184 results on '"Ogonuki N"'
Search Results
2. Germline niche transplantation restores fertility in infertile mice
- Author
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Kanatsu-Shinohara, M., Miki, H., Inoue, K., Ogonuki, N., Toyokuni, S., Ogura, A., and Shinohara, T.
- Published
- 2005
3. Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells
- Author
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Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Ogura, A., Toyokuni, S., and Shinohara, T.
- Published
- 2003
4. Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination
- Author
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Shinohara, T., Inoue, K., Ogonuki, N., Kanatsu-Shinohara, M., Miki, H., Nakata, K., Kurome, M., Nagashima, H., Toyokuni, S., Kogishi, K., Honjo, T., and Ogura, A.
- Published
- 2002
5. Resistin-like molecule β is abundantly expressed in foam cells and is involved in atherosclerosis development
- Author
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Kushiyama, A., Sakoda, H., Oue, N., Okubo, M., Nakatsu, Y., Ono, H., Fukushima, Toshiaki, Kamata, H., Nishimura, F., Kikuchi, T., Fujishiro, M., Nishiyama, K., Aburatani, H., Kushiyama, S., Iizuka, M., Taki, N., Encinas, J., Sentani, K., Ogonuki, N., Ogura, A., Kawazu, S., Yasui, W., Higashi, Y., Kurihara, H., Katagiri, H., and Asano, T.
- Subjects
Male ,Vasculitis ,Foam Cells/immunology/*metabolism/pathology ,Macrophages, Peritoneal/immunology/metabolism/pathology ,Primary Cell Culture ,Inflammation ,Biology ,Cell Line ,Mice ,Immune system ,Apolipoproteins E ,Homologous chromosome ,medicine ,Macrophage ,Animals ,Humans ,Aorta ,Mice, Knockout ,Atherosclerosis/immunology/*metabolism/pathology ,Vasculitis/immunology/metabolism/pathology ,Fatty Acids ,Antibodies, Monoclonal ,Hormones, Ectopic/genetics/immunology/*metabolism ,Aorta/immunology/metabolism/pathology ,Intercellular Signaling Peptides and Proteins/immunology/*metabolism ,Atherosclerosis ,Antibodies, Monoclonal/pharmacology ,Fatty Acids/pharmacology ,Cell biology ,CD11c Antigen ,Antigens, CD11c/metabolism ,Secretory protein ,Immunology ,Hormones, Ectopic ,Macrophages, Peritoneal ,Intercellular Signaling Peptides and Proteins ,Resistin ,Female ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,Apolipoproteins E/genetics ,Foam Cells - Abstract
Objective— Resistin-like molecule (RELM) β is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELMβ and thus investigated the role of RELMβ in the development of atherosclerosis. Approach and Results— It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELMβ. RELMβ knockout ( −/− ) and wild-type mice were mated with apolipoprotein E–deficient background mice. RELMβ −/− apolipoprotein E–deficient mice exhibited less lipid accumulation in the aortic root and wall than RELMβ +/+ apolipoprotein E–deficient mice, without significant changes in serum lipid parameters. In vitro, RELMβ −/− primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide-induced nuclear factor-κB classical pathway activation and inflammatory cytokine secretion than RELMβ +/+ , whereas stimulation with RELMβ upregulated inflammatory cytokine expressions and increased expressions of many lipid transporters and scavenger receptors in PCPMs. Flow cytometric analysis revealed inflammatory stimulation–induced RELMβ in F4/80(+) CD11c(+) PCPMs. In contrast, the expressions of CD11c and tumor necrosis factor were lower in RELMβ −/− PCPMs, but both were restored by stimulation with recombinant RELMβ. Conclusions— RELMβ is abundantly expressed in foam cells within plaques and contributes to atherosclerosis development via lipid accumulation and inflammatory facilitation.
- Published
- 2013
6. 154 Offspring production with sperm grown in vitro from cryopreserved testis tissues
- Author
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Yokonishi, T., primary, Sato, T., additional, Komeya, M., additional, Katagiri, K., additional, Ogonuki, N., additional, Inoue, K., additional, Ogura, A., additional, Hata, K., additional, Kubota, Y., additional, and Ogawa, T., additional
- Published
- 2014
- Full Text
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7. Embryonic Rather than Extraembryonic Tissues Have More Impact on the Development of Placental Hyperplasia in Cloned Mice
- Author
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Miki, H., Wakisaka, N., Inoue, K., Ogonuki, N., Mori, M., Kim, J.-M., Ohta, A., and Ogura, A.
