scholarly journals Developmental potential of cloned mouse embryos reconstructed by a conventional technique of nuclear injection

Reproduction ◽  
2002 ◽  
pp. 197-207 ◽  
Author(s):  
A Rybouchkin ◽  
B Heindryckx ◽  
J Van der Elst ◽  
M Dhont
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Somatic Cells ◽  
Cumulus Cell ◽  
Conventional Technique ◽  
Mouse Embryos ◽  
Full Term ◽  
Cell Nuclei ◽  
Developmental Potential

The fact that most of the advances in mouse cloning by nuclear transfer originate from research in a limited number of laboratories demonstrates the complexity of the reported technologies. The development of alternative and more simple techniques of nuclear transfer may therefore be of interest. Furthermore, the preimplantation biology of cloned mouse embryos originating from somatic cells has not yet been studied in detail. In the present study, a modified conventional injection (mCI) technique for cloning mice from somatic cells is described. The preimplantation development and morphology of the resulting nuclear transfer embryos in comparison with parthenogenetic embryos and embryos obtained by intracytoplasmic sperm injection (ICSI) under comparable conditions was also studied. Finally, the capacity of nuclear transfer embryos for full-term development was investigated. Eighty-nine per cent of oocytes injected with cumulus cell nuclei under mCI conditions survived and formed zygotes. However, the rate of development of these zygotes to the blastocyst stage was significantly lower (29%) than that of ICSI or parthenogenetic zygotes (95 and 92%, respectively). Cloned blastocysts had a significantly lower mean number of cells in the inner cell mass (9) and trophectoderm (52) and a lower inner cell mass:total cell ratio (14%) than did their counterparts (31, 143 and 18% for ICSI and 21, 92 and 18% for parthenogenetic blastocysts, respectively). This correlated with a significantly higher proportion of dead cells in the cloned blastocysts. The poor quality of cloned blastocysts may explain the low rate of full-term fetal development of somatic mouse clones.

139 INJECTION OF SOMATIC CELL CYTOPLASM INTO OOCYTES BEFORE ICSI IMPAIRED FULL-TERM DEVELOPMENT AND INCREASED PLACENTA WEIGHT IN MICE

2006 ◽  
Vol 18 (2) ◽  
pp. 178
Author(s):  
V. T. Nguyen ◽  
S. Wakayama ◽  
S. Kishigami ◽  
H. Ohta ◽  
T. Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cumulus Cell ◽  
Full Term ◽  
Developmental Competence ◽  
Mouse Strains ◽  
Control Group ◽  
Cell Cytoplasm ◽  
Intact Mouse

During the process of somatic cell nuclear transfer, cytoplasm is introduced into the enucleated oocytes, in addition to the genomic material, regardless of the electrofusion methods (Wilmut et al. 1997) or direct injection of somatic nucleus by the Honolulu method (Wakayama et al. 1998). Only 1 to 2% of cloned embryos, however, develop to term with many incidences of developmental anomalies. These cloning failures may be explained by incomplete reprogramming of the donor cell genome, although it is not yet clear whether cytoplasmic materials of the somatic cell also have an affect on development of the cloned embryo. In an attempt to answer this question, this study investigates the effects of somatic cytoplasm of different mouse strains and cytoplasm of fertilized embryos at different stage by injecting them into intact mouse oocytes before intracytoplasmic sperm injection (ICSI). Mature oocytes collected from B6D2F1 female after 14 to 16 h of hCG injection were injected with (1) B6D2F1 cumulus cell cytoplasm with different volumes (collected by 2 to 3 �m of injection pipette and piezo pulses), (2) cumulus cell cytoplasm from different mouse strains (B6D2F1, ICR, C57BL/6), (3) cytoplasm of 1- to 8-cell embryos. After subsequent culture for 1 h, B6D2F1 sperm were injected into those oocytes and examined for preimplantation developmental competence. The total number of cells, inner cell mass (ICM), and expression of Oct4 in expanded blastocysts were also examined. In order to examine the effects of somatic cytoplasm on full-term development, we transferred 2-cell embryos at 24 h or morula and blastocysts at 72 h after ICSI to the oviduct or uterus of surrogate mothers (ICR) on Day 1 or 3 of pseudopregnancy. The control group received a sham injection with PVP before ICSI. The results showed that an increase the volume of cytoplasm from 1-fold to 4-fold (equivalent with the volume of 1 cumulus cell) resulted in impairing full-term development (28 and 7%, respectively, vs. 56 to 63% in the control group, P < 0.01). There was no difference in the frequency of embryos developing to the blastocyts stage between B6D2F1 and ICR somatic cytoplasms at the same volume. However, C57BL/6 somatic cytoplasm induced the 2-cell block to B6D2F2 embryos. Fertilized embryo cytoplasm did not reduce the frequency of blastocyst stage and full-term development. Interestingly, we found that somatic cytoplasm increased the placenta weight of ICSI embryo (0.2002 � 0.03, n = 32; vs. 0.1198 � 0.02 in control group, n = 87; P < 0.01). We also obtained placenta with no fetus when the volume of somatic cytoplasm was the same size as cumulus cell. We found that an increase in the volume of somatic cytoplasm led to low expression of Oct4 in expanded blastocysts. These findings indicated that injection of somatic cytoplasm into oocytes before ICSI decreased the preimplantation development, clearly impaired full-term development, and caused placental overgrowth in fertilized embryos. This study suggested that somatic cell cytoplasmic material is one cause of the low rate of full-term development of cloned animals.

