scholarly journals 64 OOPLASMIC TRANSFER AFTER INTERSPECIES NUCLEAR TRANSFER: PRESENCE OF FOREIGN MITOCHONDRIA, PATTERN OF MIGRATION, AND EFFECT ON EMBRYO DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Sansinena ◽  
J. Lynn ◽  
R. Denniston ◽  
R. Godke
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Molecular Probes ◽  
Sequence Of Events ◽  
Chi Square Analysis ◽  
Donor Oocytes ◽  
Developmental Block

Interspecies somatic cell nuclear transfer (iSCNT) using the bovine cytoplast as universal recipient has potential applications in the conservation of exotic species. However, an in vitro developmental block has been observed using this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could be a cause for the embryonic developmental arrest. A series of experiments were conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of cloned embryos. In Experiment 1, we examined the effect of combining the technique of ooplasmic transfer (OT) with somatic cell nuclear transfer (SCNT) in the bovine model. In addition, presence and pattern of migration of foreign mitochondria after OT were examined by MitoTracker® (Molecular Probes, Inc., Eugene, OR, USA) staining. In Experiment 2, we examined the effect of transferring caprine ooplasm into bovine enucleated oocytes (iOT) used as recipients for goat iSCNT. Ooplasm from donor oocytes was aspirated until the oolema was ruptured and filled about 200 μm of the micropipette. Aspirated ooplasm was injected into recipient oocyte; the oolema of the recipient oocyte was also ruptured by partial aspiration into the micropipette to ensure mixing. Mean cleavage rates and embryo development were compared by chi-square analysis. Percentages (except for parthenogenic controls) were calculated from number of fused couplets. In Experiment 1, there was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst (BLST), or hatched blastocyst (HCHD) embryos (Table 1). MitoTracker Green FM staining of donor oocytes used for OT revealed foreign mitochondria were introduced by the procedure. Their pattern of distribution remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. However, when goat ooplasm was injected into bovine enucleated oocytes used for iSCNT, there was a significant reduction in fusion (52 vs. 82%) and cleavage rates (55 vs. 78%) (P < 0.05). In addition, the procedure of iOT prior to iSCNT was not effective in overcoming the 8- to 16-cell in vitro developmental block and only parthenogenic cow and goat controls reached blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively (Table 1). This study demonstrates that foreign mitochondria are introduced at the time of OT and these mitochondria remain in a cluster without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at genomic activation. To our knowledge, this is the first scientific report of iOT used in combination with iSCNT in an attempt to overcome the in vitro developmental block. Further research is needed to determine characteristics of foreign mitochondrial dynamics as well as replication of foreign mitochondria introduced into NT embryos. Table 1. Intraspecies (cow) and interspecies (goat-cow) ooplasmic transfer and nuclear transfer

Ooplasm transfer and interspecies somatic cell nuclear transfer: heteroplasmy, pattern of mitochondrial migration and effect on embryo development

Zygote ◽  
2010 ◽  
Vol 19 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Marina J. Sansinena ◽  
John Lynn ◽  
Kenneth R. Bondioli ◽  
Richard S. Denniston ◽  
Robert A. Godke
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Sequence Of Events ◽  
Distinct Cluster ◽  
Potential Applications ◽  
Developmental Block

SummaryAlthough interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

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

scholarly journals Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

2007 ◽  
Vol 22 (7) ◽  
pp. 1982-1990 ◽  
Author(s):  
B. Heindryckx ◽  
P. De Sutter ◽  
J. Gerris ◽  
M. Dhont ◽  
J. Van der Elst
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle

scholarly journals Development of Intergeneric Canine Embryo Using Bovine and Porcine Oocyte as Cytoplasm Recipient

2015 ◽  
Vol 10 (1) ◽  
pp. 801
Author(s):  
Yuda Heru Fibrianto
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Cloning ◽  
Embryo Production ◽  
Porcine Oocyte ◽  
Development In Vitro

This study wa conducted to increase the efficiency of canine embryo production by intergeneric somatic cell nuclear transfer (SCNT) technology. The effect of oocyte recipient for development of intergeneric canine somatic cell cloning embryos were examined. Bovine and porcine cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in TCM-199 medium depend on species. Adult dog fibroblasts collected from 3.5 years old Afghandhound dog, and cell between passage 1 and passage 10 used for intergeneric somatic cell cloning using bovine and porcin oocytes as oocyte cytolplasm donor. The result showed that oocytes from bovine and porcine can de-differentiated canine nucleus and no different between bovine and porcine oocyte in fusion and embryo development in vitro. Canine intergeneric cloned embryos developed to morula stages in vitro.

