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

Mitochondrion 12 (2012) 506–513

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Segregation of donor cell mitochondrial DNA in gaur–bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring Sumeth Imsoonthornruksa a, Kanokwan Srirattana a, Wanwisa Phewsoi a, Wanchai Tunwattana b, Rangsun Parnpai a,⁎, Mariena Ketudat-Cairns a,⁎ a b

Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand Surin Elephant Kingdom Project, Zoological Park Organization, Surin, 32120, Thailand

a r t i c l e

i n f o

Article history: Received 24 February 2012 received in revised form 5 May 2012 accepted 13 July 2012 Available online 21 July 2012 Keywords: Endangered species Bovine cytoplasm Gaur Mitochondrial DNA Heteroplasmy

a b s t r a c t The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur–bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Interspecies somatic cell nuclear transfer (iSCNT) has been used to study the basic fundamental properties of embryo development. It can also be used as an alternative method for the preservation of endangered species whose oocytes are difficult to obtain (Dominko et al., 1999). The success of iSCNT embryo production has been achieved in several species, including gaur–bovine (Lanza et al., 2000), panda–rabbit (Chen et al., 2002), macaque–rabbit (Yang et al., 2003), human–rabbit (Chen et al., 2003), goat–sheep (Ma et al., 2008) and cat–bovine (Imsoonthornruksa et al., 2011). Furthermore, live gaur (Srirattana et al., 2012; Vogel, 2001), mouflon (Loi et al., 2001), wildcat (Gómez et al., 2004, 2008) and wolf (Oh et al., 2008) iSCNT offspring have been obtained. However, the efficiency of iSCNT is low, and the underlying causes for this low efficiency are not well understood. One of the obvious reasons might be linked to the incomplete epigenetic modifications of the donor cell nucleus, resulting in aberrant gene expression throughout development (Daniels et al., 2000; Han et al., 2003; Imsoonthornruksa et al., 2010; Rideout et al., 2001). Many abnormalities observed in cloned embryos, fetuses and offspring might also result from deficiencies in mitochondrial functions; however, the mechanisms leading to this condition are still controversial.

⁎ Corresponding authors. Tel.: +66 4422 4355; fax: +66 4422 4150. E-mail addresses: [email protected] (R. Parnpai), [email protected] (M. Ketudat-Cairns).

In mammals, during normal fertilization, the oocyte contributes all of the mitochondria to the developing embryo, as sperm mitochondria are actively eliminated during early development through oocytedriven ubiquitination, which results in the generation of a homogeneous maternally inherited mitochondria population (Sutovsky et al., 1999). Conversely, in iSCNT, whole donor cells, including the cytoplasm, are electro-fused into enucleated oocyte cytoplasts. The resulting heteroplasmic mixture contains both the donor cell and the enucleated oocyte mitochondria. The inheritance patterns of the heteroplasmic mitochondria are not yet clear. Moreover, it is not known whether different species follow different modes of mitochondria DNA mtDNA inheritance. In most studies iSCNT embryos and early fetuses contained heteroplasmic mtDNA populations (Chen et al., 2002, 2003; Ma et al., 2008; Yang et al., 2003). However, homoplasmic mtDNA populations resulting from oocyte or donor cell domination were also observed (Chen et al., 2002; Lanza et al., 2000; Oh et al., 2008). The gaur (Bos gaurus) is listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix I and considered to be an endangered species in Thailand. Previous studies showed that cloned gaur fetuses and live offspring derived from iSCNT can be obtained, although the calf died 2 days after birth (Lanza et al., 2000; Vogel, 2001). Lanza et al. (2000) also used PCR-RFLP to demonstrate that only the mtDNAs derived from oocytes were detected in several organs of the 3 cloned gaur fetuses. To characterize the distribution and transmission of mtDNA in iSCNT, the copy number of both gaur and bovine mtDNA at various stages of pre-implantation in the development of gaur–bovine embryos and in several tissues from a calf and aborted

