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Regular Article |
a Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, D-85764 Oberschleissheim, Germany
b Bavarian Research Center for Biology of Reproduction (BFZF), D-85764 Oberschleissheim, Germany
c Bavarian Research Station for Animal Breeding, D-85586 Grub, Germany
d Department of Statistics, Ludwig-Maximilian University, D-80539 Munich, Germany
e Institute of Animal Breeding and Genetics, University of Veterinary Medicine, 1210 Vienna, Austria
f Department of Animal Breeding and Genetics and Central Biotechnical Unit, Strahlenzentrum, Justus-Liebig-University, D-35392 Giessen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, granulosa cells, ovum pick-up, pregnancy, reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study demonstrates the feasibility of nuclear transfer using cultured somatic donor cells and recipient oocytes derived by OPU. Thus, nuclear transfer embryos, fetuses, and offspring with defined constellations of nDNA and mtDNA can be generated. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic genetic components as well as intrauterine environment on embryonic, fetal, and postnatal development [14]. Significant contributions of mtDNA genotype to variation in several postnatal traits, such as milk yield, carcass composition, and reproductive performance, have already been detected by quantitative genetic analysis [21–23]. Experimental confirmation of these findings requires clones with defined constellations of nDNA and mtDNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and mtDNA Lineages
The 13 donor animals used for OPU were Simmental Fleckvieh heifers or cows after first gestation and lactation. Oocyte donors originated from 2 unrelated cows (Hinka, maternal lineage A; Suleika, maternal lineage B) and were produced within a multiple ovulation/embryo transfer (MOET) program. Donors from maternal lineage A were 5 heifers and 2 cows, and those of maternal lineage B were 3 heifers and 3 cows. Mitochondrial DNA genotype of both lineages was identified by sequence analysis of the complete control region (CR). The CRs were amplified from total genomic DNA with the primers mtDA (5'- CTCACCATCAACCCCCAAAGCT-3') and mtDB (5'-TCATCTAGGCATTTTCAGTG-3') as described previously [16] and sequenced by standard procedures on a LICOR 4200 (MWG Biotech, Ebersberg, Germany). Two clones each, derived from independent polymerase chain reaction (PCR) of a single individual of both lineages, were sequenced with identical results. Sequences have been submitted to GenBank with accession numbers AF386912 and AF386913. Cytoplasmic genetic identity of all individuals in the 2 lineages was confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using diagnostic NlaIII restriction sites.
Ovum Pick-Up
The OPU was performed in 5 sessions once weekly (16 March 2000 to 13 April 2000) and then in 12 sessions twice weekly (4 May 2000 to 3 July 2000). Two heifers belonging to maternal lineage B (no. 745 and 751) (see Fig. 2) were culled after the 2nd and 15th OPU session, respectively. For heifer no. 400 from lineage A, OPU started with the third session. Besides these exceptions, all animals, irrespective of maternal lineage or reproductive status, were punctured in parallel.
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The OPU was carried out as described previously [18] using an Aloka 500 ultrasound monitor and a UST-974-5 5-MHz, sector-scanning transducer (Aloka, Tokyo, Japan). The transducer was positioned into a 60-cm long, specifically manufactured grip (Scanning Animal Systems, Stuttgart, Germany) to facilitate intravaginal manipulation of the transducer tip. A guidance canal (length, 48 cm) on the backside of the grip of the transducer enabled sliding of the puncture needle through the transducer.
The aspiration line consisted of a single lumen needle (diameter, 17 G; length, 70 cm; William Cook Europe GmbH, Mönchengladbach, Germany) connected to a 50-ml Falcon tube by 100-cm Teflon tubing. Vacuum pressure was provided by a regulated vacuum pump (K-MAR-5000B; William Cook Europe) and adjusted to create a flow rate of 16–20 ml/min. The collection medium (CM) consisted of 100 ml of Tyrode lactate solution buffered with 10 mM Hepes supplemented with 2% of fetal calf serum (FCS; heat-inactivated; Life Technologies, Karlsruhe, Germany) and 6 mg of sodium heparin (175.5 IU; Kraeber, Hamburg, Germany). Collected cumulus-oocyte complexes (COCs) were washed with preincubated (39°C) PBS enriched with 0.2% of FCS and transferred into CM without sodium heparin but supplemented with 10% FCS for transport. Oocytes were kept in this medium at room temperature for approximately 1 h.
