Purpose: Long-term preservation of mammalian sperm is needed worldwide for the strain maintenance. However, cryopreservation using liquid nitrogen is expensive to maintain and there is a risk of sperm loss due to accidents or disasters. Freeze-drying (FD) technology can easily preserve sperm at room temperature for more than one year. However, the birth rate of FD sperm is about 20%, which is significantly lower than that of fresh sperm. It is necessary to select FD sperm which has the normal developmental ability for the improvement of the experimental efficiency, but no such method exist yet. Recently, we identified a number of spermatozoa whose heads and tails had been separated by FD treatment (Yang, Ito et al., JRD 2023), but their fertilization and developmental ability have not yet been compared between separated and intact sperm. In this study, we examined whether the sperm head separated from the tail by FD treatment had normal fertility and developmental potential after ICSI.
Methods: Sperm were collected from ICR and B6 strains, and FD sperm were prepared using conventional method. First, the rates of tail-separated (head-only) sperm and tail-connected (intact) sperm in fresh, freeze-thawed (FT), and FD sperm were compared. 0.5 M trehalose was added in a part of the sperm suspension to investigate whether the difference of sperm suspension affect those rates and sperm quality. ICSI was performed using head-only FD sperm or intact FD sperm. To examine the extent of DNA damage of FD sperm after fertilization, the brightness of γ-H2Ax expressed in the male pronuclei were measured by immuno-staining. To examine the quality of embryos, the frequency of abnormal chromosome segregation (ACS) was observed at the 2-cell stage, or the 2-cell stage embryos were transferred into recipient female mice and examined offspring rate.
Results: The rates of head-only sperm in FD group (ICR:29% and B6:45%) were higher than those in fresh group (ICR:2% and B6:18%). In γ-H2Ax assay and ACS frequency, there was no significant difference between head-only FD sperm and intact FD sperm. When trehalose was added, the rate of head-only FD sperm decreased but DNA damage was not improved. The head-only sperm rate was affected by the difference of sperm suspension though trehalose did not have a role as FD protectant. Next, the fertilization rate and the developmental rate to the 2-cell stage of embryos derived from head-only sperm were also similar with those of intact sperm. The offspring rates of embryos fertilized with head-only sperm were comparable to those of intact sperm (ICR:21% vs. 33%; B6 31% vs. 31%, respectively).
Discussion: The higher rate of head-only sperm in B6 than in ICR indicated that the tolerance to physical damage was different between mouse strains. However, regardless of mice strains, this damage is likely only in the neck region since birth rates were comparable between head-only sperm and intact sperm. On the contrary, in general of mouse ICSI, the tail must be cut off and only the sperm head are injected into oocytes. Therefore, the efficiency of ICSI can be greatly improved if sperm tail has been cut beforehand. In this study, we concluded that head-only FD sperm is viable for obtaining next generation and the separation phenomena of the sperm head and tail proved to be of value for a variety of uses including the improvement of ICSI efficiency.