- Published
- 2009
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8. t-SNARE Syntaxin2 (STX2) Is Implicated in Intracellular Transport of Sulfoglycolipids During Meiotic Prophase in Mouse Spermatogenesis
- Author
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Fujiwara, Y., primary, Ogonuki, N., additional, Inoue, K., additional, Ogura, A., additional, Handel, M. A., additional, Noguchi, J., additional, and Kunieda, T., additional
- Published
- 2013
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9. Poor centrosomal function of rabbit immature spermatogenic cells following microinsemination with sperm and spermatids
- Author
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Tachibana, M., primary, Terada, Y., additional, Ogonuki, N., additional, Ogura, A., additional, Yaegashi, N., additional, and Okamura, K., additional
- Published
- 2007
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10. 377 SPERMATOZOA RETRIEVED FROM MALE MICE FROZEN FOR 15 YEARS CAN PRODUCE NORMAL OFFSPRING
- Author
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Ogonuki, N., primary, Mochida, K., additional, Miki, H., additional, Inoue, K., additional, Iwaki, T., additional, Morozumi, K., additional, Yanagimachi, R., additional, and Ogura, A., additional
- Published
- 2007
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11. 47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS
- Author
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Inoue, K., primary, Ogonuki, N., additional, Miki, H., additional, Noda, S., additional, Inoue, S., additional, Katayama, K., additional, Mekada, K., additional, Miyoshi, H., additional, and Ogura, A., additional
- Published
- 2007
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12. 355 IMPROVEMENT OF CUMULUS-FREE IN VITRO MATURATION AND ITS APPLICATION TO MICROINSEMINATION WITH PRIMARY SPERMATOCYTES IN MICE
- Author
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Miki, H., primary, Ogonuki, N., additional, Inoue, K., additional, Baba, T., additional, and Ogura, A., additional
- Published
- 2006
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13. 358 MICROINSEMINATION USING MALE GERM CELLS FROM EPIDIDYMIDES AND TESTES STORED IN FREEZERS WITHOUT CRYOPROTECTANT
- Author
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Ogonuki, N., primary, Mochida, K., additional, Shinmen, A., additional, Ohkawa, M., additional, Miki, H., additional, Inoue, K., additional, Fray, M., additional, Moriwaki, K., additional, Obata, Y., additional, and Ogura, A., additional
- Published
- 2006
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14. 322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS
- Author
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Ogonuki, N., primary, Inoue, K., additional, Miki, H., additional, Hirose, Y., additional, Okada, H., additional, Shimozawa, N., additional, Takeiri, S., additional, Nagashima, H., additional, Sankai, T., additional, and Ogura, A., additional
- Published
- 2005
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15. Pregnancy by the tubal transfer of embryos developed after injection of round spermatids into oocyte cytoplasm of the cynomolgus monkey (Macaca fascicularis)
- Author
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Ogonuki, N., primary
- Published
- 2003
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16. New microinsemination techniques for laboratory animals
- Author
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Ogura, A, primary, Ogonuki, N, additional, Inoue, K, additional, and Mochida, K, additional
- Published
- 2003
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17. Phenotypic Effects of Somatic Cell Cloning in the Mouse
- Author
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Ogura, A., primary, Inoue, K., additional, Ogonuki, N., additional, Lee, J., additional, Kohda, T., additional, and Ishino, F., additional
- Published
- 2002
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18. Short-term nonfrozen storage of mouse epididymal spermatozoa
- Author
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Sankai, T., primary, Tsuchiya, H., additional, and Ogonuki, N., additional
- Published
- 2001
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19. Comparison of two methods of assisted fertilization in cynomolgus monkeys (Macaca fascicularis): intracytoplasmic sperm injection and partial zona dissection followed by insemination
- Author
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Ogonuki, N., primary, Sankai, T., additional, Cho, F., additional, Sato, K., additional, and Yoshikawa, Y., additional
- Published
- 1998
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20. 355 IMPROVEMENT OF CUMULUS-FREE IN VITRO MATURATION AND ITS APPLICATION TO MICROINSEMINATION WITH PRIMARY SPERMATOCYTES IN MICE
- Author
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Miki, H., Ogonuki, N., Inoue, K., Baba, T., and Ogura, A.
- Abstract
Manipulation of immature oocytes requires removal of cumulus cells, which may compromise ooplasmic maturation. This problem can be overcome by cytoplasm exchange at the MII stage, but this requires a second micromanipulative step. This study was undertaken to determine whether cumulus-free IVM can be improved by modifying IVM medium. We assessed the quality of oocytes by several cytochemical parameters, and by the subsequent development of embryos constructed by IVF, parthenogenetic activation, or microinsemination using primary spermatocytes. GV-stage oocytes were collected from superovulated B6D2F1 females. After freeing the oocytes from cumulus cells, they were subjected to IVM in either MEM or TYH media, or a 1:1 mixture of the two (termed TaM). IVM was performed under chemically semidefined conditions by using BSA as the only protein source. Oocytes that reached the MII stage were analyzed for distribution of mitochondria, activity of the catalytic p34cdc2 kinase subunit of the maturation promoting factor (MPF), and chromosomal alignment within the MII spindle. Microinsemination with primary spermatocytes was performed in oocytes at the MI stage as described previously (Ogura et al. 1998 PNAS 95, 5611-5615), but without MII chromosome exchange. TYH and MEM media had opposing effects on several parameters. TYH resulted in a better maturation rate (181/196, 92.3%) than MEM (184/257, 71.6%). In contrast, MEM supported better embryo development to the morula/blastocyst stage than TYH following IVF (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse in MEM, but aggregated in TYH. MPF activity in MII oocytes was significantly higher in TYH than in MEM (P < 0.05). Oocytes derived from TaM had intermediate characteristics between TYH- and MEM-oocytes and essentially resembled in vivo-matured oocytes, with the mitochondrial distribution pattern being most typically intermediate. By using TaM, 23.8% of GV oocytes developed into full-term fetuses following IVF and embryo transfer (compared to TYH: 6.3%, and MEM: 18.2%). This IVM system was then applied to oocytes to be injected with spermatocytes when in the MI stage. Approximately 60% of the oocytes survived injection. After 127 two-cell embryos were transfered, two normal-looking offspring were obtained. This is the first successful birth of pups derived from fertilization with spermatocytes by single injection, without serial nuclear transfer. Chromosomal analysis at MII revealed that the low developmental efficiency after embryo transfer could be attributed mainly to premature sister chromatid separation, as reported previously (Ogura et al. 1998). These results demonstrate that optimization of IVM media can improve the quality of cumulus-free IVM oocytes in mice, although it cannot overcome defects in the kinetics of injected spermatocyte chromosomes.
