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.     

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.     

Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells

This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%). Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.     

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.     

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.     

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: Effect on developmental potential

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm. Mol. Reprod. Dev. 48:332–338, 1997. © 1997 Wiley-Liss, Inc.     

The monitoring of bovine pregnancies derived from transfer of in vitro produced embryos

Both an increased rate of embryonic, foetal and perinatal losses, and the occurrence of deviations in foetal and placental development are associated with bovine pregnancies obtained from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of foetal and maternal functions during pregnancies. Such approaches will enable to establish the period during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF embryos.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.     

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.     

The influence of nuclear content on developmental competence of gaur x cattle hybrid in vitro fertilized and somatic cell nuclear transfer embryos

In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle x cattle, gaur x cattle, hybrid x cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.     

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.     

Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum-starved skin cells were electrofused to enucleated oocytes, activated 1-5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1-2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6-56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus-like structure (PN), whereas, 64.5-78.3% of embryos activated 1-2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3-21.7%) compared to those of others (0-8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos.     

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.