Sayaka Wakayama

University of Yamanashi

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 m...

Live-cell imaging 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 live-cell imaging system using our...

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 Polyc...

Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprogramming research. However, the standard mouse SCNT protocol remains expensive, labor-intensive, and requires hard work for many hours. Therefore, we have been trying to reduce the cost and simplify the mouse SCNT protoco...

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant...

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 stu...

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS...

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, w...

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 dyna...

Maintaining biodiversity is an essential task, but storing germ cells as genetic resources using liquid nitrogen is difficult, expensive, and easily disrupted during disasters. Our aim is to generate cloned mice from freeze-dried somatic cell nuclei, preserved at −30 °C for up to 9 months after freeze drying treatment. All somatic cells died after...

Recently, mice have been produced from Freeze-drying (FD) somatic cells by nuclear transfer, but the success rate was very low compared to FD spermatozoa. Since spermatozoa, unlike somatic cells, are haploid cell and their nuclei were hardened by protamine, it is unclear which is responsible for tolerance to FD treatment. Here, we attempt to produc...

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 p...

De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell l...

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS...

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 −8...

Conventional in vitro culture and manipulation of mouse embryos require a CO 2 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 CO 2 incubator-free embryo culture system and transported embryos using this system....

The preservation of mammalian freeze-dried (FD) spermatozoa is commonly performed using small glass ampules; however, they are bulky and breakable. In this study, we present a protocol to prepare and preserve mouse FD sperm using thin plastic sheets. This approach allows storing thousands of mouse strains in a card folder. We can also send the FD s...

Non-alcoholic fatty liver disease (NAFLD), which includes the subtype non-alcoholic steatohepatitis (NASH), is a major complication of type 2 diabetic mellitus (T2DM), even among non-obese patients. However, the exact cause of NAFLD/NASH in non-obese patients with T2DM is unclear. We studied a non-obese mouse model of T2DM created through the malno...

Parental pronuclei (PN) are asymmetrical in several points but the underlying mechanism for this is still unclear. Recently, a theory has been become broadly accepted that sperm are more than mere vehicles to carry the paternal haploid genome into oocytes. Here, in order to reveal the formation mechanisms for parental asymmetrically relaxed chromat...

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 mate...

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 po...

In mammalian cloning by somatic cell nuclear transfer (SCNT), treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives—such as trichostatin A—characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency...

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 low success rate of animal cloning. However, there is currently no method to manipulate the volume of RRFs in oocytes....

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...

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offsprings 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 inject...

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ioni...

Freeze-drying has been frequently used to preserve food at room temperature for extended periods of time; however, its application to mammalian spermatozoa is difficult because it is impossible to preserve spermatozoa at room temperature for long periods. It is known that trehalose is one of the best cryoprotectant agents, but not yet clear whether...

After fertilisation, mammalian oocytes resume meiosis, leading to pronuclear (PN) formation. It has been known that the mitogen-activated protein kinase pathway negatively regulates PN formation. However, it remains largely unknown how the mitogen-activated protein kinase pathway is involved in the dynamic organisation of chromatin structure in PNs...

Purpose: This study aims to demonstrate vitrification methods that provide reliable cryopreservation for embryos with compromised cryotolerance. Methods: Two-cell stage mouse embryos and in vitro produced porcine embryos were vitrified using the hollow fiber vitrification (HFV) and Cryotop (CT) methods. The performance of these two methods was c...

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 i...

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...

Placental development is a key event in mammalian reproduction and embryogenesis. However, the molecular basis underlying placental development is not fully understood. Here, we conduct a forward genetic screen to identify regulators for extraembryonic development and identify Zfp281 as a key factor. Zfp281 overexpression in mouse embryonic stem ce...

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),...

Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprograming research. The original SCNT was performed using cell fusion between the donor cell and oocyte. This method remains very popular, but we have recently developed an alternative method that relies on nuclear injectio...

Cloning animals using nuclear transfer (NT) provides the opportunity to preserve endangered species. However, there are risks associated with the collection of donor cells from a body, which may cause accidental death of the animal. Here, we tried to collect faeces-derived cells and examined the usability of those nuclei as a donor for NT. A relati...

Mammalian oocytes and zygotes have nucleoli that are transcriptionally inactive and structurally distinct from nucleoli in somatic cells. These nucleoli have been termed nucleolus precursor bodies (NPBs). Recent research has shown that NPBs are important for embryonic development, but they are only required during pronuclear formation. After fertil...

Live-cell imaging of NPB fusion during pronuclear formation of an ICSI embryo. Maximum intensity Z-projection images of EGFP-NPM2 (green, NPB) and H2B-mCherry (red, chromosomes) are shown. The time after the start of imaging (h:mm) is shown. The scale bar is 20 μm. (AVI)

High viscosity medium maintains the structure of NPBs for a long time. (A) Isolated NPBs were disassembled in the culture medium. The scale bar is 50 μm. (B) The NPB structure could be maintained in the high viscosity medium. The scale bar is 50 μm. (C) An NPB was injected into each MII oocyte. Arrows indicate the injected NPB. (TIF)

Live-cell imaging of NPB fusion during pseudo-pronuclear formation of extra-NPB-injected SCNT embryos. Maximum intensity Z-projection images of EGFP-NPM2 (green, NPB) and H2B-mCherry (red, chromosomes) are shown. The time after the start of imaging (h:mm) is shown. The scale bar is 20 μm. (AVI)

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....

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 lo...

Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral inf...

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR outbred mouse strain. Cu...

Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasur...

The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental peri-centromeric DNAs, which led to a s...

Significance Radiation on the International Space Station (ISS) is more than 100 times stronger than at the Earth’s surface, and at levels that can cause DNA damage in somatic cell nuclei. The damage to offspring caused by this irradiation in germ cells has not been examined, however. Here we preserved mouse spermatozoa on the ISS for 9 mo. Althoug...

Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is...

Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic-cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasib...

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear-transfer ES cells (ntESCs) from both MEFs and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with i...

Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloprot...

Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for...

Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-der...

The germline reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germline reprogramming that are then transmitted to germ cells and consequently, to offspring. We addressed this questio...

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the u...

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained...

Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced...

Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used...

Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here we examined whether neurons from adult mice could be cloned, using a combination of two epigenetic approaches. First, we used a specific antibody to discov...

Significance Antibodies produced by B cells provide a protective barrier to our organism against the penetration and dissemination of microorganisms. Each antibody recognizes a specific antigen through variable (V) region domains of pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In mammals, VDJ recombination generates a highly diversi...

The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adu...