Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.     

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.     

Chromosomal aneuploidy in African Wildcat somatic cells and cloned embryos

In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.     

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.     

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.     

Bovine oocyte cytoplasm supports nuclear remodeling but not reprogramming of murine fibroblast cells

Nuclear transfer (NT) is used to elucidate fundamental biological issues such as reversibility of cell differentiation and interactions between the cytoplasm and nucleus. To obtain an insight into interactions between the somatic cell nucleus and oocyte cytoplasm, nuclear remodeling and gene expression were compared in bovine oocytes that had received nuclei from bovine and mouse fibroblast cells. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from mouse fibroblasts did not develop beyond the 8-cell stage. Similar nuclear remodeling procedures were observed in oocytes reconstructed with mouse and bovine fibroblast cells. Foreign centrosomes during NT were introduced into embryos reconstructed with both fibroblast cell types. A number of housekeeping mouse genes (hsp70, bax, and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells.     

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.     

Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.     

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。     

Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.     

Development of bovine-ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine-ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.     

Nuclear and microtubule reorganization in nuclear-transferred bovine embryos

We studied the nuclear and microtubule dynamics in nonactivated and pre-activated chromatin-removed oocytes following transfer of nuclei from bovine fibroblast cells. Immediately after fusion between membranes of oocytes and fibroblasts, a microtubule aster containing a gamma-tubulin spot was seen near the transferred nucleus in most oocytes regardless of activation conditions. Most fibroblast nuclei transferred into nonactivated oocytes underwent premature chromosome condensation (PCC) and divided into two masses of chromosomes. In contrast, fibroblast nuclei in pre-activated oocytes rarely underwent PCC, but formed a swelled pronuclear-like structure. Under nonactivation condition, microtubular spindles surrounded condensed chromosomes during the division of two nuclear structures. Gamma-tubulins were detected in the vicinity of condensed chromosomes, suggesting transient spindle formation. Two pronuclear-like structures near the microtubular aster containing gamma-tubulin spot(s) later formed a syngamy-like nuclear structure. While 20% of reconstructed oocytes under nonactivated conditions developed to morulae and blastocysts, only 4% of reconstructed oocytes under pre-activated conditions developed to morulae and blastocysts. These results suggest introduction of a foreign centrosome during somatic cell nuclear transfer, which probably plays a role in nuclear remodeling and subsequent development.     

Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos.

The present study characterized the profile of nuclear remodeling in nuclear transplant rabbit embryos and investigated the relationship between chromatin behavior after transfer and embryo development. The developmental potential and pattern of remodeling of donor nuclei from cleavage-, morula-, and blastocyst- (inner cell mass ICM, and trophectoderm, TE) stage donors were evaluated. In addition, we determined whether a modification in the synchrony between blastomere fusion and oocyte activation altered the profile of nuclear remodeling and affected development of reconstituted embryos. Development to blastocysts was similar with 8- and 32-cell-stage donor nuclei (42% and 33%, respectively, p greater than 0.1). However, it was reduced with ICM transplants (17%, p less than 0.05), and development of TE transplants did not progress beyond the 8-cell stage. Upon blastomere fusion into nonactivated oocyte cytoplasm, nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by pronuclear (PN) formation and swelling. PCC occurred synchronously within 1.2-1.5 h post-fusion with all stages of donor nuclei (p greater than 0.1). PN formation in 8- and 32-cell transplants occurred approximately 4 h after fusion, and was synchronous to that of female pronuclei in activated oocytes; however, it was delayed in ICM and TE transplants (p less than 0.01). With all stages of donor nuclei, final nuclear diameter was similar to, or larger than, that of female pronuclei. Fusion to activated oocyte cytoplasm, as opposed to nonactivated cytoplasm, prevented PCC and extensive nuclear swelling (16.0 +/- 0.7 vs. 30 +/- 0.7 microns, respectively, p less than 0.01). Nuclear diameter in early embryos was smaller (p less than 0.01), and development to blastocysts was reduced (p less than 0.05). The results indicate that remodeling of the donor nucleus is not essential for development to blastocysts; however, it is beneficial. Furthermore, complete reprogramming seems possible only after remodeling of the donor nucleus, i.e., PCC in nonactivated cytoplasm, followed by nuclear swelling upon activation of the oocyte.     

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.     

Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.