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Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats
Hai‑jun Liu*1, Jun Xue†, Kui Li‡, Zheng‑Zhou Ying‡, Zi Zheng* and Ru Wang*
*Tianjin Institute of Animal Science and Veterinary Medicine, Tianjin 300112, Peoples Republic of China, †Tianjin Research Center of Agricultural Biotechnology, Tianjin 300192, Peoples Republic of China, and ‡Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing 100094, Peoples Republic of China
We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.
Key words: genetic background, goat; mitochondria origin, nuclear–cytoplasmic interactions, nuclear transfer, oocyte donor
Abbreviations: FCS, foetal calf serum, FSH, follicle-stimulating hormone, I.U., international units, mtDNA, mitochondrial DNA
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Since the birth of Dolly cloned from adult sheep mammary gland cells (Wilmut et al., 1997), many other mammals have been cloned with somatic cells. However, the reprogramming process of somatic cell is not perfect and embryos produced by nuclear transfer often show abnormal development, therefore cloning mammals by nuclear transfer is still highly inefficient (Wilmut et al., 2002). Most of the efforts to improve the efficiency of nuclear transfer were focused on the donor cell treatments and nuclear transfer process itself. It is also clear that oocyte recipients play an important role in donor cell nuclear reprogramming and early embryo development. Much less research data were reported on the effect of genetic background of oocyte recipient on nuclear transfer efficiency. In bovine, Bruggerhoff et al. (2002) demonstrated that the proportion of transferable embryos on day 7 was significantly influenced by maternal lineage of oocyte donors. Nucleocytoplasmic interaction was investigated in bovine by Hiendleder et al. (2004) and indicated that the proportion of viable foetuses were affected by cytoplasts of the oocytes. In goats, the resources of oocyte cytoplasts have been studied on the nuclear transfer efficiency, including in vivo and in vitro matured (Ohkoshi et al., 2003; Liu et al., 2008), oocytes from FSH (follicle-stimulating hormone) pre-treated and non-treated females (Reggio et al., 2001). The effect of genetic background of oocyte recipients on nuclear transfer efficiency remains unclear. Chen et al. (2007) showed that the somatic cell nuclei derived from Boer goats were reprogrammed incompletely in the cytoplasts of Sannen goats. Embryo development after nuclear transfer was improved when cytoplasts derived from cross-breeding goats was used.
mtDNA (mitochondrial DNA) of donor cells would be inevitably transferred into the oocytes during electrofusion with donor cells and enucleated oocytes, which results in heteroplasmy. Steinborn et al. (2000) reported the origins of 10 nuclear-transfer cattle mtDNA and detected the heteroplasmy, in which mtDNA derived from donor cells constituted 0.4–4%. However, no detectable mtDNA was found from the respective somatic donor cells in 10 nuclear-transfer sheep by Evans et al. (1999). The results implied differences among species in the origin of mtDNA of nuclear transfer-derived cloned mammals.
In the present study, the nuclear donor cells were derived from adult Boer goats, and the effect of cytoplasts on the developmental competence of reconstructed embryos was investigated.
2. Materials and methods
All chemicals were purchased from Sigma unless otherwise stated.
2.1. Donor cell preparation
All procedures were approved by the Animal Ethics and Welfare Committee, Animal Holding Unit, Tianjin Institute of Animal Science and Veterinary Medicine. The somatic cells used in this experiment were isolated from 2 adult female and male Boer goats. Skin samples taken from the ear were processed with 0.25% trypsin (Hyclone). Growing cells were passaged twice and washed with DMEM (Dulbecco's modified Eagle's medium; Gibco)+10% FCS (foetal calf serum; Hyclone). A portion of the cells was used immediately for nuclear transfer and the remainder cultured for later use. Cells used for nuclear transfer were passaged 2–14 times.
2.2. Oocyte collection
Boer goats and cross-breeding goats (Boer♂×Huanghuai♀) were pretreated with progesterone-impregnated intravaginal sponge (New Zealand) for 18 days. FSH (Ningbo) was administrated twice a day at 200–220 I.U. (international units) daily from day 16 to day 18. The vaginal sponge was removed on the last day soon after FSH injection. Goats in heat were injected with LH (luteinizing hormone) (Ningbo) at 100 I.U. Oocytes were collected surgically by flushing the oviducts with PBS (Gibco)+5% FCS.