- Published
- 2005
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21. 358 MICROINSEMINATION USING MALE GERM CELLS FROM EPIDIDYMIDES AND TESTES STORED IN FREEZERS WITHOUT CRYOPROTECTANT
- Author
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Ogonuki, N., Mochida, K., Shinmen, A., Ohkawa, M., Miki, H., Inoue, K., Fray, M., Moriwaki, K., Obata, Y., and Ogura, A.
- Abstract
Cryopreservation of male germ cells is a strategy for the conservation of species and strains valuable to biomedical researchers. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of the male germ cell are usually employed. This study was undertaken to see whether mouse male germ cells could be safely cryopreserved for later use by freezing the whole epididymides and testes without cryoprotectant. Furthermore, we examined whether frozen male germ cells maintained their fertilization ability after international transportation on dry ice. Epididymides and testes were collected from sexually mature male ICR and C57BL/6Cr mice and placed in polypropylene cryotubes. The cryotubes were frozen at -80C with or without a freezing container, or were plunged directly into liquid nitrogen (LN2). They were stored at -80C or in LN2 from between one week and one year. Epididymides and testes were thawed by placing the cryotubes in a water bath at room temperature. B6D2F1 and C57BL/6Cr oocytes were microinseminated with either epididymal and testicular spermatozoa or round spermatids. After embryo transfer into pseudopregnant females, normal pups were obtained irrespective of the method of cryopreservation and cell type used. However, their birth rates (2-33%) were lower than those of our conventional microinsemination using fresh sperm or spermatids (20-60%). For transportation experiments, testes were collected from C57BL/6J mice and placed in a cryotube. The cryotubes were frozen at -80C in a freezing container. On the day of transportation, the cryotubes were placed in a polystyrene foam case filled with dry ice and were transported from Harwell (UK) to Tsukuba (Japan) by air and land. After three days, the samples were delivered to the recipient facility and were stored at -80C until use (about 1 month). After thawing and collection of spermatogenic cells, C57BL/6J oocytes were microinseminated with either testicular spermatozoa or elongated spermatids. After embryo transfer, 24 (34% per transfer) and 8 (16%) offspring, respectively, were obtained from the two groups. These results indicate that mouse male germ cells retain their nuclear integrity even after freezing epididymides or testes in freezers without cryoprotectant. Since this cryopreservation technique is very simple and allows storage at -80C for at least several months, it may enable transportation of mouse male germ cells internationally on dry ice, even when the senders are not specialized in cryopreservation.
- Published
- 2005
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22. Obox4 promotes zygotic genome activation upon loss of Dux .
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Guo Y, Kitano T, Inoue K, Murano K, Hirose M, Li TD, Sakashita A, Ishizu H, Ogonuki N, Matoba S, Sato M, Ogura A, and Siomi H
- Subjects
- Animals, Mice, Embryonic Development genetics, Gene Expression Regulation, Developmental, Genome, Mice, Knockout, Homeodomain Proteins metabolism, Homeodomain Proteins genetics, Zygote metabolism
- Abstract
Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux -knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here, we present multiple lines of evidence that the multicopy homeobox gene, Obox4 , encodes a transcription factor that is highly expressed in mouse two-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4 / Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to preimplantation development., Competing Interests: YG, TK, KI, KM, MH, TL, AS, HI, NO, SM, MS, AO, HS No competing interests declared, (© 2024, Guo, Kitano, Inoue et al.)
- Published
- 2024
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23. Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation.
- Author
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Morimoto H, Ogonuki N, Matoba S, Kanatsu-Shinohara M, Ogura A, and Shinohara T
- Subjects
- Humans, Mice, Male, Animals, Testis metabolism, Fertility, Stem Cell Transplantation, Spermatogenesis, Spermatogonia metabolism, Semen
- Abstract
Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients. Pre-transplantation treatment with WIN18,446 induced abnormal claudin protein expression, which comprises the blood-testis barrier and impedes SSC colonization. Consequently, WIN18,446 increased colonization efficiency by 4.6-fold compared with untreated host. WIN18,446-treated testes remained small despite the cessation of WIN18,446, suggesting its irreversible effect. Offspring were born by microinsemination using donor-derived sperm. While WIN18,446 was lethal to busulfan-treated mice, cyclophosphamide- or radiation-treated animals survived after WIN18,446 treatment. Although WIN18,446 is not applicable to humans due to toxicity, similar ALDH1A2 inhibitors may be useful for SSC transplantation into nonablated testes, shedding light on the role of retinoid metabolism on SSC-niche interactions and advancing SSC research in animal models and humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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24. Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.
