Language：Japanese   Publisher：(一社)日本生殖医学会  

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

Is it possible to produce offspring after sperm chromosome screening?
It is possible to produce zygotes after examining the genome of individual spermatozoa prior to embryo production.
Chromosomal aberrations in gametes are a major cause of pregnancy loss in women treated with assisted reproductive technology. However, to our knowledge, there are no reports on the successful genomic screening of spermatozoa, although some attempts have been made using the mouse as a model.
To prevent the transmission of chromosomal aberrations from fathers to offspring, we performed sperm chromosome screening (SCS) prior to fertilization using the mouse as a model. The production of offspring after SCS consists of (i) replication of the sperm chromosomes, (ii) analysis of one copy of the replicated sperm chromosomes, (iii) construction of a zygote using another set of chromosomes and (iv) production of a transferable embryo.
A single spermatozoon of a male mouse, with or without a Robertsonian translocation, was injected into an enucleated oocyte to allow the replication of sperm chromosomes. One of the sister blastomeres of a haploid androgenic 2-cell embryo was used for chromosome analysis. The other blastomere was fused with an unfertilized oocyte, activated and allowed to develop to a blastocyst before transfer to a surrogate mother.
With high efficiency, we were able to analyze sperm chromosomes in a blastomere from the androgenic 2-cell embryos and culture zygotes, with and without aberrant chromosomes, to the blastocyst stage before embryo transfer. The karyotypes of the offspring faithfully reflected those of the blastomeres used for SCS.
This study was conducted using a mouse model; whether or not the method is applicable to humans is not known.
This study has shown that it is possible to produce zygotes without any paternally inherited aberrations by examining the genome of individual spermatozoa prior to embryo production.
This study was supported by a Grants-in-Aid for Scientific Research (228495 and 23890013 to H.W.) from the Japan Society for the Promotion of Science (JSPS). There are no conflicts of interest to be declared.

DOI： 10.1093/humrep/des388

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER/PLENUM PUBLISHERS  

Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy.
Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72 h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72 h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos.
DNA synthesis in the PB2s continued until 22 h after fertilization. At 4-6 h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72 h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72 h. Regardless of the fusion with the PB2, more than 90 % of the embryos developed to 4-cell stage, and over 80 % of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy.
The PB2 is viable for at least 72 h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

DOI： 10.1007/s10815-012-9899-3

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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Other Link： http://search.jamas.or.jp/link/ui/2013338948

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

DOI： 10.1007/s10815-012-9899-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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

DOI： 10.1093/mutage/ger003

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

Purpose: To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from gamma-irradiated spermatozoa.
Materials and methods: Mature males were exposed to 2 Gy or 4 Gy of (137)Cs gamma-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos. In the chromosome preparations from two-cell and four-cell embryos, the separation of the sister blastomeres was precluded by treatment of the embryos with concanavalin A. The incidence of embryos with structural chromosomal aberrations, aneuploidy, or mosaicism was estimated. The fates of the different types of gamma-ray-induced structural chromosomal aberrations were also investigated in those embryos.
Results: The exposure of spermatozoa to 2 Gy or 4 Gy gamma-rays caused structural chromosomal aberrations in 25.9% and 35.7% of the resultant one-cell embryos, respectively. At two-cell embryonic stage, the incidence of structural chromosomal aberrations was 17.4% in the 2 Gy group and 27.1% in the 4 Gy group. At the four-cell embryonic stage, although the incidence of control embryos with structural chromosomal aberrations was considerably high, the net incidence of embryos with radiation-induced structural chromosomal aberrations was similar to that at the one-cell stage. The incidence of aneuploidy was high in two-cell and four-cell embryos after both doses of gamma-rays. The incidence of mosaicism increased significantly in dose- and embryonic-stage-dependent manners. Anaphase lag, and the degeneration and non-disjunction of the aberrant chromosomes were frequently observed in aneuploid and mosaic embryos.
Conclusions: Mouse sperm DNA is highly vulnerable to gamma-rays. The structural chromosomal aberrations of sperm origin are unstable in their behaviour and structure during cleavage, and therefore cause secondary aneuploidy and mosaicism in the early cleavage embryos.

