DNase I activity in pig MII oocytes:implications in transgenesis

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNAundergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated inthe zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of aDNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in ratimmature oocytes and the presence of nuclease activities has been shown in avian oocytes.The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this,oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenceda 33 kDa, Ca21/Mg21 dependent DNase I-like activity that was inhibited by Zn21. A further identification of DNase Iwas achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activitywas confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNAtransferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protectedfrom DNase I degradation and to persist in the ooplasm till 6 h.These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoaprotects exogenous DNA from nuclease activities[...]