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Updated: Aug 23, 2025
Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
Published on: April 2, 2020
Production of mouse offspring from zygotes fertilized with freeze-dried spermatids
Sayaka Wakayama1,2, Daiyu Ito3, Masatoshi Ooga3,4
1Faculty of Life and Environmental Science, University of Yamanashi, Kofu, 400-8510, Japan. sayakaw@yamanashi.ac.jp.
Abstract:
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to produce offspring from FD spermatid, a haploid sperm progenitor cell whose nuclei, like somatic cells, have not yet been replaced by protamine.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.
Insights
Freeze-drying somatic cells for mouse cloning is less successful than using sperm. This study found nuclear protaminization, not haploid nuclei, is key for freeze-drying tolerance in reproductive cells.
Area of Science:
- Reproductive Biology
- Cell Biology
- Cryobiology
Background:
- Nuclear transfer (NT) using freeze-dried (FD) somatic cells enables mouse cloning but with low efficiency.
- FD spermatozoa exhibit higher tolerance to freeze-drying than somatic cells, but the reasons remain unclear.
Purpose of the Study:
- To investigate the role of nuclear protaminization in the tolerance of sperm cells to freeze-drying (FD) treatment.
- To assess the feasibility of producing offspring from FD spermatids via nuclear transfer.
Main Methods:
- Developed a method to collect FD spermatids from testicular suspensions.
- Performed nuclear transfer by injecting FD spermatids into oocytes.
- Collected FD spermatids from both mature and immature male mice.
Main Results:
- Healthy offspring were successfully produced from FD spermatids, albeit with a lower success rate than FD spermatozoa.
- Offspring were obtained from FD spermatids derived from immature mice lacking mature spermatozoa.
- These findings indicate that nuclear protaminization is crucial for FD tolerance.
Conclusions:
- Nuclear protaminization, a process where protamine replaces histones in sperm nuclei, is a key factor conferring tolerance to freeze-drying.
- Haploid status alone does not guarantee tolerance to FD treatment.
- Spermatids, which are haploid but lack protamine, can be used for cloning after FD, supporting the protaminization hypothesis.
