PARENT SESSION
TESTIS

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(741) SPERMATOCYTE CHROMATIN MODIFICATION AFTER INDUCED DNA DAMAGE AND INHIBITION OF TOPOISOMERASE II.

Matulis, Shannon¹, Handel, Mary Ann², ¹ University of Tennessee, Knoxville, TN² The Jackson Laboratory, Bar Harbor, ME

ABSTRACT- Spermatocytes normally sustain many meiotically induced double-strand breaks (DSBs) early in meiotic prophase; these are rapidly repaired by recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after their recombination-related DSBs have been repaired. Two DNA-damaging agents were used in this study of the response of pachytene spermatocytes: -irradiation (20Gy or 60Gy) and etoposide (0.1uM-40uM), a topoisomerase II inhibitor that causes persistent un-ligated DSBs. Chromatin modifications after exposure were monitored by staining surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and H2AX (which recognizes chromatin associated with DSBs). RAD51 was rapidly recruited to irradiation- or etoposide-damaged chromatin, and spermatocytes treated with etoposide showed a dose-dependent increase in the number of chromatin-associated RAD51 foci, with a 45-fold increase in cells with greater than 10 foci after 40uM etoposide treatment. Likewise, H2AX associated with chromatin increased in an etoposide dose-dependent manner. Both chromatin modifications were seen after irradiation, but with no apparent dose response, suggesting severe acute damage at even the low dose rate or rapid and efficient repair. These chromatin modifications imply that spermatocytes recruit an active DNA damage response and thus the effect on cell viability is of interest. To assess cell death, binding of FITC-labeled annexin V to the spermatocyte cell surface was monitored. Comparing control to 10uM etoposide treatment, there was a 2.5-fold increase in percentage of early apoptotic cells. Interestingly, this increase in apoptotic cells was not seen 3 hours following irradiation. Taken together, these results suggest more rapid repair of irradiation-induced DNA damage than of etoposide-induced damage. (Supported by NIH HD33816 to MAH.)

KEY WORDS: DNA damage, spermatocyte