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1.
Genome Res ; 32(5): 945-955, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35534232

RESUMO

De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell- and somatic cell-specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell-specific lineages, 10-15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.


Assuntos
Células Germinativas , Taxa de Mutação , Animais , Linhagem da Célula/genética , Camundongos , Mutação , Zigoto
2.
Development ; 149(18)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35993297

RESUMO

Round spermatid injection (ROSI) results in a lower birth rate than intracytoplasmic sperm injection, which has hampered its clinical application. Inefficient development of ROSI embryos has been attributed to epigenetic abnormalities. However, the chromatin-based mechanism that underpins the low birth rate in ROSI remains to be determined. Here, we show that a repressive histone mark, H3K27me3, persists from mouse round spermatids into zygotes in ROSI and that round spermatid-derived H3K27me3 is associated with less accessible chromatin and impaired gene expression in ROSI embryos. These loci are initially marked by H3K27me3 but undergo histone modification remodelling in spermiogenesis, resulting in reduced H3K27me3 in normal spermatozoa. Therefore, the absence of epigenetic remodelling, presumably mediated by histone turnover during spermiogenesis, leads to dysregulation of chromatin accessibility and transcription in ROSI embryos. Thus, our results unveil a molecular logic, in which chromatin states in round spermatids impinge on chromatin accessibility and transcription in ROSI embryos, highlighting the importance of epigenetic remodelling during spermiogenesis in successful reproduction.


Assuntos
Histonas , Espermátides , Animais , Cromatina/genética , Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Oócitos/metabolismo , Herança Paterna , Sêmen/metabolismo , Espermátides/metabolismo
3.
Nucleic Acids Res ; 51(13): 6668-6683, 2023 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-37283086

RESUMO

Sperm chromatin retains small amounts of histones, and chromatin states of sperm mirror gene expression programs of the next generation. However, it remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here, we present a novel mouse model of paternal epigenetic inheritance, in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology using testicular sperm, we rescued infertility of mice missing Polycomb protein SCML2, which regulates germline gene expression by establishing H3K27me3 on bivalent promoters with other active marks H3K4me2/3. We profiled epigenomic states (H3K27me3 and H3K4me3) of testicular sperm and epididymal sperm, demonstrating that the epididymal pattern of the sperm epigenome is already established in testicular sperm and that SCML2 is required for this process. In F1 males of X-linked Scml2-knockout mice, which have a wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in F0 sperm. Further, dysregulation of gene expression was observed in the mutant-derived wild-type F1 preimplantation embryos. Together, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin.


Assuntos
Cromatina , Epigênese Genética , Animais , Masculino , Camundongos , Cromatina/genética , Epigenômica , Histonas/genética , Histonas/metabolismo , Camundongos Knockout , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo
4.
Cell ; 137(3): 571-84, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19410550

RESUMO

Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.


Assuntos
Proteína Morfogenética Óssea 4/fisiologia , Linhagem da Célula/fisiologia , Células Germinativas , Transdução de Sinais/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/fisiologia , Células Germinativas/citologia , Células Germinativas/fisiologia , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas de Ligação a RNA , Células-Tronco/citologia , Células-Tronco/fisiologia , Testículo/citologia , Testículo/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Wnt/fisiologia
5.
Biochem Biophys Res Commun ; 680: 119-126, 2023 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-37738901

RESUMO

Round spermatid injection (ROSI) is the last resort and recourse for men with nonobstructive azoospermia to become biological fathers of their children. However, the ROSI-derived offspring rate is lower than intracytoplasmic sperm injection (ICSI) in mice (20% vs. 60%). This low success rate has hindered the spread of ROSI in ART (Assisted Reproductive Technology). However, the cause of the ROSI-zygote-derived low offspring rate is currently unknown. In the previous studies, we reported that H3K9me3 and H3K27me3 exhibited ectopic localizations in male pronuclei (mPN) of ROSI-zygotes, suggesting that the carried over histone to zygotes conveys epigenetic information. In this study, we analyzed other histone modifications to explore unknown abnormalities. H3K36me3 showed an increased methylation state compared to ICSI-derived embryos but not for H3K4me3. Abnormal H3K36me3 was corrected until 2-cell stage embryos, suggesting a long window of reprogramming ability in ROSI-embryos. Treatment with TSA of ROSI-zygotes, which was reported to be capable of correcting ectopic DNA methylation in ROSI-zygotes, caused abnormalities of H3K36me3 in male and female PN (fPN) of the zygotes. In contrast, round spermatid TSA treatment before ROSI, which was reported to improve the preimplantation development of ROSI-zygotes, showed beneficial effects without toxicity in fPN. Therefore, the results suggest that TSA has some negative effects, but overall, it is effective in the correction of epigenetic abnormalities in ROSI-zygotes. When attempting to correct epigenetic abnormalities, attention should be paid to epigenomes not only in male but also in female pronuclei.


