Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from −196 °C to 150 °C

Abstract

It has long been believed that tolerance against extreme environments is possible only for ‘lower’ groups, such as archaea, bacteria or tardigrades, and not for more ‘advanced’ species. Here, we demonstrated that the mammalian sperm nucleus also exhibited strong tolerance to cold and hot temperatures. When mouse spermatozoa were freeze-dried (FD), similar to the anhydrobiosis of Tardigrades, all spermatozoa were ostensibly dead after rehydration. However, offspring were obtained from recovered FD sperm nuclei, even after repeated treatment with conditions from liquid nitrogen to room temperature. Conversely, when FD spermatozoa were heated at 95 °C, although the birth rate was decreased with increasing duration of the treatment, offspring were obtained even for FD spermatozoa that had been heat-treated for 2 h. This period was improved up to 6 h when glucose was replaced with trehalose in the freeze-drying medium, and the resistance temperature was extended up to 150 °C for short periods of treatment. Randomly selected offspring grew into healthy adults. Our results suggest that, when considering the sperm nucleus/DNA as the material that is used as a blueprint of life, rather than cell viability, a significant tolerance to extreme temperatures is present even in ‘higher’ species, such as mammals.

Effect of trehalose on the preservation of freeze-dried mice spermatozoa at room temperature

“…1e, 2c, d; Table 4). Very recently, we reported that trehalose could increase the tolerance of FD sperm against extremely high temperatures by protecting FD sperm from burning [15]. Those results suggest that trehalose has the potential to protect several factors (such as the integrity of acrosome or cytoplasm) of FD sperm but not DNA, and therefore, even though the birth rate was increased, DNA damage was not reduced by trehalose treatment.…”

“…However, the gametes of inbred mouse strains are sensitive to assisted reproductive technology, and the production rate of offspring from inbred strains was reported to be lower than that of hybrid strains [9, 33]. Because DNA was common in all organisms, those differences were likely caused by the tolerance of the cell membrane or some organelle rather than the DNA [15, 34]. Therefore, trehalose could work more effectively in the “weaker” spermatozoa of inbred strains compared with the “stronger” spermatozoa of outbred or hybrid strains.…”

“…reported that 0.3 M trehalose had the potential to protect evaporatively dried spermatozoa, as live offspring were obtained from spermatozoa preserved at RT for 2 years [14]. We also reported that trehalose can increase tolerance of FD sperm to extremely high and low temperatures [15]. Those reports suggest that when the optimal concentration and appropriate method of trehalose are discovered, the success rate of offspring from FD sperm will improve, even after preservation at RT for many years.…”

Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions

“…These cryoprotectants remain liquid, and toxic, at room temperature and are therefore unsuitable for use in dry storage. Consequently, scientists started investigating solutions to this problem using milder cold temperatures to solidify water prior to sublimation (Restrepo et al 2019;Wakayama et al 2019;Palazzese et al 2020) with better results. After all, the aforementioned anhydrobiotes do not resort to freezing before dehydration; simply put, once a reduction in environmental water is sensed, they activate a timely, regulated gene expression leading to the accumulation of xeroprotectants in their cells.…”

Sperm Preservation: A Review on Current Methods and a Unique Approach

“…The lyophilization method allows storage of sperm at 4°C or room temperature for a long time, does not require regular replenishment of a freezing agent, and is relatively easy to manage. Mouse spermatozoa after freeze-drying can be stored at room temperature for more than one year [5], and can withstand rapid temperature change [6]. In addition, sperm processed by the lyophilization method can withstand storage in space, which is a high radiation environment [7].…”