Nuclear cytoplasmic interactions following nuclear transplantation in mouse embryos

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 915-923 ◽  
Author(s):  
S.K. Howlett ◽  
S.C. Barton ◽  
M.A. Surani
Keyword(s):  
Cell Nucleus ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Cell Donor ◽  
Donor Nucleus ◽  
Cell Embryo

We have investigated the development of reconstituted embryos in which enucleated 1- or 2-cell embryos received various advanced nuclei. Enucleated 1-cells developed to the blastocyst stage only when an early 2-cell donor nucleus was transferred but very rarely if the donor nucleus was derived from a late 2-cell, early 4-cell or mid 8-cell embryo. Although an 8-cell nucleus could only support development of an enucleated zygote to the 2-cell stage, it did express the hsp 68/70 X 10(3) Mr proteins that are characteristic of the first embryonic gene activity. These polypeptides were absent in enucleated zygotes that did not receive a donor nucleus. Moreover, an 8-cell nucleus transferred to an enucleated late 2-cell blastomere could also support preimplantation development provided that the nuclear:cytoplasmic ratio was maintained as in intact 2-cell blastomeres. 8-cell nuclei transferred to zygotes that retained at least one pronucleus were able to support development to the blastocyst stage provided that the pronucleus was both fully transcriptionally active and present beyond the late 1-cell stage. This study suggests an active and continued helper role of the resident pronucleus for the participation by an 8-cell nucleus in reconstituted eggs.

scholarly journals Role of glucose in cloned mouse embryo development

2008 ◽  
Vol 295 (4) ◽  
pp. E798-E809 ◽  
Author(s):  
Zhiming Han ◽  
Rita Vassena ◽  
Maggie M. Y. Chi ◽  
Santhi Potireddy ◽  
Miriam Sutovsky ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Beneficial Effect ◽  
Embryo Development ◽  
Glucose Transporter ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Positive Effects

Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one- and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria.

Nuclear transplantation using bovine primordial germ cells from male fetuses

1995 ◽  
Vol 7 (5) ◽  
pp. 1217 ◽  
Author(s):  
F Delhaise ◽  
FJ Ectors ◽  
Roover R de ◽  
F Ectors ◽  
F Dessy
Keyword(s):  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

scholarly journals Genetic and Molecular Studies on Om, a Locus Controlling Mouse Preimplantation Development

1996 ◽  
Vol 45 (1-2) ◽  
pp. 3-14 ◽  
Author(s):  
M. Cohen-Tannoudj ◽  
P. Balducci ◽  
C. Kress ◽  
V. Richoux-Duranthon ◽  
J.P. Renard ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Female Parent ◽  
Blastocyst Stage ◽  
Nuclear Transplantation ◽  
Genetic Constitution ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Female Pronucleus ◽  
Haploid Embryos ◽  
Transplantation Experiments

Several lines of evidence have accumulated in recent years indicating that nuclear cytoplasmic interactions play an important role in the formation and fate of the developing mouse embryo. Early nuclear transplantation experiments indicated that the ability of nuclei to direct cleavage after transfer into enucleated zygotes falls abruptly with nuclei from more advanced preimplantation stages [1]. Transcriptional activation of the nuclei, which occurs during the second cell cycle probably precludes the reprogramming of nuclei from later cleavage stages [2]. Thus, when an 8-cell nucleus is transferred to an enucleated zygote, such a reconstituted zygote is blocked at the 2-cell stage. However, when identical 8-cell nuclei were transferred into both blastomeres of enucleated 2-cell embryos, they were able to support development to the blastocyst stage and even gave rise to live offspring [2-4]. This indicated the importance of the cytoplasmic environment for the ability of the incoming nucleus to support development. It should be noted that in these experiments, the nuclear cytoplasmic ratio was also an important factor in determining the development of the reconstituted embryos [2]. Similar observations were also made when monitoring the development of haploid embryos [5]. In another study, Latham and Solter [6] examined the ability of androgenones, obtained by replacing the female pronucleus of a zygote by the male pronucleus, to develop to the blastocyst stage. Androgenones generated from C57B1/6 eggs were found to be much more competent to give rise to blastocysts than were DBA/2 androgenones. However, when androgenones were constructed from (DBA/2×C57B1/6)F1, zygotes (genetic constitution of the embryos will hereafter be indicated with the female parent coming first followed by the male parent), by replacing the DBA/2 female pronucleus with a C57B1/6 pronucleus, they also developed poorly. This was not simply due to the lack of some component in DBA/2 cytoplasm, since the impaired development was also observed when C57B1/6 male pronuclei from pairs of (DBA/2×C57B1/6) F1, were transferred to an enucleated C57B1/6 egg.

scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
W. J. D. Reeve ◽  
C. A. Ziomek
Keyword(s):  
Ligand Binding ◽  
Single Cells ◽  
Progressive Increase ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Characteristic Distribution ◽  
Surface Polarization ◽  
Cell Embryo ◽  
Fluorescent Ligand ◽  
Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 169-182
Author(s):  
Kerry B. Clegg ◽  
Lajos Pikó
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Average Length ◽  
State Level ◽  
Mouse Embryos ◽  
High Rate ◽  
Rna Sequences ◽  
Cell Stage ◽  
New Synthesis ◽  
Cell Embryo

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)− RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3′-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0·2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1·5 pg/embryo in the 1-cell embryo and about 3·0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)− RNA, assumed to be mRNA, at a rate of about 0·3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0·4 pg/embryo·h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0·045 pg/embryo/h, but the rate increases to about 0·2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2·5 × 106 tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0·8 × 106tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

Nuclear transplantation of male primordial germ cells in the mouse

Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Y. Tsunoda ◽  
T. Tokunaga ◽  
H. Imai ◽  
T. Uchida
Keyword(s):  
Germ Cells ◽  
Inner Cell Mass ◽  
Primordial Germ Cells ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Nuclear Transplantation ◽  
Embryonic Genome ◽  
Donor Nucleus ◽  
Implantation Sites ◽  
Genome Activation

We examined the developmental ability of enucleated eggs receiving embryonic nuclei and male primordial germ cells (PGCs) in the mouse. Reconstituted eggs developed into the blastocyst stage only when an earlier 2-cell nucleus was transplanted (36%) but very rarely if the donor nucleus was derived from a later 2-cell, 8-cell, or inner cell mass of a blastocyst (0–3%). 54–100%, 11–67%, 6–43% and 6–20% of enucleated eggs receiving male PGCs developed to 2-cell, 4-cell, 8-cell and blastocyst stage, respectively, in culture. The overall success rate when taking into account the total number of attempts at introducing germ cells was actually 0–6%. Live fetuses were not obtained after transfer of reconstituted eggs to recipients, although implantation sites were observed. The developmental ability of reconstituted eggs in relation to embryonic genome activation and genomic imprinting is discussed.

scholarly journals The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

1994 ◽  
Vol 14 (10) ◽  
pp. 6655-6662 ◽  
Author(s):  
N Yamauchi ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Monoclonal Antibody ◽  
Signaling Pathway ◽  
Antisense Oligonucleotides ◽  
Subsequent Development ◽  
Inhibitory Effect ◽  
Mouse Embryos ◽  
Dominant Negative ◽  
Cell Stage ◽  
Cell Embryo ◽  
And Function

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

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