Alkaline-elution measurement of DNA breakage and repair in isolated pachytene spermatocytes and spermatids after exposure to X rays

To further study the effects of X rays on the induction of DNA strand breaks in germ cells, single-cell suspensions made from mouse testes were irradiated with 800 R of X rays. Following irradiation, pachytene spermatocytes or early spermatids were isolated from the cell suspensions using centrifugal elutriation. This procedures separates cells on the basis of their size. After isolation, the pachytene spermatocytes or early spermatids were maintained for 1 h at either 330C (the approximate temperature of the mouse testis) or at 00C. Following this 1 h period, the cells were lysed on polyvinyl filters, and the alkaline elution procedure was carried out to monitor DNA breakage. The results of this study showed that there was increased strand breaks from both pachytene spermatocytes and early spermatids exposed to 800 R of X rays, but this breakage was measurable only when the cells were maintained at 00C. After 1 h posttreatment incubation at 330C, no measurable DNA breakage could be detected with the authors alkaline elution procedures. Thus, these experiments have now shown that DNA breaks do occur in pachytene spermatocytes and early spermatids of the mouse after X-ray exposure, but that the breaks are normally rejoined very rapidly (in less than 1 h)