DOI： 10.3109/09553002.2011.530334

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MEDKNOW PUBLICATIONS & MEDIA PVT LTD  

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H2O2), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (. pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time. Asian Journal of Andrology (2011) 13, 172-174; doi: 10.1038/aja.2010.105; published online 8 November 2010

DOI： 10.1038/aja.2010.105

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (conference, symposium, etc.)   Publisher：John Wiley and Sons Ltd  

Freeze-drying technology may one day be used to preserve mammalian spermatozoa indefinitely without cryopreservation. Freeze-dried mouse spermatozoa stored below 4°C for up to 1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures. Freeze-drying, per se, may reduce the integrity of chromosomes in freeze-dried mouse spermatozoa, but induction of chromosomal damage is suppressed if spermatozoa are incubated with divalent cation chelating agents prior to freeze-drying. Nevertheless, chromosomal damage does accumulate in spermatozoa stored at temperatures above 4°C. Currently, no established methods or strategies can prevent or reduce damage accumulation, and damage accumulation during storage is a serious obstacle to advances in freeze-drying technology. Chromosomal integrity of freeze-dried human spermatozoa have roughly background levels of chromosomal damage after storage at 4°C for 1 month, but whether these spermatozoa can produce healthy newborns is unknown. The safety of using freeze-dried human spermatozoa must be evaluated based on the risks of heritable chromosome and DNA damage that accumulates during storage. © Japan Society for Reproductive Medicine 2011.

DOI： 10.1007/s12522-011-0092-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：THE SOCIETY OF CHROMOSOME RESEARCH  

To evaluate cytogenetic validity of a simple vitrification technique for embryo cryopreservation, mouse 1-cell embryos were vitrified 4, 6, and 8 h after <i>in vitro</i> fertilization (IVF). In addition, chromosomal damage of spermatozoa treated with methyl methanesulfonate (MMS) was estimated using vitrified 1-cell embryos. More than 90% of embryos survived vitrification regardless of the time after IVF. In the 4-h and 6-h groups, some of the surviving embryos swelled after recovery. The incidence of structural chromosome aberrations and aneuploidy in embryos with morphologically normal features did not significantly increase in any group. The vitrification technique preserved 1-cell embryos derived from MMS-treated spermatozoa without alteration of chromosome damage. This technique will enable us to manage the optimal time for chromosome preparation of mouse 1-cell embryos.

DOI： 10.11352/scr.13.45

CiNii Books

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Other Link： http://amcor.asahikawa-med.ac.jp/modules/xoonips/detail.php?id=CS13-3-45

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

出版社版

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Other Link： http://search.jamas.or.jp/link/ui/2009233001

Authorship：Lead author   Language：Russian   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Background: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. Methods: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated. Results: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3-7 days before freeze-drying, 85-95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P&lt; 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution. conclusions: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

DOI： 10.1093/humrep/dem252

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

Prior to attempting the in vitro production of embryos in the Bryde&apos;s whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde&apos;s whale spermatozoa are competent to support embryonic development.

DOI： 10.1017/S0967199406003923

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Authorship：Lead author   Language：English   Publisher：2  

DOI： 10.1016/j.mrgentox.2004.08.005

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Authorship：Lead author   Language：English   Publisher：ELSEVIER SCIENCE BV  

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to (CS)-C-137 gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution. (C) 2004 Elsevier B.V. All fights reserved.

DOI： 10.1016/j.mrfmmm.2004.08.001

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Authorship：Lead author   Language：English   Publisher：ELSEVIER SCIENCE INC  

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCI buffered solution (ETBS: 50 mM NaCl, 50 MM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (I)DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P &lt; 0.001) and 2-4 days (P &lt; 0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2-4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants. (c) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2003.12.008

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

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze-drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be "dead" in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

DOI： 10.1095/biolreprod.103.025957

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

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at VC. Samples frozen without cryoprotection were maintained at -196degreesC. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P &gt; 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P &gt; 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P &lt; 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P &lt; 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P &gt; 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 15 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then he stored at 4degreesC and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

DOI： 10.1095/biolreprod.103.020529

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

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6), commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2H. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.

DOI： 10.1095/biolreprod.102.006106

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

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated an nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J x DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.

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

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%). Anilines and phenols tended to induce only structural CA. p-tert-Butylphenol had a peculiar feature in inducing not only structural CA but also polyploidy at considerably high frequency (93.2%) after continuous treatment for 48 h, posing an aneugenic potential. Not all, but six of 11 carboxylic acids or esters also showed the simultaneous induction of structural CA and polyploidy. The majority of organic phosphates, alcohols or ethers, alkyl benzenes and non-cyclic alkanes had no CA induction activity. For chemicals which were negative in the bacterial reverse mutation assay (Ames test), the proportion of the chemicals that induced CA at a severely cytotoxic dose (doses manifesting more than 50% cytotoxicity) was similar to that of the CA-negative chemicals manifesting severe cytotoxicity, suggesting that severely cytotoxic chemicals do not always induce CA. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1383-5718(02)00062-1

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Authorship：Lead author,　Corresponding author   Language：Japanese  

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

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered. Copyright (C) 2002 S. Karger AG, Basel.

