Fertilization and Early Embryogenesis
(M331) TOPOISOMERASE II AND ASSOCIATED NUCLEASE ACTIVITY IN MATURE MOUSE SPERMATOZOA.
Shaman, Jeffrey 1, Prisztoka, Renata1, Ward, W. Steven1, 1 University of Hawaii, Honolulu, HI
ABSTRACT- The DNA in spermatozoa is so tightly packed that electron micrographs of nuclei are seen as homogenous, electron-dense masses. To arrange the DNA in this almost crystalline structure the DNA is bound to protamines, which condense it into toroids that contain about 50 kb of DNA. We have shown that sperm DNA is also arranged into loop domains, also about 50 kb, attached at their bases to the nuclear matrix. Our lab has proposed a model of this tertiary structure termed the Donut-Loop model in which each loop domain is one protamine-toroid. In this model, the protamine-bound DNA toroids are connected by short histone-bound toroid linker regions attaching to the nuclear matrix. We have previously reported that human, mouse, and hamster spermatozoa incubated solely with Mg2+ and/or Ca2+ are able to cleave their DNA into loop-sized fragments of about 50 kb. Here, we first demonstrate that this nuclease activity remains even when the nuclei are extracted with 2 M NaCl and 2 mM dithiothreitol suggesting that the nuclease(s) in question is bound to the insoluble nuclear matrix. We found that manganese activates the nuclease activity much more than magnesium. Interestingly, we now have evidence that this nuclear matrix associated activity results from at least two distinct nucleases that interact. The first is topoisomerase II (Topo II) – an enzyme that allows for the controlled nicking, relaxation, and religation of DNA and that has been shown to be at the base of loop domains in somatic cells. This sperm nuclear Topo II is able to cut the DNA into ∼50 kb segments which is the same size as one loop domain. Subsequent digestion of DNA into pieces < 50 kb is achieved by a second, calcium-dependent nuclease. It is associated with the sperm nuclear matrix, yet not bound to the DNA prior to activation. Our evidence suggests that Topo II regulates the second nuclease activity in two ways: it can inhibit the nuclease under certain conditions, but the nuclease activity may also require Topo II to be activated. We present a model in which Topo II is a regulator of chromatin integrity in the mature spermatozoon.
KEY WORDS: spermatozoa, topoisomerase ii, nuclease, chromatin integrity