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Reproductive Technology |
a Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, D-85764 Oberschleissheim, Germany
b Department of Animal Breeding and Genetics and Central Biotechnical Unit Strahlenzentrum, Justus-Liebig-University, D-35392 Giessen, Germany
c Bavarian Research Center for Biology of Reproduction (BFZF), D-85764 Oberschleissheim, Germany
d Bavarian Research Station for Animal Breeding, D-85586 Grub, Germany
e Agrobiogen GmbH, D-86567 Hilgertshausen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.
assisted reproductive technology, developmental biology, embryo, placenta, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is different from the situation encountered in embryos, fetuses, and offspring reconstructed by nuclear transfer and related techniques, in which mtDNA introduced into the recipient oocyte by nuclear donor cell cytoplasm can be replicated [10–19]. The amounts of nuclear donor mtDNA detected in mouse embryos and mice produced by karyoplast transfer [10–13] have suggested a species-specific [10] or general preferential replication of nuclear donor mtDNA [11] and have shown strong tissue-specific and age-related directional selection of specific mtDNA genotypes [12]. More recently, a replicative advantage and tissue-specific segregation of one mtDNA genotype, regardless of nuclear background, was observed in heteroplasmic mice produced by cytoplast transfer [20].
Data concerning mitochondrial genotypes of somatic cell nuclear transfer animals are limited and controversial [18, 21], and to our knowledge, specifically designed experiments with defined mtDNA types have not been performed. Varying levels of heteroplasmy have been described in bovine embryos [14, 17, 19], a fetus [19], and offspring [15–17, 19] produced by cytoplast-blastomere fusion. The degree of heteroplasmy in some of these cloned cattle [15, 16] has, reminiscent of the situation in mice, suggested a replicative advantage of certain mtDNA genotypes, perhaps caused by nuclear DNA-mtDNA interactions in specific combinations of mitochondrial and nuclear genomes [16].
To address these points, we generated transmitochondrial clones by fusing Bos taurus cumulus cells (mtDNA type A) with oocytes from the same (mtDNA types B and C) or a different B. primigenius subspecies (B. indicus) [22]. Reconstructed embryos were transferred to B. taurus recipient heifers with known mtDNA genotype, and Day 80 fetuses were recovered and analyzed for mtDNA heteroplasmy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and mtDNA Genotypes
Nuclear donor granulosa cells were obtained from a Braunvieh cow with B. taurus mtDNA genotype A. Oocytes with B. taurus mtDNA genotype B or C were collected from two groups of maternally related Fleckvieh cows and heifers by ultrasound-guided follicle aspiration, ovum pick-up (OPU). The genotypes A, B, and C were assigned arbitrarily to distinguish among the different B. taurus mitochondrial genomes used in the present study. The B. indicus oocytes were recovered from ovaries of slaughtered Zwergzebu cattle. Fleckvieh heifers with B. taurus mtDNA genotype B were used as recipients.
Different mitochondrial DNA genotypes were initially identified by NlaIII (B. taurus lineages) or BstNI and DdeI (B. indicus lineage) restriction fragment length polymorphism (RFLP) analysis (see below). For subsequent sequence analyses, the complete mtDNA (CR) was amplified from total genomic DNA isolated from blood leukocytes by standard proteinase K/phenol-chloroform procedures [23] with the primers mtDA (5'-CTCACCATCAACCCCCAAAGCT-3') and mtDB (5'-TCATCTAGGCATTTTCAGTG-3') using Pwo Polymerase (Hybaid, Heidelberg, Germany) as described previously [16]. Both strands of two clones, each derived from independent polymerase chain reactions (PCRs) of a single individual, were sequenced by standard procedures on a LICOR 4200 (MWG Biotech, Ebersberg, Germany). Nucleotide sequence data for mtDNA genotypes B. taurus A, B, and C and B. indicus have been submitted to GenBank with accession no. AF492432, AF386912, AF386913, and AF492437, respectively.
Before oocyte collection, cytoplasmic genetic identity of all individuals in the two B. taurus oocyte donor lineages was confirmed, as described previously [24], by PCR-RFLP analysis of the CR using diagnostic NlaIII restriction sites. Mitochondrial genotype of recipient heifers was confirmed in an identical manner. The B. indicus oocyte donors were verified by digesting CR PCR product with BstNI and DdeI according to the manufacturer's recommendations (New England Biolabs GmbH, Frankfurt, Germany). The two enzymes recognize restriction sites that discriminate between B. taurus and B. indicus mtDNA (Fig. 1).
