Freeze-dried Mouse Spermatozoa Research Articles

A novel, simplified method to prepare and preserve freeze-dried mouse sperm in plastic microtubes.

Although freeze-drying sperm can save space, reduce maintenance costs, and facilitate the transportation of genetic samples, the current method requires breakable, custom-made, and expensive glass ampoules. In the present study, we developed a simple and economical method for collecting freeze-dried (FD) sperm using commercially available plastic microtubes. Mouse epididymal sperm suspensions were placed in 1.5 ml polypropylene tubes, frozen in liquid nitrogen, and dried in an acrylic freeze-drying chamber, after which they were closed under a vacuum. The drying duration did not differ between the microtube and glass ampoule methods (control); however, the sperm recovery rate was higher using the microtube method, and the physical damage to the sperm after rehydration was also reduced. Intracytoplasmic sperm injection (ICSI) using FD sperm stored in microtubes at -30°C yielded healthy offspring without reducing the success rate, even after 9 months of storage. Air infiltration into all microtubes stored at room temperature (RT) within 2 weeks of storage caused a drastic decrease in the fertilization rate of FD sperm; underwater storage did not prevent air infiltration. RT storage of FD sperm in microtubes for 1 week resulted in healthy offspring after ICSI (5-18%), but the addition of silica gel or CaCl2 did not improve the success rate. Our novel microtube method is currently the simplest and most effective method for treating FD sperm, contributing to the development of alternative low-cost approaches for preserving and transporting genetic resources.

40 Comparison of slow and rapid freezing in freeze-dried ram spermatozoa

Semen lyophilisation is an interesting technique that might be a cheap alternative to long-term storage in liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 and demonstrated, for the first time, the birth of healthy mouse pups from epididymal freeze-dried (mouse) spermatozoa. The authors follow the lyophilisation technique, commonly used in the pharmaceutical and food industries, namely, deep freezing, which requires direct immersion of the semen sample into liquid nitrogen before vacuum drying. In this work, we focused on the freezing phase to improve and make the technique more reliable. We compared two protocols: 1) rapid freezing, where the semen is plunged directly into liquid nitrogen (LN group), and 2) slow freezing, where the sample is frozen with a freezing rate of 1°C min−1 until −50°C (SL group). Then, both frozen samples were lyophilized. Subsequently, after an interval ranging between 1 and 3 months, dry spermatozoa from LN and SL groups were used for intracytoplasmic sperm injection (ICSI), and the embryo development was evaluated at 24h (2-cell stage) and 7 days (expanded blastocyst) post-ICSI. Moreover, acrosome integrity was evaluated with Pisum sativum agglutinin (PSA) staining on part of the semen, immediately after freezing. The LN-group semen showed the acrosome completely melted, whereas the SL group showed better integrity of the acrosome, which was comparable to that of the normal frozen (vital) spermatozoa. At 24h post-ICSI the number of cleaved embryos in the SL group was higher than in the LN group (42/100 (42%) vs. 19/75 (25.3%), SL and LN, respectively; P=0.0253). The blastocyst rate 7 days after ICSI in the SL group was higher (7/100 (7%) than that in the LN group (2/75 (2.7%); P=0.0238). Our data show that lyophilisation can be conveniently achieved in ram spermatozoa without liquid nitrogen, thus simplifying the procedure. These data support the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen.

Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from \u2212196\u2009\xb0C to 150\u2009\xb0C

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.

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4°C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4°C for 3years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.

Chromosomal integrity and DNA damage in freeze-dried spermatozoa.

Freeze-drying technology may one day be used to preserve mammalian spermatozoa indefinitely without cryopreservation. Freeze-dried mouse spermatozoa stored below 4°C for up to 1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures. Freeze-drying, per se, may reduce the integrity of chromosomes in freeze-dried mouse spermatozoa, but induction of chromosomal damage is suppressed if spermatozoa are incubated with divalent cation chelating agents prior to freeze-drying. Nevertheless, chromosomal damage does accumulate in spermatozoa stored at temperatures above 4°C. Currently, no established methods or strategies can prevent or reduce damage accumulation, and damage accumulation during storage is a serious obstacle to advances in freeze-drying technology. Chromosomal integrity of freeze-dried human spermatozoa have roughly background levels of chromosomal damage after storage at 4°C for 1 month, but whether these spermatozoa can produce healthy newborns is unknown. The safety of using freeze-dried human spermatozoa must be evaluated based on the risks of heritable chromosome and DNA damage that accumulates during storage.

Characterization of chromosomal damage accumulated in freeze-dried mouse spermatozoa preserved under ambient and heat stress conditions

Structural chromosome aberrations and DNA damage generated in freeze-dried mouse spermatozoa were investigated. Freeze-dried sperm samples were preserved at 4, 25 and 50°C for short duration (1 day to 2 months) and at 25°C for long duration (2 years). The spermatozoa were injected into mouse oocytes to analyse the chromosomes of the zygotes at the first cleavage metaphase. Chromosome break of the chromosome-type aberrations was the most common type of structural chromosome aberrations observed in all freeze-dried samples. The frequency of chromatid exchanges rapidly increased in freeze-dried spermatozoa preserved at 50°C for 1-5 days. The frequency of chromatid-type aberrations (break and exchange) gradually increased in freeze-dried spermatozoa preserved at 25°C for up to 2 months. Alkaline comet assay revealed significant migration of damaged DNA accumulated in freeze-dried spermatozoa preserved at 50°C for 3 days and 25°C for 2 years. However, no DNA damage was detected using the same sperm samples by neutral comet assay, which can detect mostly DNA double-strand breaks in cellular DNA. These results suggest that DNA single-strand breaks were accumulated in freeze-dried spermatozoa preserved under ambient or heat conditions, and then chromatid-type aberrations, especially the chromatid exchanges, were formed via post-replication repair system in zygotes.

