Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Reprograming of murine blastocoele formation

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

Nuclear transplantation in mouse embryos: assessment of recipient cell stage

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos. Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes. Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation. Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos. No development was observed beyond Day 12 of gestation. These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

Nuclear transplantation of mouse fetal germ cells into enucleated two-cell embryos

Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F(1) (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.     

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.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

Participation of the paternal genome is not required before the eight-cell stage for full-term development of mouse embryos

Differential expression of the paternal and maternal genomes during mouse embryonic development is considered a reason for both genomes being required for development to term. Extending previous studies performed on two-cell embryos, we show here that diploid embryos reconstituted at the four-cell stage from uniparental haploid blastomeres can produce living offspring. This result shows that for normal development to occur, a paternal genome does not need to be associated with a maternal genome within the same nucleus before the eight-cell stage.     

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.     

Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos

The nuclei from four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos. It was found that such embryos not only developed to the blastocyst stage in vitro (72% and 35%), but also developed to full term (22% and 8%) after transfer to recipient mice. However, development of embryos which contained nuclei from the inner cell mass was not observed. Since the development of enucleated zygotes which contain advanced nuclei is limited (the present study; McGrath and Solter: Science, 226:1317-1319, '84; Robl, Gilligan, Critser, and First: Biol. Reprod., 34:733-739, '86), it appears that cytoplasmic factors are important for the development of nuclei from advanced cells.     

Development of enucleated mouse oocytes reconstituted with embryonic nuclei

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.