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Regular Article |
a Animal Resources Research Center, Konkuk University, Kwangjin-gu, Seoul 143-702, Korea
b Infertility Clinic, Department of OB & GYN, Kyung Hee University Medical Center, Dongdaemun-gu, Seoul 130-702, Korea
c Catholic Institutes of Medical Science, The Catholic University of Korea, Seocho-gu, Seoul 137-701, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There have been several reports on the inheritance of mitochondria and mtDNA following NT or oocyte manipulation. Cloned sheep produced by somatic-cell NT have inherited their mitochondria entirely from the oocyte and not from the donor cell [2]. In NT using blastomeres, both homoplasmy [3] and heteroplasmy [4] of mtDNA were examined. Recently, Steinborn et al. [5] reported mtDNA heteroplasmy in cloned cattle generated from fetal and adult donor cells. These authors also reported that the donor-to-recipient ratios of parental mtDNA remained the same throughout embryogenesis and postembryonic development. In our previous study [6], we reported that donor mitochondria (labeled with a molecular probe) disappeared before the 16-cell stage following nuclear injection of cumulus cells into bovine enucleated oocytes. In humans, ooplasmic transfer from fertile donor oocytes into oocytes from idiopathically infertile patients has led to the birth of healthy babies [7-9]. Heteroplasmy of mtDNA from 1-yr-old children has been identified (in 2 out of 15) following ooplasmic transfer using direct polymerase chain reaction (PCR) product sequencing and chromatography [10]. These authors suggested that the transferred mitochondria can be replicated and maintained in the offspring so as to remain functional.
In the present study, the fate of mtDNA was determined in bovine NT embryos by detecting polymorphism in the displacement loop (D-loop) using allele-specific PCR (AS-PCR) analyses. DNA sequencing and DNA chromatography were performed to confirm the results.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recipient oocytes were collected from Holstein cattle ovaries obtained from a slaughterhouse. Each pair of ovaries was separated individually from other pairs to facilitate the following experiments. Follicles (2–10 mm in diameter) were aspirated and the cumulus-oocyte complexes (COCs) with compacted cumulus cells and evenly pigmented cytoplasm were collected and provided for this study.
Collected oocytes were washed three times in Tyrode-Hepes medium (TL Hepes; [11]) and cultured in tissue culture medium (TCM-199; Gibco BRL/Life Technologies, Gaithersburg, MD) supplemented with 2.2 g/L sodium bicarbonate (NaHCO3), 10% (v/v) heat-treated fetal bovine serum (FBS; Gibco BRL/Life Technologies), 0.22 µg/ml sodium pyruvate, 25 µg/ml gentamycin sulphate, 1 µg/ml FSH-p (Schering, West Sussex, UK), and 1 µg/ml estradiol-17ß (BCE-8875, Sigma Chemical Company, St. Louis, MO). The oocytes were cultured in 50-µl drops of TCM-199 under mineral oil (Sigma) for 20–22 h at 39°C, 5% CO2 in a humidified atmosphere. The ovaries were separately preserved to test whether the recipient oocyte had different DNA sequences from the donor-specific sequences.