A Study on Freeze-Drying as a Method of Preserving Mouse Sperm

This review describes the study of freeze-dried mouse sperm for practical application in preserving and transporting genetic resources. Freeze-dried sperm can be used to preserve and transport genetic resources; however, there still remain many areas which need to be studied. In particular, it is essential to assure long-term preservation over several decades or centuries. Recently, the theory of accelerated degradation kinetics to freeze-dried mouse sperm has been applied, and found that long-term preservation by conventional methods requires temperatures lower than -80 C. When the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse sperm was examined, a pressure of 0.37 mbar at primary drying significantly improved the developmental rate to the blastocyst stage. In addition, it has been shown that freeze-dried sperm stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. Sperm chromatin structure assay (SCSA) was applied to mouse sperm freeze-dried under several conditions and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. Furthermore, SCSA might be useful for estimation of developmental potential of fertilized eggs derived from ICSI using freeze-dried sperm in mice.

Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than −80 °C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and −80 °C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.

Assessment of three generations of mice derived by ICSI using freeze-dried sperm

Although the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 degrees C for 1-2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocysts in vitro (C57BL/6J and B6D2F1/J) and liveborn pups in vivo (B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.

Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential

Freeze-dried spermatozoa are capable of participating in normal embryonic development after injection into oocytes and thus useful for the maintenance of genetic materials. We recently reported that long-term preservation of freeze-dried mouse spermatozoa by conventional methods requires temperatures lower than -80 degrees C. Successful permanent preservation of mouse spermatozoa at much higher temperatures requires thorough investigation of the freeze-drying procedure. Thus, we examined the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse spermatozoa. Three different primary drying pressures were applied to evaluate the effect of pressure on freeze-dried spermatozoa under varying storage conditions and the rate of development measured. The developmental rate of embryos to the blastocyst stage from intracytoplasmic sperm injection by freeze-dried spermatozoa at pressures of 0.04, 0.37, and 1.03 mbar without storage were 59% (337/576), 71% (132/187), and 33% (99/302) respectively. When stored at 4 degrees C for 6 months, the rate was 13% (48/367), 50% (73/145), and 36% (66/182) respectively. These results show that primary drying pressure is an influential factor in the long-term preservation of freeze-dried mouse spermatozoa.

Transportation of Freeze-Dried Mouse Spermatozoa Under Different Preservation Conditions

Freeze-dried mouse spermatozoa can be used for normal embryonic development after injection into oocytes, thus indicating that freeze-drying is a useful method for the storage and transportation of genetic materials from animals. We recently reported that storage of freeze-dried mouse spermatozoa requires maintenance at temperatures lower than -80 C for long-term preservation and a pressure of 0.37 mbar at primary drying and that these conditions significantly improve the developmental rate to the blastocyst stage. In this study, we examined the influence of transportation and preservation conditions on freeze-dried spermatozoa. Freeze-dried spermatozoa stored for 2 or 2.5 years at 4 or -80 C were transported round trip overland between Shizuoka and Hokkaido prefectures in Japan or by air between Japan and Belgium. The freeze-drying conditions consisted of primary drying at pressures of 0.04, 0.37 and 1.03 mbar and secondary drying at a pressure of 0.001 mbar. Embryos (2-cell stage) from freeze-dried spermatozoa dried at 0.04 mbar and stored at 4 C for 2 years with and without overland transportation did not develop to term. The development rates of embryos from spermatozoa stored at -80 C for up to 2 years and transported overland, by air and without transportation were 8, 1 and 28%, respectively. The development rates of embryos from spermatozoa without transportation were significantly higher than with transportation (P<0.05). These data indicate that freeze-dried spermatozoa stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. However, there are limitations to be considered in the transportation of freeze-dried spermatozoa at ambient temperature.

Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent

This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 °C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris–HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 °C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10 mM Tris–HCl and 1 mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris–HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.

Freeze-drying in Mouse Spermatozoa

ABSTRACT Cryopreservation of mouse spermatozoa has been widely applied for maintenance of genetically modified mouse strains. Although cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than of embryos for maintaining transgenic or gene-disrupted mouse strains, maintenance of cryopreserved spermatozoa still has high running costs because of the need for a constant supply of liquid nitrogen. It has been reported that freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes, and so they have attracted a great deal of attention as storable gene resources. However, it is particularly essential to assure long-term preservation for several decades or centuries. The application of the determination of accelerated degradation kinetics calculated by extrapolation of Arrhenius plots to the preservation of freeze-dried mouse spermatozoa is a possible solution. This theory also is being applied to long-term stability of drug...

Mammalian freeze-dried sperm can maintain their calcium oscillation-inducing ability when microinjected into mouse eggs

Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 °C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.

Possibility of Long-Term Preservation of Freeze-Dried Mouse Spermatozoa1

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs γ-ray irradiation

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs γ-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with γ-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from γ-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Development of normal mice from oocytes injected with freeze-dried spermatozoa.

Freeze-dried mouse spermatozoa are all motionless and dead in the conventional sense. When injected into oocytes, however, their nuclei can support normal embryonic development even after three month preservation in a dried state. Although the freeze-drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genomes at room temperature.