- Author
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Töpf A, Cox D, Zaharieva IT, Di Leo V, Sarparanta J, Jonson PH, Sealy IM, Smolnikov A, White RJ, Vihola A, Savarese M, Merteroglu M, Wali N, Laricchia KM, Venturini C, Vroling B, Stenton SL, Cummings BB, Harris E, Marini-Bettolo C, Diaz-Manera J, Henderson M, Barresi R, Duff J, England EM, Patrick J, Al-Husayni S, Biancalana V, Beggs AH, Bodi I, Bommireddipalli S, Bönnemann CG, Cairns A, Chiew MT, Claeys KG, Cooper ST, Davis MR, Donkervoort S, Erasmus CE, Fassad MR, Genetti CA, Grosmann C, Jungbluth H, Kamsteeg EJ, Lornage X, Löscher WN, Malfatti E, Manzur A, Martí P, Mongini TE, Muelas N, Nishikawa A, O'Donnell-Luria A, Ogonuki N, O'Grady GL, O'Heir E, Paquay S, Phadke R, Pletcher BA, Romero NB, Schouten M, Shah S, Smuts I, Sznajer Y, Tasca G, Taylor RW, Tuite A, Van den Bergh P, VanNoy G, Voermans NC, Wanschitz JV, Wraige E, Yoshimura K, Oates EC, Nakagawa O, Nishino I, Laporte J, Vilchez JJ, MacArthur DG, Sarkozy A, Cordell HJ, Udd B, Busch-Nentwich EM, Muntoni F, and Straub V
- Subjects
- Animals, Humans, Male, Connectin genetics, Connectin metabolism, Muscle, Skeletal, Mutation, Muscular Diseases genetics, Muscular Diseases metabolism, Muscular Diseases pathology, Zebrafish genetics
- Abstract
In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3
-/- ; ttn.1+/- ) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases., (© 2024. The Author(s).)- Published
- 2024
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25. Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.
- Author
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Kanatsu-Shinohara M, Shiromoto Y, Ogonuki N, Inoue K, Hattori S, Miura K, Watanabe N, Hasegawa A, Mochida K, Yamamoto T, Miyakawa T, Ogura A, and Shinohara T
- Subjects
- Humans, Male, Animals, Mice, Sperm Injections, Intracytoplasmic adverse effects, Sperm Injections, Intracytoplasmic methods, Semen, Fertilization in Vitro methods, Infertility, Neoplasms etiology
- Abstract
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.
- Published
- 2023
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26. Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in vitro.
- Author
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Miyazaki T, Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, Yabe-Nishimura C, Zhang H, Pommier Y, Trumpp A, and Shinohara T
- Subjects
- Male, Mice, Animals, Reactive Oxygen Species metabolism, Cell Proliferation, Stem Cells, Cells, Cultured, Spermatogonia metabolism, Glutamine metabolism
- Abstract
Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)
- Published
- 2023
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27. Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.
- Author
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Ihashi S, Hamanaka M, Kaji M, Mori R, Nishizaki S, Mori M, Imasato Y, Inoue K, Matoba S, Ogonuki N, Takasu A, Nakamura M, Matsumoto K, Anzai M, Ogura A, Ikawa M, and Miyamoto K
- Subjects
- Animals, Mice, Cell Differentiation, Cell Nucleus, Gene Knockout Techniques, Apoptosis, Blastocyst
- Abstract
Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog , whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos., (© 2023 Ihashi et al.)
- Published
- 2023
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28. UHRF1 is essential for proper cytoplasmic architecture and function of mouse oocytes and derived embryos.
- Author
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Uemura S, Maenohara S, Inoue K, Ogonuki N, Matoba S, Ogura A, Kurumizaka M, Yamagata K, Sharif J, Koseki H, Ueda K, Unoki M, and Sasaki H
- Subjects
- Animals, Mice, Cytosol, Endoplasmic Reticulum, Mitochondria, CCAAT-Enhancer-Binding Proteins genetics, Ubiquitin-Protein Ligases genetics, Proteomics, Oocytes
- Abstract
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a protein essential for the maintenance of DNA methylation in somatic cells. However, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos, where it may play a role unrelated to the nuclear function. We herein report that oocyte-specific Uhrf1 KO results in impaired chromosome segregation, abnormal cleavage division, and preimplantation lethality of derived embryos. Our nuclear transfer experiment showed that the phenotype is attributable to cytoplasmic rather than nuclear defects of the zygotes. A proteomic analysis of KO oocytes revealed the down-regulation of proteins associated with microtubules including tubulins, which occurred independently of transcriptomic changes. Intriguingly, cytoplasmic lattices were disorganized, and mitochondria, endoplasmic reticulum, and components of the subcortical maternal complex were mislocalized. Thus, maternal UHRF1 regulates the proper cytoplasmic architecture and function of oocytes and preimplantation embryos, likely through a mechanism unrelated to DNA methylation., (© 2023 Uemura et al.)
- Published
- 2023
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29. Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.
- Author
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Ogonuki N, Kyogoku H, Hino T, Osawa Y, Fujiwara Y, Inoue K, Kunieda T, Mizuno S, Tateno H, Sugiyama F, Kitajima TS, and Ogura A
- Subjects
- Animals, Humans, Male, Meiosis, Mice, Oocytes, Spermatids, Azoospermia, Spermatocytes
- Abstract
Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure., (© 2022 The Authors.)
- Published
- 2022
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30. Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.
- Author
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Kanatsu-Shinohara M, Ogonuki N, Matoba S, Morimoto H, Shiromoto Y, Ogura A, and Shinohara T
- Subjects
- Animals, Male, Mice, Organoids, Rats, Spermatogenesis genetics, Spermatogonia transplantation, Stem Cell Transplantation, Hematopoietic Stem Cell Transplantation, Testis
- Abstract
Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 10
4 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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31. Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.