Classification of COCs
Based on cytoplasmic appearance and cumulus characteristics, COCs were classified into 3 different categories: class I, homogeneous oocyte cytoplasm and complete, compact, and multilayered cumulus oophorus; class II, overall homogeneous oocyte cytoplasm with few areas of irregular pigmentation and 3–5 layers of compact cumulus cells; and class III, partially or completely denuded oocytes and/or oocytes with abnormally pigmented or vacuolated cytoplasm.
In Vitro Maturation of Oocytes
After classification, all oocytes recovered from the 13 donor animals were pooled according to reproduction status (heifer or cow) and maternal lineage (A or B) and used for nuclear transfer experiments without any preselection. In vitro maturation (IVM) of recovered oocytes was carried out as described previously [24]. Briefly, COCs were maintained in TCM 199 (Life Technologies) supplemented with NaHCO3 (3 g/L), Hepes (1400 mg/L), pyruvate (250 mg/L), L-lactic-Ca-salt (600 mg/L), gentamicin (110 µl/L), 10% estrous cow serum (ECS), and bovine (b) FSH (10 U/L) and bLH (10 U/L; Sioux Biochem., Sioux Center, IA) for 18–20 h in an atmosphere of 5% CO2 in air at 39°C and maximum humidity.
Karyoplasts
A primary culture was established from granulosa cells collected by OPU of a 12-yr-old Brown Swiss cow [25]. Sequence analysis of the mtDNA CR revealed 5 nucleotide substitutions compared with lineage A and five substitutions compared with lineage B. The corresponding restriction profile of the karyoplast donor is shown in Figure 2. The collected cells were centrifuged 2 times at 160 x g for 5 min and incubated in 0.25% trypsin and 0.2% EDTA solution for 5 min. In addition, the cell adhesions were dissociated by gentle pipetting to make single-cell suspensions. The cells were cultured in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% FCS, 1% L-glutamine, 1% nonessential amino acids (all Life Technologies), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), and 1% sodium pyruvate at 39°C in a humidified atmosphere of 5% CO2 in air. The cells were frozen in dimethyl sulfoxide supplemented with 10% (v/v) FCS and stored in liquid nitrogen. For experiments, the cells were thawed and cultured for 3–6 passages. Cells were used as nuclear donors directly or after serum starvation. The nonstarved cells, cultured until subconfluence, were used for nuclear transfer 2–4 days after passaging. The culture medium was removed 1 day after passaging to start serum starvation. The cells were washed with PBS, and fresh medium containing 0.5% FCS was added. The cells were then cultured for another 4–7 days before use in nuclear transfer.
Nuclear Transfer and Embryo Culture
The nuclear transfer procedure was essentially that described by Zakhartchenko et al. [6]. Briefly, 18–20 h after IVM, metaphase II oocytes were stripped from cumulus cells and enucleated within 2 h. Individual starved or cycling adult cells were transferred into the perivitelline space of enucleated oocytes at 20–22 h after maturation. 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) to initiate their fusion [26]. The KCCs were placed in the incubator in Ham F-12 medium supplemented with 4 mg/ml of BSA (fraction V). Fusion rates were determined 30–60 min after the fusion pulse. At 24 h after maturation (2 h postfusion), the fused KCCs were activated by a 5-min incubation in Ham F-12 supplemented with 7% (v/v) ethanol followed by a 5-h culture in 10 µg/ml of cycloheximide and 5 µg/ml of cytochalasin B [27]. The KCCs were transferred into 100-µl drops of synthetic oviduct fluid medium supplemented with 10% ECS, covered by paraffin oil, and cultured for 7 days at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.
Embryo Transfer
At Day 7 after nuclear transfer, blastocysts were transferred nonsurgically to synchronous recipients of 7 days after standing estrus. Pregnancies were confirmed on Day 28 by ultrasonographic examination. Recipients were slaughtered on Day 80 for further investigations.
