55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

2009 ◽  
Vol 21 (1) ◽  
pp. 128 ◽  
Author(s):  
N. A. Wani ◽  
J. A. Skidmore ◽  
U. Wernery
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Light Exposure ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Reconstructed Embryos

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P < 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

76 PRODUCTION OF CLONED BOVINE TRANSGENIC EMBRYOS WITH VARIOUS TYPES OF MONO-COLONY CELLS AND OVUM PICKUP

2005 ◽  
Vol 17 (2) ◽  
pp. 188
Author(s):  
J.G. Zhao ◽  
X.Y. Yang ◽  
Y. Huang ◽  
H.F. Liu ◽  
H. Li ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Nuclear Transfer ◽  
Genetic Manipulation ◽  
Blastocyst Stage ◽  
High Tech ◽  
Cleavage Rate ◽  
Exogenous Dna ◽  
Developmental Potential ◽  
Reconstructed Embryos

The objective of this study was to determine the effects of genetic manipulation, cell type, and culture conditions on developmental potential of bovine nuclear transfer (NT) embryos. Ovum pickup (OPU) technology was developed to obtain the oocytes for NT. A total 4044 cumulus-oocyte complexes (COCs) were obtained during 492 OPU sessions, with an average of 8.2 COCs recovered each session. Cultured granulosa cells (CGC), bovine fetal (150 days) oviduct epidermic cells (FOEC), and adult ear skin fibroblasts (ASFC) were used as donor cells for NT and were transfected with the expression vector including human FIX coding sequence directed by goat β-casein promoter and neomycin gene. The cells were screened under 800 μg mL−1 G418 for 10–14 days until the apperance of a “mono-colony” of cells which were then picked. Each cell population was expanded by consecutive passage culture under 300 μg mL−1 G418 until used for NT, ensuring that the majority of cells were transgenic. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were co-cultured with vero cells in B2 medium for 7 days. NT efficiency between primary granulosa cells (PGC) without in vitro culture and CGC, as well as among CGC, FOEC and ASFC that were transfected with exogenous DNA (named TCGC, TFOEC, TASFC, respectively), were compared (Table 1). Differences between groups were verified by chi-square test using SAS 6.12 (SAS Institute, Inc., Cary, NC, USA) program. CGCs presented a higher fusion rate (P < 0.01) for reconstructed embryos and higher development to the blastocyst stage for NT embryos than did PGC (67% vs. 54% and 41% vs. 21%, respectively). There were no significant differences (P > 0.05) in cleavage rate (65%, 71%, and 69%, respectively) and development to the blastocyst stage for NT embryos (36%, 30% and 40%, respectively) for TCGC, TFOEC, and TASFC. A total of 86 blastocysts were selected for transfer into uteri of 86 cows, resulting in 26 pregnancies (30%) at 60 days by ultrasound scanning. Among these, 12 cows remain pregnant and 14 have aborted. The results indicated that oocytes recovered from OPU can be successfully used for NT with development to the blasocyst stage. PGC, CGC, FOEC, and ASFC can all be used for generating transgenic cattle by NT, although this needs to be verified by the birth of live calves. Table 1. Nuclear transfer efficiency with various cell types This work was supported by the Chinese “863” High-Tech Plan Program (Grant No. 2002AA206201).

scholarly journals 61 CLONED MOUSE PRODUCED USING A ZONA FREE METHOD OF NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 180
Author(s):  
R. Ribas ◽  
B. Oback ◽  
J. Taylor ◽  
A. Maurício ◽  
M. Sousa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Strontium Chloride ◽  
Embryonic Cells ◽  
Reconstructed Embryos

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.

58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann
Keyword(s):  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (7) ◽  
pp. 1002 ◽  
Author(s):  
Qing Guo ◽  
Long Jin ◽  
Hai-Ying Zhu ◽  
Xiao-Xu Xing ◽  
Mei-Fu Xuan ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Kinase Inhibitor ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Cyclin Dependent Kinase ◽  
Parthenogenetic Activation ◽  
Cyclin Dependent Kinase Inhibitor

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10 µM JNJ-7706621 for 4 h compared with embryos exposed to 5 µg mL−1 cytochalasin B for 4 h (P < 0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P < 0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P < 0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P < 0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P < 0.05). In conclusion, these results reveal that exposure to 10 µM JNJ-7706621 for 4 h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

81 COMPARISON OF DEVELOPMENTAL ABILITY AMONG CLONED EMBRYOS WITH VARIOUS INDIVIDUAL RECIPIENT CYTOPLASMS IN BOVINE

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. G. Zhao ◽  
X. Y. Yang ◽  
H. F. Liu ◽  
H. Li ◽  
S. Z. Huang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Vero Cells ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Chi Square ◽  
Reconstructed Embryos ◽  
Maturation Rate ◽  
Chi Square Test

