Coexistence of Bos taurus and B. indicus mitochondrial DNAs in nuclear transfer-derived somatic cattle clones

We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using "amplification refractory mutation system (ARMS) allele-specific real-time PCR." We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of approximately 1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.     

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.     

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.     

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

Background  

The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts.     

Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle

To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2–6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA‐types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast‐cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes. Mol. Reprod. Dev. 54:24–31, 1999. © 1999 Wiley‐Liss, Inc.     

Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.     

Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.     

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.     

Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer

Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes ("therapeutic cloning").     

Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer

In the process of nuclear transfer, heteroplasmic sources of mitochondrial DNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. The distribution of mitochondrial DNA heteroplasmy in nuclear transfer bovine embryos and resultant offspring was investigated by measuring polymorphism in the displacement loop region of mitochondrial DNA using PCR-mediated single-strand conformation polymorphism. Most offspring (20 of 21 calves) from recipient oocytes of undefined mitochondrial DNA genotypes showed different genotypes from the mitochondrial DNA of donor cells. The single calf that was an exception showed heteroplasmy, including the donor mitochondrial DNA genotype. Six cloned calves were produced from oocytes of a defined mitochondrial DNA genotype. All of these clonal members and various tissues showed only the mitochondrial DNA genotype derived from the oocyte. The mitochondrial DNA from donor cells appeared to be eliminated during early embryonic development; it gradually decreased at the early cleavage stages and was hardly detectable by the blastocyst stage. These results indicate that the genotype of mitochondrial DNA from recipient oocytes may become the dominant category of mitochondrial DNA in calves resulting from nuclear transfer.     

Segregation of donor cell mitochondrial DNA in gaur-bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring

The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur-bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Estradiol regulation of luteinizing hormone secretion in heifers of two breed types that reach puberty at different ages

The working hypothesis was that 17 beta-estradiol (E(2)) negative feedback on the hypothalamic-pituitary axis in regulation of LH secretion decreases during peripuberty in heifers of 2 different genotypes. We investigated whether Bos indicus heifers had a period postpuberty, as compared with prepuberty, of greater E(2) inhibition of LH secretion at a time when heifers of this genotype have been reported to have a period of anestrus. Prepubertal heifers 9 mo of age of 2 genotypes (B. indicus and B. taurus) were assigned to 3 groups (6 animals/group) to either remain intact (control), be ovariectomized, or be ovariectomized and implanted with E(2). Variables evaluated from 10 to 28 mo of age were circulating concentrations of progesterone (P(4)), presence of corpora lutea, and pulsatile pattern of LH release. Results confirmed that B. taurus heifers attained puberty at younger ages (P < 0.001) and at lower live weights (P = 0.015) than did B. indicus heifers (507 +/- 37 days of age vs. 678 +/- 7 days of age; 259 +/- 14 kg vs. 312 +/- 11 kg; respectively). There was cessation of E(2) inhibition of LH pulses coincident with the onset of puberty in heifers of both breed types but at a much younger age in B. taurus heifers. There was no evidence of enhanced negative feedback of E(2) on LH secretion subsequent to puberty in B. indicus heifers nor was there cessation of estrous cycles in control heifers of either breed type after puberty.     

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.     

Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus x Bos taurus, and Bos taurus heifers

The objective of this study was to evaluate the effects of treatment with an intravaginal progesterone-releasing device (CIDR) and estradiol benzoate (EB) on follicular dynamics in Bos indicus (n=23), Bos taurus (n=25), and cross-bred (n=23) heifers. To assess the influence of reduced serum progesterone concentrations during 8 days of treatment with a progesterone-releasing device on follicular dynamics, half of the heifers received PGF at CIDR insertion (Day 0; 3 x 2 factorial design). Mean (+/-S.E.M.) serum progesterone concentrations during CIDR treatment varied (P<0.05) among genetic groups: B. indicus (5.4+/-0.1 ng/mL), B. taurus (3.3+/-0.0 ng/mL), and cross-bred (4.3+/-0.1 ng/mL). Maximum diameter of the dominant follicle (DF) was smaller (P<0.01) in B. indicus heifers (9.5+/-0.5 mm) than in cross-bred (12.3+/-0.4 mm) or B. taurus heifers (11.6+/-0.5 mm). B. indicus experienced lower (P<0.01) ovulation rate (39.1%) than did B. taurus (72.7%) and cross-bred (84.0%). Heifers treated with PGF on Day 0 had lower (P<0.05) serum progesterone concentrations during progesterone treatment. The PGF treatment on Day 0 increased (P<0.01) the diameter of the DF (11.9+/-0.4 mm vs. 10.5+/-0.4 mm). Moreover, greater (P=0.02) ovulation rates (78.8 vs. 54.0%) occurred in heifers treated with PGF on Day 0. In summary, B. indicus heifers had greater serum progesterone concentrations, smaller DF diameter, and a lower ovulation rate compared to B. taurus heifers. Prostaglandin treatment on the day of CIDR insertion reduced serum progesterone during treatment, and resulted in increased maximum DF diameter and ovulation rate.     

