47 ACTIVATION WITH IONOMYCIN FOLLOWED BY DEHYDROLEUCODINE AND CYTOCHALASIN B OF CLONED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 181
Author(s):  
N. Canel ◽  
R. Bevacqua ◽  
D. Salamone
Keyword(s):  
Cytochalasin B ◽  
Combined Treatment ◽  
Cumulus Cells ◽  
Normal Embryo ◽  
Intracytoplasmic Injection ◽  
Glass Slides ◽  
Cloned Bovine ◽  
Pronuclear Formation ◽  
Vitro Development

A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy. Table 1.In vitro development of bovine cloned embryos activated with DhL and CB

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 262
Author(s):  
W. Fujii ◽  
H. Funahashi
Keyword(s):  
Chemical Activation ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Female Rats ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Pronuclear Formation ◽  
Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

scholarly journals Effect of granulosa and cumulus cells on in vitro development of the bovine follicular oocytes

1995 ◽  
Vol 8 (4) ◽  
pp. 317-320
Author(s):  
K. S. Im ◽  
H. J. Kim ◽  
K. M. Chung ◽  
H. S. Kim ◽  
K. W. Park ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
In Vitro Development ◽  
Vitro Development

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
F. Forell ◽  
J. C. Mezzalira ◽  
F. Zago ◽  
F. K. Vieira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Rate ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning ◽  
Activation Intervals

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Lalantha R. Abeydeera ◽  
Wei-Hua Wang ◽  
Thomas C. Cantley ◽  
Randall S. Prather ◽  
Billy N. Day
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Thiol Compound ◽  
Nuclear Maturation ◽  
Mean Differences ◽  
Pronuclear Formation

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, β-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 μM BME, 0.5 μg/ml LH, 0.5 μg/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20–22 h and then without hormonal supplements for an additional 20–22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5–6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78–89%). The mean differences in penetration rate (69–77%), polyspermy rate (31–40%), male pronuclear formation rate (93–96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32–39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13–15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.

scholarly journals Improved in vitro development of cloned bovine embryos using S -adenosylhomocysteine, a non-toxic epigenetic modifying reagent

2011 ◽  
Vol 78 (8) ◽  
pp. 576-584 ◽  
Author(s):  
Shahram Jafari ◽  
Morteza S. Hosseini ◽  
Mahdi Hajian ◽  
Mohsen Forouzanfar ◽  
Farnoosh Jafarpour ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development

scholarly journals In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

2010 ◽  
Vol 9 (1) ◽  
pp. 295-302 ◽  
Author(s):  
R.P.C. Gerger ◽  
E.S. Ribeiro ◽  
F. Forell ◽  
L.R. Bertolini ◽  
J.L. Rodrigues ◽  
...  
Keyword(s):  
Cell Culture ◽  
Somatic Cells ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning

4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN BOVINE ZYGOTES

2015 ◽  
Vol 27 (1) ◽  
pp. 94
Author(s):  
Y. S. Bogliotti ◽  
M. Vilariño ◽  
J. L. Chitwood ◽  
J. Wu ◽  
A. Mutto ◽  
...  
Keyword(s):  
Gene Disruption ◽  
Laser System ◽  
Cumulus Cells ◽  
Easy Method ◽  
Intracytoplasmic Injection ◽  
Developmental Rates ◽  
Glass Needle ◽  
Biallelic Mutations ◽  
Knockout Animals

The CRISPR/Cas9 system is a fast, effective, and easy method for gene disruption, allowing generation of knockout animals by direct zygote injection. To date there is no report on the efficiency of this microinjection system in bovine zygotes and its effects on early development. The aim of this study was to compare 2 microinjection methods on developmental rates and efficiency to induce gene disruption. Microinjection effects on embryo development were evaluated by blastocyst (BL) formation rates at Day 8 of culture and by the proportion of lysed embryos (damaged during injection); while the efficiency of CRISPR/Cas9 RNA to create targeted mutations was studied by sequencing resulting blastocysts. Three groups were evaluated: (1) noninjected (control), (2) direct intracytoplasmic injection (direct-ICI), and (3) laser-assisted ICI (laser-ICI). Direct-ICI was performed with a beveled spiked glass needle (5 μm ID; Origio, Måløv, Denmark) to pass the zona pellucida (ZP) and deliver CRISPR/Cas9 RNA as earlier described (Ross et al. 2008 BMC Dev. Biol. 8, 16). For laser-ICI, a Research Instruments (RI) Saturn 5 Active™ laser system (Research Instruments Ltd., Falmouth, United Kingdom) was used to perforate the ZP, and a blunt-end glass needle (5–6 μm ID) used to deliver CRISPR/Cas9 RNA. In both cases, cytoplasm was aspirated into the pipette to disrupt the plasma membrane and the aspirated cytoplasm and CRISPR/Cas9 injected back into the embryo. Embryos were obtained by IVF of in vitro-matured oocytes aspirated from abattoir ovaries. At 18 h post-IVF (hpf), zygotes were denuded from cumulus cells and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 of BSA. Zygotes were injected at 20 to 22 hpf. Four biological replicates were assessed for BL rates (258 embryos total). A 2-tailed t-test was used to evaluate statistically significant differences (P ≤ 0.05) between groups. Direct-ICI had a greater proportion of lysed embryos (29.5 ± 10.6%) compared with laser-ICI (15.6 ± 5%; P = 0.056). BL development was significantly lower on direct-ICI (15.5 ± 8%) compared with laser-ICI (31.4 ± 5.9%; P = 0.02) and control groups (32.8 ± 6.6%; P = 0.01). These results indicate that laser-ICI causes less damage and results in normal BL rates after microinjection (P = 0.24). Because laser-ICI had normal BL rates and less embryo lysis, we used this method to evaluate efficiency of the CRISPR/Cas9 system to induce genomic mutations. Twelve BL were sequenced. Analysis of the CRISPR targeted region showed that 50% of the embryos had biallelic mutations, 33% monoallelic mutations, and 17% were wild type. These results show that laser-ICI method was very efficient for injecting CRISPR/Cas9 RNA into zygotes, resulting in normal developmental rates and a high amount of mutations in BL. The authors thank Research Instruments Ltd. for providing the laser used in this study.

scholarly journals Effect of Cumulus Cells and Exposure Period to Ethanol on In Vitro Development of Mouse Diploid Parthenogenones

1994 ◽  
Vol 56 (2) ◽  
pp. 379-380
Author(s):  
Anuchai PINYOPUMMIN ◽  
Yoshiyuki TAKAHASHI ◽  
Hee Tae CHEONG ◽  
Mitsugu HISHINUMA ◽  
Hiroshi KANAGAWA
Keyword(s):  
Cumulus Cells ◽  
Exposure Period ◽  
In Vitro Development ◽  
Vitro Development

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

Sign in / Sign up

Export Citation Format

Share Document