Language：English   Publishing type：Research paper (scientific journal)  

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 protocol. This chapter describes the methods to use low-cost mouse strains and steps from the mouse cloning procedure. Although this modified SCNT protocol will not improve the success rate of mouse cloning, it is a cheaper, simpler, and less tiring method that allows us to perform more experiments and obtain more offspring with the same working time as the standard SCNT protocol.

DOI： 10.1007/978-1-0716-3064-8_7

PubMed

Language：Japanese  

J-GLOBAL

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：nature publishing group  

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to produce offspring from FD spermatid, a haploid sperm progenitor cell whose nuclei, like somatic cells, have not yet been replaced by protamine.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.

DOI： 10.1038/s41598-022-22850-5

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PLOS ONE  

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 without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

DOI： 10.1371/journal.pone.0270781

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s42003-022-03623-2

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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 freeze drying, and nucleic DNA damage significantly increased. However, after nuclear transfer, we produced cloned blastocysts from freeze-dried somatic cells, and established nuclear transfer embryonic stem cell lines. Using these cells as nuclear donors for re-cloning, we obtained healthy cloned female and male mice with a success rate of 0.2-5.4%. Here, we show that freeze-dried somatic cells can produce healthy, fertile clones, suggesting that this technique may be important for the establishment of alternative, cheaper, and safer liquid nitrogen-free bio-banking solutions.

DOI： 10.1038/s41467-022-31216-4

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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 lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell- and somatic cell-specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell-specific lineages, 10-15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.

DOI： 10.1101/gr.276363.121

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1262/jrd.2021-115

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1262/jrd.

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

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.

DOI： 10.1371/journal.pone.0260645

PubMed

Language：English   Publisher：Research Square Platform LLC  

<title>Abstract</title>
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 chromatin structure in zygotes, we investigated histone mobility in parthenogenetic-, androgenic-, ROSI-, ELSI-, tICSI-, and ICSI-zygotes with several numbers of PNs with the use of zygotic fluorescence recovery after photobleaching, a method previous established by our group. The results showed that sperm played a role to cause chromatin compaction in both parental PNs. Interestingly, during spermiogenesis, male germ cells acquired this ability and its resistance. On the other hand, oocytes harbored chromatin relaxation ability. Furthermore, the chromatin relaxation factor was competed for between PNs. Thus, these results indicated that the parental asymmetrically relaxed chromatin structure was established as a result of a competition between the PNs for the chromatin relaxation factor that opposed the chromatin compaction effect by sperm. Together, it was suggested that parental germ cells cooperated for their just arisen newborn zygotes by playing a distinct role in the regulation of chromatin structure.

DOI： 10.21203/rs.3.rs-806059/v1

Other Link： https://www.researchsquare.com/article/rs-806059/v1.html

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.isci.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

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.

DOI： 10.1016/j.isci.2021.102815

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/sciadv.abg5554.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

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.

DOI： 10.1126/sciadv.abg5554

PubMed

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-87

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-95

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-47

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-53

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_or-43

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-24

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-39

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-69

CiNii Research

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-86

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：J Reprod Dev  

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.

DOI： 10.1262/jrd.2020-059

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/dev.190777.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

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.

DOI： 10.1242/dev.190777

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1530/REP-20-0054.

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1530/REP-20-0054

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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 ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

DOI： 10.1038/s41467-019-13830-x

PubMed

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.113.0_p-85

CiNii Research

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.113.0_p-54

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

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 compared by the viability of the vitrified-rewarmed embryos. Results: Regardless of the method used, 100% of the mouse 2-cell embryos developed successfully after vitrification-rewarming into the blastocyst stage, whereas vitrification tests using porcine morulae with the HFV method produced significantly better results. The developmental rates of vitrified porcine morula into the blastocyst stage, as well as blastocyst cell number, were 90.3% and 112.3 ± 6.9 in the HFV group compared with 63.4% and 89.5 ± 8.1 in the CT group (P < .05). Vitrification tests using 4- to 8-cell porcine embryos resulted in development into the blastocyst stage (45.5%) in the HFV group alone, demonstrating its better efficacy. The HFV method did not impair embryo viability, even after spontaneous rewarming at room temperature for vitrified embryos, which is generally considered a contraindication. Conclusion: Vitrification test using embryos with compromised cryotolerance allows for more precise determining of effective cryopreservation methods and devices.