- Author
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Hada M, Miura H, Tanigawa A, Matoba S, Inoue K, Ogonuki N, Hirose M, Watanabe N, Nakato R, Fujiki K, Hasegawa A, Sakashita A, Okae H, Miura K, Shikata D, Arima T, Shirahige K, Hiratani I, and Ogura A
- Subjects
- Animals, Cell Differentiation genetics, Female, Mammals, Mice, Placenta, Pregnancy, Stem Cells, Histones genetics, Histones metabolism, Trophoblasts metabolism
- Abstract
The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5 , and up-regulation of the pluripotent marker Oct3/4 , indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function., (© 2022 Hada et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2022
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32. Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.
- Author
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Kamimura S, Inoue K, Mizutani E, Kim JM, Inoue H, Ogonuki N, Miyamoto K, Ihashi S, Itami N, Wakayama T, Ito A, Nishino N, Yoshida M, and Ogura A
- Subjects
- Animals, Histone Deacetylase Inhibitors classification, Mice, Peptides, Cyclic chemistry, Histone Deacetylase Inhibitors chemistry, Nuclear Transfer Techniques instrumentation, Oocytes chemistry
- Abstract
In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)
- Published
- 2021
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33. Spermatogonial stem cell transplantation into nonablated mouse recipient testes.
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Morimoto H, Ogonuki N, Kanatsu-Shinohara M, Matoba S, Ogura A, and Shinohara T
- Subjects
- Animals, Apoptosis, Biomarkers metabolism, Busulfan pharmacology, Claudins metabolism, Cytokines metabolism, Germ Cells drug effects, Germ Cells metabolism, Glial Cell Line-Derived Neurotrophic Factor metabolism, Male, Mice, Knockout, Regeneration drug effects, Spermatogenesis, Mice, Spermatogonia cytology, Spermatogonia transplantation, Stem Cell Transplantation, Testis cytology
- Abstract
Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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34. Tsga8 is required for spermatid morphogenesis and male fertility in mice.
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Kobayashi Y, Tomizawa SI, Ono M, Kuroha K, Minamizawa K, Natsume K, Dizdarević S, Dočkal I, Tanaka H, Kawagoe T, Seki M, Suzuki Y, Ogonuki N, Inoue K, Matoba S, Anastassiadis K, Mizuki N, Ogura A, and Ohbo K
- Subjects
- Animals, Female, Histone-Lysine N-Methyltransferase genetics, Histone-Lysine N-Methyltransferase metabolism, Infertility, Male genetics, Infertility, Male metabolism, Male, Mice, Mice, Knockout, Myeloid-Lymphoid Leukemia Protein genetics, Myeloid-Lymphoid Leukemia Protein metabolism, Nucleoproteins genetics, Spermatogonia metabolism, Fertility, Nucleoproteins metabolism, Spermatids metabolism, Spermatogenesis, Stem Cells metabolism
- Abstract
During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)
- Published
- 2021
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35. OGG1 protects mouse spermatogonial stem cells from reactive oxygen species in culture†.
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Mori Y, Ogonuki N, Hasegawa A, Kanatsu-Shinohara M, Ogura A, Wang Y, McCarrey JR, and Shinohara T
- Subjects
- Animals, DNA Breaks, Double-Stranded, DNA Glycosylases genetics, DNA Repair, Gene Expression Regulation, Genome, Hydrogen Peroxide toxicity, Male, Mice, Mutation, Adult Germline Stem Cells metabolism, DNA Glycosylases metabolism, Reactive Oxygen Species metabolism
- Abstract
Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2021
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36. An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia.
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Morimoto H, Yamamoto T, Miyazaki T, Ogonuki N, Ogura A, Tanaka T, Kanatsu-Shinohara M, Yabe-Nishimura C, Zhang H, Pommier Y, Trumpp A, and Shinohara T
- Subjects
- Animals, Cell Division genetics, Cell Proliferation genetics, Cells, Cultured, DNA Topoisomerases, Type I genetics, Gene Expression Regulation, Developmental, Hypoxia-Inducible Factor 1, alpha Subunit deficiency, Mice, Mice, Knockout, Mitochondria physiology, NADPH Oxidase 1 metabolism, Adult Germline Stem Cells cytology, Cell Hypoxia physiology, Oxygen metabolism, Reactive Oxygen Species metabolism
- Abstract
Reactive oxygen species (ROS) produced by NADPH1 oxidase 1 (NOX1) are thought to drive spermatogonial stem cell (SSC) self-renewal through feed-forward production of ROS by the ROS-BCL6B-NOX1 pathway. Here we report the critical role of oxygen on ROS-induced self-renewal. Cultured SSCs proliferated poorly and lacked BCL6B expression under hypoxia despite increase in mitochondria-derived ROS. Due to lack of ROS amplification under hypoxia, NOX1-derived ROS were significantly reduced, and Nox1 -deficient SSCs proliferated poorly under hypoxia but normally under normoxia. NOX1-derived ROS also influenced hypoxic response in vivo because Nox1 -deficient undifferentiated spermatogonia showed significantly reduced expression of HIF1A, a master transcription factor for hypoxic response. Hypoxia-induced poor proliferation occurred despite activation of MYC and suppression of CDKN1A by HIF1A, whose deficiency exacerbated self-renewal efficiency. Impaired proliferation of Nox1 - or Hif1a -deficient SSCs under hypoxia was rescued by Cdkn1a depletion. Consistent with these observations, Cdkn1a -deficient SSCs proliferated actively only under hypoxia but not under normoxia. On the other hand, chemical suppression of mitochondria-derived ROS or Top1mt mitochondria-specific topoisomerase deficiency did not influence SSC fate, suggesting that NOX1-derived ROS play a more important role in SSCs than mitochondria-derived ROS. These results underscore the importance of ROS origin and oxygen tension on SSC self-renewal., (© 2021 Morimoto et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2021
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37. Germ cell depletion in recipient testis has adverse effects on spermatogenesis in orthotopically transplanted testis pieces via retinoic acid insufficiency.
