Chromatin structure in totipotent mouse early preimplantation embryos.

Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes) undergo reprogramming, which results in the acquisition of totipotency in zygotes. Since the 1990s, numerous studies have focused on the mechanisms of totipotency. With the emergence of the concept of epigenetic reprogramming, which is important for the undifferentiated and differentiated states of cells, the epigenomes of germ cells and fertilized oocytes have been thoroughly analyzed. However, in early immunostaining studies, detailed epigenomic information was difficult to obtain. In recent years, the explosive development of next-generation sequencing has made it possible to acquire genome-wide information and the rise of genome editing has facilitated the analysis of knockout mice, which was previously difficult. In addition, live imaging can effectively analyze zygotes and 2-cell embryos, for which the number of samples is limited, and provides biological insights that cannot be obtained by other methods. In this review, the progress of our research using these advanced techniques is traced back from the present to its earliest years.

Time-lapse observation of mouse preimplantation embryos using a simple closed glass capillary method.

Time-lapse observation is a popular method for analyzing mammalian preimplantation embryos, but it often requires expensive equipment and skilled techniques. We previously developed a simply and costly embryo-culture system in a sealed tube that does not require a CO2 incubator. In the present study, we developed a new time-lapse observation system using our previous culture method and a glass capillary. Zygotes were placed in a glass capillary and sunk in oil for observation under a stereomicroscope. Warming the capillary using a thermoplate enabled most of the zygotes to develop into blastocysts and produce healthy offspring. This time-lapse observation system captured images every 30 min for up to 5 days, which confirmed that the developmental speed and quality of the embryos were not affected, even with fluorescence. Overall, this new system is a simple time-lapse observation method for preimplantation embryos that does not require dedicated machines and advanced techniques.

Aberrant histone methylation in mouse early preimplantation embryos derived from round spermatid injection.

Round spermatid injection (ROSI) is the last resort and recourse for men with nonobstructive azoospermia to become biological fathers of their children. However, the ROSI-derived offspring rate is lower than intracytoplasmic sperm injection (ICSI) in mice (20% vs. 60%). This low success rate has hindered the spread of ROSI in ART (Assisted Reproductive Technology). However, the cause of the ROSI-zygote-derived low offspring rate is currently unknown. In the previous studies, we reported that H3K9me3 and H3K27me3 exhibited ectopic localizations in male pronuclei (mPN) of ROSI-zygotes, suggesting that the carried over histone to zygotes conveys epigenetic information. In this study, we analyzed other histone modifications to explore unknown abnormalities. H3K36me3 showed an increased methylation state compared to ICSI-derived embryos but not for H3K4me3. Abnormal H3K36me3 was corrected until 2-cell stage embryos, suggesting a long window of reprogramming ability in ROSI-embryos. Treatment with TSA of ROSI-zygotes, which was reported to be capable of correcting ectopic DNA methylation in ROSI-zygotes, caused abnormalities of H3K36me3 in male and female PN (fPN) of the zygotes. In contrast, round spermatid TSA treatment before ROSI, which was reported to improve the preimplantation development of ROSI-zygotes, showed beneficial effects without toxicity in fPN. Therefore, the results suggest that TSA has some negative effects, but overall, it is effective in the correction of epigenetic abnormalities in ROSI-zygotes. When attempting to correct epigenetic abnormalities, attention should be paid to epigenomes not only in male but also in female pronuclei.

Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin.

Sperm chromatin retains small amounts of histones, and chromatin states of sperm mirror gene expression programs of the next generation. However, it remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here, we present a novel mouse model of paternal epigenetic inheritance, in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology using testicular sperm, we rescued infertility of mice missing Polycomb protein SCML2, which regulates germline gene expression by establishing H3K27me3 on bivalent promoters with other active marks H3K4me2/3. We profiled epigenomic states (H3K27me3 and H3K4me3) of testicular sperm and epididymal sperm, demonstrating that the epididymal pattern of the sperm epigenome is already established in testicular sperm and that SCML2 is required for this process. In F1 males of X-linked Scml2-knockout mice, which have a wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in F0 sperm. Further, dysregulation of gene expression was observed in the mutant-derived wild-type F1 preimplantation embryos. Together, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin.

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.

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

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.

Paternally inherited H3K27me3 affects chromatin accessibility in mouse embryos produced by round spermatid injection.

Round spermatid injection (ROSI) results in a lower birth rate than intracytoplasmic sperm injection, which has hampered its clinical application. Inefficient development of ROSI embryos has been attributed to epigenetic abnormalities. However, the chromatin-based mechanism that underpins the low birth rate in ROSI remains to be determined. Here, we show that a repressive histone mark, H3K27me3, persists from mouse round spermatids into zygotes in ROSI and that round spermatid-derived H3K27me3 is associated with less accessible chromatin and impaired gene expression in ROSI embryos. These loci are initially marked by H3K27me3 but undergo histone modification remodelling in spermiogenesis, resulting in reduced H3K27me3 in normal spermatozoa. Therefore, the absence of epigenetic remodelling, presumably mediated by histone turnover during spermiogenesis, leads to dysregulation of chromatin accessibility and transcription in ROSI embryos. Thus, our results unveil a molecular logic, in which chromatin states in round spermatids impinge on chromatin accessibility and transcription in ROSI embryos, highlighting the importance of epigenetic remodelling during spermiogenesis in successful reproduction.

Parental competition for the regulators of chromatin dynamics in mouse zygotes.