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S. Imsoonthornruksa et al. / Mitochondrion 12 (2012) 506–513

iSCNT and mummified fetuses was examined using quantitative real-time PCR (qPCR). 2. Material and method All approved animal experiments were performed according to the guidelines of the Ethics Committee of the Laboratory Animal Care of Suranaree University of Technology. The chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise stated. 2.1. iSCNT embryo production 2.1.1. Preparation of donor cells Fibroblast cells were established from the biopsied skin tissue of adult female and male gaur from the Khao Kheow Open Zoo, Chonburi, Thailand according to the protocol of Srirattana et al. (2012). Briefly, skin tissues were dissected into small pieces and placed onto glass cover slips. The tissues were cultured in alpha modified Eagle's minimum essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, NY, USA) at 37 °C in a humidified atmosphere of 5% CO2 until the cells formed a subconfluent fibroblast monolayer. The fibroblasts were subsequently passaged three times, frozen in αMEM containing 10% dimethyl sulfoxide and 10% FBS and stored in liquid nitrogen. Prior to iSCNT, fibroblasts at passage 4 were thawed and cultured until reaching 80% confluence (2–3 days). Subsequently, the cells were dissociated into single cells with trypsin and used as donor cells. 2.1.2. Preparation of recipient cytoplasts Bovine ovaries were collected from a slaughterhouse and processed as previously described (Srirattana et al., 2012). Briefly, cumulus oocyte complexes were aspirated from 2 to 6 mm follicles and cultured in TCM 199 maturation medium supplemented with 10% FBS, 50 IU/ml human chorionic gonadotropin (Intervet, Boxmeer, Netherlands), 0.02 AU/ml follicle-stimulating hormone (Antrin, Kawasaki Seiyaku K.K., Kawasaki, Japan) and 1 μg/ml 17β-estradiol for 21 h at 38.5 °C in a humidified atmosphere of 5% CO2. The cumulus cells were removed with gentle pipetting in 0.2% hyaluronidase. The oocytes were washed 5 times in Emcare holding medium (ICP bio, Auckland, New Zealand) to inactivate the hyaluronidase. The matured oocytes with the first polar body were enucleated in Emcare holding medium supplemented with 5 μg/ml cytochalasin B. A small amount of cytoplasm from the area beneath the polar body was collected through micromanipulation methods using the sharp edge of a micropipette (Narishige, Tokyo, Japan). The cytoplasm was subsequently stained with 5 μg/ml Hoechst 33342 and visualized under UV light to confirm successful enucleation. 2.1.3. Nuclear transfer, parthenogenetic activation (PA) and embryo culture Individual nonquiescent fibroblasts were transferred into the perivitelline space of enucleated oocytes. The fibroblast–cytoplasm couplets were placed in Zimmermann fusion medium, and cell fusion was induced with a DC double pulse of 24 V for 15 μs generated using a SUT-F1 fusion machine (Suranaree University of Technology, Nakhon Ratchasima, Thailand). The fused couplets and matured oocytes (PA, control group) were activated upon incubation in 7% ethanol for 5 min followed by culture in modified oviduct synthetic fluid with amino acid (mSOFaa) medium containing 10 μg/ml cycloheximide and 1.25 μg/ml cytochalasin D for 5 h at 38.5 °C in a humidified atmosphere of 5% CO2. The activated embryos were cultured in mSOFaa for two days in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 °C. Subsequently, embryos at the eight-cell stage were selected and co-cultured with bovine oviductal epithelium cells in mSOFaa for five days at 38.5 °C in a humidified atmosphere of 5% CO2. Embryo development was monitored, and half of the culture medium was replaced daily.