DOI： 10.1002/rmb2.12312

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1262/jrd.2019-043

PubMed

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_p-96

CiNii Research

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_p-120

CiNii Research

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_p-66

CiNii Research

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_p-128

CiNii Research

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_p-115

CiNii Research

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1262/jrd.2019-058

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41598-019-42062-8

PubMed

Other Link： http://www.nature.com/articles/s41598-019-42062-8

Language：English  

DOI： 10.1007/978-1-4939-8831-0_12

PubMed

Language：English  

DOI： 10.1038/s41598-018-33304-2

PubMed

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.111.0_p-67

CiNii Research

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.111.0_p-75

CiNii Research

J-GLOBAL

Authorship：Lead author   Language：Japanese  

J-GLOBAL

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-018-28896-8

PubMed

Language：Japanese   Publisher：(一社)日本卵子学会  

J-GLOBAL

Language：Japanese   Publisher：(一社)日本卵子学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

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. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/ embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

DOI： 10.1530/REP-17-0372

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/nature23286.

Language：English   Publisher：NATL ACAD SCIENCES  

DOI： 10.1073/pnas.1711468114

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Inhibitors of Mek1/2 and Gsk3 beta, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents(1,2). 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 erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

DOI： 10.1038/nature23286

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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 degrees 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.

DOI： 10.1073/pnas.1701425114

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

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 embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells2017;35:1189-1196

DOI： 10.1002/stem.2601

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

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 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.

DOI： 10.1089/cell.2016.0026

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PubMed

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

The germ line 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 germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

DOI： 10.1095/biolreprod.116.138677

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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 urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

DOI： 10.1038/srep23808

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Language：Japanese  

J-GLOBAL

Authorship：Lead author   Language：Japanese  

J-GLOBAL

Authorship：Lead author   Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

DOI： 10.14882/jrds.108.0_P-82

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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 during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

DOI： 10.1371/journal.pone.0138854

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PubMed

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Part of collection (book)   Publisher：Springer New York  

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 adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

DOI： 10.1007/978-1-4939-1594-1_8

Scopus

PubMed

Language：Japanese  

DOI： 10.14882/jrds.107.0_OR1-25

J-GLOBAL

Language：Japanese  

CiNii Books

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Inc.  

Since the first cloned mammal, Dolly the sheep, the success rates of somatic cell nuclear transfer (SCNT) have been extremely low. This inefficiency has been limiting practical applications of SCNT. In the past decade, many trials aimed to enhance SCNT have led to the development of new modified SCNT methods, resulting in an increased success rate. These trials can be mainly divided into four types: type 1, improvement by optimizing SCNT protocols in each condition such as the methods of oocyte activation
type 2, improvement by modified donor cell preparation, including optimization of the cell cycle
type 3, improvement by chemical treatment of reconstructed oocytes
and type 4, improvement by gene manipulation of donor cells.In 2006, treatment of SCNT embryos with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), was found to improve significantly the development of SCNT embryos in mice
this was the first example of success (of type 3) using chromatin remodeling agents. Since then, a variety of modified SCNT protocols with HDACs have been examined in many species. Thus, the success of significant improvement in SCNT with HDACi has not only contributed to development of new method of SCNT, but also provided a lot of insights into nuclear reprogramming. In this chapter, we review recent advances in modified SCNT methods using chemical agents for chromatin remodeling, focusing particularly on modified SCNT with HDACi in mice. © 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/B978-0-12-386541-0.00011-4

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

DOI： 10.1371/journal.pone.0078380

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Language：Japanese  

DOI： 10.14882/jrds.106.0.P-97.0

J-GLOBAL

Authorship：Lead author   Language：Japanese  

DOI： 10.14882/jrds.106.0.P-102.0

J-GLOBAL

Language：English   Publishing type：(MISC) Other article   Publisher：WILEY-BLACKWELL  

