Diploid Oocyte Formation and Tetraploid Embryo Development Induced by Cytochalasin B in Bovine

2011 ◽  
Vol 13 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Chunling Bai ◽  
Hui Liu ◽  
Ying Liu ◽  
Xia Wu ◽  
Lei Cheng ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cytochalasin B ◽  
Tetraploid Embryo

Effect of glutathione on tetraploid embryo development in the pigs

2016 ◽  
Vol 31 (3) ◽  
pp. 207-213
Author(s):  
Hwa-Young Kim ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Seung Tae Lee ◽  
Eunsong Lee ◽  
...  
Keyword(s):  
Embryo Development ◽  
Tetraploid Embryo

scholarly journals Postimplantation development of CB-induced triploid mouse embryos

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 81-89
Author(s):  
Anna Niemierko
Keyword(s):  
Embryo Development ◽  
Cytochalasin B ◽  
Polar Body ◽  
Mouse Embryos ◽  
Foetal Membranes ◽  
Polar Body Extrusion

By subjecting A strain eggs at the time of fertilization and polar body extrusion to 5 µg/ml cytochalasin B, digynic triploidy was produced in 80% fertilized eggs. Triplonucleate eggs were transplanted to recipients and examined between 9–1lth day of pregnancy. Development of triploid mouse embryos up to day 7 is normal and most embryos form early egg cylinder. At day 8 the embryonic part of the cylinders is under-developed and later development fails to form an embryo. Development of foetal membranes is much less affected, CB-induced triploids survive to 10th- day of pregnancy.

176 CYTOCHALASIN PREVENTS SPERM HEAD INCORPORATION INTO FERTILIZED PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 200
Author(s):  
Z. Machaty ◽  
C. Wang ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Control Group ◽  
Wild Type ◽  
Sry Gene ◽  
Cell Stage ◽  
Developmental Potential

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.

Development of cytochalasin B-induced tetraploid and diploid/tetraploid mosaic mouse embryos

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 47-64 ◽  
Author(s):  
A. K. Tarkowski ◽  
A. Witkowska ◽  
J. Opas
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cytochalasin B ◽  
Primitive Streak ◽  
Cell Number ◽  
Mouse Embryos ◽  
Proliferation Rate ◽  
Foetal Membranes ◽  
Diploid Cells

By subjecting F1 (CBA × C57/BL) × A eggs at the time of 2nd cleavage to 10 μg/ml of cytochalasin B (CB), tetraploidy was produced in 52 % of 2-cell eggs and 35 % of 3-cell eggs. 2n/4n mosaic embryos were produced from 2-, 3- and 4-cell eggs and amounted to 20 % of all treated eggs. 80 % of tetraploid embryos developed in vitro into regular blastocysts with half the cell number of control diploids. The effectiveness of CB in producing tetraploid embryos is limited by the asynchrony of 2nd cleavage, both between eggs and between sister blastomeres. Two-cell presumed tetraploids were transplanted to recipients and examined between the 6th and 11th day of pregnancy. Up to 6½ days development is normal and most embryos form egg-cylinders. At 7½ days the embryonic part of the cylinders is underdeveloped and in later development fails to form an embryo. Development of foetal membranes is much less affected and in the most successfully developing egg-cylinders their formation can be fully accomplished. Failure of embryonic development appears to be due to subnormal activity of the primitive streak, resulting in shortage of mesoderm. Postimplantation development of 2n/4n mosaics was normal. While in embryos tetraploid cells were either absent or in very low proportion (below 4 %), their contribution to the foetal membranes amounted in some cases to up to 50 %. Elimination of tetraploid cells from mosaic embryos suggests that they have a lower proliferation rate than diploid cells.

133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
A. J. Pearks Wilkerson ◽  
R. D. Landry ◽  
C. R. Long
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Culture Media ◽  
Domestic Cat ◽  
Bovine Embryo ◽  
Development Rates ◽  
L1 And L2

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P < 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P > 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.

Fine Structure of Cytochalasin Induced Polyploid Cells

1968 ◽  
Vol 26 ◽  
pp. 122-123
Author(s):  
Awtar Krishan ◽  
Nestor Bohonos
Keyword(s):  
Fine Structure ◽  
Cytochalasin B ◽  
Present Report ◽  
Cell Monolayer ◽  
Polyploid Cell ◽  
Specific Cell ◽  
Nuclear Surface ◽  
Unsuccessful Attempt ◽  
Daughter Cells ◽  
Polyploid Cells

Cytochalasin B, a mould metabolite from Helminthosporium dermatioideum has been shown to interfere with specific cell activities such as cytoplasmic cleavage and cell movement. Cells undergoing nuclear division in the presence of cytochalasin B are unable to complete the separation of the resulting daughter cells. In time-lapse studies, the daughter cells coalesce after an initial unsuccessful attempt at separation and form large multinucleate polyploid cells. The present report describes the fine structure of the large polyploid cells induced in Earle's L-cell monolayer cultures by exposure to cytochalasin B (lγ/ml) for 92 hours.In the present material we have seen as many as 7 nuclei in these polyploid cells. Treatment with cytochalasin B for longer periods of time (6 to 7 days, with one medium change on the 3rd day) did not increase the number of nuclei beyond the 7 nuclei stage. Figure 1 shows a large polyploid cell with four nuclei. These nuclei are indistinguishable in their fine structure from those of the cells from control cultures but often show unusually large numbers of cytoplasmic invaginations and extensions of the nuclear surface (Figure 2).

Rho-GTPase effector ROCK regulates mouse oocyte maturation and embryo development

2014 ◽  
Author(s):  
Xing Duan ◽  
Zhen-Bo Wang ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim ◽  
Shao-Chen Sun
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Rho Gtpase ◽  
Mouse Oocyte
Sign in / Sign up

Export Citation Format

Share Document