Assuntos
Histonas , Espermátides , Humanos , Criança , Masculino , Feminino , Camundongos , Animais , Espermátides/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Sêmen/metabolismo , Blastocisto/metabolismo , Metilação de DNA
6.
Development ; 147(15)2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32665239

RESUMO

The reason for the poor development of cloned embryos is not yet clear. Several reports have suggested that some nuclear remodeling/reprogramming factors (RRFs) are removed from oocytes at the time of enucleation, which might cause the low success rate of animal cloning. However, there is currently no method to manipulate the amount of RRFs in oocytes. Here, we describe techniques we have developed to gradually reduce RRFs in mouse oocytes by injecting somatic cell nuclei into oocytes. These injected nuclei were remodeled and reprogrammed using RRFs, and then RRFs were removed by subsequent deletion of somatic nuclei from oocytes. The size of the metaphase II spindle reduced immediately, but did recover when transferred into fresh oocytes. Though affected, the full-term developmental potential of these RRF-reduced oocytes with MII-spindle shrinkage was not lost after fertilization. When somatic cell nuclear transfer was performed, the successful generation of cloned mice was somewhat improved and abnormalities were reduced when oocytes with slightly reduced RRF levels were used. These results suggest that a change in RRFs in oocytes, as achieved by the method described in this paper or by enucleation, is important but not the main reason for the incomplete reprogramming of somatic cell nuclei.


Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Clonagem de Organismos , Metáfase , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos ICR
7.
Nature ; 548(7666): 224-227, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28746308

RESUMO

Inhibitors of Mek1/2 and Gsk3ß, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.


Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Células-Tronco Embrionárias/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Impressão Genômica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Fator Inibidor de Leucemia/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL
8.
J Reprod Dev ; 69(4): 198-205, 2023 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-37357399

RESUMO

Although freeze-drying sperm can save space, reduce maintenance costs, and facilitate the transportation of genetic samples, the current method requires breakable, custom-made, and expensive glass ampoules. In the present study, we developed a simple and economical method for collecting freeze-dried (FD) sperm using commercially available plastic microtubes. Mouse epididymal sperm suspensions were placed in 1.5 ml polypropylene tubes, frozen in liquid nitrogen, and dried in an acrylic freeze-drying chamber, after which they were closed under a vacuum. The drying duration did not differ between the microtube and glass ampoule methods (control); however, the sperm recovery rate was higher using the microtube method, and the physical damage to the sperm after rehydration was also reduced. Intracytoplasmic sperm injection (ICSI) using FD sperm stored in microtubes at -30°C yielded healthy offspring without reducing the success rate, even after 9 months of storage. Air infiltration into all microtubes stored at room temperature (RT) within 2 weeks of storage caused a drastic decrease in the fertilization rate of FD sperm; underwater storage did not prevent air infiltration. RT storage of FD sperm in microtubes for 1 week resulted in healthy offspring after ICSI (5-18%), but the addition of silica gel or CaCl2 did not improve the success rate. Our novel microtube method is currently the simplest and most effective method for treating FD sperm, contributing to the development of alternative low-cost approaches for preserving and transporting genetic resources.


Assuntos
Plásticos , Preservação do Sêmen , Animais , Camundongos , Masculino , Sêmen , Liofilização/métodos , Espermatozoides , Injeções de Esperma Intracitoplásmicas/métodos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos
9.
J Reprod Dev ; 69(1): 48-52, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36529517

RESUMO

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.


Assuntos
Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide , Camundongos , Masculino , Ratos , Animais , Injeções de Esperma Intracitoplásmicas/métodos , Tripsina , Sêmen , Espermatozoides/fisiologia , Oócitos
10.
J Reprod Dev ; 68(4): 262-270, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35676029

RESUMO

Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.


Assuntos
Preservação do Sêmen , Injeções de Esperma Intracitoplásmicas , Animais , DNA , Desenvolvimento Embrionário , Liofilização/métodos , Masculino , Camundongos , Oócitos , Sêmen , Preservação do Sêmen/métodos , Espermatozoides , Vácuo
11.
J Reprod Dev ; 68(2): 118-124, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-34980785

RESUMO

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at -80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at -80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at -80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.


Assuntos
Injeções de Esperma Intracitoplásmicas , Vitrificação , Animais , Criopreservação/métodos , Embrião de Mamíferos , Fertilização in vitro/métodos , Mamíferos , Camundongos , Concentração Osmolar , Injeções de Esperma Intracitoplásmicas/métodos
12.
Biol Reprod ; 105(2): 543-553, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-33982061

RESUMO

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.