DOI： 10.1002/stem.1346

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Language：Japanese  

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Previous studies of serial cloning in animals showed a decrease in efficiency over repeated iterations and a failure in all species after a few generations. This limitation led to the suggestion that repeated recloning might be inherently impossible because of the accumulation of lethal genetic or epigenetic abnormalities. However, we have now succeeded in carrying out repeated recloning in the mouse through a somatic cell nuclear transfer method that includes a histone deacetylase inhibitor. The cloning efficiency did not decrease over 25 generations, and, to date, we have obtained more than 500 viable offspring from a single original donor mouse. The reprogramming efficiency also did not increase over repeated rounds of nuclear transfer, and we did not see the accumulation of reprogramming errors or clone-specific abnormalities. Therefore, our results show that repeated iterative recloning is possible and suggest that, with adequately efficient techniques, it may be possible to reclone animals indefinitely.

DOI： 10.1016/j.stem.2013.01.005

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Language：Japanese   Publisher：日本繁殖生物学会  

【目的】我々は既にマウスおよびブタ胚のガラス化保存において，中空糸法により高い生存性が得られることを報告した。本研究は，中空糸法の融解条件が胚生存性に及ぼす影響を検証することを目的とした。 【方法】過排卵処理したBDF1マウスから採取した2細胞期胚を実験に用いた。既報(Matsunariら，2012)に従い，7.5% DMSO，7.5% エチレングリコール(EG)を含む平衡液中で，セルローストリアセテート製中空糸内に10個の胚を収容した。5–7分の平衡後，中空糸を15%DMSO，15%EG，0.5M sucroseを含むガラス化液に1分間維持し，その後液体窒素(LN)中に投入した。3種の融解条件を比較した。通常法：LNから取り出した中空糸を37.5℃に温めた融解液(1M sucrose含)に素早く投入した。加温盤法：中空糸を37.5℃に温めた加温盤上に約3秒静置して融解し，その後室温の融解液に投入した。空気中法：中空糸を室温の空気中に5秒間保持して融解し，その後室温の融解液に投入した。融解液投入以降の凍害保護剤の段階的希釈および洗浄は常温で行った。胚の生存判定は培養および移植試験により行った。 【結果】通常法，加温盤法，空気中法および非ガラス化区の胚盤胞形成率には，有意差は見られなかった(105/110, 95.5% vs. 107/110, 97.3% vs. 94/100, 94.0% vs. 109/110, 99.1%)。各区のガラス化胚を発情同期化したレシピエント雌に移植した結果，妊娠率はいずれも100% (4/4；非ガラス化区のみ2/2)であり，移植胚の胎仔への発達率は，55/80(68.8%)，56/80(70.0%)，55/80(68.8%)，35/40(87.5%)であった(有意差なし)。 【考察】一般にガラス化法においては，超急速な融解条件が必須と考えられている。中空糸ガラス化法では，比較的緩除な条件で融解が行われた場合にも，胚の生存性が高く保たれることが明らかとなった。