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Tsuchimoto A, Tone M, Ogonuki N, Hada M, Ogura A, and Takashima S
- Subjects
- Aldehyde Dehydrogenase 1 Family biosynthesis, Animals, Gene Expression Regulation, Enzymologic, Humans, Male, Mice, Retinal Dehydrogenase biosynthesis, Retinoic Acid 4-Hydroxylase biosynthesis, Organ Transplantation, Spermatogenesis, Spermatogonia enzymology, Testis enzymology, Testis transplantation, Tretinoin metabolism
- Abstract
Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.
- Published
- 2020
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38. A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.
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Kazuki Y, Gao FJ, Li Y, Moyer AJ, Devenney B, Hiramatsu K, Miyagawa-Tomita S, Abe S, Kazuki K, Kajitani N, Uno N, Takehara S, Takiguchi M, Yamakawa M, Hasegawa A, Shimizu R, Matsukura S, Noda N, Ogonuki N, Inoue K, Matoba S, Ogura A, Florea LD, Savonenko A, Xiao M, Wu D, Batista DA, Yang J, Qiu Z, Singh N, Richtsmeier JT, Takeuchi T, Oshimura M, and Reeves RH
- Subjects
- Animals, Brain pathology, Disease Models, Animal, Female, Heart Defects, Congenital genetics, Humans, Male, Mice, Mice, Inbred C57BL, Trisomy genetics, Whole Genome Sequencing, Chromosomes, Human, Pair 21 genetics, Down Syndrome genetics, Mice, Transgenic genetics
- Abstract
Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research., Competing Interests: YK, FG, YL, AM, BD, KH, SM, SA, KK, NK, NU, ST, MT, MY, AH, RS, SM, NN, NO, KI, SM, AO, LF, AS, MX, DW, DB, JY, ZQ, NS, JR, TT, MO, RR No competing interests declared, (© 2020, Kazuki et al.)
- Published
- 2020
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39. Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas.
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Inoue K, Ogonuki N, Kamimura S, Inoue H, Matoba S, Hirose M, Honda A, Miura K, Hada M, Hasegawa A, Watanabe N, Dodo Y, Mochida K, and Ogura A
- Subjects
- Animals, Cellular Reprogramming genetics, Cellular Reprogramming physiology, Female, Genomic Imprinting, Mice, Multigene Family genetics, Pregnancy, RNA, Untranslated genetics, RNA, Untranslated metabolism, Histones metabolism, MicroRNAs genetics, Placenta metabolism, Repressor Proteins genetics
- Abstract
Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.
- Published
- 2020
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40. Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice.
- Author
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Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, and Shinohara T
- Subjects
- Animals, Disease Models, Animal, Fertility physiology, Humans, Infertility genetics, Infertility pathology, Male, Mice, Spermatogenesis genetics, Spermatogonia growth & development, Spermatozoa growth & development, Spermatozoa transplantation, Stem Cells cytology, Transplantation, Autologous methods, Fertility genetics, Infertility therapy, Spermatogonia transplantation, Stem Cell Transplantation
- Abstract
The blood-testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11 , a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11 -deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11 -deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11 -deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11 -deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11 -deficient testes, those from WT or Cldn11 -deficient testes regenerated sperm in Cldn11 -deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3 / 5 regenerated endogenous spermatogenesis in Cldn11 -deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue., Competing Interests: The authors declare no competing interest.
- Published
- 2020
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41. Early production of offspring by in vitro fertilization using first-wave spermatozoa from prepubertal male mice.
- Author
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Mochida K, Hasegawa A, Ogonuki N, Inoue K, and Ogura A
- Subjects
- Animals, Culture Media pharmacology, Embryo Transfer, Female, Fertilization, Glutathione metabolism, Male, Mice, Mice, Inbred C57BL, Oocytes cytology, Sperm Motility, Time Factors, Epididymis cytology, Fertilization in Vitro methods, Spermatozoa cytology
- Abstract
Mature male mice (aged 10-12 weeks or older) are conventionally used for in vitro fertilization (IVF) in order to achieve high fertilization rates (e.g., > 70%). Here, we sought to determine the earliest age at which male mice (C57BL/6J strain) can be used efficiently for producing offspring via IVF. Because we noted that the addition of reduced glutathione (GSH) to the IVF medium significantly increased the fertilizing ability of spermatozoa from prepubertal males, we used this IVF protocol for all experiments. Spermatozoa first reached the caudal region of the epididymides at day 35; however, they were unable to fertilize oocytes. Caudal epididymal spermatozoa first became competent for oocyte fertilization at day 37, albeit at a low rate (2.9%). A high fertilization rate (72.0%) was obtained at day 40, and 52.4% of the embryos thus obtained developed into offspring after embryo transfer. Moreover, we found that corpus epididymal spermatozoa in prepubertal mice could fertilize oocytes; however, the fertilization rates were always < 50%, regardless of the age of the males. Caput epididymal spermatozoa failed to fertilize oocytes irrespective of the age of the males. Therefore, we propose that caudal epididymal spermatozoa from male mice aged 40 days can be efficiently used for IVF, to obtain offspring in the shortest attainable time. This protocol will reduce the turnover time required for the generation of mice by ~1 month compared with that of the conventional IVF protocol.