Preparation of Cumulus Cells as Donor Cells
Cumulus cells collected from Korean native cattle (Hanwoo) were used as adult donor cells. Twenty hours after the onset of maturation, cumulus cells were isolated by pipetting in phosphate-buffered saline (PBS) supplemented with 2.5 µg/ml hyaluronidase (Sigma) and transferred to TCM-199 for use as donor cells. The fresh cumulus cells were cultured several passages and cryopreserved for future use. Cumulus cells were cryopreserved by a slow freezing method described by Tani et al. [12]. Briefly, Dulbecco Modified Eagle Medium (D-MEM; Sigma) supplemented with 10% FBS was used for the cell culture. Cumulus cells were cultured in culture dishes that were previously coated with 0.1% gelatin (Costar, Cambridge, MA). After 7-10 days, cells were disaggregated for subculture using Trypsin EDTA (Gibco BRL). Cells were placed in fresh dishes at concentrations of 7.5 x 104 to 3.6 x 105/ml. Cells were passaged every 3–4 days and cells passaged 2–7 times were cultured in D-MEM supplemented with 0.5% FBS for 10–15 days. Cumulus cell-derived cultured cells were mixed in PBS supplemented with 5% FBS and 5% DMSO (Sigma), and the concentration of cells was 7.5 x 104/ml. The mixture was transferred to 2-ml cryotubes (NUNC, Roskilde, Denmark) and cooled in a programmable freezer (Planer, Middlesex, UK). The cooling rate was 2.1°C/min from 0 to -60°C and 0.6°C/min from -60 to -70°C. For thawing, cryotubes were plunged into water at 36°C until ice melted. Frozen-thawed cells were stained with 0.4% trypan blue to test the viability. Thawed cumulus cells were cultured for 6 days and then made quiescent by culturing in D-MEM containing 0.5% FBS for 3–5 days.
Oocyte Enucleation and Donor Cell Microinjection
The enucleation of recipient oocytes and the injection of cumulus cells were performed as previously described [13] with some modification. Denuded oocytes were enucleated through a slit previously cut in the zona pellucida by transpiercing it at two adjacent points with a finely drawn glass microneedle and opening it by rubbing the area between the two puncture sites against the holding pipette. Oocytes were enucleated with a 25-µm glass pipette by aspirating the first polar body and MII plate in a small volume of surrounding cytoplasm. Successful enucleation was confirmed by Hoechst 33342 (Sigma) fluorescent staining of the pushed-out karyoplasts. Stained karyoplasts with observable chromatin were considered enucleated.
At 1 h postnucleation, prepared cumulus cells were placed in a 2-µl drop of PBS under mineral oil. Enucleated oocytes were placed in an adjacent drop of PBS containing 10% FBS (v/v), and individual cumulus cells were picked up with the injection pipette and the pipette moved to the injection drop. A 7- to 8-µm pipette (inner diameter) containing the cumulus cell was introduced through the slit in the zona pellucida made during enucleation and injected into the cytoplast. A small amount of cytoplasm was then drawn into the micropipette and the cytoplasm together with the cumulus cell and a small amount of medium expelled into the enucleated oocyte. Immediately after injection, the injecting micropipette was quickly withdrawn and the oocytes were released from the holding pipette to reduce the intracytoplasmic pressure exerted on the oocyte.
Activation and In Vitro Culture of Nuclear Transfer Embryos
After the injection, embryos were transferred to a drop of CR1aa supplemented with 5 µM ionomycin (Sigma) for 4 min at 37°C for activation. Then the same volume (50 µl) of PBS containing bovine serum albumin (BSA; 6 mg/ml) was added to stop the activation process [14]. Embryos were then extensively washed in PBS containing 30 mg/ml fatty-acid-free (FAF) BSA for 5 min before culturing for 3 h in 2 mM 6-dimethylaminopurine (6-DMAP; Sigma) in CR1aa containing 10% FBS. Activated embryos were transferred to drops of embryo culture medium, CR1aa [15] supplemented with 3 mg/ml FAF BSA, 20 µl/ml MEM essential amino acids, 10 µl/ml MEM nonessential amino acids, 0.44 µg/ml sodium pyruvate, 1.46 µg/ml glutamine, and 25 µg/ml gentamycin.
All animal experiments were approved and performed under the guidelines of the Monash University Animal Experiment Ethics Committee.
Preparation of DNA from a Single Nuclear Transferred Embryo
Nuclear transfer bovine embryos were collected at different developmental stages and the zona pellucida removed using 0.5% proteinase (Sigma). Each of the zona-free embryos was isolated and placed in 10 µl of distilled water and stored at -40°C. The frozen materials were thawed and warmed at 95°C for 10 min to inactivate proteinase.
