GAMETE BIOLOGY AND GAMETOGENESIS - C
Wednesday, August 4, 2004
10:30 AM–12:30 PM
(710) HUMAN SPERM CHROMATIN CONTAIN NUCLEASE ACTIVITY.
Sotolongo, Barbara1, Huang, Thomas 1, Ward, W. Steven1, 1 Insitute for Biogenesis Research, Honolulu, HI
ABSTRACT- We have previously provided evidence that hamster epididymal spermatozoa has an endogenous nuclease activity that cleaves sperm DNA into 50 kb fragments after overnight incubation in Triton X-100 (Biol. Repro. 69:2029, 2003). This activity was dependent on MgCl2 and inhibited by EDTA. The overnight digestion, however, suggested the possibility that some exogenous contaminant may have contributed the nuclease. In this work, we investigated the possible presence of an endogenous nuclease in human spermatozoa, because the presence of an endogenous nuclease in human spermatozoa would have obvious implications for human fertility practices. Also, human spermatozoa are obtained as ejaculates and may have different properties than epididymal spermatozoa. Ejaculates obtained from a human fertility clinic were either centrifuged twice (total spermatozoa) or purified by swim-up in Percoll gradients, and frozen. Samples were resuspended in HTF media, counted, and incubated at 37°C, in 0.25% Triton X-100, 5 mM MgCl2, with or without varying concentrations of DNase I. The spermatozoa were then plugged in agarose, and analyzed by pulse field gel electrophoresis. We found that the human sperm chromatin was digested to 50 kb fragments in the absence of exogenous DNase I in as little as 15 minutes. DNase I concentrations as low as 0.01 g/ml stimulated the digestion of human sperm DNA into much smaller fragments. These smaller fragments could only be seen on conventional gel electrophoresis, and are similar to those reported by Lamond, et al. (Reprod Biomed Online 7:407, 2003). Percoll gradient purified spermatozoa were more susceptible to DNA degradation than total spermatozoa. These data suggest the possibility that human sperm chromatin contains a mechanism to digest its own DNA under certain conditions. We are currently testing possible mechanisms for this activity. Possible functions of nucleases in spermatozoa include the rapid degradation of the bulk of ejaculated spermatozoa that does not fertilize oocytes, and the protection against passing potentially damaged paternal DNA to the newly developing oocyte.
KEY WORDS: nuclease, DNA, spermatozoa, human