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1.
The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.  相似文献   

2.
The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.  相似文献   

3.
In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0‐phase (G0‐SCNT group) or early G1‐phase (eG1‐SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0‐phase and eG1‐phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake‐off method). The fusion rate in the G0‐SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1‐SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1‐SCNT groups (12.7%) per recipient cow was higher than that in G0‐SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0‐SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1‐SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1‐phase using the shake‐off method improved the overall production efficiency of the clone offspring per transferred embryo.  相似文献   

4.
Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.  相似文献   

5.
本研究采用爪蟾卵母细胞抽提物处理和牛胎儿成纤维细胞(JBCFF),并以发生重编程的细胞为核供体进行体细胞核移植,研究其对克隆效率的影响.分别超排3只爪蟾,收集卵母细胞后提取其抽提物,用BCA蛋白浓度测定试剂盒确定其蛋白含量,SDS-PAGE分析其中所含蛋白的种类.应用细胞膜透化剂Digitonin对建立的JBCFF进行通透处理和PI染色,筛选最适浓度;并用获得的抽提物处理牛体细胞,获得重编程细胞.以发生重编程的体细胞为供体进行核移植,同时比较离子霉素+6-DMAP和A23187+6-DMAP两种组合激活后克隆胚的体外发育能力.结果,3个爪蟾卵母细胞抽提物样品的蛋白浓度分别为56.2255、64.6570和71.2158 μg/mL,其中所含蛋白种类一致,主要集中在40~55、70~100 ku之间.经过连续的筛选,透化剂Digitonin对JBCFF通透处理的最适浓度为7 μg/mL,PI染色显示透化效率为55.44%.爪蟾卵母细胞抽提物与体细胞共孵育后继续培养6~7 d,细胞聚集形成"克隆簇".分别以"克隆簇"细胞和未处理细胞为核供体,制备的重构胚在融合率(92.83%和96.04%)、卵裂率(89.64%和89.78%)和囊胚率(24.06%和23.12%)均无显著差异(P>0.05);离子霉素+6-DMAP和A23187+6-DMAP两种激活方式对克隆胚胎的分裂率(92.16%和92.28%)和囊胚率(23.21%和24.18%)均无显著影响(P>0.05).爪蟾卵母细胞抽提物诱导和牛体细胞发生重编程,并恢复到较低的分化状态,但没有显著促进克隆胚胎的体外发育,可见缺少对胚胎发育起重要作用的重编程过程,还需要继续深入研究.  相似文献   

6.
To artificially activate embryos in somatic cell nuclear transfer (SCNT), chemical treatment with ionomycin has been used to induce transient levels of Ca(2+) and initiate reprogramming of embryos. Ca(2+) oscillation occurs naturally several times after fertilization (several times with 15- to 30-min intervals). This indicates how essential additional Ca(2+) influx is for successful reprogramming of embryos. Hence, in this report, the experimental design was aimed at improving the developmental efficiency of cloned embryos by repetitive Ca(2+) transients rather than the commonly used ionomycin treatment (4 min). To determine optimal Ca(2+) inflow conditions, we performed three different repetitive ionomycin (10 μM) treatments in reconstructed embryos: Group 1 (4-min ionomycin treatment, once), Group 2 (30-sec treatment, 4 times, 15-min intervals) and Group 3 (1-min treatment, 4 times, 15-min intervals). Pronuclear formation rates were checked to assess the effects of repetitive ionomycin treatment on reprogramming of cloned embryos. Cleavage rates were investigated on day 2, and the formation rates of blastocysts (BLs) were examined on day 7 to demonstrate the positive effect of repeated ionomycin treatment. In Group 3, a significant increase in BL formation was observed [47/200 (23.50%), 44/197 (22.33%) and 69/195 (35.38%) in Groups 1, 2 and 3, respectively]. Culturing embryos with different ionomycin treatments caused no significant difference among the groups in terms of the total cell number of BLs (164.3, 158.5 and 145.1, respectively). Additionally, expression of the anti-apoptotic Bcl-2 gene and MnSOD increased significantly in Group 3, whereas the expression of the pro-apoptotic Bax decreased statistically. In conclusion, the present study demonstrated that repeated ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.  相似文献   

