| Abstrakt: |
The frequency of spermatozoa binding exogenous DNA after sperm/DNA co-culture is a key to a successful sperm mediated-gene transfer. In the study, the influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin labeled DNA of pEGFP-N1 expressive vector as trace, and the acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained. Results showed that goat spermatozoa could spontaneously take up exogenous DNA, and which was initially bound to the outer sperm membrane at postacrosomal region. In 27 goat samples, binding rates varied between 3.2%–48.4%, and there were significant differences in the capability of spermatozoa from different donors to bind exogenous DNA (p<0.01). The spermatozoa from the same goat were incubated with exogenous DNA for 30, 45, 60, 90 and 120 min in BO medium at 17°C, 27°C, 37°C, respectively. The result suggested that the rate of spermatozoa binding exogenous DNA was increasing with the extending co-incubated times. Under the 27°C and 37°C temperature condition, the rate of spermatozoa binding exogenous DNA would not increase after the incubation of 60 min, similar to the result of 90 min treatment under 17°C. To obtain the most efficient transfection, the influence of four factors such as epidermal growth factor (EGF), Insulin, Heparin and insulin-transferrin-selenium mix (ITS) required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into goat spermatozoa was found to be efficient using the BO medium plus 10 ng/ml EGF, and the rate of spermatozoa binding exogenous DNA was significant difference between the experimental group (30.5%) and control group (19.5%), but not in the ratio of sperm capacitation between the experimental and control group (88.0% vs. 86.1%). The similar results were found in the Insulin treatment group, and it was significant difference between the 5 μg/ml Insulin experimental group (32.5%) and control groups (22.3%), but not in the sperm capacitation ratio between the experimental and control group (88.2% vs. 82.7%). Plusing 5 μg/ml Heparin in BO medium, the rate of spermatozoa binding exogenous DNA was significant difference between the experimental and control group (32.9% vs. 22.3%), but not in the sperm capacitation ratio between the experimental and control group (86.6% vs. 85.6%). The ability of insulin-transferrin-selenium mix (5 mg/ml, 5 mg/ml, and 5 ng/ml) to facilitate the association between DNA and spermatozoa was evaluated with final ITS concentrations ranging from 0.5 to 5%. In the 0.5% ITS concentration group, the transfection rate of exogenous DNA was a significant difference between the control group (21.9%) and experimental groups (0.5%, 16.2%; 1%, 12.1%; 2%, 9.5%; 5%, 9.4%, respectively) (p<0.05). It was significant difference for the sperm capacitation ratio between the 5% ITS experimental and control group (64.8% vs. 82.3%), but not in other experimental groups (0.5%, 87.9; 1%, 85.8%; 2%, 82.8%). Furthermore, the ability of EGF (10 ng/ml), Insulin (5 μg/ml) and Heparin (10 μg/ml) to facilitate the association between DNA and spermatozoa was evaluated, and it was a significant difference in the transfection efficiencies recorded for the experimental and control group (28.0% vs. 19.3%)(p<0.05). However, BO medium plus LipefectaminTM- 2000 restrained the efficiency of the three factors for the transfection rate of exogenous DNA into goat spermatozoa (15.3% vs. 27.9%)(p<0.01). (poster) |
