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

Abstract Recently, mice have been produced from Freeze-drying (FD) somatic cells by nuclear transfer, but the success rate was very low compared to FD spermatozoa. Since spermatozoa, unlike somatic cells, are haploid cell and their nuclei were hardened by protamine, it is unclear which is responsible for tolerance to FD treatment. Here, we attempt to produce offspring from FD spermatid, a haploid sperm progenitor cell, but nuclei were not yet replaced by protamine as same as somatic cells. We developed the method to collect FD spermatids from testicular suspension. When FD spermatids were injected into oocytes, healthy offspring were obtained, despite the significantly reduced success rate compared to FD spermatozoa. Offspring were also obtained from FD spermatids derived from immature male mice, which have not yet produced spermatozoa. These results indicate that nuclear protaminization, rather than haploid nuclei, is the key process responsible for tolerance to FD treatment.