Wu Yi, Liu Cheng-Jun, Wan Peng-Cheng, Hao Ze-Dong, Zeng Shen-Ming
Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, PR China.
Anim Reprod Sci. 2009 Jul;113(1-4):156-66. doi: 10.1016/j.anireprosci.2008.08.014. Epub 2008 Aug 12.
This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 microg/mL, 0.05 microg/mL or 0.1 microg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 microg/mL. When oocytes were microinjected on a warm stage at 30 degrees C, the percentage of EGFP-expressing embryos was higher than that at 38.5 degrees C (40.1% vs. 20.9%, P<0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P<0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P<0.01). However, neither incubation temperatures of spermatozoa and DNA (4 degrees C, 24 degrees C or 39 degrees C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.
本研究旨在探究影响猪胚胎在胞浆内精子注射（ICSI）介导的DNA转移后囊胚发育效率和增强型绿色荧光蛋白（EGFP）表达的因素。将冻融的死精子暴露于不同浓度（0.01μg/mL、0.05μg/mL或0.1μg/mL）的EGFP DNA溶液中，然后显微注射到体外成熟的卵母细胞中。所得胚胎EGFP表达的最佳浓度为0.05μg/mL。当卵母细胞在30℃的温暖阶段进行显微注射时，表达EGFP的胚胎百分比高于在38.5℃时（40.1%对20.9%，P<0.01）。使用线性EGFP DNA暴露精子进行ICSI后胚胎中EGFP表达的效率高于使用环状DNA（40.8%对28.2%，P<0.05）。电激活后用6-二甲基氨基嘌呤（6-DMAP）处理的ICSI卵母细胞中表达EGFP的胚胎百分比高于未处理的（52.5%对26.3%，P<0.01）。然而，精子和DNA的孵育温度（4℃、24℃或39℃）以及在孵育培养基中添加牛血清白蛋白（BSA）均不影响产生表达EGFP胚胎的效率。此外，精子与DNA预孵育后用脱氧核糖核酸酶I（DNase I）处理可阻止胚胎表达EGFP。ICSI卵母细胞的EGFP表达既不受使用精子头部或完整精子进行胞浆内注射的影响，也不受预孵育后精子洗涤的影响。除电激活后6-DMAP处理外，上述因素均不影响胚胎发育能力。总之，DNA与精子孵育后，大多数外源DNA分子紧密结合在精子头部膜上，ICSI过程中的温度、6-DMAP处理、外源DNA浓度和构建体可显著影响猪胚胎在ICSI介导的DNA转移后EGFP的表达。