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Oocyte Recovery and In Vitro Maturation
Ovaries of Zwergzebu heifers and cows with B. indicus mtDNA were collected during slaughter in different locations and transported (0.5–2.5 h) to the laboratory in PBS at 25–30°C. Cumulus-oocyte complexes (COCs; n = 84) were obtained by aspiration and slicing of ovaries.
The COCs with B. taurus mtDNA A (n = 968) or B (n = 374) were collected by OPU as described previously [24]. The collection medium consisted of 98 ml of Tyrode lactate solution buffered with 10 mM Hepes supplemented with 2 ml of fetal calf serum (FCS; heat-inactivated; Life Technologies, Karlsruhe, Germany) and 6 mg of sodium heparin (175.5 IU; Kraeber, Hamburg, Germany). Collected COCs were washed with PBS enriched with FCS (5 ml/L) and transferred into collection medium without sodium heparin but supplemented with 10% (v/v) FCS for transport. Oocytes were kept in this medium at room temperature for approximately 1 h.
All recovered oocytes were used for maturation without any preselection. The COCs were washed three times in the culture-medium TCM 199 (Seromed, Berlin, Germany) supplemented with L-glutamine (100 mg/L), NaHCO3 (3 g/L), Hepes (1400 mg/L), pyruvate (250 mg/L), L-lactic-Ca-salt (600 mg/L), and gentamicin (55 mg/L; Seromed). Oocytes were transferred to four-well plates (Nunc, Roskilde, Denmark) with 400 µl of TCM 199 containing 0.01 U of b-FSH and b-LH (Sioux Biochemical, Sioux Center, IA) supplemented with 10% estrous cow serum (ECS). After 18 h of maturation at 39°C in an atmosphere of 5% CO2 and maximum humidity, oocytes were exposed for 5 min to modified PBS (mPBS; PBS plus 3 mg/ml of bovine serum albumin) containing 3 mg/ml of hyaluronidase, vortexed for 4 min, and stripped by gentle pipetting. Maturation rate for B. indicus oocytes and for B. taurus oocytes with mtDNA genotypes B and C were 48%, 53%, and 59%, respectively.
Karyoplasts
Granulosa cell primary cultures were established from a 12-yr-old Braunvieh cow. Cells obtained after OPU were washed twice in saline solution and dispersed by exposure to 0.1% (w/v) trypsin (Gibco, Grand Island, NY). The cell suspension was then transferred into 5-cm culture dishes containing Dulbecco modified Eagle medium (Gibco) supplemented with 10% (v/v) FCS (Biochrom, Berlin, Germany), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 2 mM nonessential amino acids, 100 IU/ml of penicillin, and 100 µg/ml of streptomycin. The cells were cultured until subconfluence at 37°C in a humidified atmosphere of 5% CO2 in air and then frozen in 10% (v/v) dimethyl sulfoxide in FCS and stored in liquid nitrogen. For experiments, the cells were thawed and cultured for three to six passages. All viable fetuses and one fetus in resorption were obtained using nuclear donor cells after serum starvation. To initiate serum starvation, the culture medium was removed 1 day after passaging. Cells were washed with PBS, and fresh medium containing 0.5% (v/v) FCS was added. The cells were then cultured for another 4–7 days before use in nuclear transfer experiments. One fetus in resorption was produced with a nonstarved nuclear donor cell from a culture that had been grown to subconfluence.
Nuclear Transfer
All matured oocytes were used for nuclear transfer without further selection. The nuclear transfer procedure was essentially as described previously [25] but with minor modifications. Briefly, oocytes were exposed for 15 min to 5 µg/ml of cytochalasin B (Sigma) and 5 µg/ml of Hoechst 33342. The metaphase chromosomes were removed within 1 h in mPBS containing 5 µg/ml of cytochalasin B. Oocytes were visualized under an epifluorescence microscope (Zeiss, Jena, Germany) to confirm the absence of chromatin. Single donor cells were transferred into the perivitelline space of enucleated oocytes at 19–21 h after initiating maturation, and the karyoplast-cytoplast complexes (KCCs) were exposed to a double electric pulse of 2.1 kV/cm for 10 µsec using the Zimmermann Cell Fusion Instrument (Bachofer, Reutlingen, Germany). The KCCs were placed in the incubator in Ham F-12 medium supplemented with 0.3% (v/v) bovine serum albumin.