DOI： 10.14882/jrds.106.0.OR2-26.0

Other Link： http://search.jamas.or.jp/link/ui/2014046938

Language：Japanese   Publisher：日本繁殖生物学会  

【目的】人類は宇宙で繁栄できるだろうか。これまでの研究から，両生類や鳥類の胚は宇宙でも発生できることが確認されている。しかし，肝心の哺乳動物を用いた繁殖実験はすべて失敗に終わっている。これは動物を宇宙で飼育することが困難なことや，宇宙飛行士にとって初期胚を使った実験は難しすぎることが原因だと思われる。我々が行った疑似微小重力発生装置を用いた実験でも，マウス胚は無重力環境下で発育できない可能性を示していた（Wakayama et al. Plos One 2009）。だがシミュレーションでは本当のことはわからない。宇宙での本当の実験が不可欠である。【方法】我々は2009年にJAXAが公募していた国際宇宙ステーション「きぼう」第二次後期利用実験計画に，フリーズドライにしたマウス精子を宇宙で長期間保存し，生殖細胞への宇宙放射線の影響を調べるというテーマで応募した。この方法ならロケットの打ち上げ時と回収時を常温で行え，また宇宙飛行士は容器を運ぶだけなので難しい技術の習得も時間も必要ない。これらの利点が評価され，我々のテーマは2010年に「打ち上げ候補」として採択された。次に我々は1．実際の打ち上げを想定した温度変化を与えても生まれることの確認，2．振動・衝撃が加わっても精子の保存が可能な容器の決定3．エックス線やガンマ線照射を行い，生まれる限界放射線量を決定，4．詳細な実験計画とその評価方法の技術的確立など膨大な量の実験を行い，2012年についに打ち上げ決定となった。実験ではBDF1やB6など4系統のマウスそれぞれ3−4個体からフリーズドライ精子を作成し，宇宙ステーションと地上（コントロール）でそれぞれ3か月間，1年間および2年間保存する。回収後，精子のDNAダメージ度の測定や顕微授精による産仔作出を試み，生殖細胞への宇宙放射線の影響を明らかにする。現在，打ち上げ用として最も出産率の高い試料を選定中である。【結果】2013年8月4日，種子島からH2Bロケット4号機で打ち上げ予定。

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.

DOI： 10.1095/biolreprod.111.098764

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

DOI： 10.1371/journal.pone.0031638

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PubMed

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P &lt; 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in MI6 or CZB media (P &lt; 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2011.06.028

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Language：Japanese  

CiNii Books

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (research society, symposium materials, etc.)  

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

The successful generation of cloned animals and the establishment of embryonic stem (ES) cell lines from somatic cells suggest that these techniques may be used in human regenerative medicine. However, the fact that oocytes must be donated by women undergoing infertility treatment remains a fundamental ethical objection, as they might be concerned about the potential exploitation of their genome. Here, we investigated the reprogramming potential of enucleated and cryopreserved oocytes for the development of full-term cloned mice. BDF1 strain mouse oocytes were cryopreserved at metaphase II, before and after enucleation. After thawing, cumulus cell nuclei were microinjected to generate clones. Although the rate of development of cloned embryos to the blastocyst stage using the treated oocytes was lower than that obtained using fresh oocytes, three live pups were delivered after embryo transfer into pseudopregnant females (0.4% of the oocytes used). Thus, although cryo-preservation reduces the potential of oocytes, these cells retain the ability to support the full-term development of cloned embryos. In addition, the removal of DNA from human oocytes may alleviate the ethical and psychological problems for women who are undergoing infertility treatment and are considering oocyte donation for research or therapeutic purposes.

DOI： 10.1089/cell.2010.0059

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis. Reproduction (2011) 141 67-77

DOI： 10.1530/REP-10-0109

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results. in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

DOI： 10.1095/biolreprod.109.083337

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Mammalian parthenogenetic embryos invariably die in mid-gestation from imprinted gene defects and placental hypoplasia. Based on chimera experiments, trophoblastic proliferation is supposed to be inhibited in the absence of a male genome. Here, we show that parthenogenetic mouse embryonic cell nuclei can be reprogrammed by serial rounds of nuclear transfer without using any genetic modification. The durations of survival in uteri of cloned foetuses derived from green fluorescent protein (GFP)-labelled parthenogenetic cell nuclei were extended with repeated nuclear transfers. After five repeats, live cloned foetuses were obtained up to day 14.5 of gestation; however, they did not survive longer even when we repeated nuclear transfer up to nine times. All foetuses showed intestinal herniation and possessed well-expanded large placentas. When embryonic stem (ES) cells derived from fertilised embryos were aggregated with the cloned embryos, full-term offspring with large placentas were obtained from the chimeric embryos. Those placentas were derived from parthenogenetic cell nuclei, judging from GFP expression. The patterns of imprinted gene expression and methylation status were similar to their parthenogenetic origin, except for Peg10, which showed the same level as in the normal placenta. These results suggest that there is a limitation for foetal development in the ability to reprogramme imprinted genes by repeated rounds of nuclear transfer. However, the placentas of parthenogenetic embryos can escape epigenetic regulation when developed using nuclear transfer techniques and can support foetal development to full gestation.