In vitro culture and mtDNA fate of ibex-rabbit nuclear transfer embryos

Rabbit oocyte can be used as the recipient in interspecies somatic cell nuclear transfer (iSCNT). This work was undertaken in order to study the developmental competence of Capra ibex somatic cells reprogrammed by rabbit oocytes and the fate of mitochondria in iSCNT embryos. Metaphase II (MII) oocytes from superovulated rabbit were used as nuclear recipients. The nuclear donors were Capra ibex somatic cells with different proliferative status: population doubling time (PDL) = 15 +/- 2 (group 1), 35 +/- 2 (group 2), 55 +/- 2 (group 3) and 70 +/- 2 (group 4). Oocytes reconstructed with electrical pulses (2.1kV/cm, 10 micros, 2 times) were activated (1.4kV, 20 micros, 2 times) and then cultured in Medium199 containing 10% fetal bovine serum at 38.5 degrees C, 5% CO2 in air. In groups 1, 2, 3 and 4, the fusion rates were 35.83%, 66.03%, 65.40% and 35.35%, respectively. Similar cleavage rates were observed among the four groups. However, the developmental potential to morula/blastocyst from early nuclear donor embryos (16.42%/10.45%) was significantly higher (p < 0.05) than in terminal donor embryos (9.52%/3.81%). Polymerase chain reaction analysis of the mitochondrial (mt) DNA cytb gene demonstrated that mtDNAs from ibex and rabbit could be detected at various developmental stages before implantation. In conclusion, our results provide some original information about rescuing Capra ibex using the iSCNT technique. These results indicate that: (1) enucleated rabbit oocytes make Capra ibex fibroblast nuclei reprogramme; (2) the proliferative status of donor cells affects the efficiency of iSCNT; and (3) rabbit ooplasm rescues the donor-derived mtDNAs, resulting in mtDNA heteroplasmy before implantation.     

Bovine mtDNA D-loop haplotypes exceed mutations in number despite reduced recombination: an effective alternative for identity control

Mitochondrial (mt) DNA D-loop heterogeneity, haplotype distribution and possible sub-population structures within the relevant populations are important for DNA-based traceability. To gain insight into this distribution, we compared 1515 Bos taurus mtDNA D-loop sequences available from GenBank to 219 sequences that we sequenced de novo. A pronounced ambiguous trace typical of C-track length heteroplasmy was encountered in 5% of the samples, which were excluded from the analysis. Previously undescribed mutations and haplotypes were observed in 6% and 63% of the sequences, respectively. B. taurus haplotypes divided into the taurus, indicus and grunniens types and 302 variable sites formed the 858 taurus haplotypes detected. Fifty-five sites displayed a complex level of variation. As each level represents an independent mutation event, a total of 399 mutations were traced, which could potentially explain independent formation of less than half (47%) of the haplotypes encountered: most haplotypes were derived from different combinations of these mutations. We suggest that a mutational hotspot may explain these results and discuss the usefulness of mtDNA for identity and maternity assurance.     

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

Background  

The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.     

Tissue-specific distribution of donor mitochondrial DNA in cloned mice produced by somatic cell nuclear transfer

Highly diverse results have been reported for mitochondrial DNA (mtDNA) hetero-plasmy in nuclear-transferred farm animals. In this study, we cloned genetically defined mice and investigated donor mtDNA inheritance following somatic cell cloning. Polymerase chain reaction (PCR) analysis with primers that were specific for either the recipient oocytes or donor cells revealed that the donor mtDNA coexisted with the recipient mtDNA in the brain, liver, kidney, and tail tissues of 96% (24/25) of the adult clones. When the proportion of donor mtDNA in each tissue was measured by allele-specific quantitative PCR and subjected to ANOVA analysis, a tissue-specific mtDNA segregation pattern (P < 0.05) was observed, with the liver containing the highest proportion of donor mtDNA. Therefore, the donor mtDNA was inherited consistently by the cloned offspring, whereas donor mtDNA segregation was not neutral, which is in accordance with previous notions about tissue-specific nuclear control of mtDNA segregation.