Abstract: 【Objective】 Sperm-mediated gene transfer technique is simple, it has been recognized by many scientists as a way to produce transgenic animals, however, the results from different laboratories showed poor stability and reproducibility, transgenic efficiency is also significantly different. The aim of this study is to clarify the main factors causing this phenomenon.【Method】Capacitated sperms were incubated with 0, 15, 30 and 300 nmol·L^-1 Cy-3 labeled DNA (Cy-3-DNA）at 37℃ for 30 min. After incubation, sperms viabilities were detected by CBC board. Sperms incubated by DNA were used to direct smeared, smeared after washing by DPBS and smeared after digested by Dnase I, sperm smears were placed under fluorescent microscope. The total number of sperms and the number of sperms which show Cy-3 signal in the vision were recorded, the records were used for the efficiency statistics of sperms binding and uptaking exogenous DNA. 50µmol·L^-1 P4 were used for inducing sperms acrosome reaction which were incubated with 300 nmol·L^-1 Cy-3-DNA. The proportion of sperms showing Cy-3 signals were checked before and after the acrosome reaction. Sperms incubated with 15 and 300 nmol·L^-1 Cy-3-DNA were used for IVF, the presence of Cy-3-DNA in the zygotes was detected under fluorescence microscopy. According to the optimized conditions for these experiments, sperms incubated with 0, 15 and 300 nmol·L^-1 pEGFP-C1 were used for getting transgenic embryos, and oocyte fertilization rate and embryonic development rate in experimental group and control group were compared, PCR and RT-PCR were used to detect the presence and expression of pEGFP in the blastocyst.【Result】Sperms after capacitation were incubated with 0, 30, 15, 30 and 300 nmol·L^-1 Cy-3-DNA, the sperm viability was 82.21%, 73.63%, 77.38%, 76.33% and 77.80%, respectively. The rate of positive sperms was 76%, 94%, 99% and 100% before washing , and that in 15，30 and 300 nmol·L^-1 treatment groups had more efficiency than 3 nmol·L^-1 (P＜0.05) group. The rate of positive sperms was 45%, 66%, 84%, 87% after washing and 44%, 56%, 71%, 76% after digestion, and that in 300 and 30 nmol·L^-1 treatment groups had more efficiency than the other two groups, and 15 nmol·L^-1 treatment group is had more effiency than 3 nmol·L^-1 (P＜0.05) group. The result showed that the amount of uptaking DNA increased significantly as the increasing of DNA concentration by ImageJ analysis(P＜0.01). After acrosome reaction, DNA attached on the acrosome would be lost, but DNA exist in the rear of the sperm head still retained, so the ratio of positive sperms did not decrease. Sperms incubated with 15 and 300 nmol·L^-1 DNA were used for IVF. There were exogenous DNA fluorescence signals distributed in zygotic embryos under fluorescence microscopy, embryos which fluorescence signals densely distributed in male pronuclear were recorded and considered as positive embryos. The positive rate of zygotic embryos derived from sperm incubated with 300 nmol·L^-1 DNA was 27.89%, which was significantly higher than that incubated with 15 nmol·L^-1 (P＜0.05). Sperms were incubated with pEGFP-C1 plasmid were used for IVF. The result showed that incubation did not affect the oocyte fertilization rate，and the embryonic development rate compared with the control group. Single blastocyst PCR detection showed that the transgenic blastocyst rate derived from sperms incubated with 15 and 300 nmol·L^-1 pEGFP-C1 was 8.72% and 21.50%, respectively. RT-PCR detection of EGFP expression in the blastocysts derived from 300 nmol·L^-1 DNA showed positive results but no protein expression was detected by fluorescence microscopy.【Conclusion】Sperms have the ability of binding and uptaking exogenous DNA, and sperms can carry exogenous DNA into oocytes. Acrosome reaction causes part loss of the DNA, but in the rear zone of the sperm head, DNA is still retained. The key factors restricting the efficiency of sperm-mediated gene transfer might be integration of exogenous DNA into the genome and expression activation of the exogenous genes during fertilization.

Key words: sperm-mediated gene transfer, concentrations of DNA, acrosome reaction, exogenous genes integration and expression, mouse