METHOD FOR UTILIZATION OF SOLUBILIZED ZONAE PELLUCIDAE IN SPERM ZONA-ADHESION TEST
RUMIANA GANEVA*, MIRYANA GOSPODINOVA, IVKA IVANOVA, IVAYLO RANGELOV, MAGDALENA VASILEVA, KRISTINA NIKOLOVA, DIMITAR PARVANOV, GEORGI STAMENOV, TODOR CHAUSHEV
Nadezhda, Women’s Health Hospital, 3 “Blaga vest” Street, Sofia, Bulgaria
*Corresponding author: rum.ganeva@gmail.com
Keywords: zona pellucida, spermatozoa, adhesion test
Abstract: Glycoproteins of zona pellucida are natural ligands for human spermatozoa and they play crucial role in sperm maturation during fertilization process in vivo. The utilization of zona pellucida ligands on polystyrene surfaces can be used for developing of method for physiological separation of human spermatozoa, based on their zona pellucida adhesion abilities.In this article we propose a simple and effective procedure for utilization of native zona pellucida glycoproteins in the development of sperm-zona adhesion test. The most important checkpoints were to choose the appropriate concentration of HCl for zona solubilisation and to mark down the most convenient conditions for zona glycoproteins immobilization. We report the optimal concentration of the zona ligands, immobilization buffer, support material, adsorption method, incubation temperature and shape of the adhesion area.
This study was conducted with 180 zonae pellucidae from 36 healthy donors. Zonae pellucidae were solubilised in five concentrations of HCl (2.5 mM, 5 mM, 10 mM and 20 mM) and were used in two-fold serial dilutions in three buffers: carbonate buffer (CB), phosphate buffer (PBS) and Tris buffer (TBS). The zona glycoproteins were physically adsorbed in solution or allowed to dry on glass and polystyrene at 4ºC, 25ºC and 37ºC. In addition, two types of dried adhesion area shapes were examined: dot and line.The rate of sperm adhesion was assessed by placing 0.5x106/ml motile spermatozoa from 46 fertile men on the immobilized zona proteins and counting the attached spermatozoa. Sperm-zona adhesion score was calculated as number of attached spermatozoa per mm2. The results from the conducted experiments showed that optimal sperm adhesion is reached by solubilisation of the zonae by 10 mM HCl followed by dilution into CB in concentration 50 μg zonae proteins/ml and immobilizing the solution by air drying 0.1 μl as dots on polystyrene plates at 25ºC.Here we describe a useful method for utilization of zona pellucida glycoproteins that may be applicable both in developing new diagnostic tests for human sperm quality assessment and designing functional sperm selection procedure for IVF in the future.