Exogenous DNA uptake by South American catfish (Rhamdia quelen) spermatozoa after seminal plasma removal.

摘要

Sperm from different species shows biological differences, determining the success or failure of the sperm-mediated gene transfer (SMGT) technique. There is evidence that exogenous DNA uptake by the spermatozoa is a species-specific and highly regulated phenomenon. Problems involving SMGT procedures might be related to activation of defenses in spermatozoa and in seminal plasma such as DNase enzymes. The objective in the present study was to transfect South American catfish spermatozoa after seminal plasma removal. Seminal plasma had a strong DNase activity that is reduced after sperm washes in isosmotic solution, in which Western blot analysis demonstrated a reduction in the DNase content after washes and Southern blot evaluations show the presence of plasmid after sperm washes. The seminal plasma DNase digests exogenous DNA in a few minutes and has an optimal activity at 43 degrees C. Also, EDTA at 30 mM concentration inhibits the DNase activity. Using PCR the pEGFP vector was internalized by sperm cells even at lesser concentrations (5-40 ng/106 spermatozoa) without motility loss after seminal plasma removal. Conversely, using greater pEGFP concentrations (100 ng/106 spermatozoa), there were no motile cells, suggesting toxicity of exogenous DNA for sperm cells. These results are interpreted to provide information that can improve the protocol for generation of transgenic South American catfish.