 10.1111/j.1439-0272.1997.tb00324.x. [DOI] [PubMed] [Google Scholar]
- 17.Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril. 1985;44:493–498. doi: 10.1016/s0015-0282(16)48918-1. [DOI] [PubMed] [Google Scholar]
- 18.Hammit DG, Syrop CH, Walker DL, Bennet MR. Conditions of oocyte storage and use of non-inseminated as compared with inseminated, nonfertilized oocytes for the hemizona assay. Fertil Steril. 1993;60:131–136. doi: 10.1016/s0015-0282(16)56050-6. [DOI] [PubMed] [Google Scholar]
- 19.Morales P, Cross NL, Overstreet JW, Hanson FW. Acrosome-intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. Dev Biol. 1989;133:385–392. doi: 10.1016/0012-1606(89)90042-0. [DOI] [PubMed] [Google Scholar]
- 20.Morales PJ, Cross NL. A new procedure for determining acrosomal status of very small numbers of human sperm. J Histochem Cytochem. 1989;37:1291–1292. doi: 10.1177/37.8.2474023. [DOI] [PubMed] [Google Scholar]
- 21.Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA, Nicolson GL. Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril. 1979;31:562–567. doi: 10.1016/s0015-0282(16)44004-5. [DOI] [PubMed] [Google Scholar]
- 22.Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay: Development of a diagnostic test for the binding of human spermatozoa to the human zona pellucida to predict fertilization potential. Fertil Steril. 1988;49:688–693. [PubMed] [Google Scholar]
- 23.Franken DR, Oosthuizen WT, Cooper S, Kruger TF, Burkman LJ, Coddington CC, Hodgen GD. Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human zonae pellucidae after storage in a buffered salt solution. Andrologia. 1991;23:205–208. doi: 10.1111/j.1439-0272.1991.tb02538.x. [DOI] [PubMed] [Google Scholar]
- 24.Brewis IA, Clayton R, Barratt CLR, Hornby DPJ, Moore HDM. Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Hum Reprod. 1996;2:583–589. doi: 10.1093/molehr/2.8.583. [DOI] [PubMed] [Google Scholar]
- 25.Chapman NR, Barrattt LR. The role of carbohydrate in sperm-ZP3 adhesion. Mol Hum Reprod. 1996;2:767–774. doi: 10.1093/molehr/2.10.767. [DOI] [PubMed] [Google Scholar]
- 26.Coddington CC, Fulgham DC, Alexander NJ, Johnson D, Herr JC, Hodgen GD. Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil Steril. 1990;54:504–508. [PubMed] [Google Scholar]
- 27.Mortimer D. The essential partnership between diagnostic andrology and modern assisted reproductive technologies. Hum Reprod. 1994;9:1209–1213. doi: 10.1093/oxfordjournals.humrep.a138680. [DOI] [PubMed] [Google Scholar]