PCR and Sequencing of Mitochondrial D-Loop Region
The first round of PCR employed the D-loop region of mtDNA. PCR primers specific for mtDNA were synthesized based on the reference genotypes of the Holstein cow [16]. The D-loop regions of the mtDNA were used in this study. Forward primer KNCF1 corresponds to the sequence from 15767 to 15790 and reverse primer KNCR2000 was complementary to the sequence from 365 to 388 (Fig. 2).
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PCR cycles were as follows: denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 74°C for 1 min in a thermo cycler (TwinBlock EasyCycler; Ericomp, San Diego, CA). KNCF1 primer was 5'-GAAGTTCTATTTAAACTATTCCCT-3' and the KNCR2000 primer was 5'-TTGCTTTGGGTTAAGCTACATCAA-3'. The D-loop fragments were sequenced using an ABI 377 automated DNA sequencer (Retrogen, San Diego, CA).
AS-PCR and Oligonucleotides
The second round of PCR, or AS-PCR, used 2 µl of the first PCR mixture as a template. Allele-specific primers were designed to exclude a possible amplification of a false mitochondrial allele from the oocyte that did not contain donor mtDNA. Therefore, an additional mismatch was introduced in allele-specific primer, DO1. Details concerning the design of allele-specific primer are given in Figure 2. The AS-PCR steps were denaturation at 94°C for 1 min, annealing at 44°C for 30 sec, and extension at 74°C for 30 sec in a thermo cycler. After AS-PCR, 15 µl of the reaction mixture was applied to a 1% agarose gel stained with ethidium bromide (0.1 µg/ml) and electrophoresed in 0.5x TBE buffer.
Direct mtDNA Sequencing and DNA Chromatography
The D-loop region of the mitochondrial genome was amplified from donor, recipient, and NT-derived blastocysts using the PCR. These PCR-amplified mtDNA fragments were used for direct DNA sequencing and DNA chromatography. Primer for DNA sequencing corresponds to the sequence from 15855 to 15872, 5'-CAATAACTCAACACAGAA-3'. An additional PCR product sequencing was performed on the AS-PCR products from the donor and NT-derived blastocysts to confirm the result of AS-PCR. The 387-base pair (bp) AS-PCR products were sequenced using a primer from 16 142 to 16 159, 5'-TACCATTAGATCACGAGC-3'. The direct PCR product DNA sequencing and DNA chromatography were performed on the PCR and AS-PCR-amplified mtDNA fragment using an ABI 377 automated DNA sequencer (Retrogen, Foster City, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the fate of donor mitochondrial DNA during preimplantation development after NT in cattle, mitochondrial DNA heteroplasmy in the NT embryos was analyzed by AS-PCR and by direct DNA sequencing and chromatography. The D-loop region of mtDNA was used for the analysis of mtDNA heteroplasmy. In the preliminary experiments, 960-bp D-loop regions of mtDNA could be amplified from ovarian tissue (in both Holstein cattle and Korean native cattle), a cumulus cell, and an oocyte (data not shown). The primers for D-loop amplification thus had specificity that was sufficient to allow detection of the D-loop region of mtDNA in very small quantities. The same region of the D-loop was amplified from the DNA of several additional cow ovaries. These PCR-amplified D-loop samples were sequenced and compared (Fig. 2b). Sequence comparisons in the D-loop regions of mtDNA revealed no species-specific differences between Holstein cattle and Korean native cattle (Fig. 2b). The diversity in D-loop sequences seems instead to be related to individual specificity. The D-loop products from ovary tissues, oocytes, and the reconstructed embryos were used for AS-PCR.
The mtDNA content during preimplantation development was determined by AS-PCR. The oligonucleotides used in the AS-PCR analysis and their localization are shown in Figure 2, a and b. The donor-specific PCR-amplified product was 387 bp long. The 387-bp donor-specific product could be detected exclusively in positive control D1, which was from the donor cells (Fig. 3). The mtDNA genotype from each of the eight ovaries supplying recipient oocytes was compared with the mtDNA genotype of the donor cell; in all cases, the genotypes of recipient oocytes (R1–R8) were different from that of the donor cumulus cell, D1 (Fig. 3). This means that, if the 387-bp donor-specific product were obtained from other ovaries, the oocytes obtained from the ovary could not be used as recipient oocytes for NT.
