Sperm nucleases

While a nuclease could be detrimental to the integrity of the paternal genome, they are necessary for proper spermatogenesis. As discussed earlier, negative supercoils must be removed from the DNA when histones are replaced by protamines during spermiogenesis. To remove these supercoils, nicks must be introduced into the DNA by a nuclease, at specific stages of spermiogenesis, as discussed above (Marcon and Boissonneault, 2004). Topoisomerase has been implicated in this process as it allows for controlled nicking, increase in linking number and subsequent DNA relaxation, and religation of the DNA. The TUNEL assay does detect in situ DNA strand breaks in the early stages of spermatogenesis (Marcon and Boissonneault, 2004). However, the lack of proper elimination of errant cells during spermatoge-nesis could allow cells with DNA strand breaks to continue along the maturation pathway thus causing positive TUNEL assays in epididymal and ejaculated sperm.

The studies described above provide evidence for nucleolytic activity by topo-isomerases during spermiogenesis, but at least three separate laboratories have shown that mature spermatozoa may also have active nucleases. Spadaforra and colleagues suggested that the mouse spermatozoon digests a discrete portion of its histone-bound DNA when challenged with exogenous DNA (Maione et al., 1997). A portion of exogenous DNA is internalized into the nuclei triggering nuclease(s)

that cleave the exogenous and the genomic DNA. The nuclease response requires challenges with much higher DNA concentrations in ejaculated sperm compared to that in epididymal spermatozoa, eventually leading to cell death in both groups (Spadafora, 1998). Yanagimachi and colleagues have shown that ethylene diamine tetracetic acid (EDTA) and ethylene glycol bis-2-aminoethyl ether-N,N',N", n'-tetraacetic acid (EGTA) treatment of spermatozoa prior to intracytoplasmic sperm injection can prevent paternal chromosomal damage (Kaneko et al., 2003; Kusakabe et al., 2001; Tateno et al., 2000), suggesting that some endogenous nuclease does exist in spermatozoa. Finally, our laboratory has shown that fully mature spermatozoa from hamster, mouse, and human have the ability to digest their DNA into loop-sized fragments of 50 kb or so (Sotolongo et al., 2003,2005). More recent, unpublished data from our laboratory suggests that these nucleases are associated with the sperm nuclear matrix, and can be activated even more completely than we have previously shown. These data suggest that mature spermatozoa do contain some type of nuclease. We propose that these will be similar to the apoptotic related nucleases in somatic cells, including topoisomerase II, but that they might not necessarily function for that purpose. Just as the proteolytic apoptotic machinery was 'borrowed' for normal spermiogenesis functions in Drosophila (Arama et al., 2003), so might the nucleolytic functions be used for processes other than classical apoptosis in spermatozoa.

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100 Pregnancy Tips

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