Nuclei from fertilized mouse embryos have calcium-releasing activity

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1123-1128 ◽  
Author(s):  
T. Kono ◽  
J. Carroll ◽  
K. Swann ◽  
D.G. Whittingham
Keyword(s):  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Nuclear Envelope Breakdown ◽  
Mammalian Embryos ◽  
Sperm Factor ◽  
Physical Stimuli ◽  
Mammalian Fertilization ◽  
Parthenogenetic Embryos

During mammalian fertilization, the sperm triggers a series of intracellular Ca2+ oscillations which initiate oocyte activation and the formation of pronuclei. Oocyte activation can be induced artificially by a variety of chemical and physical stimuli which elevate intracellular calcium. We show that the transfer of nuclei from 1- and 2-cell-stage fertilized mouse embryos to unfertilized oocytes stimulates the completion of meiosis and the formation of pronuclei. Nuclei from embryos that had developed to the 4-cell stage did not stimulate meiotic resumption. The ability to cause oocyte activation was specific to nuclei transferred from fertilized embryos as nuclei from parthenogenetic embryos or cytoplasts from fertilized or parthenogenetic embryos did not induce activation. Nucleus-induced oocyte activation was associated with the generation of intracellular Ca2+ transients, which were seen after nuclear envelope breakdown of the transferred nuclei. Treatment of the oocyte with the intracellular Ca2+ chelator, BAPTA, prior to nuclear transfer inhibited intracellular Ca2+ transients and oocyte activation. The specific Ca(2+)-releasing activity of the nucleus was not caused by sperm-induced protein synthesis since similar activity was present in nuclei originating from embryos exposed to cycloheximide throughout fertilization. The specific ability of nuclei from fertilized embryos to stimulate Ca2+ transients and oocyte activation was also found in nuclei from embryos parthenogenetically activated by the injection of a partially purified cytosolic sperm factor. The results suggest that the fertilizing sperm introduces Ca(2+)-releasing activity which becomes associated with the nucleus of early mammalian embryos.

57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

2008 ◽  
Vol 20 (1) ◽  
pp. 109
Author(s):  
O. Svarcova ◽  
A. Dinnyes ◽  
Z. Polgar ◽  
S. Bodo ◽  
M. Adorjan ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Ultrastructural Changes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Stage Of Development ◽  
Nucleolar Proteins ◽  
Genome Activation ◽  
Different Origin

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).

scholarly journals Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution

2019 ◽  
Vol 25 (11) ◽  
pp. 729-744 ◽  
Author(s):  
E Casser ◽  
S Wdowik ◽  
S Israel ◽  
A Witten ◽  
S Schlatt ◽  
...  
Keyword(s):  
De Novo ◽  
Monozygotic Twins ◽  
Super Resolution ◽  
Culture Conditions ◽  
Mouse Embryos ◽  
Gene Products ◽  
Cell Stage ◽  
Mammalian Embryos ◽  
Cytoplasmic Organization ◽  
Diverse Culture

Abstract It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere’s ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

scholarly journals Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. 325-333 ◽  
Author(s):  
Rodrigo C Bohrer ◽  
Limei Che ◽  
Paulo B D Gonçalves ◽  
Raj Duggavathi ◽  
Vilceu Bordignon
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chloride Ion ◽  
Dna Integrity ◽  
Strontium Chloride ◽  
Oocyte Activation ◽  
Dna Double Strand Breaks ◽  
Cell Stage

Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (ION+Sr2+). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.

