Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.     

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

Background

Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes.

Results

Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors.

Conclusions

Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.     

Chromosomes in the Porcine First Polar Body Possess Competence of Second Meiotic Division within Enucleated MII Stage Oocytes

To determine whether chromosomes in the porcine first polar body (PB1) can complete the second meiotic division and subsequently undergo normal pre-implantation embryonic development, we examined the developmental competence of PB1 chromosomes injected into enucleated MII stage oocytes by nuclear transfer method (chromosome replacement group, CR group). After parthenogenetic activation (PA) or in vitro fertilization (IVF), the cleavage rate of reconstructed oocytes in the IVF group (CR-IVF group, 36.4 ± 3.2%) and PA group (CR-PA group, 50.8 ± 4.2%) were significantly lower than that of control groups in which normal MII oocytes were subjected to IVF (MII-IVF group, 75.8 ± 1.5%) and PA (MII-PA group, 86.9 ± 3.7%). Unfertilized rates was significantly higher in the CR-IVF group (48.6 ± 3.3%) than in the MII-IVF group (13.1 ± 3.4%). The blastocyst formation rate was 8.3 ± 1.9% in the CR-PA group, whereas no blastocyst formation was observed in the CR-IVF group. To produce tetraploid parthenogenetic embryos, intact MII stage oocytes injected with PB1chromosomes were electrically stimulated, treated with 7.5 μg/mL cytochalasin B for 3 h (MII oocyte + PB1 + CB group), and then cultured without cytochalasin B. The average cleavage rate of reconstructed oocytes was 72.5% (48 of 66), and the blastocyst formation rate was 18.7% (9 of 48). Chromosome analysis showed similar proportions of haploid and diploid cells in the control (normal MII oocytes) and CR groups after PA; overall, 23.6% of blastocysts were tetraploid in the MII oocyte + PB1 + CB group. These results demonstrate that chromosomes in PB1 can participate in normal pre-implantation embryonic development when injected into enucleated MII stage oocytes, and that tetraploid PA blastocysts are produced (although at a low proportion) when PB1 chromosomes are injected into intact MII stage oocytes.     

Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts

The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Buballus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblasts, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 +/- 6.2 vs. 68.2 +/- 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2-4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18-24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments.     

Ethylenediamine-N,N,N',N'-tetraacetic acid induces parthenogenetic activation of porcine oocytes at the germinal vesicle stage, leading to formation of blastocysts

The present study showed that treatment with a cell membrane-impermeable metal ion chelator, EDTA, of porcine oocytes at the germinal vesicle (GV) stage collected from follicles 2-6 mm in diameter induced artificial activation followed by formation of a pronucleus (PN). When the oocytes were cultured for 48 h in medium containing 0.1 to 2 mM EDTA disodium salt (Na-EDTA), they were activated to form PN, and the maximum PN formation rate (63%, n = 68) was achieved in oocytes cultured with 1 mM Na-EDTA. More than 90% of oocytes activated by 1 mM Na-EDTA treatment formed 1 PN without emission of the first and the second polar bodies (PB). This result suggests that EDTA at 1 mM may force the maturing (meiosis I) oocytes to form a PN without chromosome segregation. When oocytes at the GV stage that had been cultured with 1 mM Na-EDTA for 48 h were further cultured in 0.4% BSA-containing NCSU23 medium for 144 h, blastocysts that appeared to be morphologically normal were formed at the rate of 10%, whereas no blastocysts were formed from oocytes that had not been cultured with Na-EDTA. Next we examined the effects of Ca2+, Zn2+, Fe3+, or Cu2+-saturated EDTA (Ca-EDTA, Zn-EDTA, Fe-EDTA, and Cu-EDTA, respectively), and a Ca2+-specific chelator, EGTA, at a concentration of 1 mM. The Ca-EDTA, Fe-EDTA, and Cu-EDTA, but not Zn-EDTA or EGTA, had the ability to activate the oocytes. From these results, it is suggested that extracellular chelation of Zn2+ with EDTA of maturing (meiosis I) porcine oocytes results in parthenogenetic activation of the oocytes, which induces PN formation followed by development to blastocysts.     