Activation and Embryo Culture
At 23–24 h after in vitro maturation (2 h postfusion), the fused KCCs were activated by initiating a 5-min incubation in 7% (v/v) ethanol followed by 5-h culture in 10 µg/ml of cycloheximide and 5 µg/ml of cytochalasin B [26]. Activated embryos were washed three times in culture medium before transfer into 100-µl drops of synthetic oviductal fluid medium [27] supplemented with 2% (v/v) BME amino acids (Gibco), 1% (v/v) MEM nonessential amino acids (Gibco), and 10% (v/v) ECS; covered by paraffin oil (Merck, Darmstadt, Germany); and then cultured at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.
Embryo Transfer
At Day 7 after nuclear transfer, embryos were transferred to recipient heifers as described previously [24].
Determination of Nuclear Donor Cell and Recipient Cytoplast Cytoplasmic Volumes
Trypsinized nuclear donor cells (n = 30) were stained with 2 µg/ml of 33342 Hoechst dye, and the diameters (2r) of cells and nuclei were measured with an ocular scale. Assuming a perfectly spherical cell and nucleus, volumes were calculated as 4/3
r3 and subtracted to derive nuclear donor cell cytoplasmic volume. The diameter and volume of cytoplasts (n = 30) were also determined with the aid of an ocular scale.
Recovery of Fetuses and Collection of Tissue Samples
Recipient heifers diagnosed as pregnant were killed in a local slaughterhouse on Day 80 after nuclear transfer by standard procedures. The uterus was transferred to a clean tray, and the fetus was removed for sample collection. Precautions were taken to prevent contamination of the fetus with recipient tissue. Fetal blood was either collected directly from the umbilical cord with a syringe or the umbilical cord was cut, the blood collected in a sterile kidney dish, and then transferred with a syringe. Tissue samples (200 mg) were obtained from fetal skin, biceps femoris muscle, brain, lung, heart, rumen, jejunum, liver, spleen, kidney, and from a cotyledon. Samples were frozen at -20°C until further processing.
DNA Isolation and mtDNA Genotype Analysis of Fetal Tissue
Total cellular DNA was isolated from tissue samples and samples of nuclear donor cells with the E.Z.N.A. Tissue DNA Kit II (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The DNA from blood was extracted by the standard proteinase K/phenol-chloroform procedure [23]. The DNA extractions were performed in a laminar flow hood on tissues of a single fetus at a time, with extensive cleaning and ultraviolet irradiation between DNA extractions from tissues of different fetuses. The mtDNA CRs were PCR amplified in a volume of 25 µl as described above, and 10 µl of the PCR product were digested with 16 U of NlaIII as recommended by the manufacturer (New England Biolabs GmbH). The NlaIII restriction fragment patterns, which discriminate between all four investigated mtDNA genotypes, were analyzed in 2% agarose (BIOzym Diagnostic GmbH, Hessisch Oldendorf, Germany) gels using Tris-borate-EDTA (TBE) buffer according to standard procedures [23].
To determine the sensitivity of the agarose gel assay for heteroplasmic mtDNA fragments, PCR product of B. taurus mtDNA genotype A was mixed with PCR product of B. taurus mtDNA genotype B in proportions ranging from 2.5% to 40%, showing that the assay conditions could consistently detect heteroplasmy at a proportion of >2% (see Fig. 4c).
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A more sensitive detection method involving RFLP analysis of cloned CR PCR fragments [16, 21] was performed on tissues of a fetus reconstructed from an oocyte with B. indicus mtDNA. The PCR products were purified from 1% agarose gels with the Qiaex II Kit (Qiagen, Hilden, Germany) and cloned into the plasmid pCR-TOPO-BluntII with the TOPO-BluntII cloning Kit (Invitrogen, Groningen, The Netherlands). Plasmid DNA for RFLP analysis of cloned CR fragments was prepared as described previously [16]. Restriction enzyme digestion of plasmid DNA with BstNI was performed as recommended by the manufacturer (New England Biolabs GmbH). The resulting restriction fragments were separated in 1.6% agarose gels using TBE buffer according to standard procedures [23]. To preclude preferential amplification or cloning of recipient oocyte B. indicus mtDNA over nuclear donor cell B. taurus mtDNA, equal amounts of total cellular DNA of individuals with B. taurus mtDNA and B. indicus mtDNA were mixed and used for PCR amplification and cloning of fragments containing the CR. The RFLP analysis of 96 plasmid clones from this experiment did not show evidence for preferential amplification or cloning of B. indicus mtDNA.
