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.     

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.     

克隆动物研究进展

本文介绍了世界上和中国采用细胞核移植技术克隆动物的研究历史。综述了细胞核移植的程序、方法和影响因素,包括受体卵母细胞的去核、供体细胞核的制备、核移植、激活、受体细胞与供体细胞的融合、重组胚的体内和体外培养以及胚胎移植产生克隆动物。对克隆动物研究和应用前景进行了讨论。近期的研究结果表明,多代克隆可产生大量遗传性相同的动物,不久的将来克隆技术在商业上的应用将成为现实。     

Two-staged nuclear transfer can enhance the developmental ability of goat�Csheep interspecies nuclear transfer embryos in vitro

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.     

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, gamma-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. gamma-tubulin translocated into the two poles of the transient spindles, while no accumulated gamma-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, gamma-tubulin was translocated to the spindle poles. The distribution of gamma-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. gamma-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.     

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.     

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos.     

核移植后的细胞核再程序化机制研究进展

目前动物克隆技术体系极待完善,其极低的成功率及克隆动物普遍存在的早衰、早天现象是阻碍研究深入进行的首要问题,其突破的关键在于对核移植后的细胞核再程序化机制的阐明。从移植核在结构上的重塑、移植核与受体卵细胞质所处的细胞周期及其相互作用、重构胚与两性胚在分子水平的变化等多方面研究表明：受体细胞质的环境对于细胞核的再程序化至关重要,处于有丝分裂各时期的细胞作为核供体一旦移植到卵母细胞后,移植核在卵质环境里将出现结构上的重塑和分子的再程序化;移植核与受体卵问细胞周期的相容性、重构胚的染色体倍性的正确与否,可能是决定重构胚发育率高低的重要因素;合子型基因激活是基因表达再程序化的关键事件之一;印记基因对于体细胞克隆动物移植核的再程序化过程可能起着非常独特的作用。     

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

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

Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine

The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M-phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28-day fetuses and cultured for 1-30 days prior to NT. Fibroblast cultures were treated with 0.05 microg/ml of demecolcine for 3 hr or overnight (14-16 hr) after various times in culture to determine the optimal conditions for M-phase synchronization. The percentage of G2/M-phase cells in demecolcine-treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M-phase fibroblasts were collected by mitotic shake-off and evaluated for their effectiveness in NT. M-phase somatic cell-derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M-phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis.     

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.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.     

Full-term development of mouse eggs transplanted with male pronuclei derived from round spermatids: the effect of synchronization between male and female pronucleus on embryonic development

Pronucleus transplanted mice have been produced, but their donor male pronuclei were derived from mature sperm and were completely synchronous with female pronuclei because both male and female pronuclei came from the same fertilized oocyte. The present study firstly produced male pronuclei by introducing round spermatids into enucleated mouse oocytes, then transferred the male pronuclei into mouse oocytes at three activation stages and finally compared the effect of three kinds of oocytes on the development of reconstructed embryos. Our results indicate that, in enucleated oocytes, mouse round spermatid nuclei can transform to male pronuclei in a higher proportion, and the synchronization between male and female pronucleus does not significantly influence the early cleavage but the later and full-term development of reconstructed embryos.     

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

Background

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

Results

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

Conclusions

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

Oocyte mitochondria: Strategies to improve embryogenesis

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

Aberrant heteroplasmic transmission of mtDNA in cloned pigs arising from double nuclear transfer

Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.     

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho⁺-Hd⁻) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho⁻-Hd⁺) or non-heated (Ho⁻-Hd⁻) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6-32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9-33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.