Development of pig embryos by nuclear transfer of cultured fibroblast cells

Pig fibroblast cells were transferred to enucleated oocytes by micromanipulation and electrofusion. The donor cells used for nuclear transfer were synchronized in presumptive G0 by serum starvation. In the first experiment, nuclear transfer was performed with fibroblasts that had either a smooth or a rough surface. A significant difference (p < 0.05) in the percentage of chromosome condensation (39.5%, 15/38 and 16.6%, 5/30) and nuclear formation (36.8%, 14/38 and 16.3%, 8/49) was found between the reconstructed embryos derived from the cells with smooth surface and with rough surfaces, respectively. The percentage of chromosome condensation (42.5%, 17/40 and 19.6%, 11/56) and nuclear formation (38.3%, 23/60 and 18.8%, 9/48) were higher (p < 0.05) in reconstructed embryos derived from small (15 microm) donor cells compared to large donor cells (20 microm), respectively. The percentage nuclei at 3 different time points (3, 6, and 9 hours in culture medium) was higher (p = 0.003) in the reconstructed embryos activated by thimerosal and dithiothreitol (20%, 36%, and 41.3%) compared to those without activation treatment (0%, 11.8%, and 22.2%). In addition, there was an increased percentage with nuclei as the time in culture increased from 3 to 9 hours (p = 0.029). The percentages of chromosome condensation (34.6%; 9/26) and nuclear formation (33.3%; 9/27) in nuclear transfer embryos were similar. The rate of blastocysts/morulae development (14.0%; 6/43) was low. However, 2 cavitated embryos (presumptive blastocysts) with 14 and 11 nuclei and 1 morula with 8 nuclei were obtained. This together with the above evidence indicate that the nuclei from pig fibroblast cells can be partially reprogrammed, which suggests that transfer of nuclei from fibroblast cells to in vitro matured oocytes resulting in production of identical or genetically altered pigs may be possible.     

A simple method for producing tetraploid porcine parthenogenetic embryos

The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.     

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.     

Developmental capacity of ferret embryos by nuclear transfer using G0/G1-phase fetal fibroblasts

With the ultimate goal of establishing experimental protocols necessary for cloning ferrets, the present study has established parameters for the reconstruction of ferret embryos by nuclear transfer (NT) using G0/G1-phase donor fetal fibroblasts. Cumulus-oocyte complexes were harvested from superovulated ferrets and cultured in maturation medium for 24 h. Matured oocytes were then enucleated and injected with the fibroblast nuclei derived from 14-16-h serum-starved cells. Reconstructed embryos were then activated by a combination of electric pulses and chemical stimulations. Subsequently, the reconstructed and activated embryos were either cultured in vitro or transferred to pseudopregnant ferrets to evaluate their developmental capacity in vitro and in vivo. Our results demonstrated that 56.3% of reconstructed embryos (n = 187) cleaved, while 26.0% and 17.6% developed to morula and blastocyst phases in vitro, respectively. The blastocysts derived from NT embryos demonstrated normal morphology by differentially staining as compared to normal blastocysts developed in vivo following fertilization. In vivo developmental studies at 21 days posttransplantation demonstrated 8.8% of reconstructed embryos (n = 91) implanted into the uterine lining of recipients, while 3.3% formed fetuses. However, reconstructed embryos (n = 387) failed to develop to term (42 days). These results demonstrate donor nuclei of G0/G1-phase fetal fibroblast cells can be reprogrammed to support the development of reconstructed ferret embryos in vitro and in vivo; however, a significant third-trimester block occurs preventing full-term development.     

Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein

A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.     

Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).     

Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 microg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.     

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus)

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.     

6-DMAP和胚胎干细胞代数对异种重构卵体外发育的影响(简报)

核移植技术已经广泛应用于动物克隆,但是克隆动物的成活率仍然很低。许多克隆胚胎死于妊娠期,少部分能发育到期,正常出生,但多数在出生后由于心肺和消化道的问题,很快就夭折,有些克隆动物有异常表型,如出生时体重和胎盘过大等。研究发现,在同种克隆动物实验中用胚胎干细胞(Embryonic stem cell,ES细胞)作为核供体,发育到期的克隆动物比例明显高于体细胞,并且用杂交一代的小鼠ES细胞为核供体,绝大多数克隆仔     

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.     

Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage

The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P?相似文献   

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1．088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41．03％(128／312),桑葚胚和囊胚发育率分别为9．29％(29／312),1．92％(6／312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0．64％(2／312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1.088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl_2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41.03%(128/312),桑葚胚和囊胚发育率分别为9.29%(29/312),1.92%(6/312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0.64%(2/312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.