Sperm penetration in parthenogenetic mouse embryos triggers a plasma membrane block to polyspermy

Zygote ◽  
1993 ◽  
Vol 1 (3) ◽  
pp. 237-242 ◽  
Author(s):  
Marek Maleszewski ◽  
Anna Bielak
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Sperm Cell ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Cell Cycles ◽  
Sperm Penetration ◽  
Membrane Components ◽  
Parthenogenetic Mouse Embryos ◽  
Parthenogenetic Embryos

SummaryMouse oocytes activated parthenogenetically do not generate a plasma membrane block against spermatozoa over the first three cell cycles. We show that they lose this fusibility spontaneously at the 8-cell stage. Insemination of 1-cell parthenogenetic embryos induces loss of fusibility earlier, at the 2-cell stage. This observation suggests that incorporation of the sperm cell membrane components into the oolemma may be responsible for the development of the membrane block.

scholarly journals 166MOUSE EMBRYONIC DEVELOPMENT FROM ONE-CELL STAGE TO BLASTOCYST, UTILIZING KSOM SUPPLEMENTED WITH AMINO ACIDS IN A MICROCHANNEL DEVICE

2004 ◽  
Vol 16 (2) ◽  
pp. 205
Author(s):  
P.H.C. Lopes ◽  
S.A. Malusky ◽  
A.S. Lima ◽  
D.J. Beebe ◽  
M.B. Wheeler
Keyword(s):  
Amino Acids ◽  
Embryonic Development ◽  
Mouse Strain ◽  
Mouse Embryos ◽  
Type I ◽  
Alternative Technique ◽  
Cell Stage ◽  
Mammalian Embryos ◽  
Microchannel Device

Great efforts have been made to improve in vitro culture for enhancement of embryonic development. However, in vitro development of mammalian embryos still remains a challenge for the scientific community. Recently, the use of microfluidic culture devices, as an alternative technique compared to the standard drop, has allowed mammalian embryos to develop to the hatched blastocyst stage. With the use of a different medium, mouse strain, and microchannel device than previously reported (Raty S et al., 2001 Theriogenology 55, 241 abst), this study was undertaken to determine if a microchannel device fabricated from borosilicate and poly-dimethylsiloxane would support development of mouse embryos from one-cell to blastocyst, as an alternative to standard microdrop culture. Mice (F1 inbred C57BL/6CRL X SJL) from 3 to 8 weeks old were superovulated with 5IU of PMSG and 5IU of hCG. The female SJL strain of the mice has demonstrated low reproductive performance. One-cell embryos were collected in M2 medium (Sigma, St. Louis, MO, USA.). For each treatment, 240 embryos in 24 replicates were cultured. Groups of 10 embryos were cultured in the microchannel device using 500μL of KSOM with amino acids (MR-106-D, Speciality Media, Phillipsburg, NJ, USA.); no additional supplements were added. Groups of 10 embryos were cultured in standard microdrops (control) using 30μL of the same medium covered with mineral oil. Embryos were cultured in a 100% humidified, 5% CO2 in air atmosphere at 37°C for 96h. Embryos were allocated to the control treatment or the microchannel device treatment using a randomized block design. The percentage of embryos at each stage of development was evaluated at 24-h intervals. The stage of embryo development at each observation was analyzed by ANOVA using the general linear model in SAS (PROC GLM, type I sum of squares). Blastocyst development in the microchannel device was not different when compared to results obtained in the standard drop. The percentage of blastocysts developing, when analyzed from one-cell stage, was 29±5% for the control and 26±6% for the microchannel. The percentage of blastocysts, when analyzed from cleavage, was 35±5% for the standard drop and 31±7% for the microchannel device. The results obtained are encouraging, when considering the non-optimized medium and mouse strain utilized in this experiment. In conclusion, the results show the microchannel device may be considered an alternative technique for use in embryo culture as it supports development of mouse embryos from one-cell stage to blastocyst.

58 EFFECTS OF CAFFEINE AND VANADATE ON NUCLEAR REMODELING AND MICROTUBULE DISTRIBUTION OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
D. J. Kwon ◽  
C. K. Park ◽  
B. K. Yang ◽  
C. I. Kim ◽  
H. T. Cheong
Keyword(s):  
Nuclear Transfer ◽  
Laser Scanning ◽  
Morphological Changes ◽  
Laser Scanning Confocal Microscopy ◽  
Blastocyst Stage ◽  
Scanning Confocal Microscopy ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Remodeling ◽  
Parthenogenetic Embryos

