Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.     

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.     

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post co?tum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.     

Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer

Parthenogenetic development (PA) is often used as a model to investigate activation protocols for nuclear transfer (NT) embryos. The objective of this study was to compare the development, as well as the dynamics of the nuclear materials and microtubules of PA and NT embryos following similar activation treatment. Our results demonstrate that, during parthenogenesis, activation through either electrical pulses or chemical stimulation alone resulted in low cleavage rates and compromised development. A combination of two sets of electrical pulses and a 2-h-exposure to chemical activation medium (5 microg/ml cycloheximide (CHX) and 2 mM 6-dimethylaminopurine (6-DMAP) in KSOM+0.1% BSA) could effectively activate rabbit oocytes, and resulted in a 99% (n = 73) cleavage rate with greater than 60% (n = 73) developing to blastocysts at day 4. However, the same activation protocol following NT resulted in only 65-72% of oocytes cleaved (depending on donor cell type), with less than 20% developing to the blastocyst stage. The differences observed between NT and PA embryos subjected to the same activation protocol were also evident in terms of the time required for their development to the blastocyst stage, as well as the cell numbers present in blastocysts at day 6. Furthermore, laser confocal microscopy revealed that pronuclear formation in the NT embryos was delayed by comparison to that in the parthenotes. In conclusion, our study suggests that an effective protocol for parthenogenesis cannot promise a comparable outcome for NT embryos.     

Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.     

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.     

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.     

Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer

Epigenetic modifications of the genome play a significant role in the elaboration of the genetic code as established at fertilisation. These modifications affect early growth and development through their influence on gene expression especially on imprinted genes. Genome-wide epigenetic reprogramming in germ cells is essential in order to reset the parent-of-origin specific marking of imprinted genes, but may have a more general role in the restoration of totipotency in the early embryo. In a similar way, on somatic nuclear cloning, a differentiated cell must become 'reprogrammed' restoring totipotency in order to undergo development. Here we discuss the dynamic epigenetic reprogramming that takes place during normal development and highlight those areas with relevance to somatic nuclear cloning and the possibility of improving the efficiency of this process. We propose the concept of 'epigenetic checkpoints' for normal progression of development and the loss of totipotency.     

Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2, 3, 4, or 5 (day 0 = day of breeding) can be transferred with +/- 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2, 2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P less than .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P less than .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral environment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P less than .01) and young born (56% vs. 19%, P less than .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Improved postimplantation development of rabbit nuclear transfer embryos by activation with inositol 1,4,5-trisphosphate

Cloned rabbit embryos are characterized by their extremely poor postimplantation development, despite their high survivability until the blastocyst stage in vitro. This study examined whether the developmental failure of cloned rabbit embryos in vivo can be overcome by technical improvements to the activation protocol. Freshly collected cumulus cells were transferred into enucleated oocytes by intracytoplasmic injection. One to two hours later, the oocytes were activated by electroporation with Ca(2+) or inositol 1,4,5-trisphosphate (IP3), which is known to induce repeated rises in intracellular Ca(2+), as in normal fertilization. After transfer of embryos at the two- to four-cell stages, well-defined implantation sites with remnant fetal tissue were observed at term (day 28) only in the IP3-stimulation groups (0.9% and 5.8% per transferred embryo for single and triple stimulation groups, respectively). When some recipients in the same group were examined at days 16-20, a viable cloned fetus (day 19) with normal organogenesis was obtained. These findings clearly demonstrate that the oocyte activation protocol using IP3 enhances the postimplantation development of nuclear-transferred rabbit embryos.     

Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro

Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to demecolcine at various times post-activation. Onset and duration of demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in demecolcine-induced enucleation.     

In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal

Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.     

Optimisation of porcine oocyte activation following nuclear transfer

Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development of the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca(2+)-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 micrograms/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.     

Adult murine neurons: their chromatin and chromosome changes and failure to support embryonic development as revealed by nuclear transfer

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered.     

Development of pig embryos by nuclear transfer of cultured fibroblast cells

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