The underlying mechanism for parental asymmetric chromatin dynamics is still unclear. To reveal this, we investigate chromatin dynamics in parthenogenetic, androgenic, and several types of male germ cells-fertilized zygotes. Here we illustrate that parental conflicting role mediates the regulation of chromatin dynamics. Sperm reduces chromatin dynamics in both parental pronuclei (PNs). During spermiogenesis, male germ cells acquire this reducing ability and its resistance. On the other hand, oocytes can increase chromatin dynamics. Notably, the oocytes-derived chromatin dynamics enhancing ability is dominant for the sperm-derived opposing one. This maternal enhancing ability is competed between parental pronuclei. Delayed fertilization timing is critical for this competition and compromises parental asymmetric chromatin dynamics and zygotic transcription. Together, parental competition for the maternal factor enhancing chromatin dynamics is a determinant to establish parental asymmetry, and paternal repressive effects have supporting roles to enhance asymmetry.

Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development.

Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and -80°C preservation than IVF or ICSI embryos.

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at -80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at -80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at -80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator.

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%-5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

Mailing viable mouse freeze-dried spermatozoa on postcards.

Freeze-drying techniques allow the preservation of mammalian spermatozoa without using liquid nitrogen. However, the current method requires the use of glass ampoules, which are breakable, expensive, and bulky to store or transport. In this study, we evaluated whether mouse freeze-dried (FD) spermatozoa can be preserved and transported on thin materials. In this study, we demonstrated that FD sperm can be preserved in thin plastic sheets. Its DNA integrity was comparable to that of glass ampoule spermatozoa, and healthy offspring were obtained after preservation at -30°C for more than 3 months. We attached preserved FD sperm to postcards, and transported these to other laboratory inexpensively at room temperatures without any protection. This method will facilitate the preservation of thousands of mouse strains in a single card holder, promote collaboration between laboratories, conservation of genetic resources, and assisted reproductive technology.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Removal of remodeling/reprogramming factors from oocytes and the impact on the full-term development of cloned embryos.

The reason for the poor development of cloned embryos is not yet clear. Several reports have suggested that some nuclear remodeling/reprogramming factors (RRFs) are removed from oocytes at the time of enucleation, which might cause the low success rate of animal cloning. However, there is currently no method to manipulate the amount of RRFs in oocytes. Here, we describe techniques we have developed to gradually reduce RRFs in mouse oocytes by injecting somatic cell nuclei into oocytes. These injected nuclei were remodeled and reprogrammed using RRFs, and then RRFs were removed by subsequent deletion of somatic nuclei from oocytes. The size of the metaphase II spindle reduced immediately, but did recover when transferred into fresh oocytes. Though affected, the full-term developmental potential of these RRF-reduced oocytes with MII-spindle shrinkage was not lost after fertilization. When somatic cell nuclear transfer was performed, the successful generation of cloned mice was somewhat improved and abnormalities were reduced when oocytes with slightly reduced RRF levels were used. These results suggest that a change in RRFs in oocytes, as achieved by the method described in this paper or by enucleation, is important but not the main reason for the incomplete reprogramming of somatic cell nuclei.

Improvement of a twice collection method of mouse oocytes by surgical operation.

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

Production of mouse offspring from inactivated spermatozoa using horse PLCζ mRNA.

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52-53% or 48-51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

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

Freeze-drying of spermatozoa is a convenient and safe method to preserve mammalian genetic material without the use of liquid nitrogen or a deep freezer. However, freeze-dried spermatozoa (FD sperm) are not frequently used because of the low success rate of offspring after intracytoplasmic spermatozoa injection (ICSI). In this study, we determined the optimal concentration and a point of action of trehalose as a protectant for the preservation of FD sperm from different mouse strains at room temperature (RT). Although trehalose demonstrated no potential to protect the FD sperm of ICR mice against the freeze-drying procedure itself, the blastocyst rate was significantly improved when FD sperm was preserved for more than 1 month at RT (56-63% vs. 29% without trehalose). The optimal concentration of trehalose was 0.5 M. Importantly, remarkable results were obtained when spermatozoa of inbred mouse strains (C57BL/6N, C3H/He, and 129/Sv) were used, and many offspring were obtained when FD sperm that was preserved for 3 months at RT (26-28% vs. 6-11% of without trehalose) was used. However, when DNA damage in FD sperm was examined by gamma-H2Ax assays, it was found that trehalose failed to protect the FD sperm from DNA damage. These results suggest that trehalose has the potential to protect other sperm factors rather than sperm DNA during preservation at RT for longer periods and trehalose is more effective for inbred mouse strains.

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

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.

Assessing the tolerance to room temperature and viability of freeze-dried mice spermatozoa over long-term storage at room temperature under vacuum.

Freeze-drying has been frequently used to preserve food and microorganisms at room temperature (RT) for extended periods of time; however, its application to mammalian species is difficult. Here, we developed a method to prolong the stability of freeze-dried (FD) mice spermatozoa at RT for more than one year without using any cryoprotectant agents. Our data showed that maintaining a vacuum in ampoules is critical to ensuring the viability of FD spermatozoa, as the stability of spermatozoa DNA increased when imperfectly vacuumed ampoules were detected using a non-destructive test and eliminated. Finally a large number of healthy offspring were obtained from mice oocytes fertilized with FD spermatozoa stored at RT for more than one year. Although the birth rate from three-month stored spermatozoa was lower than that from one-day stored spermatozoa, no further reduction was observed even in one-year stored spermatozoa. Therefore, FD spermatozoa preserved in this study were highly tolerant to warm temperatures. This method of storage shows a great potential for the preservation of genetic resources of mammalian species, such as genetically-modified mouse strains, without the use of electric power.