Adhesion of Funa Spermatozoa with Protamine to Achieve Polyspermy

Abstract

Artificial polyspermy using adhesive spermatozoa can be used as a method of chromosome manipu-lation. In this study, adhesion of funa spermatozoa was attempted as a basis of the polyspermy procedure by using the basic protein protamine of the cell nucleas, and its influence on adherence and motility was examined. Protamine was added to sperm diluted to about 1:100 to make final concentrations of 1, 5, 10, 20, and 30μg/ml, and each of these mixtures was allowed to react for 10 minutes. The rate of adhesion of the spermatozoa (the proportion of adherent spermatozoa to the total number tested) increased as the concentration increased; from 4.8% (at 1μg/ml) to 40.1% (at 30μg/ml). When the treatment was made for 1, 5, 10, 20, and 30 minutes at l0μg/ml and 30μg/ml, the rate of adhesion increased as the reaction time increased; at 10μg/ml it increased from 17.0% (1 min) to 57.2% (30 min), and at 30μg/ml, it increased from 32.5% to 63.0%. When application to chromosome manipulation is considered, the rate of adhesion between two spermatozoa becomes important (based on the diameter of the micropylar canal, it appears that only adherent pairs of spermatozoa can enter the egg), but the rate of adhesion between two spermatozoa did not increase very markedly even when the concentration was increased; at 1, 5, 10, 20, and 30μg/ml, the rates were 4.8, 13.6, 14.1, 13.9, and 18.1%, respectively. Even in the study using different reaction times, the rates of adhesion were almost unchanged: at 10μg/ml the rats ranged from 14.7% to 18.1%, and at 30μg/ml they ranged from 15.5% to 17.0%. After the adhesion treatment, sperm motility lasted 76.4, 73.6, 50.0, 32.0, and 18.2 seconds at 10μg/ml at reaction times of 1, 5, 10, 20, and 30 minutes, respectively. At 30μg/ml, the corresponding values were 52.9, 37.9, 22.7, 26.8, and 5.6 seconds, respectively. The results suggest that reaction for 5 minutes at 10μg/ml is adequate for adhesion between two spermatozoa.