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2.2. DNA preparation Total DNA was extracted from individual iSCNT embryos at the 2-, 4-, 8-cell, morula and blastocyst stages and from fibroblast– cytoplasm couplets (prior to fusion). Single embryos were collected into microcentrifuge tubes containing 10 μl lysis buffer (10 mM Tris–Cl, pH 8.0, 1 mM EDTA and 100 μg/ml proteinase K). The samples were incubated at 56 °C for 30 min, heated to 95 °C for 5 min and stored at −20 °C until further use. Total DNA was also extracted from frozen tissue samples from an aborted and mummified female fetuses and a male calf offspring (died at 12 h after birth), including brain, heart, muscle, lung, stomach, liver, kidney and intestine (Srirattana et al., 2012) and donor fibroblasts, using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The DNA concentrations were quantified using Quant-iT PicoGreen (Invitrogen, CA, USA). 2.3. Absolute quantitative analysis of mtDNA copy number 2.3.1. Primer design Total DNA from Bos indicus, Bos taurus and B. gaurus (donor cells) were used as templates to amplify the entire displacement loop (D-loop) region of the mtDNA using the forward primer 5′-CTCATCCTAGTGCTA ATACC-3′ and the reverse primer 5′-AGTTGGGAGACTCATCTAGG-3′. The PCR products were purified and ligated into the pGEM-T-Easy cloning vector (Promega, WI, USA) to generate species-specific D-loop plasmids. The DNA sequences of the inserts were identified and submitted to GenBank. Primers specific to gaur and bovine mtDNAs were synthesized based on the D-loop sequence region of the species-specific D-loop plasmids. The primers for the gaur-specific mtDNA were 5′-CTCCCTGATCTAA ACTATTTCC-3′ and 5′-GAAGAGTATATTCTGTAGTGG-3′. The primers for the bovine-specific mtDNA were 5′-CTATTTAAACTATTCCCTGAACAC-3′ and 5′-GGTAATTCATTCTGTGGTC-3′. The specificities of the primers were determined using PCR. 2.3.2. External standard calibration curve The concentration of species-specific D-loop plasmids was measured using Quant-iT PicoGreen kit (Invitrogen), and the corresponding copy number was calculated using the following equation: DNA (copy)= 6.02×1023 (copy/mol)×DNA amount (g)/DNA length (bp)×660 (g/ mol/bp). The plasmids were diluted to 10–107 copies per 2 μl and used as species-specific standard plasmids for qPCR. 2.3.3. qPCR The mtDNA copy number was evaluated using a Bio-Rad Chromo4 real-time PCR detection system (Bio-Rad, CA, USA). Each 20 μl reaction contained 10 μl of 2× SsoFast EvaGreen Super mix (Bio-Rad), 0.25 μM of either gaur-specific D-loop primers or bovine-specific D-loop primers and 2 μl of embryo DNA templates, 10 ng of tissue DNA templates or 2 μl of standard plasmid of a known copy number. The PCR reaction was initiated with denaturation at 98 °C for 2 min, followed by 40 cycles of denaturation at 98 °C for 5 s, annealing at 56 °C for 15 s and extension at 72 °C for 15 s. The EvaGreen fluorescence signal was read at the end of each extension step. Subsequently, a melting curve was generated by slowly heating (0.2 °C/s) the PCR products from 55 °C to 95 °C to determine the specificity of the PCR products. The quantification was performed in duplicate for each embryo sample using at least 18 samples for each embryo stage and in triplicate for the tissue and standard plasmid samples to obtain an average copy number for the mtDNA. The raw data from the embryo samples were multiplied 5-fold to determine the total mtDNA copy number in each oocyte or embryo. 2.4. Statistical analysis The iSCNT embryo development experiments were repeated at least four times for each donor fibroblast. The data were analyzed using ANOVA in the Statistical Analysis Systems (SAS) software. Duncan's

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Multiple Range Test (DMRT) was used to determine the difference between mean at P b 0.05. 3. Results and discussion The development of gaur iSCNT embryos reconstructed with bovine oocytes is summarized in Table 1. The fusion and cleavage rates were significantly (Pb 0.05) higher in the embryos reconstructed with male fibroblasts (MF) than those with female fibroblasts (FF) (93.2 vs. 89.5% and 97.3 vs. 92.9%, respectively). However, the proportion of reconstructed embryos developed to 8-cell, morula and blastocyst stages were not significantly different (P>0.05) when using either MF or FF as donor cells. Approximately 30% of the gaur iSCNT embryos developed to the blastocyst stage, which was higher than previously reported by Lanza et al. (2000) and Mastromonaco et al. (2007) at 12% and 18%, respectively. The development of the gaur iSCNT embryos was not significantly different (P>0.05) compared with the PA control group (Table 1). No difference was observed when the efficiency of the development of iSCNT embryos in vitro was compared between the two genders of donor cell. This result was similar and consistent with the studies of bovine, domestic cat, leopard cat and sheep SCNT (Hosseinia et al., 2008; Kato et al., 2000; Yin et al., 2005, 2006). However, the donor tissue gender affected mouse and banteng SCNT efficiencies (Sansinena et al., 2005; Wakayama and Yanagimachi, 1999). It is also important to consider not only the gender but also the ability of each cell line to develop. In previous studies, both Lanza et al. (2000) and Mastromonaco et al. (2007) worked on gaur– bovine iSCNT, but different embryo development and pregnancy rates were observed. The differences in embryo development were potentially due to the variation of procedures and the donor cells used to produce the SCNT embryos; therefore, the SCNT success between experiments or laboratories cannot be compared. Furthermore, the epigenetic event and recipient cytoplasm could also potentially influence the developmental efficiency of iSCNT embryos (Gómez et al., 2008). Other possible reasons for the low SCNT efficiency include pathological mitochondrial distribution that could lead to deficiencies in mitochondrial function. It has been suggested that mtDNA heteroplasmy might induce incompatibility between the nucleus and cytoplasm, as mitochondria are involved in energy production, and the functions of these organelles depend on orchestrated communication between the nuclear DNA and several copies of the 16.5 kb mtDNA contained within the organelles. Many abnormalities observed in SCNT embryos, fetuses, and offspring might result from abnormalities in mitochondrial functions (St John et al., 2010), but the mechanisms underlying these conditions are still unknown. In this study, the transmission of donor cell mtDNA in gaur–bovine iSCNT embryos produced from two different genders was quantified throughout pre-implantation development. The D-loop regions have been widely used in the study of mtDNA heteroplasmy due to their highly variable sequences. In this study, new D-loop species-specific primers were designed because the available primer sequences and the sequences in GenBank were not able to distinguish B. gaurus from B. indicus and B. taurus (data not shown). We sequenced the mtDNA D-loop of the B. gaurus donor cells (GU324988 and GU324986) and recipient cytoplasts (B. indicus and B. taurus). The sequences were analyzed and aligned to use as templates to design the species-specific primers, and the primers were chosen