24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS

2008 ◽  
Vol 20 (1) ◽  
pp. 92
Author(s):  
X. J. Bai ◽  
J. L. Yu ◽  
M. Murakami ◽  
Y. Zhang ◽  
Y. J. Dong
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cultured Cells ◽  
Es Cells ◽  
Donor Cells ◽  
The Mean ◽  
Development In Vitro ◽  
Chi Square Analysis

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P < 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time. Table 1. Development in vitro of bovine NT embryos produced from different donor cell types

scholarly journals 140 EXPRESSION OF LEPTIN LIGAND AND RECEPTOR AND EFFECT OF EXOGENOUS LEPTIN SUPPLEMENTATION ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO FERTILIZED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 220
Author(s):  
H.S. Kim ◽  
G.S. Lee ◽  
J.H. Kim ◽  
S.K. Kang ◽  
B.C. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Differential Staining ◽  
Staining Procedure ◽  
Receptor Proteins ◽  
Arcsine Transformation

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro-fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. In Exp. 1, in vitro-matured porcine oocytes and embryos in 2-, 4-, and 8-cell as well as morula and blastocyst stages derived from IVF or SCNT were immunostained for leptin ligand and receptor with their specific antibodies. The expression of leptin ligand and receptor proteins was detected in oocytes and all stages of IVF and SCNT embryos. The IVF (Exp. 2; n = 635, 630, 633, 635, respectively) or SCNT oocytes (Exp. 3; n = 256, 258, 251, 258, respectively) were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (1, 10, 100, or 1000 ng/mL) of leptin. For the control group, IVF (n = 635) or SCNT embryos (n = 249) were cultured without leptin supplementation (0 ng/mL leptin). Embryo development and cell number in blatoscysts after differential staining according to a modified staining procedure (Thouas et al. 2000 Reprod. Biomed. Online 3, 25–29) were evaluated. The IVF or SCNT embryos were randomly distributed, and experiments were replicated at least 11 times. The differences in embryo development among experimental groups were analyzed using one-way ANOVA after arcsine transformation (without arcsine transformation for cell number of blastocysts) to maintain homogeneity of variance. Post hoc analyses to identify between-group differences were performed using the LSD test. In SCNT embryos, the cleavage rate was not different in leptin-treated groups (73.8, 77.5, 75.7, or 78.7%, respectively) compared to the control (76.3%). The rate of blastocyst formation (at 166 h after the day of injection of donor cells) in SCNT embryos was significantly increased (P < 0.05) in 1000 ng/mL leptin-supplemented group (20.2%) compared with the control (12.9%) and 1 ng/mL leptin-supplemented (12.5%) groups. Supplementing mNCSU-23 with 1000 ng/mL leptin also significantly increased (P < 0.05) the number of total cells (54.6) and trophectoderm (TE) cells (39.1) in SCNT blastocysts (n = >25) compared with the control [45.1 (total cells) and 31.6 (TE cells)] and 10 ng/mL leptin-supplemented group [44.4 (total cells) and 31.7 (TE cells)]. In IVF embryos, leptin supplementation did not affect pre-implantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor proteins in porcine in vitro matured oocytes, IVF and SCNT embryos, and the embryotropic role of leptin in the SCNT embryo development. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1) and the Korean Ministry of Science and Technology (M10310060000-03B4606-00000).

scholarly journals Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs

2016 ◽  
Vol 30 (4) ◽  
pp. 585-592 ◽  
Author(s):  
Jae-Gyu Yoo ◽  
Byeong-Woo Kim ◽  
Mi-Rung Park ◽  
Deug-Nam Kwon ◽  
Yun-Jung Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell

scholarly journals Effects of manganese on maturation of porcine oocytes in vitro and their subsequent embryo development after parthenogenetic activation and somatic cell nuclear transfer

2019 ◽  
Vol 65 (3) ◽  
pp. 259-265 ◽  
Author(s):  
Muhammad QASIM ◽  
Jun-Xue JIN ◽  
Sanghoon LEE ◽  
Anukul TAWEECHAIPAISANKUL ◽  
Erif Maha Nugraha SETYAWAN ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Parthenogenetic Activation

scholarly journals A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Xiangpeng Dai ◽  
Jie Hao ◽  
Qi Zhou
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Culture Method ◽  
Early Embryo ◽  
Blastocyst Development

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and αMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10–30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

Sign in / Sign up

Export Citation Format

Share Document