7.
Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6‐dimethylaminopurine (6‐DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6‐DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71–72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6‐DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 μg/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1–8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)‐expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM‐AA, MEM‐α and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM‐α medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6‐DMAP following embryo transfer. In conclusion, treatment with 6‐DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full‐term development of the transgenic SCNT cloned embryos.  相似文献   

8.
本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。  相似文献   

9.
The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.  相似文献   

10.
Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium‐3 (mPZM‐3) with increased NaCl to 138 mmol/L (mPZM‐3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM‐3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM‐3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM‐3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM‐3+NaCl for the first 2 days and then cultured in mPZM‐3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM‐3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.  相似文献   

11.
The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μm Io for 4 min followed by 5 μm DhL concentration and after 3‐h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6‐dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI‐embryos or SCNT‐embryos is reported here for first time.  相似文献   

12.
The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.  相似文献   

13.
The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B4 isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.  相似文献   

14.
This experiment aimed to study the effect of brilliant cresyl blue (BCB) on in vitro maturation of pig oocytes and the developmental capacity of pig SCNT embryos.The cumulus-oocyte complexes (COCs) were stained with different concentrations of BCB (13,26,39 and 52 μmol/L) for 90 min,and then we divided the COCs into BCB+ and BCB- for in vitro culture 42 to 44 h.The results showed that,with the concentration of BCB increased,the staining rate (20.00%,46.39%,51.66% and 59.03%) raised gradually while the maturation rate of oocytes (74.03%,72.16%,70.53% and 48.61%) reduced,the percentages of oocytes staining by 26 μmol/L BCB for 90 min were higher than that of other groups in staining rate and maturation rate.However,the nuclear maturation rate of BCB+ groups were higher than that of BCB- group.Therefore,26 μmol/L BCB was selected as the most effective concentration dying the oocytes (BCB+),which were used as parthenogenetic activation and nuclear transfer embryos.The cleavage and blastocyst rates of parthenogenetic activation and SCNT embryos in BCB+ group were significantly higher than that of BCB- group (P<0.05),but there were no significant differences between the cleavage and blastocyst rates in the groups of BCB+ and control (P>0.05).Reconstructed embryos derived from the COCs stained with BCB were transferred to five surrogates,and six cloned piglets were obtained from one of the two pregnant pigs.These results showed that COCs stained with BCB was an effective method to select high-quality oocytes,which could improve the efficiency of in vitro embryo production.  相似文献   

15.
试验旨在研究亮甲酚蓝(brilliant cresyl blue,BCB)染色对卵母细胞体外成熟及后期胚胎发育潜力的影响。本研究利用13、26、39 、52 μmol/L BCB对成熟培养前的卵丘－卵母细胞复合体(cumulus-oocyte-complexes,COCs)染色90 min,比较各组卵母细胞的着色率、成熟率及孤雌激活胚胎和核移植胚胎的发育情况。结果表明,随着BCB浓度的增加,COCs着色率依次增加(20.00%、46.39%、51.66%和59.03%),但猪卵母细胞体外成熟率逐渐降低(74.03%、72.16%、70.53%和48.61%);不同浓度BCB染色后所得BCB+组卵的成熟率均明显高于BCB-组。试验结果发现,BCB浓度在26 μmol/L时,经染色的COCs既有较高的着色率,且不影响其体外成熟的效率。基于此,研究选取26 μmol/L BCB作为最佳浓度对猪卵母细胞进行染色筛选,然后进行体外培养、孤雌激活及核移植试验。结果显示,筛选的BCB+组卵母细胞的孤雌胚和核移植胚的卵裂率和囊胚率均显著高于BCB-组(P<0.05),而与对照组间无显著差异(P>0.05)。胚胎移植试验挑选BCB+组中发育较好的1-2细胞期重组胚对5头代孕母猪进行了移植,其中2头怀孕,1头顺利产下了6头健康胎儿。综合以上试验结果表明,利用BCB染色可作为一种有效的方法筛选体外成熟质量较高的猪卵母细胞,同时提高胚胎体外生产效率。  相似文献   

