Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley-Liss, Inc.     

Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos.

We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with G1, early S, and late S phase donor nuclei (G1, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G1, early S, and late S transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in G1 transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in G1 and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Premature chromosome condensation is not essential for nuclear reprogramming in bovine somatic cell nuclear transfer

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.     

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.     

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.     

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming

Nuclei of differentiated cells can acquire totipotency following transfer into the cytoplasm of oocytes. While the molecular basis of this nuclear reprogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuclei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear reprogramming, we investigated the link between cell-cycle state and depletion of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, telophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was lost more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly when the recipient cytoplast was at metaphase and most slowly when the recipient was at interphase. When the recipient oocyte was not enucleated, however, immunoreactive H1 remained in the donor nucleus. The phosphorylation inhibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreactive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 was depleted from mouse blastomere nuclei following transfer into bovine oocytes. Finally, expression of the developmentally regulated gene, eIF-1A, but not of Gapdh, was extinguished in metaphase recipients but not in interphase recipients. These results indicate that evolutionarily conserved cell-cycle-regulated activities, nuclear elements, and phosphorylation-linked events participate in the depletion of immunoreactive histone H1 from blastomere nuclei transferred in oocyte cytoplasm and that this is linked to changes in gene expression in the transferred nucleus.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

Factors affecting the efficiency of nuclear transplantation in the rabbit embryo

Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).     

Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin

In preliminary experiments, the treatment of donor somatic cells with beta-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitro-development of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P < 0.05) effect was found after the combined treatment of cloned embryos with Hb (1 microg/ml) and ME (10 microM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142-154 vs. 123 cells/embryo), inner cell mass (ICM) (39-41 vs. 27), and trophectoderm (TE) (103-114 vs. 98), and the ratio of ICM to TE cell number (0.26-0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056-0.069). When embryos cloned with ME-treated cells were cultured in Hb + ME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with Hb + ME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in Hb + ME-containing medium yielded the optimal results for promoting the production of blastocysts with improved quality.     

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.     

Effects of the removal of cytoplasm on the development of early cloned bovine embryos

Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.     

Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.     

Nuclear reprogramming in nuclear transplant rabbit embryos

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.     

Blastomeres from somatic cell nuclear transfer embryos are not allocated randomly in chimeric blastocysts

A marker has been developed to allow detection of blastomeres that originate from embryos produced by nuclear transfer (NT) of genetically engineered fetal fibroblasts. A plasmid (phEFnGFP) was constructed with a G418 resistance cassette for selection in fibroblasts and a nuclear localized green fluorescent protein (nGFP) expression cassette that expresses in every cell of day-6, -7, and -8 bovine embryos. This construct was utilized to follow the blastomere distribution in aggregation chimeras produced from fertilized embryos (in vitro produced, IVP) or parthenotes and NT embryos. Fluorescent and nonfluorescent NT embryos were aggregated early on day 4 and evaluated on day 8. Nuclei of blastomeres that carried the transgene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluorescence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the inner cell mass (ICM) or trophectoderm in NT<>NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres in the trophectoderm when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was significantly less (p < 0.05) when aggregated with parthenotes as compared to aggregation with IVP embryos. In NT<>NT aggregates, no chimeric embryos were produced that had an ICM composed of blastomeres from a single origin. However, in NT<>Parthenote aggregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 0% to 83% of the ICM that expressed nGFP. Similarly, in NT<>IVP aggregates 50% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We conclude that production of divaricated chimeras from NT origin is feasible. Other applications of this technology are discussed.     

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.