Assuntos
Inibidores de Histona Desacetilases/química , Técnicas de Transferência Nuclear/instrumentação , Oócitos/química , Animais , Inibidores de Histona Desacetilases/classificação , Camundongos , Peptídeos Cíclicos/química
13.
Mol Cell ; 52(3): 380-92, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24120664

RESUMO

Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential role in different types of multipotent stem cells, raising the possibility that Sox2 governs the common stemness phenotype. Here we show that Sox2 is a critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal of trophoblast stem cells (TSCs). Sustained expression of Sox2 together with Esrrb or Tfap2c can replace FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and TSCs combined with inducible knockout systems, we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct sets of genes with unique functions in these two different yet developmentally related types of stem cells. Our findings provide insights into the functional versatility of transcription factors during embryogenesis, during which they can be recursively utilized in a variable manner within discrete network structures.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Trofoblastos/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/imunologia , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator de Transcrição AP-2/metabolismo , Trofoblastos/citologia
14.
Biosci Biotechnol Biochem ; 85(5): 1215-1226, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33587104

RESUMO

We examined whether peripheral leukocytes of mice derived from in vitro αMEM-cultured embryos and exhibiting type 2 diabetes had higher expression of inflammatory-related genes associated with the development of atherosclerosis. Also, we examined the impact of a barley diet on inflammatory gene expression. Adult mice were produced by embryo transfer, after culturing two-cell embryos for 48 h in either α minimal essential media (α-MEM) or potassium simplex optimized medium control media. Mice were fed either a barley or rice diet for 10 weeks. Postprandial blood glucose and mRNA levels of several inflammatory genes, including Tnfa and Nox2, in blood leukocytes were significantly higher in MEM mice fed a rice diet compared with control mice. Barley intake reduced expression of S100a8 and Nox2. In summary, MEM mice exhibited postprandial hyperglycemia and peripheral leukocytes with higher expression of genes related to the development of atherosclerosis, and barley intake reduced some gene expression.


Assuntos
Aterosclerose/dietoterapia , Blastocisto/efeitos dos fármacos , Dieta/métodos , Hordeum/química , Hiperglicemia/dietoterapia , Efeitos Tardios da Exposição Pré-Natal/dietoterapia , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Blastocisto/metabolismo , Blastocisto/patologia , Glicemia/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Transferência Embrionária , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Compostos Orgânicos/efeitos adversos , Oryza/química , Período Pós-Prandial , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
15.
Reproduction ; 160(2): 319-330, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585638

RESUMO

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.


Assuntos
Embrião de Mamíferos/fisiologia , Técnicas de Transferência Nuclear/estatística & dados numéricos , Oócitos/fisiologia , Fosfoinositídeo Fosfolipase C/metabolismo , RNA Complementar/administração & dosagem , Espermátides/fisiologia , Zigoto/fisiologia , Animais , Animais Recém-Nascidos , Embrião de Mamíferos/citologia , Feminino , Masculino , Camundongos Endogâmicos ICR , Oócitos/citologia , Fosfoinositídeo Fosfolipase C/administração & dosagem , Fosfoinositídeo Fosfolipase C/genética , Gravidez , Espermátides/citologia , Zigoto/citologia
16.
Nature ; 505(7485): 641-7, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24476887

RESUMO

Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.


Assuntos
Ácidos/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Animais , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células , Quimera/metabolismo , Metilação de DNA/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator 3 de Transcrição de Octâmero/metabolismo , Especificidade de Órgãos
17.
Nature ; 505(7485): 676-80, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24476891

RESUMO

We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency.


Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Placenta/citologia , Trofoblastos/citologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Fator 4 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Placenta/efeitos dos fármacos , Gravidez , Trofoblastos/efeitos dos fármacos
18.
J Reprod Dev ; 66(5): 427-433, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-32507772

RESUMO

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.


Assuntos
Desenvolvimento Embrionário , Recuperação de Oócitos/métodos , Oócitos/citologia , Animais , Blastocisto , Butorfanol/administração & dosagem , Transferência Embrionária , Feminino , Fertilização in vitro/métodos , Masculino , Medetomidina/administração & dosagem , Camundongos , Midazolam/administração & dosagem , Oócitos/metabolismo , Ovulação , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/citologia , Fatores de Tempo
19.
J Reprod Dev ; 66(1): 67-73, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31852860

RESUMO

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52-53% or 48-51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.


Assuntos
Desenvolvimento Embrionário/fisiologia , Oócitos/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/metabolismo , Animais , Feminino , Cavalos , Masculino , Camundongos , Fosfoinositídeo Fosfolipase C/genética , Injeções de Esperma Intracitoplásmicas
20.
Proc Natl Acad Sci U S A ; 114(23): 5988-5993, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533361

RESUMO

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.


Assuntos
Dano ao DNA/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Espermatozoides/efeitos da radiação , Animais , Transferência Embrionária/métodos , Transferência Embrionária/mortalidade , Feminino , Liofilização/métodos , Células Germinativas/efeitos da radiação , Tamanho da Ninhada de Vivíparos/efeitos da radiação , Masculino , Camundongos , Oócitos , Técnicas de Reprodução Assistida , Voo Espacial , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/fisiologia
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