54 CHEMICAL ENUCLEATION OF IN VITRO-MATURED BUFFALO (BUBALUS BUBALIS) OOCYTES INDUCED BY DEMECOLCINE FOR HANDMADE SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
R. A. Shah ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
S. K. Singla
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Uv Light ◽  
Genetic Material ◽  
Bubalus Bubalis ◽  
Significant Loss ◽  
Control Group ◽  
Cone Formation ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer requires enucleation of the recipient oocyte to remove its genetic material. In handmade cloning, a simplified cloning procedure (Vajta et al. 2001 Cloning 3, 89–95), zona-free oocytes are manually bisected, stained, and exposed to UV light for selection of demi-oocytes devoid of chromosomes. Development of procedures for enucleation which avoid exposure to UV light and conserve most of the cytoplasmic volume are necessary for improving the efficiency of handmade cloning. Chemically assisted enucleation protocols involve treating cumulus–oocyte complexes (COCs) during IVM with cytoskeleton-modifying agents, like demecolcine, which induce protrusion cone formation on the surface of the oocytes (Tani et al. 2006 Cloning Stem Cells 8, 61–66). Such a cone-like structure can then be easily excised with a microblade and enucleation of the oocyte achieved without a significant loss of cytoplasm. The aim of the present study was to establish an efficient protocol for demecolcine treatment of buffalo COCs during IVM to obtain the maximal proportion of oocytes where chromosomes are either expelled into the surface protrusion cone or completely enucleated. In Experiment I, COCs (n = 244), obtained from slaughterhouse buffalo ovaries and matured in vitro in TCM-199 (containing 10% FBS, 5 µg mL–1 pFSH, and 0.81 mm sodium pyruvate) at 38.5�C (in 5% CO2, 90–95% relative humidity), were treated in two groups with demecolcine (0.5 µg mL–1) beginning at either 15 h or 19 h from the start of IVM up to the end of IVM at 22 h, and compared to a control group (without demecolcine). Data were analyzed using ANOVA. The proportion of oocytes where a protrusion cone was observed was greatest (P < 0.05) in the 15-h treatment group (84%, n = 72/86) compared to those in either the 19-h or control groups (58%, n = 46/78, and 60%, n = 48/80, respectively). The presence of demecolcine for the last 7 h of IVM appears to have a significant effect on protrusion cone formation in buffalo oocytes. In Experiment II, COCs (n = 276)were divided into four groups, matured in vitro this time for 24 h and treated with demecolcine (0.5 µg mL–1) from the onset of IVM and up to 18, 21, or 24 h, and compared to a control group without demecolcine. Protrusion cone formation was observed in 72% (52/72), 66% (40/60), 62% (40/64), and 70% (56/80) of oocytes, respectively, in these groups. These percentages did not differ significantly (P < 0.05). However, the 21-h treatment resulted in complete enucleation in 32% (20/64) of oocytes, which was significantly greater (P < 0.05) than that in the other three groups where no such enucleation was observed. It can be hypothesized that demecolcine-free treatment from 21 to 24 h may have assisted in inducing complete enucleation of a significant number of treated oocytes. In conclusion, these results show that demecolcine-assisted and induced enucleation procedures can be used for increasing the efficiency of oocyte enucleation in handmade cloning and other nuclear transfer procedures in buffalo.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

54 IN VITRO DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY HANDMADE CLONING FROM DISTINCT CELL CULTURE CONFLUENCES AND AGGREGATION SCHEMES

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
E. S. Ribeiro ◽  
J. C. Mezzalira ◽  
L. U. Olwheiler ◽  
F. Forell ◽  
...  
Keyword(s):  
Cell Culture ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Plateau Phase ◽  
In Vitro Development ◽  
Distinct Cell ◽  
Aggregation Scheme ◽  
Vitro Development ◽  
Handmade Cloning

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitro development of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) >90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1 than cycling cells at the log phase (>91% v. 59%; Sun et al. 2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1 proportion in that cell population. Table 1.In vitro development of bovine cloned embryos from distinct cell culture confluences and aggregation scheme This study was supported by FAPESP and CAPES/Brazil.