Statistical Analysis
The OPU data were statistically analyzed by a linear mixed model [28] using the procedure MIXED (SAS Release 8.01 [29]). The model included reproductive status, maternal lineage, aspiration frequency, and the interaction maternal lineage x reproductive status as fixed effects, whereas individual donor was included as a random effect. The numbers were transformed by n to square root of (n + 0.5).
To estimate effects of maternal lineage, reproductive status, aspiration frequency, and starvation method on success rates of the various steps of nuclear transfer, logistic regression was used. Due to lack of data, logistic regression was not applicable for the pregnancy rates; therefore, the Fisher exact probability test was applied.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A comparison of both CR sequences is shown in Figure 1. Overall, 11 variable nucleotide positions were found. These consist of 4 insertions/deletions and 7 transitions. Three insertions are found in a cytosine run at the 3' end of the CR, a region prone to replication slippage. When this region is excluded, the 2 maternal lineages display a sequence divergence of 0.89%. A C/T polymorphism is located in the center of conserved sequence block (CSB) 1, and a "C" deletion is found within CSB 2+3. Two A/G polymorphisms are located 11 and 15 nucleotides upstream of the origin of H-strand replication. The identity of mtDNA in all individuals used for OPU in both maternal lineages was confirmed by a diagnostic PCR-RFLP that covers 3 polymorphic NlaIII restriction sites (Fig. 2).
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Ovum Pick-Up
The OPU was performed over a period of 3.5 mo. In 17 sessions, a total of 1342 COCs were recovered, of which 637 were classified as class I or II. In lineage A, 364 of 968 (38%) COCs were recovered in the period with weekly aspiration sessions. The corresponding proportion in lineage B was 34% (126 of 374) and was not significantly different from that in lineage A (
2 = 1.782).
The numbers of total COCs recovered per aspiration session were significantly (P < 0.001) affected by maternal lineage and aspiration frequency, whereas maternal lineage represented the only factor significantly (P < 0.001) influencing the number of class I/II COCs (Table 1). Overall, the numbers of total COCs and class I/II COCs (mean ± SEM) recovered per session from donors of lineage A were higher than those obtained from lineage B (8.2 ± 0.8 vs. 4.5 ± 0.5 and 4.2 ± 0.5 vs. 1.7 ± 0.3, respectively). Whereas more COCs were recovered from cows than from heifers in lineage A, the latter were the more efficient donors of COCs in lineage B (Fig. 3).
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Overall, follicle aspiration once per week yielded a significantly (P < 0.001) higher total number of COCs per session than aspiration twice weekly (7.9 ± 1.2 vs. 6.1 ± 0.6). This effect was more pronounced in lineage A (10.7 ± 1.6 vs. 7.2 ± 0.6) than in lineage B (4.7 ± 0.5 vs. 4.5 ± 0.7). The numbers of class I/II COCs per session were 3.0 ± 0.6 (once weekly) and 3.2 ± 0.39 (twice weekly) and were not significantly (P > 0.05) influenced by aspiration frequency.
Reproductive status of donor animals had no effect on the total numbers of COCs (heifers, 6.5 ± 0.6; cows, 6.6 ± 1.7) and class I/II COCs (heifers, 3.0 ± 0.5; cows, 3.1 ± 0.9) recovered per session.
In addition, the individual donor within maternal lineage had a significant (P < 0.01) effect on the total number of recovered COCs. The total numbers of COCs obtained from individual donors per session ranged from 5.2 ± 0.7 to 12.3 ± 1.7 in lineage A and from 2.8 ± 0.4 to 6.4 ± 0.9 in lineage B. The numbers of class I/II COCs varied from 3.1 ± 0.5 to 6.2 ± 0.9 in lineage A and from 1.0 ± 0.0 to 3.1 ± 0.7 in lineage B.
Efficiency of Nuclear Transfer and Embryonic Development
The results of the statistical analysis of embryo developmental rates after nuclear transfer are summarized in Table 2.