DOI： 10.1242/dev.051375

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PubMed

Authorship：Lead author   Language：Japanese   Publisher：エヌ・ティー・エス  

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY UPV-EHU PRESS  

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

DOI： 10.1387/ijdb.103205sw

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PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach'' and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold.

DOI： 10.1016/S0076-6879(10)76009-2

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Authorship：Lead author   Language：Japanese  

CiNii Books

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (mu G) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under mG. However, although we obtained 75 healthy offspring from mu G-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under mG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under mG environment in a mammal, but normal preimplantation embryo development might require 1G.

DOI： 10.1371/journal.pone.0006753

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIO SCIENTIFICA LTD  

Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos. Reproduction (2009) 138 309-317

DOI： 10.1530/REP-08-0299

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PubMed

Language：Japanese  

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)  

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-59745-471-1_13

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

In mammalian cloning, evidence suggests that genomic reprogramming factors are located in the nucleus rather than the cytoplasm of oocytes or zygotes. However, little is known about the mechanisms of reprogramming, and new methods using nuclear factors have not succeeded in producing cloned mice from differentiated somatic cell nuclei. We aimed to determine whether there are functional reprogramming factors present in the cytoplasm of germinal vesicle stage (GV) oocytes. We found that the GV oocyte cytoplasm could remodel somatic cell nuclei, completely demethylate histone H3 at lysine 9 and partially deacetylate histone H3 at lysines 9 and 14. Moreover, cytoplasmic lysates of GV oocytes promoted somatic cell reprogramming and cloned embryo development, when assessed by measuring histone H3-K9 hypomethylation, Oct4 and Cdx2 expression in blastocysts, and the production of cloned offspring. Thus, genomic reprogramming factors are present in the cytoplasm of the GV oocyte and could facilitate cloning technology. This finding is also useful for research on the mechanisms involved in histone deacetylation and demethylation, even though histone methylation is thought to be epigenetically stable.

DOI： 10.1242/dev.023747

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 degrees C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.

DOI： 10.1073/pnas.0806166105

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.

DOI： 10.1017/S0967199408004620

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PubMed

Language：English   Publisher：ELSEVIER INC  

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2008.01.027

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：REPRODUCTIVE HEALTHCARE LTD  

Cloning methods in mice are now well described and are becoming routine. However, the frequency at which cloned mice are produced remains below 5%, irrespective of the nucleus donor species or cell type. Only a few laboratories have made clones from adult mouse somatic cells and most strains have never produced cloned mice. On the other hand, nuclear transfer can be used to generate human embryonic stem (ntES) cell lines from a patient&apos;s own somatic cells. It has been shown that such cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. This technique could be used in regenerative medicine and, in theory, in infertility clinics to treat completely infertile individuals. However, these results suggest that the reprogramming integrity of each cloned embryo differs: some cloned embryos can be converted to ntES cells, but these embryos cannot achieve full term development. This review outlines the nature of genomic reprogramming potential and its application, and suggests new approaches to avoid the ethical problems of creating embryos by nuclear transfer.

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cell transplantation with embryonic stem (ES) cell progeny requires immunological compatibility with host tissue. 'Therapeutic cloning' is a strategy to overcome this limitation by generating nuclear transfer (nt) ES cells that are genetically matched to an individual. Here we establish the feasibility of treating individual mice via therapeutic cloning. Derivation of 187 ntES cell lines from 24 parkinsonian mice, dopaminergic differentiation, and transplantation into individually matched host mice showed therapeutic efficacy and lack of immunological response.

