Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved—thawed spermatozoa

  • Sergio Oehninger
  • Mahmood Morshedi
  • Hakan Ertunc
  • Christine Philput
  • Silvina M. Bocca
  • Anibal A. Acosta
  • Gary D. Hodgen
Animal Investigations


We compared fresh and frozen—thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 × 104 motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen—thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 × 106 motile sperm/hemizona, binding of frozen—thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.

Key words

sperm zona pellucida binding hemizona assay 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD: The hemizona assay (HZA): Development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 1988;49:688–697PubMedGoogle Scholar
  2. 2.
    Hodgen GD, Burkman LJ, Coddington CC, Franken DR, Oehninger SC, Kruger TF, Rosenwaks Z: The hemizona assay (HZA): Finding sperm that have the “right stuff.” J Vitro Fert Embryo Transfer 1988;5:311–313Google Scholar
  3. 3.
    Franken DR, Burkman LJ, Oehninger S, Coddington CC, Veeck LL, Kruger TF, Rosenwaks Z, Hodgen GD: The hemizona assay using salt-stored human oocytes: Evaluation of zona pellucida capacity for binding human spermatozoa. Gamete Res 1989;22:15–26PubMedGoogle Scholar
  4. 4.
    Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA, Micholson 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;31:562–574PubMedGoogle Scholar
  5. 5.
    Franken DR, Oehninger S, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z, Acosta AA, Hodgen GD: The hemizona assay (HZA): A predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J Vitro Fert Embryo Transfer 1989;6:44–50Google Scholar
  6. 6.
    Oehninger S, Coddington CC, Scott R, Franken DR, Burkman LJ, Acosta AA, Hodgen GD: Hemizona assay: Assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril 1989;52:665–670Google Scholar
  7. 7.
    Kreitman O, Lynch A, Nixon WE, Hodgen GD: Ovum collection, induced luteal dysfunction, in vitro fertilization, embryo development and low tubal ovum transfer in primates.In In Vitro Fertilization and Embryo Transfer, ESE Hafez, K Semm (eds). Lancaster, England, MTP Press, 1980, pp 303–317Google Scholar
  8. 8.
    Balmaceda JP, Pool TB, Arana JB, Heitman TS, Asch RH: Successfulin vitro fertilization and embryo transfer in cynomolgus monkeys. Fertil Steril 1984;42:791–795PubMedGoogle Scholar
  9. 9.
    Boatman DE, Bavister BD: Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J Reprod Fertil 1984;71:357–366PubMedGoogle Scholar
  10. 10.
    Oehninger S, Scott RT, Coddington CC, Franken DR, Acosta AA, Hodgen GD: Validation of the hemizona assay in a monkey model: Influence of oocyte maturational stages. Fertil Steril 1989;51:881–885PubMedGoogle Scholar
  11. 11.
    Veeck LL: Extracorporeal maturation: Norfolk, 1984. Ann NY Acad Sci 1985;442:357–367PubMedGoogle Scholar
  12. 12.
    Jeyendran RS, Vanderven HH, Kennedy W, Perez Pelaez M, Zaneveld LD: Comparison of glycerol and a zwitterion buffer system as cryoprotective media for human spermatozoa. J Androl 1984;5:1–5PubMedGoogle Scholar
  13. 13.
    Franken DR, Kruger TF, Menkveld R, Oehninger S, Coddington CC, Hodgen GD: Hemizona assay and teratozoospermia: Increasing sperm insemination concentration to enhance zona pellucida binding. Fertil Steril 1990;54:497–503PubMedGoogle Scholar
  14. 14.
    Franken DR, Coddington CC, Burkman LJ, Oosthuizen WT, Oehninger S, Kruger TF, Hodgen GD: Defining the valid hemizona assay: Accounting for binding variability within zona pellucidae and within semen samples from fertile males. Fertil Steril 1991;56:1156–1161PubMedGoogle Scholar
  15. 15.
    Oehninger S, Alexander NJ, Franken DR, Coddington CC, Burkman LJ, Kruger TF, Acosta AA, Hodgen GD: Antifertilization assay.In Proceedings of Contraceptive Research and Development (CONRAD) on Gamete Interaction: Prospects for Immunocontraception, Alexander NJ, Acosta A, Griffin D, Spieler J (eds). Bariloche, Argentina, Alan R. Liss, Nov 27–29, 1989Google Scholar
  16. 16.
    Roussel JD, Austin CR: Preservation of primate spermatozoa by freezing. J Reprod Fertil 1967;13:333–335PubMedGoogle Scholar
  17. 17.
    Tollner TL, VandeVoort CA, Drobuis EZ, Overstreet JW: Cryopreservation techniques for cynomolgus monkey semen. Presented at the 14th Annual Meeting of the American Society of Andrology, New Orleans, LA, Apr 13–16, 1989 (Abstr)Google Scholar
  18. 18.
    Davis RO, VandeVoort CA, Andrew JA, Overstreet JW: High-speed videomicrography and computer-aided sperm analysis (CASA) of macaque spermatozoa activated with caffeine and db cAMP. Presented at the 15th Annual Meeting of the American Society of Andrology, Columbia, SC, Apr 6–9, 1990 (abstr)Google Scholar
  19. 19.
    Bleil JD, Wassarman PM: Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol 1983;95:317–324PubMedGoogle Scholar
  20. 20.
    Keel BA, Webster BW, Roberts DK: Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil 1987;81:213–220PubMedGoogle Scholar
  21. 21.
    Critser JK, Huse Banda AR, Aaker DV, Arnescon BW, Ball GD: Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability and acrosome reaction. Fertil Steril 1987;47:656–663PubMedGoogle Scholar
  22. 22.
    Lopata A, Summers PM, Hearn JP: Births following the transfer of cultured embryos obtained byin vitro fertilization in the marmoset monkey (Callithrix jacchus). Fertil Steril 1988;50:503–509PubMedGoogle Scholar
  23. 23.
    Oehninger S, Veeck LL, Franken DR, Kruger TF, Acosta AA, Hodgen GD: Human preovulatory oocytes have a higher sperm binding ability than immature oocytes under hemizona assay conditions: Evidence supporting the concept of “zona maturation.” Fertil Steril 1991;55:1165–1170PubMedGoogle Scholar
  24. 24.
    Oehninger S, Acosta AA, Hodgen GD: Antagonistic and agonistic properties of saccharide moieties in the hemizona assay. Fertil Steril 1990;53:143–149PubMedGoogle Scholar
  25. 25.
    Coddington CC, Fulgham D, Alexander N, 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;54:504–508PubMedGoogle Scholar
  26. 26.
    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 the human hemizonae pellucida after storage in a buffered salt solution. Andrologia 1991;23:205–208PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Sergio Oehninger
    • 1
  • Mahmood Morshedi
    • 1
  • Hakan Ertunc
    • 1
  • Christine Philput
    • 2
  • Silvina M. Bocca
    • 1
  • Anibal A. Acosta
    • 1
  • Gary D. Hodgen
    • 1
  1. 1.The Jones Institute for Reproductive Medicine, Department of Obstetrics and GynecologyEastern Virginia Medical SchoolNorfolkUSA
  2. 2.Department of Radiation Oncology (Biostatistics)Eastern Virginia Medical SchoolNorfolkUSA

Personalised recommendations