- Published
- 2019
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42. Birth of a marmoset following injection of elongated spermatid from a prepubertal male.
- Author
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Ogonuki N, Abe Y, Kurotaki YK, Nakao K, Aiba A, Sasaki E, and Ogura A
- Subjects
- Animals, Callithrix, Female, Male, Pregnancy, Live Birth, Sperm Injections, Intracytoplasmic, Spermatids
- Abstract
The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene-modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene-modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage spermatids (Steps 5-7), elongated spermatids, and testicular spermatozoa from the testis of a prepubertal 11-month-old male marmoset and injected them into in vitro-matured oocytes. After 7 days in culture, two embryos from elongated spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated spermatid injection-derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a spermatid from a prepubertal male primate can support full-term development. Using this method, we can expect to obtain offspring of gene-modified males 6 months to a year earlier than with natural mating., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
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43. ROS amplification drives mouse spermatogonial stem cell self-renewal.
- Author
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Morimoto H, Kanastu-Shinohara M, Ogonuki N, Kamimura S, Ogura A, Yabe-Nishimura C, Mori Y, Morimoto T, Watanabe S, Otsu K, Yamamoto T, and Shinohara T
- Subjects
- Animals, Benzodiazepinones pharmacology, Cell Proliferation drug effects, DNA-Binding Proteins metabolism, Feedback, Physiological physiology, Gene Knockout Techniques, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Knockout, Mitogen-Activated Protein Kinase 14 genetics, Mitogen-Activated Protein Kinase 14 metabolism, Mitogen-Activated Protein Kinase 7 genetics, Mitogen-Activated Protein Kinase 7 metabolism, NADPH Oxidase 1 genetics, NADPH Oxidase 1 metabolism, Real-Time Polymerase Chain Reaction, Repressor Proteins genetics, Repressor Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Transfection, Adult Germline Stem Cells physiology, Cell Self Renewal physiology, Reactive Oxygen Species metabolism
- Abstract
Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop., (© 2019 Morimoto et al.)
- Published
- 2019
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44. Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.
- Author
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Matsuda M, Ono R, Iyoda T, Endo T, Iwasaki M, Tomizawa-Murasawa M, Saito Y, Kaneko A, Shimizu K, Yamada D, Ogonuki N, Watanabe T, Nakayama M, Koseki Y, Kezuka-Shiotani F, Hasegawa T, Yabe H, Kato S, Ogura A, Shultz LD, Ohara O, Taniguchi M, Koseki H, Fujii SI, and Ishikawa F
- Subjects
- Animals, CD56 Antigen metabolism, Female, Fetal Blood cytology, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Models, Animal, Thymus Gland cytology, Transcriptome, Transplantation, Heterologous, Cell Differentiation, Gene Knock-In Techniques, Interleukin-15 blood, Interleukin-15 genetics, Interleukin-7 blood, Interleukin-7 genetics, Killer Cells, Natural physiology
- Abstract
The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells., (© 2019 Matsuda et al.)
- Published
- 2019
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45. Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.
- Author
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Miura K, Matoba S, Ogonuki N, Namiki T, Ito J, Kashiwazaki N, and Ogura A
- Subjects
- Animals, Female, Mice, Oocytes metabolism, Embryonic Development drug effects, Oocytes drug effects, Phosphoinositide Phospholipase C metabolism, RNA, Messenger pharmacology, Sperm-Ovum Interactions drug effects
- Abstract
In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca
2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.- Published
- 2018
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46. In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses.
- Author
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Watanabe S, Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, and Shinohara T
- Subjects
- Animals, Infertility, Male pathology, Kinetics, Male, Mice, Inbred C57BL, Microinjections, Neuraminidase metabolism, Serogroup, Sertoli Cells pathology, Spermatogenesis, Spermatogonia metabolism, Spermatozoa cytology, Stem Cell Factor metabolism, Stem Cells metabolism, Testis cytology, Cellular Microenvironment, Dependovirus metabolism, Genetic Techniques, Spermatogonia cytology, Stem Cells cytology
- Abstract
Adeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile Kitl
Sl /KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2018
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47. Oocyte-activating capacity of fresh and frozen-thawed spermatids in the common marmoset (Callithrix jacchus).
- Author
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Ogonuki N, Inoue H, Matoba S, Kurotaki YK, Kassai H, Abe Y, Sasaki E, Aiba A, and Ogura A
- Subjects
- Animals, Callithrix, Female, Male, Mice, Microinjections, Oocytes cytology, Spermatids cytology, Calcium Signaling, Cell Nucleus metabolism, Cryopreservation, Nuclear Transfer Techniques, Oocytes metabolism, Spermatids metabolism
- Abstract
The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca
2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation., (© 2018 Wiley Periodicals, Inc.)- Published
- 2018
- Full Text
- View/download PDF
48. Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos.