RNA sequencing and transcriptome analysis of buffalo ( Bubalus bubalis ) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization

2019 ◽  
Vol 86 (9) ◽  
pp. 1149-1167 ◽  
Author(s):  
Tanushri Jerath Sood ◽  
Swati Viviyan Lagah ◽  
Manishi Mukesh ◽  
Suresh Kumar Singla ◽  
Manmohan Singh Chauhan ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Rna Sequencing ◽  
Transcriptome Analysis ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bubalus Bubalis ◽  
Vitro Fertilization

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

Role of linker histone H1c during the reprogramming of Chinese swamp buffalo (Bubalus Bubalis) embryos produced by somatic cell nuclear transfer

2016 ◽  
Vol 28 (3) ◽  
pp. 302
Author(s):  
Gao-Bo Huang ◽  
Li Quan ◽  
Yong-Lian Zeng ◽  
Jian Yang ◽  
Ke-Huan Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Bubalus Bubalis ◽  
Linker Histone ◽  
Control Group ◽  
Swamp Buffalo

During reprogramming, there is exchange of histone H1c and the oocyte-specific linker histone, and H1c may play a critically important role in the reprogramming process of somatic cell nuclear transfer (SCNT). The aim of the present study was to investigate the role of the H1c gene in SCNT reprogramming in Chinese swamp buffalo (Bubalus bubalis) using RNA interference (RNAi). Chinese swamp buffalo H1c gene sequences were obtained and H1c-RNAi vectors were designed, synthesised and then transfected into a buffalo fetal skin fibroblast cell line. Expression of H1c was determined by real-time polymerase chain reaction to examine the efficiency of vector interference. These cells were then used as a nuclear donor for SCNT so as to observe the further development of SCNT embryos. Inhibition of H1c gene expression in donor cells significantly improved the developmental speed of embryos from the 1-cell to 8-cell stage. Furthermore, compared with the control group, inhibition of H1c gene expression significantly reduced the blastocyst formation rate. It is concluded that linker histone H1c is very important in SCNT reprogramming in Chinese swamp buffalo. Correct expression of the H1c gene plays a significant role in preimplantation embryonic development in B. bubalis.

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

2016 ◽  
Vol 5 (12) ◽  
pp. 5124
Author(s):  
Rajesh Wakchaure ◽  
Subha Ganguly*
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Genetic Material ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Oocyte Activation

Cloning a horse means using the genetic material (DNA) from a donor horse to produce a genetically identical foal. This technique involves collecting the DNA from the donor and inserting that DNA into an egg from another mare whose, DNA content has been removed, fusing donor nucleus with enucleated recipient oocytes, which then develops as an embryo, in vitro culture of embryo and lastly transfer cultured embryo into the uterus of a recipient mare. The modification of the in vitro culture conditions which can be suitable for equine oocyte activation, oocyte maturation and embryo development are the fundamental steps for a successful in vitro procedure for somatic cell nuclear transfer (SCNT) in the horse to avoid the embryo losses. In general, few studies are available in the literature on equine in vitro embryo production and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation The present review discusses the latest developments in the field of equine cloning technique with the employment of SCNT. The basic understanding of SCNT for in vitro culture conditions is relevant to the increased efficiency of cloning. The available genotype can be used by SCNT which can enhance the vigour of a particular infertile or low fertile animal to produce normal fertility.

scholarly journals Meiotic arrest as an alternative to increase the production of bovine embryos by somatic cell nuclear transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 32-40 ◽  
Author(s):  
F.M.C. Caixeta ◽  
R.V. Sousa ◽  
A.L. Guimarães ◽  
L.O. Leme ◽  
J.F.W. Sprícigo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Meiotic Arrest ◽  
Embryo Production

SummaryThis study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal–Wallis test with a P-value <0.05 considered to be significant. Approximately 87.8% of the oocytes remained at germinal vesicle stage (GV) after 6 h of PM with 5 μM of cilostamide, confirming the meiotic block. Embryo development in IVP was similar (P > 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

Sign in / Sign up

Export Citation Format

Share Document