DOI： 10.1038/nm1732

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ALPHAMED PRESS  

Recent cloning technology has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem (ES) cells. Mice can be cloned from adult somatic cells or ES cells by NT, and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remain unclear. In this study, our goal was to clarify why the contribution rate of ES cells derived from parthenogenetic embryos (pES) cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was significantly increased in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status of U2af1-rs1 could be used as a genetic marker to investigate the effects of NT.

DOI： 10.1634/stemcells.2007-0907

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PubMed

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：Japanese  

PubMed

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2008108304

Language：Japanese  

DOI： 10.14882/jrds.100.0.20103.0

J-GLOBAL

Authorship：Lead author   Language：Japanese  

CiNii Books

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

CiNii Books

Language：English   Publisher：CELL PRESS  

DOI： 10.1016/j.cub.2006.12.020

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

DOI： 10.1262/jrd.18098

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIO SCIENTIFICA LTD  

Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting. The objective of this study was to investigate changes in histone H3 modifications in relation to chromatin/chromosome morphology in pig oocytes during their growth, maturation, and activation. During the growth phase, histone H3 was acetylated at lysines 9, 14, and 18 (K9, K14, and K18), and became methylated at K9 when the follicles developed to the antral stage (oocyte diameter, 90 gm). When the fully grown oocytes (diameter, 120 gm) started their maturation, histone H3 became phosphorylated at serine 28 (S28) and then at S10, and deacetylated at K9, K14, and K18 as the chromosomes condensed. After the electroactivation of mature oocytes, histone H3 was reacetylated and dephosphorylated concomitant with the decondensation of the chromosomes. Histone H3 kinase activity increased over a similar time course to that of the phosphorylation of histone H3-S28 during oocyte maturation, and this activity decreased as histone H3-S10 and H3-S28 began to be dephosphorylated after the activation of the mature oocytes. These results suggest that the chromatin morphology of pig oocytes is regulated by the acetylation/deacetylation and the phosphorylation/dephosphorylation of histone H3, and the phosphorylation of histone H3 is the key event in meiotic chromosome condensation in oocytes. The inhibition of histone deacetylase with trichostatin A (TSA) inhibited the deacetylation and phosphorylation of histone H3, and chromosome condensation. Therefore, the deacetylation of histone H3 is thought to be required for its phosphorylation in meiosis. Although histone H3 acetylation and phosphorylation were reversible, the histone methylation that was established during the oocyte growth phase was stable throughout the course of oocyte maturation and activation.

DOI： 10.1530/REP-06-0099

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PubMed

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：CSIRO PUBLISHING  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Recently, ES cell lines were established from single blastomeres taken from eight-cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two-cell (establishment rate, 50%-69%), early four-cell (28%-40%), late four-cell (22%), and eight-cell (14%-16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%-40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.

DOI： 10.1634/stemcells.2006-0615

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIO SCIENTIFICA LTD  

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.50%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.

DOI： 10.1530/rep.1.01010

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PubMed

Language：English  

PubMed

Language：Japanese   Publisher：共立出版  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2007144692

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ALPHAMED PRESS  

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT-ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT-ESCs, we established more than 150 NT-ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT-ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst-derived ESCs in terms of their expression of pluripotency markers in the presence of tissue-dependent differentially DNA methylated regions, in DNA microarray profiles, and in high-coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT-ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

DOI： 10.1634/stemcells.2005-0537

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：VERSITA  

Trichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantantion period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embyros generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embyros and found that TSA treatment inhibited the preimplantation development of ICSI embyros but not ROSI or parthenogenetic embyros. And, although we often observed hypomorphism following TSA treatment in embyros grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their procedure in current Assisted Reproductive Technologies.(c) Versita Warsaw and Spring-Verlag Berlin Heidelberg. All rights reserved.