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The fate of donor mtDNA in NT embryos during preimplantation development were examined. AS-PCR analysis was performed at the 1-, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages of the embryos. The mitochondrial DNA from donor cells could be detected at all developmental stages of the NT embryos (Fig. 4). This result indicates that NT resulted in mtDNA heteroplasmy representing both donor and recipient. To confirm this result, mitochondrial D-loop sequences were analyzed by direct PCR product sequencing and DNA chromatography. The sequencing was performed using fluorescence-labeled dye terminator chemistry in an automated DNA sequencer.
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The sequence of the mitochondrial D-loop region in NT-derived blastocysts was compared with that of donor and recipient (Fig. 5). Base-pair comparison was performed at positions 16 050, 16 062, and 16 135 in the mitochondrial D-loop. The mtDNA nucleotides of donor cells were presented as cytosine (C), guanine (G), and C at positions 16 050, 16 062, and 16 135, respectively. These nucleotides were compared with nucleotides thymine (T), adenine (A), and T, respectively, in recipient oocytes. Unexpectedly, the differences in mtDNA between recipient and NT-derived blastocysts were not evident in DNA chromatography. In NT-derived blastocysts, at base-pair positions 16 050, 16 062, and 16 135, the mtDNA sequence revealed the presence of T, A, and T, respectively, as in recipient oocytes. It was expected that both the C and T were detectable at base-pair positions 16 050 and 16 135 and that both the G and A were detectable at base-pair position 16 062 only in NT-derived blastocysts and not in the recipient oocytes.
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Fortunately, there were two different base-pair positions in the 387-bp sequences of the AS-PCR product (at 106 and 16 255) between recipient oocyte and donor cumulus cell (Fig. 6). The mtDNA nucleotides of the donor cell were identified as T and those of the recipient oocyte were identified as C at both positions 106 and 16 255. At both positions 106 and 16 255, distinguishing nucleotides T and T, respectively, were present in NT-derived blastocysts (Fig. 6). The nucleotides T of the NT-derived blastocysts were not in accordance with the nucleotides from the recipient oocyte but rather with those from the donor cell. DNA chromatographs from the NT-derived blastocysts showed a mixture of nucleotides C and T at each base pair, predominantly containing donor mtDNA sequence at positions 106 and 16 255 (Fig. 7). There were an additional three NT-derived blastocysts showing a AS-PCR positive band, but there was no different base-pair position in the 387-bp sequences of the AS-PCR product between recipient oocytes and donor cumulus cell. Therefore, these three AS-PCR products of NT-derived blastocysts could not be confirmed by chromatography.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we used the AS-PCR method to detect the donor mtDNA in NT-derived embryos. In addition, mitochondrial D-loop and AS-PCR products were analyzed by direct PCR product sequencing and DNA chromatography to confirm the result. The D-loop region contains the regulatory sequences responsible for replication and transcription of the mitochondrial genome, such as the origin of heavy-strand replication [20]. The D-loop region is widely used for studying the mtDNA heteroplasmy because it is the most variable region of mtDNA [21].
The NT embryos were produced using an enucleated mature oocyte and a cumulus cell of a previously defined mtDNA genotype, after which mtDNA heteroplasmy and the origin of mtDNA in the NT embryos were analyzed. The predominant contribution from the recipient's cytoplast could be anticipated since the enucleated oocyte contains most of the mtDNA in its cytoplasm whereas the cumulus cell contains only a small amount of mtDNA. When the D-loop sequence of Korean native cattle was compared with that of Holstein cattle, no unique difference was observed, as has been observed in other European cattle [22]. Therefore, the diversity of D-loop sequences appears to be unrelated to species specificity. The nucleotide diversity of D-loop sequences was estimated at approximately 1% among individuals regardless of their species.
