Extracting and analyzing micronuclei from mouse two-cell embryos fertilized with freeze-dried spermatozoa.

Abnormal chromosome segregation (ACS) in preimplantation embryos causes miscarriages. For a normal pregnancy, it is necessary to reduce ACS occurrences in embryos. However, the causes of such abnormalities are unclear because no method to extract the segregated chromosomes from the blastomeres for detailed analysis.

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.

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%).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development.

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been shown to be involved in the cellular differentiation potential of pluripotent embryonic stem cells. It was previously reported that compared with embryonic stem cells, zygotes harbor an extremely loosened chromatin structure, suggesting its association with their totipotency.

Linker histone H1FOO regulates the chromatin structure in mouse zygotes.

The chromatin structure in one-cell-stage mouse embryos is extremely loose and becomes tighter at the two-cell stage. As linker histones are involved in higher-order chromatin structure, we examined the involvement of the linker histone variant H1foo in the change of chromatin looseness between the one- and two-cell stages. H1foo knockdown causes the chromatin structure to be tighter in the pronucleus and increases deposition of the histone H3 variant H3.

Chd9 mediates highly loosened chromatin structure in growing mouse oocytes.

During oogenesis, oocytes prepare for embryonic development following fertilization. The mechanisms underlying this process are still unknown. Recently, it has been suggested that a loosened chromatin structure is involved in pluripotency and totipotency in embryonic stem (ES) cells and early preimplantation embryos, respectively.