Evaluation of Separating X-and Y-Sperms by Percoll Density Gradient Centrifugation Using Sperms of Transgenic Mice Carrying a Transgene on Y-Chromosome

Abstract

Using sperms of the transgenic mice carrying a human^Aγ /β -globin gene on Ychromosome, we attempted to separate X-and Y-bearing sperms by the Percoll density gradient centrifugation. The ratio of X- and Y-sperms was determined by DNA dot blot hybridization procedure with sperm DNA. Sperm suspension collected from cauda epididymidis was loaded on the gradient composed of 7 Percoll concentrations (35-84%) and was centrifuged at 300×g for 10, 15 or 20 minutes, respectively, at room temperature. After centrifugation, sperms were collected from each gradient fraction and washed with 0.85% saline solution. DNA was extracted from sperms, dotted and fixed on nitrocellulose filter, and was hybridized with the³²P-labeled DNA probe derived from the β-globin gene. Each DNA spot was cut out, immersed in the liquid scintillator and was counted for radioactivity. There was no difference among the radioactivities in the DNA spots, indicating that the ratio of X-and Y-sperms was the same in all the gradient fractions of three different centrifugal conditions. The result suggests to be difficult to separate X-and Y-sperms by Percoll density gradient centrifugation, at least, using sperms from cauda epididymidis of mouse.