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 DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature 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 evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from 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 spermatozoa protects exogenous DNA from nuclease activities.
Zannoni
Augusta
A
Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell' Emilia, Bologna, Italy.
Spinaci
Marcella
M
Bernardini
Chiara
C
Bacci
Maria Laura
ML
Seren
Eraldo
E
Mattioli
Mauro
M
Forni
Monica
M
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western / Q000379:methods; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D008297:Male; D009865:Oocytes / Q000201:enzymology*; D009866:Oogenesis / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic; D013552:Swine
D012333:RNA, Messenger; D004247:DNA; D003850:Deoxyribonuclease I