Production of offspring by intracytoplasmic sperm injection using sperm from deceased transgenic mice at different postmortem intervals

Successful fertilization by intracytoplasmic sperm injection (ICSI) is possible as long as the sperm genome is intact, even in the context of defective sperm or sustained adverse treatment. However, there are few reports on rescuing gene-modified mouse lines after accidental death. To investigate whether sperm from a dead transgenic mouse can fertilize an oocyte and enable embryo development into a pup, Nestin-GFP transgenic male mice were sacrificed, and sperm was collected 14 h, 24 h, and 48 h after death. The collected sperm was injected into oocytes from hybrid B6D2F1 or inbred C57BL/6 N mice. The results showed that the sperm in the three groups activated oocytes from B6D2F1 and supported embryo development to the blastocyst stage. For ICSI embryos derived from B6D2F1 mice, the cleavage and blastocyst rates were significantly lower in the three experimental groups than in the control group (0 h) (P < 0.05), and the birth rate in the 24 h and 48 h groups was significantly lower than that in the 14 h and control groups (0 h). For C57BL/6N-derived ICSI embryos, the cleavage rates were significantly lower at 24 h and 48 h than at 14 h and 0 h (control group), and the birth rate in the three experimental groups was significantly lower than that in the control group (0 h). The F0 mice derived from B6D2F1 and C57BL/6 N oocytes had normal reproductive ability, and F1 mice were successfully obtained. The characteristics of the GFP gene were preserved and inherited. The histone H2AX phosphorylation assay showed that the proportion of focus-negative embryos was markedly and significantly lower in the 14 h, 24 h, and 48 h groups than in the control group (0 h). The proportion of focus-negative embryos was significantly lower at 48 h than at 14 h or 24 h. The number of foci was significantly higher in the three experimental groups than in the control group (0 h), indicating that sperm DNA sustained more damage after death and that few sperm had an intact genome. In summary, sperm obtained from mice 14 h, 24 h, and 48 h after death is capable of activating an oocyte and supporting complete embryo development into a pup. This study provides an effective way to rescue accidently died mouse strains.