404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

Abstract

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 10⁵ spermatozoa group than in the other groups (10⁴, 10⁶, and 10⁷ spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 ± 0.2, 70.8 ± 1.8, and 68.0 ± 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 ± 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 ± 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 ± 1.1%) and control (67.8 ± 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 ± 1.3 and 11.3 ± 0.8%) than in the control group (22.2 ± 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 ± 12.0%) than in the DNA solution group (38.7 ± 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes.

This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea