Abstract

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. It has been a useful tool for the production of transgenic mice but is still rather inefficient in farm animals. The aim of this study was to assess the effect of Lysolecithin (LL), Triton X-100 (TX) and sodium hydroxide (NaOH) on the simultaneous removal of plasma membrane and acrosome from bovine sperm co-incubated with/without exogenous DNA. First, we evaluated the removal of plasma membrane and acrosome from sperm by evaluating the integrity of these membranes by SYBYR/PI and PNA/FITC, respectively. Second, we determined the percentage of DNA fragmentation from sperm (TUNEL assay). And finally, we estimated the sperm capacity to bind exogenous DNA and the location of the bound DNA by flow cytometry and confocal laser microscopy. Removal of plasma membrane and acrosome significantly increased (LL 100%, TX 100% and NaOH 94%) compared with that of the control group (3%). DNA fragmentation increased only slightly in all treatments (4%) compared to the control (2%). All treatments showed a high binding of DNA (>99%) similar to the control group. The location of the DNA was observed mainly in the post-acrosomal region of the control group, however all treatments showed binding of DNA throughout the sperm structure. These results demonstrate the altered sperm membranes facilitate exogenous DNA- spermatozoa interaction. Nevertheless, severe sperm treatments may be detrimental on embryonic development. For this reason, the next step will be to use the SMGT coupled to intracytoplasmic sperm injection (ICSI) to evaluate if these treatments are suitable to generate transgenic bovine embryos. Acknowledgments: FONDECYT 11130724 CONICYT-Chile, CONICYT Doctoral thesis Scholarship.