DOI： 10.2478/s11535-006-0023-5

Web of Science

Language：Japanese  

DOI： 10.14882/jrds.99.0.150.0

J-GLOBAL

Language：Japanese  

DOI： 10.14882/jrds.99.0.162.0

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 mu m, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

DOI： 10.1095/biolreprod.105.044677

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and fullterm development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P &lt; 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P &lt; 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

DOI： 10.1095/biolreprod.105.047803

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PubMed

Language：Japanese  

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressmg blastocysts were as low as 50%, and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (&gt; 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.02.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of historic deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.11

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Language：English  

DOI： 10.1111/j.1749-0774.2005.00001.x

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Although round spermatid injection can be used to create progeny for males who do not produce mature sperm, the rate of successful embryogenesis after such procedures is significantly lower than that for similar procedures using mature spermatozoa. The mechanisms underlying this difference are unknown. In this study, we demonstrate that, unlike the normal paternal genome, the paternal zygotic genome derived from a round spermatid is highly remethylated before first mitosis after demethylation. Genomes from elongated spermatids exhibited an intermediate level of DNA methylation, between those of round spermatids and mature spermatozoa, suggesting that the male germ cell acquires the ability to maintain its undermethylated state in the paternal zygotic genome during this phase of spermiogenesis. In addition, treatment of zygotes with trichostatin A led to a significant reduction in DNA methylation, specifically in the spermatid-derived paternal genome, except for the pericentromeric regions enriched by trimethylation of Lys9 of histone H3. These data provide insight into epigenetic errors that may be associated with the poor development of embryos generated from immature spermatozoa. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2005.10.026

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains &lt; 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo- actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell&apos;s genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in &gt;= 3 months.

DOI： 10.1038/nprot.2006.21

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PubMed

Language：English  

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.

DOI： 10.1262/jrd.17061

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PubMed

Language：Japanese   Publisher：Japanese Society of Animal Reproduction  

体細胞からのクローンマウスの成功率は低いが、ES細胞からは比較的容易に作出できる。そこで我々は体細胞核移植胚より得られた胚性幹細胞（nES細胞）を用いてクローンマウスを作成することにより、作成効率が体細胞を用いた場合より向上するか検討した。その結果、7個体のマウスの体細胞核から11匹のクローンマウスと68のnES細胞株、およびそれらのnES細胞核から41匹のクローンマウスを作出した。クローンマウスの成功率は、予想に反してnES細胞を用いた場合でも改善はみられなかったが、体細胞核移植ではクローンマウスの作出に失敗した2個体からでも、nES細胞の核移植によってクローンマウスの作出に成功した。最終的に7個体中6個体から体細胞あるいはnES細胞をドナー核として用いることでクローンマウスの作出に成功した。このように、nES細胞をドナーとしてもクローンマウスの成功率は改善されないが、重要なマウスの個体をクローン技術で維持するためには、nES細胞株も同時に樹立しておいた方が有効である。それらのnES細胞は核移植のドナーとして無限に増やすことができ、現在の核移植技術を補完するであろう。

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.

DOI： 10.1002/gene.20153

Web of Science

PubMed

Authorship：Lead author   Language：Japanese  

DOI： 10.14882/jrds.98.0.43.0

J-GLOBAL

Language：Japanese  

DOI： 10.14882/jrds.98.0.97.0

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F(1)mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.

DOI： 10.1095/biolreprod.104.035105

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27degreesC); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4degreesC, RT, or 37degreesC (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4degreesC; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20degreesC. Preservation of mouse spermatozoa at VC in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4degreesC. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20degreesC for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.

DOI： 10.1095/biolreprod.104.034678

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based. on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.

DOI： 10.1017/S0967199405003023

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Animals generated by systematic mutagenesis and routine breeding are often infertile because they lack germ cells, and maintenance of such lines of animals has been impossible. We found a hermaphrodite infertile mouse in our colony, a genetic male with an abnormal Y chromosome lacking developing germ cells. We tried to clone this mouse by conventional nuclear transfer but without success. ES cells produced from blastocysts, which had been cloned by using somatic cell nuclear transfer (ntES cells) from this mouse, were also unable to produce offspring when injected into enucleated oocytes. Although we were able to produce two chimeric offspring using these ntES cells by tetraploid complementation, they were infertile, because they also lacked developing germ cells. However, when such ntES cells were injected into normal diploid blastocysts, many chimeric offspring were produced. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome-bearing sperm. Thus, ntES cells were used to propagate offspring from infertile mice lacking germ cells.