2.3. Nuclear transfer
Oocytes were denuded by aspirating and expelling repeatedly in 0.5% hyaluronidase for 5 min. Those with first polar bodies and normal morphology were pretreated for enucleation in medium supplemented with 5 μg/ml Hoechst 33342 and 5 μg/ml cytochalasin B for 10–15 min. Enucleation was performed by removing the first polar body and small portion of ooplasm which contained the metaphase II spindle and confirmed by exposure to UV light. A single donor cell was inserted inside the perivitelline space of each enucleated oocyte. The oocyte–fibroblast couplets were washed twice in fusion medium (0.3 M mannitol, 0.1 mM CaCl2 and 0.1 mM MgSO4) and moved into the fusion chamber and aligned manually. Two electric pulses (2.0 kV/cm, 25 μs each and 1 s interval) were applied. The couplets after fusion were activated by incubating in medium supplemented with 10 μg/ml cycloheximide and 5 μg/ml cytochalasin B for 5 h and then cultured in microdrops of TCM199+10% FCS, fed with Vero cells. Cleavage and morula development was assessed on day 2 and 7 post-fusion.
2.4. Embryo transfer
Cross-breeding goats were used as embryo recipients and synchronized by pre-treating with progesterone-impregnated intravaginal sponge (same as the oocyte donors). Embryos were transferred into oviduct on the side that the corpus luteum was found on day 2 after estrus. Pregnancy was checked 45 days after transfer and foetus development was monitored with ultrasound.
2.5. DNA identification
DNA samples from each donor cell lines, cloned kids and recipient goats were processed by microsatellite analysis consisting of a multiplexed set of nine polymorphic goat loci, recommended by Food and Agriculture Organization. Length variations were assayed by PCR amplification with fluorescence labelled locus-specific primers and PAGE on an automated DNA sequencer (ABI3700).
2.6. Mitochondria DNA analysis
2.6.1 Sample collection and DNA isolation
Two cloned Boer goats were chosen to analyse the mtDNA origin. The cell lines used as nuclear donors were cultured and collected. The oocyte-donor goats were sold to a slaughterhouse after collecting oocytes, and no blood samples were retained for specific comparison. Therefore we collected the blood from 5 randomly selected goats from each oocyte donor goats group as a representative sample. In addition, the blood of 2 cloned goats and 2 surrogate goats were collected (all samples listed in Table 1). DNA isolation from cells and blood was performed using the DNA isolation kit (Tiangen Biotech).
Table 1 The source and label of each samples
2.6.2 Analysis of the mtDNA D-loop region
The goat mtDNA D-loop regions derived from each samples as described (Joshi et al., 2004) were amplified. Approximately 1.7 kb size fragments were purified with gel extracted kit (Tiangen Biotech) and inserted into pMD18-T easy vectors. After sequencing, the phylogenetic divergence between pairs of species was performed on the basis of their nucleotide sequences. Moreover, the sequences containing HVRI (hypervariable region I) were aligned by ClustalX program.
2.7. Statistical analysis
Data were analysed using the χ2 test. Differences were considered to be significant when P<0.05.
3.1. Effect of genetic background of recipient cytoplasts on in vitro development of reconstructed embryos
Embryos were reconstructed with oocyte cytoplasts that had a different genetic background (Boer background and cross-breeding background). No significant differences were found in fusion rate and cleavage rate (P>0.05) between Boer background and cross-breeding background (Table 2). The morula development rate of embryos reconstructed with Boer background cytoplasts was significantly higher than those reconstructed with cross-breeding background (34.1 versus 19.1%; P<0.05).
Table 2 Effect of genetic background of oocyte donors on in vitro development of reconstructed embryos
For each column, superscripts indicate statistical difference (P<0.05).
3.2. Effect of genetic background of recipient cytoplasts on in vivo development of reconstructed embryos
Embryos reconstructed with oocyte cytoplasts that had different genetic background (Boer background and cross-breeding background) were transferred into recipients. No significant difference in the rates of pregnancy and foetus loss between the two breeds was found (P>0.05; Table 3). However, the live-birth rate was significantly higher with Boer background than with the cross-breeding background (3.1% versus 0.8%; P<0.05; Figures 1A–1C).
Table 3 Effect of genetic background of oocyte donors on in vivo development of reconstructed embryos
For each column, superscripts indicate statistical difference (P<0.05).
3.3. Microsatellite DNA analysis
Nine microsatellite loci were analysed with samples from donor cells, clones and recipients. Genetic identification was confirmed between clones and respective donor cells.