from the divergent regions (Fig. 1A). The oocytes for this study were obtained from the ovaries of domestic cattle of unknown species (B. indicus and B. taurus). Therefore, the bovine-specific primers were designed to amplify both B. indicus and B. taurus D-loop regions. The neighbor-joining method was used to construct a phylogenetic tree, which showed that the sequences were categorized into two distinct genetic lineages between domestic (B. indicus and B. taurus) and wild (B. gaurus) cattle, (100% supported with 1000 bootstrap iterations; Fig. 1B). To validate the specificity of the primers, the templates were tested for cross-amplification. Species-specific mtDNA D-loop sequences could be amplified from either gaur or bovine DNA using only gaur or bovine species-specific primers, but no cross amplifications were observed (data not shown). The two primer pairs were species-specific and therefore were used in the reactions to construct the standard curves. The pGEM-T-Easy vectors containing either gaur or bovine D-loop sequences were constructed, and the standard curve concentrations were generated from seven points spanning the expected unknown value (Fig. 2). The copy numbers of gaur and bovine mtDNA and the proportion of gaur mtDNA to total mtDNA per embryo throughout the preimplantation development of female and male iSCNT embryos are shown in Figs. 3 and 4. The mtDNA copy numbers in the cytoplasm of donor and recipient cells were also evaluated prior to electro-fusion. The results revealed that the mean copy numbers of mtDNA from female and male gaur donor fibroblasts were 365 and 350, respectively, while there were 2.7 × 105 copies of bovine mtDNA from the recipient cytoplast. This observation was consistent with the previously reported results of Mastromonaco et al. (2007). In our study, during embryo development, the mean copy number of gaur mtDNA in iSCNT embryos derived from female gaur fibroblasts, remained relatively constant from the 2-cell to the morula stage (129 to 196 copies) and significantly increased at the blastocyst stage (354 copies) (Fig. 3A). However, the mean copy number of the mtDNA from male gaur iSCNT embryos decreased by 60% from 350 at the injected stage to approximately 204–234 at the 2-cell to blastocyst stages (Fig. 4A). Other researchers have also observed inconsistent patterns of donor cell mtDNA throughout development (Ma et al., 2008; Mastromonaco et al., 2007; Srirattana et al., 2011; Yang et al., 2004). However, the number of mtDNA in cytoplasm of bovine recipient embryos reconstructed using either female or male gaur fibroblasts was similar. These numbers decreased from 3.0 × 105 copies per embryo at the 2-cell stage to 1.0 × 105 copies per embryo at the 4- and 8-cell stages followed by a sharp increase at the morula and blastocyst stages (7.0× 105 and 1.0 × 10 6 copies per embryo, respectively; Figs. 3B and 4B). No significant changes in the percentage of the gaur mtDNA to the total amount of mtDNA per embryos were observed, ranging from 0.17 to 0.03% throughout embryo development, with the exception of the 8-cell stage in which the ratio was significantly higher due to the low number of bovine mtDNA at this stage. These data were similar between embryos derived from both female and male gaur fibroblasts. However, the results revealed that some morulae (2 and 3 of 24) and blastocysts (3 and 5 of 25) contained less than 0.01% gaur donor mtDNA, indicating that bovine mtDNA homoplasmy occurred in some of the iSCNT embryos. It has been suggested that the relatively low success rate of SCNT is associated with changes in the pattern of mtDNA transmission following SCNT (Hiendleder, 2007; Spikings et al., 2007; St John et al., 2005). It has been suggested that