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Of 1342 recovered oocytes, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) KCCs, resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 23 morulae and 61 blastocysts (4% and 11% based on fused KCCs, respectively) were obtained. Seventy-two embryos were transferred to 38 recipients in 10 single (6 lineage A and 4 lineage B), 22 double (15 lineage A and 7 lineage B), and 6 triple transfers (all lineage A). Nine recipients, including 8 double transfers (6 lineage A and 2 lineage B) and 1 triple transfer were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. One of the 2 pregnancies of maternal lineage B was aborted between Days 56 and 80, whereas 4 of the 7 pregnancies of maternal lineage A were lost by Day 80 (1 between Days 28 and 42 and 3 between Days 56 and 80).
Statistical analysis revealed significant effects of maternal lineage on the proportion of fused KCCs (lineage A, 73%; lineage B, 80%; P < 0.05) and on the proportion of transferable embryos on Day 7 (lineage A, 17%; lineage B, 11%; P < 0.05).
In addition, results of nuclear transfer were significantly influenced by frequency of oocyte recovery. The proportion of matured oocytes was higher with 2 aspiration sessions per week (63% vs. 40% once weekly, P < 0.001). In contrast, the proportion of fused KCCs was higher when oocytes were recovered once per week (81% vs. 73% twice weekly, P < 0.01). The yield of transferable embryos on Day 7 was again higher when oocytes were recovered twice weekly (18% vs. 10% once weekly, P < 0.05).
Serum starvation of donor cells significantly (P < 0.001) reduced the fusion rate (72% vs. 82% using nonstarved donor cells). Serum starvation did not affect further development of nuclear transfer embryos.
Reproductive status of oocyte donors had no significant effect on any of the investigated parameters. Pregnancy rates were not significantly affected by any of the factors investigated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Significant effects of individual oocyte donors on the number of COCs recovered by OPU, as seen in the present study, have been reported previously [30, 31]. However, to our knowledge, this study provides the first evidence for an effect of maternal lineage of oocyte donors on the efficiency of OPU, which was highly significant in spite of the large variation between individual donor animals. Although the number of COCs and the efficacy of recipient cytoplasts to reprogram introduced donor nuclei were higher in lineage A than in lineage B, animals from lineage B were superior in a MOET program. The number of transferable embryos recovered per donor of lineage B corresponded to 6.8 ± 2.8 (5 donors, first superovulation cycle), whereas lineage A yielded only 2.3 ± 0.8 transferable embryos per donor (6 donors, first superovulation cycle). Potential roles of cytoplasmic factors need to be confirmed in a larger number of cow families, including biochemical analysis of cytoplasmic components as well as morphological and metabolic parameters of recovered oocytes [24].
The effect of aspiration frequency on the efficiency of OPU was also investigated in other studies [32, 33], demonstrating an advantage of 2 versus 1 aspiration sessions per week. With average numbers of 6.1 ± 0.6 and 7.9 ± 1.2 COCs per session in the twice-weekly and once-weekly aspiration periods, respectively, 2 aspiration sessions yielded 1.5-fold more oocytes per week than 1 OPU session. In addition, the quality of oocytes was higher when OPU was performed twice weekly, as evidenced by significantly improved maturation rates. Furthermore, the development of nuclear transfer embryos to morulae and blastocysts was significantly improved when recipient oocytes were recovered by OPU twice weekly. The advantage of 2 vs. 1 aspiration sessions per week was explained by a better adaptation to the physiological hormonal changes during folliculogenesis [34].
Our data show that, in general, a combination of OPU with nuclear transfer is possible, although the efficiency of nuclear transfer was lower than previously observed with the same type of donor cells [25]. This is due to only high-quality oocytes obtained from the ovaries of slaughtered cows being used in our previous study, whereas in the present study, all available oocytes recovered by OPU were cultured for maturation and all oocytes extruding a polar body were enucleated and used as recipient cytoplasts for nuclear transfer.