In the present study, donor mtDNA was detectable in the preimplantation embryos reconstructed by NT. However, the DNA chromatographs of the D-loop region did not reveal mtDNA heteroplasmy in NT-derived blastocysts. The NT-derived blastocysts exhibited only the recipient mtDNA sequence at donor-specific base-pair positions 16 050, 16 062, and 16 135, and the mtDNA sequence revealed the presence of T, A, and T, respectively (Fig. 5). Although no sequence differences were detected between recipient and NT-derived blastocysts, if only very small amounts of product are present, any differences could remain undetected. Additional PCR product sequencing was therefore performed on the AS-PCR product of NT-derived blastocysts in order to confirm the result of the AS-PCR, or mtDNA heteroplasmy. As expected, at positions 106 and 16 255 of the chromatographs, distinguishing nucleotides T and T, respectively, were present in the AS-PCR product from NT-derived blastocysts. The nucleotides T of NT-derived blastocysts were not in accordance with the sequence of the recipient oocyte but rather with the sequence of the donor cell. This indicates that the AS-PCR product of NT-derived blastocysts came specifically from the donor cell.
Together with these results, we suggest that mtDNA originating from donor cells would not disappear during preimplantation development following NT. In addition, it is possible that direct PCR product sequencing and DNA chromatography are methods that are inappropriate for estimating the heteroplasmy after NT because these methods are unlikely to be sensitive enough to detect a very small amount of donor mtDNA. Although data concerning the transmission of foreign mtDNA after manipulation of mammalian embryos is still controversial, there have been several reports on the inheritance of mtDNA following NT or embryo manipulation. However, different methods have been used to analyze the mtDNA heteroplasmy: restriction fragment length polymorphism (RFLP) in cloned sheep [2], single-strand conformation polymorphism of PCR fragments (PCR-SSCP) in cloned cattle [3], AS-PCR in cloned cattle [4, 5], and direct PCR sequencing and DNA chromatography in ooplasm-transferred humans [9, 10]. Of these methods, AS-PCR seems to be the most sensitive and hence suitable method for the analysis of mtDNA heteroplasmy after NT because donor-cell-derived mtDNA was not detectable by PCR-SSCP [3] and DNA chromatography (this study).
Recently, NT with cultured transgenic cell lines has produced cloned transgenic livestock [23, 24]. When, e.g., a transgenic line producing a pharmaceutical protein in milk is established by NT, inheritance of mtDNA may be important because some genes in the mitochondria may be important for certain traits such as milk production in cattle [25]. Moreover, Nagao et al. [26] reported the deleterious effect of heterogeneous mtDNA on embryonic development after NT in mice. These authors also suggested that the mtDNA component of cytoplasts plays an important role in nuclear-cytoplasmic incompatibilities. However, the presence of a small degree of mtDNA heteroplasmy does not appear to have major harmful effects because it occurs spontaneously in normal humans [27, 28] and in chimpanzee species [29].
We have demonstrated that donor-cell-derived mtDNA was detectable during preimplantation development following nuclear injection of cumulus cells. This result was contrary to that of our previous study [6], in which the donor mitochondria were eliminated from the cytoplasm before the 16-cell stage. One possible explanation is that mitochondria transferred from donor cells may be broken down by cytoplasmic events, whereas mitochondrial DNA remains in the cytoplasm and is transported to endogenous mitochondria. It is also possible that the molecular probe for mitochondria disappears at the mitochondrial maturation stage during which mitochondria show extensive morphological transformations. Further studies are required to determine the mechanism of mitochondrial destruction and the mode of transmission of mtDNA in both somatic cell NT and normal fertilization.
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FOOTNOTES |
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1 Correspondence: Kil Saeng Chung, Department of Animal Science, Konkuk University, Kwangjin-gu, 93-1, Seoul 143-701, Korea. FAX: 82 2 457 8488; cks123{at}kkucc.konkuk.ac.kr 
Accepted: March 8, 2002.
Received: November 30, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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