Domingues William Borges, da Silveira Tony Leandro Rezende, Komninou Eliza Rossi, Monte Leonardo Garcia, Remião Mariana Härter, Dellagostin Odir Antônio, Corcini Carine Dahl, Varela Junior Antônio Sergio, Seixas Fabiana Kömmling, Collares Tiago, Campos Vinicius Farias
Laboratório de Genômica Estrutural,Programa de Pós-Graduação em Biotecnologia,Centro de Desenvolvimento Tecnológico,Universidade Federal de Pelotas,Pelotas,RS,Brasil.
Laboratório de Biotecnologia do Cancer,Programa de Pós-Graduação em Biotecnologia,Centro de Desenvolvimento Tecnológico,Universidade Federal de Pelotas,Pelotas,RS,Brasil.
Zygote. 2017 Aug;25(4):519-528. doi: 10.1017/S0967199417000375. Epub 2017 Jul 13.
Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.
牛性别分选精子已商业化，并通过精子介导基因转移（SMGT）技术成功用于生产所需性别的转基因胚胎。然而，性别分选精子内化外源DNA的能力有所降低。在其他物种中，已报道精子细胞与外源DNA之间的相互作用是一个依赖CD4样分子的过程。基于流式细胞术的性别分选过程使精子受到不同的应激，从而导致细胞膜发生变化。本研究的目的是阐明CD4样分子的重新分布与外源DNA与性别分选牛精子结合之间的关系。在第一组实验中，评估了膜磷脂紊乱和CD4的重新分布。进行了第二组实验，以研究CD4重新分布对性别分选牛精子中外源DNA与精子细胞结合机制以及脂质转染效率的影响。性别分选程序增加了膜磷脂紊乱并诱导了CD4样分子的重新分布。与未分选的精子相比，X分选精子和Y分选精子与膜结合的DNA均减少；然而，添加脂质体后，外源DNA的结合显著增加。此外，我们证明，当这些细胞与抗牛CD4单克隆抗体预孵育时，与精子结合的外源DNA数量减少，这支持了我们的假设，即CD4样分子确实在外源DNA/牛精子细胞相互作用过程中起关键作用。
