VALIDATION OF THE HEMIZONA ASSAY (HZA) IN A MONKEY MODEL .2. KINETICSOF BINDING AND INFLUENCE OF CRYOPRESERVED - THAWED SPERMATOZOA

We compared fresh and frozen- thawed cynomolgus monkey spermatozoa tig ht binding to the zona pellucida under hemizona assay (HZA) conditions . Monkey oocytes were recovered after superovulation and stored in sal t solution. Matching hemizonae were obtained by micromanipulation. Sem en, obtained by electroejaculation, was used fresh or was cyropreserve d, thawed, and washed by swim-up separation. At the standard initial d ilution of 500,000 motile sperm/ml (or 5 x 10(4) 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 t ightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozo a was similar to that of fresh at a standard concentration. Kinetic st udies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evalua tion of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.