Table 1 In vitro development of gaur–bovine iSCNT embryos from female (FF) and male (MF) fibroblasts and parthenogenetic (PA) embryos. Experiments

No. couplets

No. (%) fused

No. (%) cleaved/cultured

No. (%)⁎embryos developed to 8-Cell

PA FF MF

– 410 384

– 367 (89.5)a 358 (93.2)b

a

132/166 (79.5) 339/365 (92.9)b 335/344 (97.3)c

Within the same column, different letters are significantly different (P b 0.05). ⁎ The rates of development were calculated from the number of cultured embryos.

Morula a

102 (61.4) 297 (81.4)b 281 (81.7)b

Blastocyst a

83 (50.0) 154 (42.2)b 144 (41.9)b

48 (28.9) 107 (29.3) 109 (31.7)

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mtDNA heteroplasmy might also inhibit the development of SCNT embryos (Chen et al., 2002; Sansinena et al., 2005). We also observed that the recipient mtDNA decreased gradually at early stages of development and significantly increased at the morula and blastocyst stages. These results are consistent with previous reports (Ma et al., 2008; Mastromonaco et al., 2007; May-Panloup et al., 2005; Yang et al., 2004), and suggest that maternal mtDNA begin replication at the 8-cell stage, which is the time of embryonic genome activation in bovine embryos (Eystone and First, 1986). As embryonic transcriptional activities become fully functional, the mitochondria progressively undergo functional and structural changes (Khurana and Niemann, 2000; Thompson et al., 1996). Several studies have demonstrated that the replication of mammalian mtDNA is initiated at the blastocyst stage (Cummins, 1998; May-Panloup et al., 2005; Spikings et al., 2007) and does not depend on the cell cycle (St John et al., 2010). Furthermore, the replication of mtDNA could increase nuclear transcription levels, producing the energy in the form of ATP that is required for early embryonic development (Hua et al., 2011). In addition, gene reprogramming is most likely ATP dependent (Kikyo et al., 2000), which requires an increase in the level of energy production during the embryonic development. It was shown that the increased mtDNA copy numbers were closely associated with the requirement of ATP for later pre-implantation of embryo during development (Reynier et al., 2001; Stojkovic et al., 2001). ATP limitation has been reported to influence the normal cellular function and the embryo quality (Van Blerkom et al., 1998). However, the results of our study demonstrated that a low donor fibroblast mtDNA copy number, as compared with the amount of maternal mtDNA in gaur–bovine iSCNT embryos, did not affect development to the blastocyst stage in vitro. The amount of mtDNA is strongly associated with mitochondrial function (Jeng et al., 2008). The recipient mtDNA is approximately 100 to 1000 times more than that of donor fibroblasts (El-Shourbagy et al., 2006; Kameyama et al., 2007; Piko and Taylor, 1987). Approximately 0.1% of the donor fibroblast mtDNA is present in the reconstructed embryo. In iSCNT embryos, donor cell mtDNA has been detected throughout the pre-implantation development stages at varying levels of heteroplasmy, including 0.011 to 2% in macaque–rabbit (Yang et al., 2004), 0.01 to 0.6% in gaur–bovine (Mastromonaco et al., 2007), 0.1 to 1% in ovine–bovine (Hua et al., 2008), 0.012 to 2% in goat–sheep (Ma et al., 2008) and 0.07 to 0.15% in buffalo–bovine (Srirattana et al., 2011). These trends were independent of the experiments. Less than 0.1% donor cell-derived mtDNA heteroplasmy was observed, and a large amount of recipient oocyte cytoplasm-derived mtDNA was detected. Interestingly, when mitochondrial biogenesis occurs in the cloned embryos, replication of the higher number of oocyte-derived mtDNA would be selectively favored over the low number of donor cell-derived mtDNA, consequently contributing to the incompatibility of nuclear cytoplasm interactions with specific combinations of mitochondrial and nuclear genomes. Moreover, this study did not only analyze the degree of heteroplasmic mtDNA in the gaur–bovine iSCNT embryos, but rather observed the degree of heteroplasmy in the fetuses and offspring derived from the iSCNT embryos. At the blastocyst stage, the gaur–bovine iSCNT embryos from female and male gaur fibroblasts were transferred to surrogate mothers and pregnancies were detected (Srirattana et al., 2012). Aborted and mummified female fetuses were obtained from two surrogate mothers. A male gaur calf was born from another surrogate mother but died 12 h after birth. DNA samples from the tissues of several organs of the fetuses and offspring were subjected to species-specific mtDNA