Verification of Nuclear Clonal Identity of Fetuses by Microsatellite Analyses
The DNA samples of recovered fetuses were subjected to microsatellite analyses with a panel of 13 standardized microsatellites recommended by the International Society for Animal Genetics (ISAG) for parentage control in cattle.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean cytoplasmic volume of the granulosa cells used as nuclear donors was 5.1 ± 0.8 µm3 (mean ± SEM). The volume of cytoplasts was 619.1 ± 92.0 µm3. Nuclear donor cell contribution to the cytoplasm of reconstructed embryos is therefore in the range of 0.8%.
Mitochondrial DNA Genotype Differences of Nuclear Donor Cells, Oocytes, and Recipient Heifers
A comparison of the complete CR sequences of all four mitochondrial genotypes used in the experiments revealed extensive sequence polymorphism (Fig. 2). Overall, 56 variable nucleotide positions were observed in a total of 914 nucleotides. These consisted of 6 insertions/deletions, 49 transitions, and 1 transversion. Three of the insertions/deletions were found in a poly-cytosine tract at the 3' end of the CR, a region prone to replication slippage. Sequence differences among B. taurus mtDNA genotypes ranged from 6–11 nucleotides (0.7–1.2%) and between B. taurus and B. indicus mtDNA genotypes from 45–50 nucleotides (5.0–5.5%). Sequence polymorphisms are present in several major mtDNA regulatory elements, such as the conserved sequence block (CSB) 1, CSB 2+3, the D-loop strand termination signal, a termination-associated sequence, and in the H-strand origin of replication and the L-strand promoter sequences (Fig. 2).
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Mitochondrial DNA Genotype of Transmitochondrial Cloned Fetuses
Twelve reconstructed Day 80 fetuses were recovered in total. Nuclear clonal identity of fetuses was confirmed by multiplex microsatellite analyses (Fig. 3a). Ten fetuses with the mtDNA combinations B. taurus A/B (n = 7), B. taurus A/C (n = 1), and B. taurus A/B. indicus (n = 2) were intact, without any obvious abnormalities. Two fetuses with the mtDNA combinations B. taurus A/B (n = 1) and B. taurus A/C (n = 1), however, were in resorption. Agarose gel analysis of PCR-amplified and NlaIII-restricted CRs failed to detect nuclear donor B. taurus mtDNA type A in DNA isolated from skin, muscle, brain, lung, heart, rumen, jejunum, liver, spleen, kidney, and cotyledon tissue samples of all intact fetuses. The mtDNA CR fragment patterns obtained for heart samples are shown in Figure 3b. The DNA samples obtained from the two fetuses in resorption were also homoplasmic for recipient oocyte mtDNAs (data not shown). In contrast, DNA extracted from blood samples of two fetuses with the mtDNA combinations B. taurus A/C and B. taurus A/B. indicus showed clear evidence for a heteroplasmic condition (Fig. 4, a and b). This status was confirmed in additional freshly prepared DNA samples, PCR amplifications, and restriction enzyme digestions with considerable enzyme surplus. Unexpectedly, however, the additional, less abundant mtDNA genotype did not resemble nuclear donor cell B. taurus mtDNA type A (Fig. 4a) but, rather, recipient heifer mtDNA type B (Fig. 4, b and d). The amount of recipient heifer mtDNA in blood of the two fetuses was estimated at approximately 2.5% in the B. taurus A/B. indicus and approximately 5% in the B. taurus A/C fetus based on comparisons with mixed mtDNA samples (Fig. 4c).