DOI： 10.1159/000064037

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

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

DOI： 10.1073/pnas.241517598

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Authorship：Lead author   Language：Japanese   Publishing type：Doctoral thesis   Publisher：杏林医学会  

DOI： 10.11434/kyorinmed.30.573

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

To determine the fate of chromosome aberrations induced primarily by clastogenic chemicals, aberrations of chromosome 9 in cultured human peripheral blood lymphocytes were analyzed after exposure to mitomycin C (MMC) at Go phase. Chromosome 9 painting by fluorescence in situ hybridization revealed that the translocation of 9p or 9q to another chromosome and the centric fragment representing the entire length of 9p were characteristically generated from chromatid-type aberrations involving the centromeric region of chromosome 9. These changes were not observed at 48 h after culture initiation, but persistently appeared at later stages (72-120 h postinitiation). Induction of centric fragments of 9p and micronuclei without the a satellite DNA of chromosome 9 suggested that most of the breaks were induced near the ct satellite DNA locus on 9q. Modified patterns of chromosome 9 aberrations were also observed, being related to the copy number of the short or long arm of the chromosome. Such unbalanced karyotypes could remain in the lymphocyte genome over further cell divisions for at least 120 h after culture initiation, indicating that these aberrant cells can survive and that they could pose a health risk.

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

The MCL-5 cell line was established from human lymphoblastoid TK+/- cells transfected with cDNAs of human cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) and microsomal epoxide hydrolase. The TK+/- cells constitutively express a relatively high level of endogenous CYP1A1. To study metabolic activities to indirect-acting clastogens, MCL-5 cells were treated with four clastogens, i.e. aflatoxin B-1 (AFB1), diethylnitrosamine (DEN), cyclophosphamide (CPA), and 7,12-dimethylbenz[a]anthracene (DMBA). Human lymphocytes from peripheral blood were used as control cells under the assay conditions with or without induced rat liver metabolic activation (S9). All chemicals tested without S9 induced chromosomal aberrations (CA) in MCL-5 cells but not in human lymphocytes. All chemicals induced CA in both cell types in the presence of S9. (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S1383-5718(97)00170-8

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

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) 'unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy), These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy, The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

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

Mature sperm and late spermatid are known to be sensitive stages to clastogens in mammalian spermatogenesis. Certain types of chromosomal damage induced in these stages will pass to successive generations as heritable translocations. In the present study, we employed whole chromosome 2 painting with the fluorescence in situ hybridization (FISH) technique to detect the chemically induced translocations in human sperm. Mature human sperm were treated in vitro with an antitumor drug, neocarzinostatin (NCS), and fertilized in vitro with golden hamster oocytes. Sperm pronuclear chromosome slides were prepared at the first cleavage metaphase. To compare the characteristics of translocations between somatic and germ cells, human lymphocytes in peripheral blood treated with NCS in vitro were analyzed at first round metaphase after PHA-stimulation. From the analysis of translocations by whole chromosome 2 painting, frequencies of the haploid genomic translocations (F-hG) were predicted for both sperm and lymphocytes. At 1.0 mu g/ml, the actual percentages of sperm and lymphocytes with chromosome 2 translocations were almost identical (11.9% and 12.0%). At the same dose, however, the F-hG of the sperm (1.15) was considerably higher than that of the lymphocytes (0.58), indicating that complex translocations having two or more rearranged sites were induced by NCS more frequently in sperm than in lymphocytes.

DOI： 10.1016/S0165-1218(96)90047-6

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

DOI： 10.11238/jmammsocjapan.20.125

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Authorship：Lead author   Publishing type：Master’s thesis  

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Language：English   Publisher：SOC STUDY REPRODUCTION  

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at VC. Samples frozen without cryoprotection were maintained at -196degreesC. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P &gt; 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P &gt; 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P &lt; 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P &lt; 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P &gt; 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 15 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then he stored at 4degreesC and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

DOI： 10.1095/biolreprod.103.020529

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publisher：KARGER  

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered. Copyright (C) 2002 S. Karger AG, Basel.

DOI： 10.1159/000064037

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

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Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Chinese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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