scholarly journals In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer1

2000 ◽  
Vol 63 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Deog-Bon Koo ◽  
Yong-Kook Kang ◽  
Young-Hee Choi ◽  
Jung Sun Park ◽  
Sun-Kyung Han ◽  
...  
Keyword(s):  
Somatic Cell ◽  
In Vitro Development ◽  
Vitro Development

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 105
Author(s):  
E. S. Ribeiro ◽  
R. P. C. Gerger ◽  
L. U. Ohlweiler ◽  
I. Ortigari Jr ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Chemical Activation ◽  
Primary Cultures ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

47 EFFECT OF THE TYPE OF CYTOPLASTS, SOURCE OF KARYOPLASTS, AND ACTIVATION PROCESS ON IN VITRO DEVELOPMENT OF BOVINE CLONE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
L. U. Ohlweiler ◽  
J. C. Mezzalira ◽  
R. P. C. Gerger ◽  
E. S. Ribeiro ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Fluorescent Light ◽  
Activation Process ◽  
Chi Square ◽  
In Vitro Development ◽  
Polar Bodies ◽  
M Phase ◽  
Early Zygote ◽  
Vitro Development

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

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.

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.

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

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