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Because the sensitivity of the agarose gel analyses was limited to the detection of >2% of nuclear donor mtDNA, a more sensitive assay for the detection of very low levels of nuclear donor cell mtDNA was employed. This involved PCR amplification and cloning of CRs from tissues of a fetus reconstructed with a B. indicus oocyte. The RFLP analysis of 1801 plasmid clones with BstNI, which discriminates between B. taurus and B. indicus mtDNA (Figs. 1 and 5), detected either no (skin, lung, heart, rumen, spleen, and kidney) or very low levels (0.5–0.7%) of heteroplasmy in the investigated tissues with the exception of blood, in which a relatively high proportion of B. taurus mtDNA (2.0%) was found. These results confirmed the agarose gel data, because the fetus investigated in this manner was identical to the B. taurus A/B. indicus fetus that had previously shown recipient heifer-derived heteroplasmy in blood at an estimated proportion of approximately 2.5%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found no evidence for nuclear donor cell mtDNA contributions in a wide range of fetal and extraembryonic tissues of 10 intact fetuses produced by intra- and inter-subspecific nuclear transfer with an assay method that detected heteroplasmy at a level of >2%. However, a more sensitive analysis of tissues from a fetus reconstructed with a B. indicus oocyte and B. taurus nuclear donor cell revealed very low amounts of B. taurus mtDNA in several tissues, whereas other tissues lacked evidence for nuclear donor mtDNA. The observed proportion of nuclear donor mtDNA in six fetal tissues (0.5–0.7%) is very close to the detection limit of the assay and is similar to the amount of cytoplasm calculated to be contributed to reconstructed embryos by the somatic cell (0.8%). In combination with the PCR-RFLP data, this excludes an advantage for donor mtDNA and suggests neutral segregation of nuclear donor mtDNA even in inter-subspecific bovine somatic cell clones with extensive mtDNA sequence differences.
Two previous studies concerning somatic cell nuclear transfer animals either consistently detected no [21] or very low levels [18] of heteroplasmy. One study investigated 10 cloned sheep and failed to detect nuclear donor cell mtDNA at a detection limit of 0.5–0.1% in one or two tissues analyzed per individual [21]. Another study on a broader range of tissues from up to six B. taurus cattle fetuses (Days 60–212) and four calves revealed low nuclear donor cell mtDNA proportions (0.1–4%) in most investigated individuals and tissues [18]. The present data indicate that the different results obtained in previous studies could be caused by the numbers and types of tissues sampled or by the age of the investigated individuals. In addition, donor cell type and treatment or nuclear transfer protocol have also been proposed to account for differing results in sheep and cattle clones [18]. However, our results using fetuses with defined mtDNA genotype and previously obtained data [18] clearly show that somatic cell nuclear donor mtDNA is not, as suggested [21], generally eliminated in bovine embryos; rather, it is present in at least some fetal tissues in proportions reflecting the amount of cytoplasm contributed by the nuclear donor. This also corroborates data obtained for the majority of previously investigated blastomere-cloned B. taurus embryos and calves [14–16]. Considering the very low degree of heteroplasmy observed in somatic clones, it is nevertheless possible that nuclear donor mtDNA would be undetectable in offspring [21] because of loss of the rare mtDNA genotype by genetic drift.
Homoplasmy (detection limit, <1%) has recently also been reported for several tissues of a calf obtained by blastomere cloning using a B. taurus oocyte and B. indicus nuclear donor, although a Day 55 fetus of the same combination showed heteroplasmy in embryonic (0.6–3.6%) and extraembryonic (0.9–4.7%) tissues [19]. This is the reciprocal combination of donor nucleus and cytoplast of the two inter-subspecific fetuses reconstructed by somatic cell nuclear transfer in the present study. As in the present study, the average amount of nuclear donor cell-derived mtDNA in the blastomere-cloned fetus was similar to that introduced at nuclear transfer into the oocyte. These results indicate a general, nontissue-specific, neutral segregation of mitochondrial DNA genotypes in intra- and inter-subspecific bovine nuclear transfer fetuses and offspring.