16.
本研究旨在比较水牛2种体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)方法的效果以及激活前的时间间隔对全细胞胞质内注射法(Whole-Cell Intracytoplasmic Microinjection,WCICSI)核移植效果的影响.采用水牛胎儿成纤维细胞作为供核,比较了透明带下注核法(Perivitelline Microinjection,PM)和WCICSI核移植效果.另外,试验了不同类型的供体细胞进行全细胞胞质内注射后与激活前的受体胞质的最适宜作用时间.结果,WCICSI构建核移植重构胚的成功率显著高于PM(87.1%vs 81.1%,P＜0.05),虽然其重构胚的分裂率极显著低于PM(49.5%vs 71.8%,P＜0.01),但囊胚率、核移植的效率无显著差异(P＞0.05).卵丘细胞和胎儿成纤维细胞在注射后3 h激活,重构胚的囊胚发育率最高;颗粒细胞注射后与激活前的最佳时间间隔可在1.5～3 h,但3 h是最佳的作用时间.结果表明,(1)WCICSI可用于水牛体细胞核移植的研究;(2)水牛胞质内注射供体细胞后3 h进行激活,核移植重构胚的发育效果最好.  相似文献   

17.
The objective of this study was to determine the effects of various methods of sperm pre‐treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro‐matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non‐treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p < 0.05) than those activated with either ionomycin (Io) + 6‐dimethylaminopurine (6‐DMAP) or DTT + I + 6‐DMAP. In Exp. 2, the effects of sperm pre‐incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two‐time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non‐treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p < 0.05) than other groups except for freeze–thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre‐treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre‐treated and control groups. In conclusion, pre‐treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6‐DMAP after ICSI.  相似文献   

18.
Efficient Production of Cloned Bovine Embryos Using cdc2 kinase Inhibitor   总被引：2，自引：0，他引：2  
This study was carried out to compare the effects of the combination of ionomycin with a H1‐histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on the development of reconstituted bovine eggs. For this study, the enucleated bovine oocytes were injected with a presumptive primordial germ cell pre‐treated with 1% sodium citrate, and randomly allocated into three activation groups: Group 1 (ionomycin 5 μm , 5 min), Group 2 (ionomycin + DMAP 1.9 mm , 3 h), and Group 3 (ionomycin + SPP 2 mm , 3 h). The reconstituted eggs were compared on the rates of cleavage and development with the blastocyst stage and the ploidy of embryos at 96 h post‐activation. Cleavage rates and blastocyst development in Groups 1, 2 and 3 were 7 and 0%, 63 and 17%, and 53 and 14%, respectively. The chromosomal composition differed significantly (p < 0.05) among treatments. Although the embryos in Group 1 had significantly lower developments, 60% of embryos evaluated had diploid chromosomal sets. In contrast, ～60% of embryos in Group 2 had abnormal ploidy (21% polyploid and 38% mixoploid). In Group 3, the appearance of abnormal chromosome sets was reduced with the proportion of diploid embryos being increased to 86% (19 of 22), significantly higher (p < 0.05) than in Group 2. It can be concluded that the use of SPP with ionomycin reduces greatly the incidence of chromosomal abnormalities, and may be applicable for the activation of nuclear transplant bovine embryos.  相似文献   

19.
The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri- and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT.  相似文献   

20.
To determine the effect of maturational age of porcine oocytes on the induction of activation and development following nuclear transfer, we investigated maturation rate and efficacy of oocyte activation treatment with an electrical stimulus (ES) and cycloheximide (CHX) at various timing of maturation culture. Most oocytes developed to the metaphase II (MII) stage after 32 hr of maturation culture. Both in newly matured (32 and 36 hr of age) and MII-arrested (42 and 48 hr of age) oocytes, ES followed by exposure to CHX for 6 hr caused higher rates of pronuclear formation than ES alone. Effect of maturational age of oocytes on the development of parthenotes activated with ES and CHX was then examined. The highest percentage of parthenotes developed to blastocysts was obtained when ES was added at 42 hr of culture. Finally, newly matured (33 hr of age) and MII-arrested oocytes (44 hr of age) were enucleated, fused with serum-starved pig fetal fibroblasts and activated with ES and CHX around suitable timing (39 to 40 hr of age) or later (50 to 51 hr of age), respectively. The frequencies of cleavage (58.8%) and blastocyst formation (13.6%) of newly matured oocytes were significantly (P<0.05) higher than those of MII-arrested oocytes (38.9 and 1.8%, respectively). These results demonstrated that the development of porcine nuclear transfer embryos can be improved by using complete matured oocytes as cytoplasts and activation treatment with ES followed by CHX treatment.  相似文献   

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