Faithful reprogramming ensures the proper activation of genes during embryonic development of the somatic cell nuclear transfer (NT) in bovine. It is unambiguous that all these remodeling factors are presented in the oocyte cytoplasm (Du et al. 2002 Mol. Reprod. Dev. 63, 183–191). It will be interesting to determine if the recipient cytoplasms derived from individuals have different development ability and reprogramming competence during NT. Oocytes recovered by Ovum pickup from five Holstein heifers at 14 months of age were used as recipient cytoplasms. Cultured granulosa cells of the same origin were used as donor cells. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were cultured in B2 medium (Laboratoire CCD, Paris, France) on a monolayer of Vero cells for 7 days. The oocyte number, development ability, and NT efficiency of recipient cytoplasm derived from each individual were compared (Table 1). Differences among individuals were verified using a chi-square test, SAS 6.12 version (SAS Institute, Cary, NC, USA). There were significant differences of survival after fusion and the rate of development to the blastocyst stage for embryos reconstructed with recipient cytoplasm from five different individual heifers (P < 0.05). However, maturation rate, fusion rate and cleavage rate of embryos reconstructed with recipient cytoplasm from five different individual heifers presented no significant differences (P > 0.05). Reconstructed embryos with recipient cytoplasm from one heifer (03025) showed a lower survival after fusion (61% vs. 80%, 86%, 77%, 91%) but a higher ability to develop to blastocyst stage (61% vs. 24%, 31%, 52%, 31%) than the embryos from the other four heifers. The current study showed that recipient cytoplasm from various individuals may present great differences in developmental ability in nuclear transfer. This may result from different compatibility between nucleus and mitochondria or the content of maternal RNA as well as proteins in the oocyte. Further studies are needed to elucidate the genetic factors that affect the reprogramming in nuclear transfer. Table 1. Nuclear transfer efficiency with various individual recipient cytoplasms

236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Electrical Pulses ◽  
Blastocyst Rate ◽  
Activation Rates

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses

scholarly journals Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei

Reproduction ◽  
2001 ◽  
pp. 49-60 ◽  
Author(s):  
T Wakayama ◽  
R Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Cytochalasin D ◽  
Full Term ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Reconstructed Embryos

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

60 EFFECTS OF CAFFEINE ON THE IN VITRO DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 138
Author(s):  
W. E. Maalouf ◽  
J. H. Lee ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Cell Cycle Regulators ◽  
Reconstructed Embryos

Previous studies have demonstrated that treating ovine oocytes with caffeine increases the activities of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). When such oocytes are used as cytoplast recipients for nuclear transfer (NT), there is an increase in cell numbers at the blastocyst stage (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125). The objective of this study was to determine the effects of caffeine on MPF and MAPK activities and the development of bovine NT embryos. Oocytes were matured in maturation medium (MM) composed of TCM199, 10% fetal bovine serum (FBS), 5 �g mL-1 follicle-stimulating hormone FSH, 5 �g mL-1 lutcinizing hormone (LH) and 1 �g mL-1 estradiol for 24 h. Subsequently, oocytes were cultured in MM supplemented with 0, 5, 10, and 15 mM caffeine for 6 h. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye et al. 2003 Reproduction 125, 645-656). Treatment with 15 mM caffeine significantly increased the levels of MPF and MAPK activities in MII oocytes. To study development potential, oocytes at 16 h post-onset of maturation (hpm) were stripped of cumulus cells and enucleated in HSOF containing 5 �g mL-1 Hoechst 33342 and 7.5 �g mL-1 cytochalasin B; enucleation was achieved using a blunt (25-�m i.d.) pipette after cutting a hole in the zona pellucida with a XYClone laser (Hamilton Thorne Research, Beverly, MA, USA). Enucleated oocytes were then cultured in MM �15 mM caffeine for a further 6 h. For NT, quiesced primary bovine foetal fibroblasts were used. Cell fusion was induced with two DC pulses of 35 V for 65 �s at 24 hpm. At 2 h post-fusion, all reconstructed embryos were briefly exposed to ultraviolet light under a fluorescence microscope (Leica Microsystems AG, Wetzler, Germany) in order to assess nuclear morphology, and then activated in HSOF containing 5 �g mL-1 calcium ionophore (A23187), cultured in SOF with 10 �g mL-1 cycloheximide and 7.5 �g mL-1 cytochalasin B for 5 h, and transferred to mSOFaaBSA medium. On Day 2, cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed using the chi-square test. There was a significant increase in the number of reconstructed embryos that underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) when caffeine-treated cytoplast recipients were used (28.6 � 9.9% and 60.0 � 11.0% for control and caffeine groups respectively, P < 0.05). Cleavage rates (47.6 � 10.9% and 50.0 � 11.1%), development to blastocyst (20.0 � 4.0% and 30.0 � 4.6%), and mean cell number (85.0 � 7.1 and 122.5 � 3.5) were not statistically different between control and caffeine treated groups, respectively. In summary, treatment of bovine oocytes with 15 mM caffeine increased the activities of two key cell-cycle regulators MPF and MAPK, and statistically increased the occurrence of NEBD and PCC in the donor nuclei. We previously hypothesized that the occurrence and extent of NEBD and PCC may increase nuclear reprogramming in NT embryos (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125; Campbell et al. 2005 Rep. Dom. Anim. 40, 256-268); however, further studies are required to determine the developmental competence of these embryos.

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