The apparently neutral segregation of nuclear donor mtDNA observed in most nuclear transfer bovines contrasts sharply with data reported for mouse embryos and mice produced by karyoplast transplantation or related techniques [10–12, 20]. Here, species-specific [10] or general preferential amplification of nuclear donor mtDNA [11], a tissue-specific selection of mtDNA genotypes [12], and a general replicative advantage of a particular mtDNA genotype [20] have been described. Such effects are apparently not present in intra- or even inter-subspecific bovine nuclear transfer individuals, although nucleotide sequence differences in the mtDNA CR, including important regulatory elements, of B. taurus and B. indicus subspecies of cattle are similar to or even surpass the differences between the species/subspecies and strains used in mouse experiments [10, 12, 20]. However, reproductive techniques such as karyoplast or cytoplast transfer, which have been employed to investigate mtDNA segregation in mice (e.g., [11, 20]), differ considerably from nuclear transfer protocols employed in bovine (e.g., in donor and recipient cell types and the ratio of combined cytoplasms). Data obtained concerning mtDNA segregation in mice therefore might not be directly comparable to nuclear transfer ruminants.
There have nevertheless been reports of occasional, striking deviations from the segregation pattern generally observed in bovine clones, which are reminiscent of the nonneutral mtDNA segregation observed in mice [15, 16, 19]. This could reflect abnormal segregation in cloned cattle, and it has been suggested that mtDNA heteroplasmy might be responsible for the high death rate in nuclear transfer individuals [6]. Two of our fetuses that were in resorption, however, showed mtDNA homoplasmy. Heteroplasmic mice that display a tissue-specific selection for a particular mtDNA genotype in vivo [12] have recently been shown to reverse this selection in cultured cells in vitro [28]. It is therefore notable that we have not observed heteroplasmy in cardiac cells or skin fibroblasts from individuals with B. taurus mtDNA A/B or B. taurus mtDNA A/B. indicus mtDNA after two to three passages (data not shown).
To our knowledge, the present study is the first regarding mtDNA inheritance and segregation in nuclear transfer animals in which all involved mtDNA genotypes (nuclear donor cells, recipient oocytes, and recipient heifers) are known. Failure to control for mtDNA genotype could be responsible for some previously reported discordant results concerning mtDNA segregation in bovine nuclear transfer clones [15–17]. Recipient oocytes for cloning are usually collected from ovaries of slaughtered cattle with unknown mtDNA genotype, and only very recently have oocytes with defined mtDNA genotype been used in cloning experiments [24]. It is also common practice to transfer more than one cloned embryo to recipient cattle [29–32]. Some of the previously observed heteroplasmy in DNA samples extracted from blood of nuclear transfer bovines [15–17] could therefore actually have been caused by chimerism in the hematopoietic system because of fusion of the chorioallantois and placental vascular anastomoses, which is encountered in >90% of calves from multiple births [33].
One of the most interesting findings of the present study is the detection of recipient heifer mtDNA in DNA samples extracted from blood of two fetuses (both singletons), pointing to leakage of the placental barrier and mimicking heteroplasmy. This might be even more common, because in the present study, mtDNA genotype combinations of only three fetuses and heifers would have enabled detection of this phenomenon. Placental abnormalities that could cause leakage of blood cells have recently been reported in cloned animals, including improper vascular development and hemorrhagic cotyledons [34, 35]. Heteroplasmy derived from the recipient heifer could explain the unexpected mtDNA genotypes detected in three heteroplasmic cloned calves of a previous investigation [17], in which the nuclear donor mtDNA genotype was known but did not match the second mtDNA genotype in the calves. Two of these calves were from the same pregnancy and showed exactly the same single strand conformation polymorphism (SSCP) band patterns [17].
In conclusion, our data indicate that previous results on mtDNA heteroplasmy and segregation in nuclear transfer animals should be treated with caution if the mtDNA genotype was either not or only partly controlled [15–17]. Mitochondrial DNA in cattle has been associated with several traits of economic importance, including milk fat yield [36], carcass composition [37], fertility [38], and nuclear transfer efficiency [24]. The present data concerning nuclear transfer-induced heteroplasmy in intra- and inter-subspecific bovine clones shows that proposed studies on mtDNA effects in transmitochondrial somatic cell-cloned cattle will not be compromised by heteroplasmy [24]. Such studies will further clarify the role of cytoplasmic genetic components on the efficiency of nuclear transfer and on growth and metabolism of the resulting fetuses and offspring.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stefan Hiendleder, Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University Munich, Hackerstrasse 27, D-85764 Oberschleissheim, Germany.FAX: 49 89 315 2799; s.hiendleder{at}gen.vetmed.uni-muenchen.de
Received: 7 June 2002.
First decision: 26 June 2002.
Accepted: 16 July 2002.
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