The present study was conducted to control nuclear remodeling types by treatment with caffeine or vanadate, and to examine the microtubule distribution of nuclear transfer embryos (NTs) after nuclear remodeling control and the mitotic spindle assembly and its morphological changes during the first mitosis of NTs in the pig. Enucleated oocytes were treated with 5 mM caffeine or 0.5 mM sodium orthovanadate (vanadate) for 2.5 or 0.5 h to increase or decrease MPF activity before injection of fetal fibroblast cells. Reconstituted eggs were fused by an electric stimulation (ES, 1.5 kV cm-1), activated by a combination of 2 pulses of ES (1.0 kV cm-1), and cultured for 3 h with 2 mM 6-dimethylaminopurine (6-DMAP) at 1 h after fusion treatment. Some matured oocytes were also treated by the same chemicals before parthenogenetic activation under the same conditions as NTs, and cultured in vitro to evaluate the effects of these chemicals on embryo development. NTs and parthenogenetic embryos were cultured in PZM-3 for 20 h or 6 days at 39�C, 5% CO2 in air, respectively. Nuclear remodeling types of NTs were examined at 1 h after fusion (before activation) by the whole-mount method. At least 3 replicates for each experiment were performed. Microtubules and DNA of NTs that were fixed at 1 h or 20 h after fusion were detected by indirect immunocytochemical technique. Images were captured using laser scanning confocal microscopy. Caffeine and vanadate did not affect the development to the blastocyst stage of porcine parthenogenetic embryos. When a nucleus was exposed to oocyte cytoplasm treated with caffeine, premature chromosome condensation (PCC) occurred at a higher rate (82/98, 83.7%) compared to control (42/73, 57.5%) and vanadate-treated (11/91, 12.1%) groups (P < 0.05). The proportion of embryos that did not undergo nuclear envelope breakdown (NEBD) was higher in the vanadate treatment group (87.9%) compared to the caffeine and control groups (16.3 and 42.5%, respectively; P < 0.05). The frequency of embryos showing a γ-tubulin only and both γ- and β-tubulins were 3.9–9.4% and 21.9–34.6%, respectively, in NTs (total 87 embryos) at 1 h after fusion regardless of caffeine and vanadate treatments. In the majority of NTs (61.5–68.6%), microtubules were not observed. At 20 h after fusion, the frequency of the embryos undergoing normal mitosis was similar in the control (17/45, 37.8%) and caffeine (19/43, 44.2%) groups, but it was significantly lower in the vanadate group (7/37, 18.9%; P < 0.05). The present study demonstrates that the nuclear remodeling type of NTs can be controlled by treatment with MPF regulators, caffeine and vanadate, and such treatment is not related to the microtubule distribution in porcine NTs. The finding, however, that the vanadate can delay the mitotic progression of porcine NTs at the first cell cycle may be due to the lack of NEBD and PCC. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD;KRF-2005-042-F00030).

Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction

Zygote ◽  
2010 ◽  
Vol 19 (3) ◽  
pp. 271-276 ◽  
Author(s):  
Chao-Bo Li ◽  
Zhen-Dong Wang ◽  
Zhong Zheng ◽  
Li-Li Hu ◽  
Shu-Qi Zhong ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Time Course ◽  
Mouse Embryos ◽  
Apical Region ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Significant Difference ◽  
Before And After

SummaryThe events resulting in compaction have an important influence on the processes related to blastocyst formation. To analyse the quality of the embryos obtained by somatic cell nuclear transfer (SCNT) in aspects different from previous studies, not only the number of blastomeres of cloned embryos during the initiation of compaction, but also the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos before and after compaction was preliminarily investigated in mouse. Our results showed that during compaction the number of blastomeres in SCNT embryos was fewer than that in intracytoplasmic sperm injection (ICSI) embryos and, before compaction, there was a uniform distribution of microvilli over the blastomere surface, but microvilli became restricted to an apical region after compaction in the four types of embryos. We also reported here that the time course of compaction in SCNT embryos was about 3 h delayed compared with that in ICSI embryos, while there was no significant difference between SCNT and ICSI embryos when developed to the 4-cell stage. We concluded that: (i) the cleavage of blastomeres in cloned embryos was slow at least before compaction; (ii) the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos was coherent before and after compaction; and (iii) the initiation of compaction in SCNT embryos was delayed compared with that of ICSI embryos.