3.4. mtDNA analysis
All 15 DNA samples belonging to 2 different goat strains were isolated and D-loop sequences amplified. Fragments at 1.7 kb size were confirmed by nucleotide sequencing. Neighbour-joining tree was constructed, based on the sequences (Figure 2). Cloned goat labelled 070409 (Clone 1) was similar to Boer goat, whereas the cloned goat labelled 070702 (Clone 2) was similar to cross-bred goat (Boer♂×Huanghuai♀); the cloned goats were significantly different from their nuclear donor (Donor cell 1). The D-loop sequence of 864, 364 and S1854 individuals of oocytes donor goats were closer to the nuclear donor cells in neighbour-joining tree. The region between 720 and 1020 bp showed high polymorphism (Figure 3), which indicated that the most specific sequence bases of goats 864, 364, S1854 and nuclear donor cells were replaced in the 2 cloned goats, with significant difference in mitochondrial genes between the 2 cloned goats and the nuclear donor cells. PCR clones and PCR products of the D-loop sequence from the 2 cloned goats were sequenced and re-analysed, which have the same as described above. Mitochondrial genetic components of the nuclear donor cells could not be detected in the clones.
Skin fibroblast cells derived from adult Boer goats as nuclear donors were used in vitro and in vivo in the development of embryos reconstructed with cytoplasts from different genetic backgrounds (Boer and cross-breeding background). Higher in vitro and in vivo development rates of embryos reconstructed from Boer background cytoplasts and Boer donor cells were obtained compared with the embryos reconstructed from cross-breeding background cytoplasts. After embryo transfer, all five pregnant recipients carrying embryos reconstructed with Boer cytoplasts gave birth to 6 live offspring. However, only two of six pregnant recipients carrying embryos constructed with cross-breeding cytoplasts went to term with a single live offspring. The higher embryo-losing rate was observed in the recipients carrying embryos constructed with cross-breeding cytoplasts. Similar work in sheep by Dinnyes et al. (2001) showed that embryos reconstructed with Dorset somatic cells and Dorset cytoplasts had a higher blastocyst development rate both in vitro and in vivo. Chen et al. (2007) studied the effect of cytoplasts on the development of inter-subspecies nuclear transfer reconstructed goat embryos. The embryo development was improved by using the hybrid cytoplasts. The same group also reported that closer nuclear–cytoplasmic background improved the development rate of cloned inter-subspecies embryos (Sha et al., 2009). In bovine species, Bruggerhoff et al. (2002) reported that the proportion of transferable embryos was significantly influenced by maternal lineage of oocyte donors. Yang et al. (2005) also found that the use of F1 hybrid oocytes as recipient cytoplast improved in vitro development of cloned bovine embryos. Even the individual oocyte donor showed clear variation of in vitro blastocyst production (Yang et al., 2008). These investigations indicated that maternal factors, most likely mitochondria, were playing critical roles during embryo development.
Mitochondria play an important role during embryonic development. Developmental block of pre-implantation embryos and chromosome abnormality are related to the lack of sufficient ATP (mainly generated by mitochondria) or mitochondria dysfunction. We found that mtDNA origins of the clones tracked back, that the mitochondria in the clones were derived from their oocyte donors, not those of the nuclear donors. In cloned sheep, including Dolly, the first animal cloned from an established adult somatic cell line, mtDNA of each cloned sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells (Evans et al., 1999). To clarify whether this was a dilution factor or directly related to the transcriptional status of the donor cell in respect of mtDNA transcription factor, embryos were generated using donor cells that the mtDNA was depleted. The results indicated that in the donor cells used nuclear-encoded mtDNA transcription and replication factors persist even after mtDNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mtDNA replication and transcription (Lloyd et al., 2006). Those embryos produced using donor cells depleted to residual levels of mtDNA developed to term and live lambs were produced in which no donor somatic mtDNA was detected, the lambs being homoplasmic for recipient oocyte DNA (Lee et al., 2010). Most likely the donor-derived mitochondria were eliminated during early embryo development and the mitochondrial replication following nuclear transfer might occurred post-implantation. The lower morula development rate in vitro and higher embryo losing rate in vivo in the present study were clearly the consequences of mtDNA heteroplasmy and/or possible incompatibility between nuclear and mtDNA genotypes.
In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, the homologous background of cytoplast and nuclear donor supported development of embryos reconstructed.
Haijun Liu performed the design of the study, nuclear transfer, reconstructed embryos transfer and the preparation of the manuscript. Jun Xue carried out the culture of the donor somatic cells and preparation of the manuscript. Kui Li and Zhengzhou Ying investigated the mitochondria DNA origin of cloned goats. Zi Zheng performed the collection of recipient oocytes; Ru Wang was responsible for the management of the goats.
This work was supported by
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Received 25 May 2011/18 January 2012; accepted 23 February 2012
Published as Cell Biology International Immediate Publication 23 February 2012, doi:10.1042/CBI20110287
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 (A) Cloned Boer goats (clone 1, clone 3, clone 5 and clone 6) from Boer recipient cytoplasts; (B, C) Cloned Boer goats (clone 2 and clone 7) from cross-breeding recipient cytoplasts