Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

Mouse cloning by nuclear transfer using freeze-drying (FD) somatic cells is now possible, but the success rate is significantly lower than that of FD spermatozoa. Because spermatozoa, unlike somatic cells, are haploid cells with hardened nuclei due to protamine, the factors responsible for their tolerance to FD treatment remain unclear. In this study, we attempt to produce offspring from FD spermatid, a haploid sperm progenitor cell whose nuclei, like somatic cells, have not yet been replaced by protamine. We developed a method for collecting FD spermatids from testicular suspension. Despite the significantly lower success rate than that of FD spermatozoa, healthy offspring were obtained when FD spermatids were injected into oocytes. Offspring were also obtained from FD spermatids derived from immature male mice that had not yet produced spermatozoa. These results suggest that nuclear protaminization, rather than haploid nuclei, is one of the key processes responsible for tolerance to FD treatment.