Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation.
Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000 or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm.
Patil
J G
JG
Department of Zoology, National University of Singapore.
Khoo
H W
HW
D000818:Animals; D030801:Animals, Genetically Modified; D001345:Autoradiography; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D004274:DNA, Recombinant / Q000378:metabolism*; D018274:Electroporation; D005306:Fertilization; D017403:In Situ Hybridization; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids; D013081:Sperm Motility; D013094:Spermatozoa / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D015027:Zebrafish
D004274:DNA, Recombinant