Fig. 1. ClustalW alignment of mtDNA D-loop region sequences of female and male B. gaurus and B. indicus and B. taurus; gaps (−) indicate insertion/deletion, asterisks (*) indicate identical base pairs. The highlighted box indicates the primer sequences used for species-specific PCR amplification to distinguish between donor fibroblasts from B. gaurus mtDNA and recipient oocytes mtDNA (A). Neighbor-joining tree of the Bos species. The number at the node is the percentage of 1000 resampling bootstrap replicates (B).

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Fig. 2. The standard curves generated using qPCR of the gaur-specific mtDNA (A) and the bovine-specific mtDNA standard samples (B). Values are the mean ± SD of triplicates.

analysis. The mtDNA copy number in gaur donor fibroblasts and the bovine recipient cytoplasm from the tissue samples are summarized in Table 2. Varying degrees of unpredictable heteroplasmic patterns were observed in the tissue samples of the fetuses and offspring. An analysis of tissues from the female aborted fetus showed variable low percentages of donor cell fibroblast mtDNA in several tissues, including kidney (0.03%), lung (0.7%), muscle (1.1%), liver (1.2%), intestine (1.3%), heart (2.2%) and stomach (2.7%), whereas a higher percentage of donor cell mtDNA (46.4%) was observed in the brain tissue. In the female mummified fetus, a high percentage of gaur-derived mtDNA was observed in the brain (35%), liver (55%), muscle (71%), lung (91%), stomach (93%), intestine (98%), and kidney (99%), but a low percentage was detected in the heart tissue (2.1%). The samples from the gaur iSCNT calf (GM1) also showed variable percentages of gaur mtDNA with less than 13% in muscle, lung, stomach, liver, kidney and intestine. However, higher percentages of gaur mtDNA were observed in the brain (75%) and heart (23%) (Table 2). The results demonstrated that the proportion of donor cell mtDNA varied greatly according to the tissue and individual animal. Interestingly, the percentage of donor cell mtDNA was higher in the fetuses and calf as compared with the embryonic stages. These higher percentages of donor cell mtDNA might have some beneficial effect on post-implantation development. During the post-implantation stages of embryogenesis and organogenesis, the donor cell mtDNA might confer replicative advantages. It has been demonstrated that donor cell mtDNA can be transmitted to cloned offspring with varying efficiencies (Burgstaller et al., 2007; Chen et al., 2002; Inoue et al., 2004; Takeda et al., 2003, 2006). However, data presented here are in contrast with the first observations of gaur iSCNT (Lanza et al., 2000). Using PCR and ethidium bromide-stained gel electrophoresis, they demonstrated homoplasmy in the gaur iSCNT calf. The mtDNA was exclusively derived from the oocytes, and no mtDNA from the gaur was detected. These controversial results might be due to the differences in the sensitivity of the PCR product detection methods used. Hiendleder et al. (2003) showed that the detection limit of

