Expression of Foreign DNA Is Associated with Paternal Chromosome Degradation in Intracytoplasmic Sperm Injection-Mediated Transgenesis in the Mouse1

Abstract

The efficiency of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is often limited by poor embryo development. Because our previous work indicated that impairment of embryo development is frequently related to chromosomal abnormalities, we hypothesized that foreign DNA and/or conditions used to enhance integration of the DNA might induce chromosome damage. Therefore, we examined the chromosomes of mouse embryos produced by transgenesis with the EGFP gene. Spermatozoa were processed with three methods that cause membrane disruption: freeze-thawing, Triton X-100, or Triton X-100 followed by a sucrose wash. Membrane-disrupted spermatozoa were mixed with EGFP plasmids and injected into metaphase II oocytes. Three endpoints were evaluated: paternal chromosomes of the zygote, embryo capacity to develop in vitro, and expression of the transgene at the morula/blastocyst stage. In all pretreatments, we observed a significant decrease (approximately 2-fold) in the frequency of normal karyoplates when spermatozoa were incubated with exogenous DNA as compared with the treatment when no DNA was added. As predicted, embryo development was correlated with the integrity of the paternal chromosomes of the zygote. Searching for the possible mechanism of chromosome degradation, we used the ion chelators EGTA and EDTA and found that they neutralize the harmful effect of the transgene and stabilize the paternal chromosomes. In the presence of chelating agents, however, the number of embryos expressing EGFP produced with ICSI-mediated transgenesis decreased significantly. The results suggest that treatment of spermatozoa with exogenous DNA leads to paternal chromosome degradation in the zygote. Furthermore, the mechanisms of disruption of paternal chromosomes and the integration of foreign DNA may be closely related.

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

“…It is believed that EGTA, a calcium chelator, either prevents or decreases the activity of calcium-dependent endonucleases [14] by limiting the availability of the circulating calcium; therefore, EGTA is recommended to minimize chromosome breakage [26]. Conversely, trehalose, a disaccharide of glucose is present in many organisms that are able to survive complete dehydration, a phenomenon known as anhydrobiosis [20] and [27].…”

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

“…ROS are known mostly for their detrimental effects on sperm functions (Twigg et al 1998). Szczygiel et al (2003) demonstrated that incubation of spermatozoa with exogenous DNA leads to breaks in paternal chromosomes, and it is well known that spermatozoa deficiency in nuclear DNA repair systems results in their increased susceptibility to oxidative stress and DNA fragmentation (De Lamirande et al 1997).…”

Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

“…Factors that have been reported to influence the outcome of transgenesis in porcine embryos through ICSI include temperature during micromanipulation, exogenous DNA concentrations, plasmid constructs [3] and DNase I treatment [4] . Szczygiel et al [5] found that Ca 2+ chelators (EDTA or EGTA) in the suspensions of sperm cell and exogenous DNA could decrease transgenesis and affect the degree of DNA integration into chromosomes, and that this was related to a Ca 2+ /Mg 2+ -dependent enzyme in the oocyte. A Ca 2+ /Mg 2+ -dependent DNase I in pig MII oocytes has been documented which could significantly protect linearized exogenous DNA when microinjected [6] .…”