DOI： 10.1073/pnas.0408548102

Web of Science

PubMed

Language：English   Publisher：JAPANESE SOCIETY OF OVA RESEARCH  

The progress of nuclear transfer technology, we can create cloned animals. And recently from this same technology we can possible to establish nuclear transfer embryonic stem cells (ntES cell). From our experiments we can establish mouse ntES cells, which any kind of cell type and strain and sex. And ntES cell has very similar ability like ES cells that has capacities for in vitro differentiation and in vivo germline transmission. The ntES cell made from donor somatic cells, which are very useful for therapeutic cloning Because of no immune rejection. Especially for human, it have high expectation from this new opportunity for rejuvenation of the ageing or diseased body. However, even for cloned animals the efficiency is so low and that have many problems. So, it necessary to do more analysis of ntES cell can work normal and safe before clinic.<br>

DOI： 10.1274/jmor.22.152

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromo some complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or lVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.

DOI： 10.1262/jrd.50.627

Web of Science

PubMed

Language：Japanese   Publisher：Japanese Society of Animal Reproduction  

マウス未受精卵はガラス化保存などで超低温保存されているが、液体窒素などを利用するため、簡単な輸送であっても極低温を維持しなければならない。そこで我々はマウス卵母細胞の4C以上の温度下で保存の可能性を検討した。未受精卵を数種類の培地で4C、27Cおよび37Cで24時間保存し、保存後の形態を光学および共焦点顕微鏡で観察した。4Cでは約70％の卵が細胞質中に水疱を形成し、単為発生刺激を与えると5％程度が胚盤胞まで発生した。27Cでは比較的正常だったが紡錘体のα-チューブリンはほとんどの卵で異常を示した。単為発生刺激後、30％程度が胚盤胞まで発生した。37Cでは形態は正常なものが多かったが細胞質の色が濃くなり、単為発生刺激を与えても胚盤胞へはまったく発生しなかった。培地による違いは見られなかったが、HYが比較的よかったため、HYで室温保存した卵にIVFあるいはICSIをしたところ、染色体の不等分離が数多く観察されたものの、わずかながら産仔を得ることができた。成績を改善するために、保存卵の紡錘体を新鮮卵の紡錘体と置換してみたが、紡錘体異常は改善されなかった。本研究により少なくともマウス未受精卵の一部を室温で24時間保存できることが明らかになった。また、発生率の低下は細胞質だけでなく核にも原因があることが示された。

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.

DOI： 10.1017/S0967199404002965

Web of Science

PubMed

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Authorship：Lead author   Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

The injection of male haploid germ cells, such as spermatozoa and round spermatids, into preactivated mouse oocytes can result in the development of viable embryos and offspring. However, it is not clear how the timing of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) affects the production of offspring. We carried out ICSI and ROSI every 20 min for up to 4 h after the activation of mouse oocytes by Sr2+ and compared the late-stage development of ICSI- and ROSI-treated oocytes, including the formation of pronuclei, blastocyst formation, and offspring production. The rate of pronucleus formation (RPF) after carrying out ICSI started to decrease from &gt;95% at 100 min following oocyte activation and declined to &lt;20% by 180 min. In comparison, RPF by ROSI decreased gradually from &gt;70% between 0 and 4 h after activation. The RPFs were closely correlated with blastocyst formation. Offspring production for both ICSI and ROSI decreased significantly when injections were conducted after 100 min, a time at which activated oocytes were in the early G1 stage of the cell cycle. These results suggest that spermatozoa and round spermatids have different potentials for inducing the formation of a male pronucleus in activated oocytes, but ICSI and ROSI are both subject to the same time constraint for the efficient production of offspring, which is determined by the cell cycle of the activated oocyte.

DOI： 10.1095/biolreprod.103.025171

Web of Science

PubMed

Language：Japanese  

CiNii Books

J-GLOBAL

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr2+-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.

DOI： 10.1089/153623003769645848

Web of Science

PubMed