ethidium bromide-stained gel electrophoresis was approximately 2% of the donor cell mtDNA. The causes for the low proportion of cloned embryos that can develop, implant and survive the pregnancy are still unclear. The patterns of mtDNA inheritance from both the donor cell and the oocyte are still controversial. In this study, we have provided information concerning the mtDNA composition in the cloned embryos and their offspring. However, it is difficult to draw a reasonable conclusion because of differences in the experimental design involved in the generation of embryos and offspring subjected to mtDNA analysis, and studies of mitochondrial function, mtDNA expression and ATP production are lacking. To improve the efficiency of iSCNT, the knowledge of epigenetic reprogramming, and mitochondria transmission and function is still needed. 4. Conclusion iSCNT can be used to study basic developmental biology and as a tool for the preservation and propagation of endangered species. In this study, the copy number of both gaur and bovine mtDNA in embryos during pre-implantation and several tissue samples of their fetuses and offspring was investigated. The results indicated that the sex of the donor fibroblasts has no effect on the development to blastocyst stage. The mtDNA from the donor fibroblasts could be detected during all developmental stages (Figs. 3 and 4). These results indicate heteroplasmic mtDNA in the gaur–bovine iSCNT embryos. Furthermore, the fetuses and offspring of gaur–bovine iSCNT embryos transmitted both the population of mtDNA of the recipient ooplasm and the donor fibroblast cytoplasm with varying degrees of heteroplasmy. The level of heteroplasmy is independent of tissue and individual sample. However, the mechanisms leading to this controversy are still unknown. This data demonstrated that high percentages of donor cell mtDNA transmission do not guarantee the success of the iSCNT offspring. To date, the relationships and underlying mechanisms between mtDNA heteroplasmy and iSCNT efficiency have not

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Fig. 4. mtDNA copy number of male gaur–bovine iSCNT embryos during various developmental stages. The number of gaur mtDNA (A) and bovine mtDNA (B) per embryo. The percentage of gaur mtDNA to total mtDNA (C). Individual values are plotted. The bars represent the means. Statistical significance (P b 0.05) is denoted using different superscript letters when differences were observed between developmental stages. Fig. 3. mtDNA copy number in female gaur–bovine iSCNT embryos during various developmental stages. The number of gaur mtDNA (A) and bovine mtDNA (B) per embryo. The percentage of gaur mtDNA to total mtDNA (C). Individual values are plotted. The bars represent the means. Statistical significance (P b 0.05) is denoted using different superscript letters when differences were observed between developmental stages.

been fully understood. Further experiments are needed to clarify the physiological impact of mtDNA inheritance in iSCNT animals to increase our understanding of donor and recipient mitochondrial incompatibility that might contribute to iSCNT inefficiency.

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Table 2 Quantification of gaur and bovine mtDNA segregation in tissue samples of gaur fetuses cloned using iSCNT with female and male fibroblasts. Tissues Samplesb mtDNA copy number Total analyzeda mtDNA Gaur donor Bovine copy fibroblast-derived oocyte-derived number Brain

Heart

Muscle

Lung

Stomach

Liver

Kidney

Intestine

GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1 GF1 GF2 GM1

4.2 × 106 3.0 × 106 4.9 × 106 1.5 × 105 1.5 × 105 1.4 × 106 1.7 × 105 1.2 × 107 1.0 × 106 5.2 × 105 6.3 × 107 6.2 × 106 8.1 × 105 2.7 × 107 3.9 × 106 4.1 × 105 1.2 × 107 2.6 × 106 5.2 × 105 1.8 × 109 1.4 × 107 1.7 × 105 1.4 × 107 1.3 × 106

4.8 × 106 5.7 × 106 1.6 × 106 6.7 × 106 6.9 × 106 4.6 × 106 1.5 × 107 5.2 × 106 1.7 × 107 7.0 × 107 5.7 × 106 8.1 × 107 2.9 × 107 1.1 × 106 2.6 × 107 3.2 × 107 1.0 × 107 3.0 × 107 1.8 × 109 1.8 × 107 1.7 × 109 1.2 × 107 2.4 × 105 1.4 × 107

9.0 × 106 8.7 × 106 6.5 × 106 6.8 × 106 7.0 × 106 6.1 × 106 1.5 × 107 1.7 × 107 1.8 × 107 7.1 × 107 6.9 × 107 8.7 × 107 3.0 × 107 2.9 × 107 3.0 × 107 3.3 × 107 2.2 × 107 3.3 × 107 1.8 × 109 1.8 × 109 1.7 × 109 1.3 × 107 1.4 × 107 1.6 × 107

% gaur mtDNA

46.7 34.5 75.4 2.2 2.1 23.0 1.1 70.6 5.6 0.7 91.3 7.1 2.7 93.1 13.0 1.2 54.5 7.9 0.03 99.0 0.8 1.3 98.3 8.1

Values are the mean of triplicates. a 10 ng of DNA from each tissue was used as a template for qPCR. b An aborted female gaur fetus (GF1), a mummified female gaur fetus (GF2) and a live male gaur calf (GM1).

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