- Author
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Hirose M, Hada M, Kamimura S, Matoba S, Honda A, Motomura K, Ogonuki N, Shawki HH, Inoue K, Takahashi S, and Ogura A
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Transport System A genetics, Amino Acid Transport System A metabolism, Animals, Blastocyst metabolism, Blastocyst pathology, Cloning, Organism, DNA Methylation, Embryonic Stem Cells metabolism, Female, Mice, Mice, Inbred C57BL, Phosphoproteins genetics, Phosphoproteins metabolism, Placenta metabolism, Placenta pathology, Placentation, Pregnancy, Repressor Proteins, Transcription Factors genetics, Transcription Factors metabolism, Trophoblasts metabolism, Embryonic Stem Cells pathology, Epigenesis, Genetic, Genomic Imprinting, Nuclear Transfer Techniques adverse effects, Placenta abnormalities, Trophoblasts pathology
- Abstract
Although phenotypic abnormalities frequently appear in the placenta following somatic cell nuclear transfer (SCNT), mouse trophoblast stem cells (TSCs) established from SCNT embryos reportedly show no distinct abnormalities compared with those derived from normal fertilization. In this study, we reexamined SCNT-TSCs to identify their imprinting statuses. Placenta-specific maternally imprinted genes (Gab1, Slc38a4, and Sfmbt2) consistently showed biallelic expression in SCNT-TSCs, suggesting their loss of imprinting (LOI). The LOI of Gab1 was associated with decreased DNA methylation, and that of Sfmbt2 was associated with decreased DNA methylation and histone H3K27 trimethylation. The maternal allele of the intergenic differentially methylated region (IG-DMR) was aberrantly hypermethylated following SCNT, even though this region was prone to demethylation in TSCs when established in a serum-free chemically defined medium. These findings indicate that the development of cloned embryos is associated with imprinting abnormalities specifically in the trophoblast lineage from its initial stage, which may affect subsequent placental development.
- Published
- 2018
- Full Text
- View/download PDF
49. Efficient and scheduled production of pseudopregnant female mice for embryo transfer by estrous cycle synchronization.
- Author
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Hasegawa A, Mochida K, Ogonuki N, Hirose M, Tomishima T, Inoue K, and Ogura A
- Subjects
- Animals, Embryo Transfer, Female, Male, Pregnancy, Estrus Synchronization methods, Progesterone administration & dosage, Pseudopregnancy chemically induced
- Abstract
In embryo transfer experiments in mice, pseudopregnant females as recipients are prepared by sterile mating with vasectomized males. Because only females at the proestrus stage accept males, such females are selected from a stock of animals based on the appearance of their external genital tract. Therefore, the efficiency of preparing pseudopregnant females largely depends on the size of female colonies and the skill of the operators who select females for sterile mating. In this study, we examined whether the efficiency of preparing pseudopregnant females could be improved by applying an estrous cycle synchronization method by progesterone (P4) pretreatment, which significantly enhances the superovulation outcome in mice. We confirmed that after two daily injections of P4 (designated Days 1 and 2) in randomly selected females, the estrous cycles of most females (about 85%) were synchronized at metestrus on Day 3. When P4-treated females were paired with vasectomized males for 4 days (Days 4-8), a vaginal plug was found in 63% (20/32) of the females on Day 7. After the transfer of vitrified-warmed embryos into their oviducts, 52% (73/140) of the embryos successfully developed into offspring, the rate being comparable to that of the conventional embryo transfer procedure. Similarly, 77% (24/31) of females became pregnant by fertile mating with intact males for 3 days, which allowed the scheduled preparation of foster mothers. Thus, our estrous cycle synchronization method may omit the conventional experience-based process of visually observing the vagina to choose females for embryo transfer. Furthermore, it is expected that the size of female stocks for recipients can be reduced to less than 20%, which could be a great advantage for facilities/laboratories undertaking mouse-assisted reproductive technology.
- Published
- 2017
- Full Text
- View/download PDF
50. Transfer of a Mouse Artificial Chromosome into Spermatogonial Stem Cells Generates Transchromosomic Mice.
- Author
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Shinohara T, Kazuki K, Ogonuki N, Morimoto H, Matoba S, Hiramatsu K, Honma K, Suzuki T, Hara T, Ogura A, Oshimura M, Kanatsu-Shinohara M, and Kazuki Y
- Subjects
- Animals, Biomarkers, Cell Tracking, Gene Expression, Genes, Reporter, Genomic Instability, Immunophenotyping, Karyotype, Male, Mice, Mice, Transgenic, Mouse Embryonic Stem Cells cytology, Mouse Embryonic Stem Cells metabolism, Phenotype, Spermatogenesis, Chromosomes, Artificial, Gene Transfer Techniques, Spermatogonia cytology, Spermatogonia metabolism
- Abstract
The introduction of megabase-sized large DNA fragments into the germline has been a difficult task. Although microcell-mediated chromosome transfer into mouse embryonic stem cells (ESCs) allows the production of transchromosomic mice, ESCs have unstable karyotypes and germline transmission is unreliable by chimera formation. As spermatogonial stem cells (SSCs) are the only stem cells in the germline, they represent an attractive target for germline modification. Here, we report successful transfer of a mouse artificial chromosome (MAC) into mouse germline stem cells (GSCs), cultured spermatogonia enriched for SSCs. MAC-transferred GSCs maintained the host karyotype and MAC more stably than ESCs, which have significant variation in chromosome number. Moreover, MAC-transferred GSCs produced transchromosomic mice following microinjection into the seminiferous tubules of infertile recipients. Successful transfer of MACs to GSCs overcomes the problems associated with ESC-mediated germline transmission and provides new possibilities in germline modification., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
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