Identification of a 2-cell stage specific inhibitor of the cleavage of preimplantation mouse embryos synthesized by rat hepatoma cells as 5′-deoxy-5′-methylthioadenosine

Zygote ◽  
2010 ◽  
Vol 19 (2) ◽  
pp. 117-125 ◽  
Author(s):  
Masayuki Kobayashi ◽  
Koichi Saito ◽  
Shigeru Tamogami ◽  
Junko Takashima ◽  
Kano Kasuga ◽  
...  
Keyword(s):  
Biological Activities ◽  
Specific Inhibitor ◽  
Biological Properties ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Mammalian Embryos ◽  
Rat Hepatoma

SummaryRat hepatoma Reuber H-35 cells produce a unique compound designated as Fr.B-25, a 2-cell stage-specific inhibitor of the cleavage of preimplantation mouse embryos cultured in vitro. Here, we identified Fr.B-25 as a purine nucleoside, 5′-deoxy-5′-methylthioadenosine (MTA), by mass spectroscopic analysis. All of the biological activities examined of authentic MTA on the development of mouse zygotes were indistinguishable from those of Fr.B-25. The mechanism of MTA action in the development of preimplantation mouse embryos was probably different from those of hypoxanthine and adenosine, which are well-characterized purine nucleosides that act as inhibitors of the cleavage of mouse 2-cell embryos. From the shared molecular and biological properties of Fr.B-25 and MTA, we concluded that Fr.B-25 is MTA. To the best of our knowledge, this is the first delineation of the effect of MTA on the development of preimplantation mammalian embryos cultured in vitro.

The onset of phosphatase activity in early mammalian embryos

Development ◽  
1977 ◽  
Vol 42 (1) ◽  
pp. 305-308
Author(s):  
V. Ishiyama ◽  
L. Izquierdo
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Azo Dye ◽  
Mouse Embryos ◽  
Dye Coupling ◽  
Cell Stage ◽  
Acid And Alkaline Phosphatase ◽  
Mammalian Embryos

The onset of acid and alkaline phosphatase activity is determined by means of Burstone's azo dye coupling methods in oocytes and embryos of the mouse, rat and hamster, and in mouse embryos cultured in vitro. Acid phosphatase activity is detected in all cases while alkaline phosphatase activity begins during the 4-cell stage and is always present thereafter. The method is sensitive regarding detection of activity but does not permit the quantification nor a precise localization of the enzymes.

Complete preimplantation development in culture of parthenogenetic mouse embryos

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 179-190
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Haploid Nucleus ◽  
Second Polar Body ◽  
Haploid Embryos ◽  
Parthenogenetic Mouse Embryos ◽  
Parthenogenetic Embryos ◽  
Better Than

The present experiments were undertaken to determine whether, in parthenogenesis, heterozygous embryos develop better than homozygous embryos. Such experiments may provide an approach to elucidating whether fertilized embryos develop better than parthenogenetic ones because of heterozygosity, or if the sperm provides another contribution necessary for complete embryonic development. The parthenogenetic embryos studied included uniform haploids after extrusion of the second polar body, mosaic haploids in which each blastomere contained a genetically different haploid nucleus, and heterozygous diploid mouse embryos. Eggs were activated and cultured in a chemically denned medium. About three times as many mosaic haploid or heterozygous diploid eggs developed beyond the 4-cell stage after 98–100 h and to the blastocyst stage after 120 h in culture, than uniform haploid eggs. This indicates that the development of parthenogenetic embryos is probably under genetic control and that there was a better development of the heterozygous embryos. Mosaic haploid embryos showed the same high frequency of development as heterozygous diploids. The results therefore indicate that heterozygosity provided a developmental advantage even when distributed between two genetically different clones of cells in the same embryo.

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