scholarly journals Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei

Reproduction ◽  
2001 ◽  
pp. 49-60 ◽  
Author(s):  
T Wakayama ◽  
R Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Cytochalasin D ◽  
Full Term ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Reconstructed Embryos

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

72 LIMITED REPROGRAMMING OF SOMATIC CELL NUCLEI TRANSFERRED INTO MOUSE IMMATURE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 153
Author(s):  
C. Palmieri ◽  
H. Fulka ◽  
J. Fulka, Jr ◽  
P. Loi ◽  
G. Ptak ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cell Nuclei ◽  
Practical Applications ◽  
Functional Changes ◽  
Low Efficiency

Somatic cell nuclear transfer, an important approach for the analysis of certain functional changes in the genome during differentiation and for many practical applications, is in general a low-efficiency procedure, mainly due to a low effectivity in the re-establishment of the developmental program in the reconstructed embryo. The process of reprogramming is, however, poorly understood and some additional studies are clearly necessary. The aim of this study was the ultrastructural and immunofluorescent (B23-nucleophosmin) evaluation of somatic (cumulus) cell nuclei reprogramming after their transfer into intact immature mouse oocytes, kept at the germinal vesicle (GV) stage (dbcAMP) during the whole culture. Control somatic cells and nuclear transfer-reconstructed embryos (1 and 24 h after fusion induced by polyethyleneglycol) were fixed for transmission electron mcroscopy (TEM) in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated through an ethanol series, and embedded in epoxy resin. Finally, ultrathin sections were stained with uranyl acetate followed by lead citrate. The above reagents were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). In parallel, we have evaluated immunocytochemically the pattern of B23 labelling in intact and reconstructed cells. The samples were fixed in 4% paraformaldehyde, incubated with an antibody against B23 (Santa Cruz, CA, USA) and then with a biotinylated secondary antibody, and detected by fluorescein isothiocyanate (FITC)-coupled Streptavidin (Jackson ImmunoResearch, Cambridgeshire, UK). Mouse cumulus cells (12/12) contain a reticulated fibrillogranular nucleolus. The cell are also positively labeled with the anti-B23 antibody. One hour after fusion, the introduced nuclei displayed shape modifications and nuclear envelope irregularities, whereas the nucleolus still showed the typical fibrillar pattern (19/19). The volume of transferred nuclei remains unchanged. Interestingly, while the oocyte nucleolus remains negative for B23, the nucleoli in transferred somatic cells were always positively labeled (45 cells). After 24 h, the transferred nuclei increased their volume up to two-three times and displayed an irregular shape with nucleoli still possessing the unchanged reticulated pattern (17/17). As in the previous experimental interval, only the somatic cell nucleus remained labeled with the anti-B23 antibody (52 cells). The GV oocyte nucleoli remained unchanged during the whole culture period, exhibiting the typical dense-fibrillar pattern. Our results showed that the immature oocyte cytoplasm possesses a limited remodelling activity. Interestingly, the evident increase in volume of transferred somatic cells indicated some changes but TEM morphology and B23 labeling pattern remained basically unchanged. We cannot, however, exclude the beneficial effect upon reprogramming if these nuclei were subsequently used for the transfer into definitive cytoplasts. This work was supported by ESF STE/05/E004.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D Zhigalina ◽  
N Skryabin ◽  
O Kanbekova ◽  
V Artyukhova ◽  
A Svetlakov ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Preimplantation Embryos ◽  
Molecular Karyotyping ◽  
Embryonic Cells ◽  
Developmental Potential ◽  
Positive Correlation ◽  
Registration Number ◽  
Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

Changes in the properties of the developing trophoblast of preimplantation mouse embryos as revealed by aggregation studies

Development ◽  
1977 ◽  
Vol 40 (1) ◽  
pp. 143-157
Author(s):  
Paul S. Burgoyne ◽  
Thomas Ducibella
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Supporting Cell ◽  
Mouse Embryos ◽  
Mixed Cell ◽  
Time Period ◽  
Preimplantation Mouse Embryos ◽  
Wide Range ◽  
Preimplantation Mouse

Mouse embryos (8-cell to early blastocyst) were denuded with pronase, and apposed in pairs which represented a wide range of stage combinations. These pairs either formed aggregates which differentiated into double-sized blastocysts, or they failed to aggregate. The 8–16-cell stages would not envelop late morulae/early blastocysts to form layered aggregates. This must mean that as the embryo differentiates into a blastocyst, the outer surface of the trophoblast loses its capacity for supporting cell spreading. The aggregation data also demonstrate that embryos almost completely lose their potential for aggregation at a very discrete stage in development – namely, between 8 and 9 h before blastocoel formation. It is argued that this is the stage at which the zonular tight junctional seal is completed, and that it is this physical barrier which prevents aggregation. It has been argued previously that the zonular tight junctional seal allows the creation of the special microenvironment which is necessary for the determination of the inner cells as inner cell mass. The completion of this seal 8–9 h before it is required for the formation of a blastocoel would provide a suitable time period for this cell determination to occur. The results obtained also relate to the technique of chimera production. Since the aim of this technique is to generate mice with mixed cell populations, it is important that the blastocyst formed following aggregation should have both cell lines present in the inner cell mass. This can best be assured by using relatively late morula stages (75 h post-HCG injection) since these will have already segregated their inner cells, but the incomplete seal will still allow aggregation to take place.

Sign in / Sign up

Export Citation Format

Share Document