Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.     

Developmental kinetics of bovine nuclear transfer and parthenogenetic embryos

Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.     

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.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.     

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.     

Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.     

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.     

Nuclear transfer in the bovine embryo: a comparison of 5-day, 6-day, frozen-thawed, and nuclear transfer donor embryos

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.     

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.     

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.     

Porcine sperm factor supports activation and development of bovine nuclear transfer embryos

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca(2+)](i)) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca(2+)](i) and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca(2+)](i) oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60-90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3-5 h postinjection and a second injection was necessary to extend the [Ca(2+)](i) oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.     

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.     

Telomerase activity in early bovine embryos derived from parthenogenetic activation and nuclear transfer

This study examined the telomerase activity in preimplantation bovine embryos derived from either parthenogenetic activation or nuclear transfer. Telomeres are the DNA-protein structures located at the ends of eukaryotic chromosomes. Telomerase is the ribonuclear enzyme that helps to restore telomere length by synthesizing telomeric DNA repeat (5'-TTAGGG-3') from its own RNA template. Without telomerase activity, telomeres shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Previously, we reported the dynamics of telomerase activity in bovine in vitro fertilized (IVF) embryos. In the present study, we examined the telomerase activity in bovine embryos derived either from parthenogenetic activation or somatic cell nuclear transfer (i.e., cloning). Embryos from both sources were harvested at different stages, from zygote to blastocyst. Telomerase activity in embryos derived from parthenogenetic activation and nuclear transfer showed a dynamic profile similar to that of those derived from IVF. Telomerase activity was detected in embryos at all stages examined, with the highest level in the blastocyst stage, regardless of the method of embryo production.