Interestingly, we observed a significant effect of maternal lineage of oocyte donors on the proportion of transferable nuclear transfer embryos, suggesting that cytoplasmic components affect the efficiency of nuclear transfer. The regulation of mitochondrial functions, as 1 major cytoplasmic determinant, is complex and involves cooperation of the mitochondrial and the nuclear genomes (reviewed in [35]). The 16.4 kilobases of mammalian mtDNA encode 37 genes, including 13 proteins of the electron-transport chain as well as 22 tRNA and 2 rRNA species. The corresponding nuclear gene products are synthesized in the cytosol and imported into the mitochondrial matrix in an energy-dependent manner (reviewed in [36]). Thus, a tight control of species-specific nuclear-mitochondrial interactions is necessary for proper mitochondrial function, and more than 100 specific interactions between proteins encoded by nuclear and mitochondrial genomes are necessary for optimized ATP production [37]. Nevertheless, interspecific nuclear transfer experiments involving fusion of skin fibroblasts from sheep, pig, monkey, and rat with enucleated bovine oocytes resulted in early development of the reconstructed embryos, but no pregnancies, following transfer to recipients [38], indicating that the incompatibility of nuclear- and mitochondrial-encoded components from different species is likely to inhibit normal development. For instance, mitochondria from gorilla and chimpanzee, but not from the more distant orangutan, were able to restore oxidative phosphorylation in human cells [39]. Similarly, mouse xenomitochondrial cybrids harboring rat mtDNA exhibited reduced proliferation and oxidative phosphorylation [37]. In contrast, nuclear transfer in bovine subspecies using Bos indicus blastomeres as karyoplasts and B. taurus recipient cytoplasts resulted in live offspring [40].
However, an effect of maternal lineage of recipient oocytes on the efficiency of nuclear transfer in the bovine has not been described so far. This is probably because, in most studies on nuclear transfer, large numbers of oocytes with nondefined mtDNA genomes are recovered from the ovaries of slaughtered cows to enable a strong selection for high-quality oocytes. Thus, varying compatibility between nuclear and cytoplasmic genes in cloned calves has been discussed as a potential reason for the wide variation in body weight of these individuals [41]. To address this point, we combined somatic cell nuclear transfer and OPU technology for the production of cloned embryos with defined nDNA/mtDNA constellations. Two lineages of oocyte donors of the same breed were selected according to a relatively large divergence of their B. taurus mtDNA sequences. That maternal lineage of oocyte donors had a significant effect on the proportion of nuclear transfer embryos developing to morulae and blastocysts provides experimental evidence for the influence of recipient cytoplasmic background in nuclear transfer technology. At least 2 polymorphisms detected in the mtDNA molecules of the investigated maternal lineages are located in important domains of the CR, which are believed to be critical for regulating mtDNA function. One substitution is located in CSB 1, a conserved sequence with significant mtTFA (mitochondrial transcription factor A) binding, a regulatory element involved in the initiation of heavy-strand DNA synthesis. The CSB 1 is believed to signal the transition from transcription to replication of mtDNA. A single nucleotide deletion was observed in CSB 2+3, another conserved element involved in transcription and replication [42, 43]. Two additional polymorphisms were found in the vicinity of the heavy-strand origin of replication (OH) [44]. Thus, the observed sequence differences between the 2 mtDNA genotypes of oocyte donors used in our study could account for the significant maternal lineage effects on the efficiency of nuclear transfer. Besides the differences in mtDNA genotypes, segregation patterns [40, 45] and heteroplasmic ratios [15, 16] as well as intrauterine environment [14] have to be considered as factors limiting the development of nuclear transfer clones.
The findings of the present study need to be further substantiated by measurements of cell proliferation and metabolic parameters representing mitochondrial cell function. Furthermore, it will be interesting to determine the segregation patterns of the distinct mtDNA genotypes to rule out if some of these genotypes are biased, as observed in heteroplasmic constellations of different mouse strains [45]. Such studies will further clarify the role of cytoplasmic genetic components regarding both the efficiency of nuclear transfer and the growth and metabolism of the resulting fetuses and offspring.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Deutsche Forschungsgemeinschaft (WO 685/3-1, HI 503/3-1). 
2 Correspondence: Eckhard Wolf, Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Hackerstrasse 27, D-85764 Oberschleissheim, Germany. FAX: 49 89 2180 6849; ewolf{at}lmb.uni-muenchen.de
3 S.H. and E.W. contributed equally to this work.
Accepted: September 24, 2001.
Received: May 31, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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