Dominguez, Kenneth2016-05-022016-05-022013-12http://hdl.handle.net/10125/100685Ph.D. University of Hawaii at Manoa 2013.Includes bibliographical references.The goal of this work was to identify the nuclease associated with sperm DNA degradation in the sperm and surrounding fluid of the vas deferens and cauda epididymis of the mouse. Earlier work in our laboratory demonstrated sperm nucleolytic activity that cleaved chromatin to the size of 50 kbp with Mn2+ and Ca2+, which could be reversed using EDTA. We have named this activity Sperm Chromatin Fragmentation (SCF). The reversible nature and activation of SCF using Mn2+ and Ca2+ are hallmarks of the nuclease topoisomerase IIB. When SCF is active for a period of time a different activity is observed that completely and irreversibly degrades the sperm chromatin. We have named this activity Sperm DNA Degradation (SDD). The complete degradation seen in SDD is much more pronounced in sperm from the vas deferens and the activity is greatly accelerated by the use of EGTA and Ca2+. We found that this combination causes the buffer to become acidic, and it is this acidity that activates the nuclease rather than EGTA and Ca2+. We developed a purification scheme for this nuclease, and then identified it as DNAse II, a known acidic nuclease, by Western blot analysis. The cauda epididymis and vas deferens act as storage areas for sperm, but may also function as a quality control mechanism for defective sperm. This is suggested by the drop in number of defective sperm seen in the cauda epidydimis compared to the caput suggesting they are being marked for degradation. The presence of DNase II in the vas deferens has yet to be described in the literature. Here we present evidence of its presence and activity in the sperm and luminal fluid of the male reproductive tract.engsperm DNA degradationcauda epididymisThesis