We demonstrated that mouse spermatozoa cleave their DNA into approximately 50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl(2) and CaCl(2) in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl(2) alone could elicit this activity, but CaCl(2) had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by ethylene glycol tetraacetic acid (EGTA) to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn(2+), Ca(2+), or Zn(2+) could each activate SDD in spermatozoa but Mg(2+) could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca(2+) elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37 degrees C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein.
2007 Wiley-Liss, Inc.