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In vitro development of reconstructed ibex (Capra ibex) embryos by nuclear transfer using goat (Capra hircus) oocytes
Small Ruminant Research 73 (2007) 135–141
In vitro development of reconstructed ibex (Capra ibex) embryos by nuclear transfer using goat (Capra hircus) oocytes Liang Wang a,c,d , Tao Peng b , Hai Zhu b , Zili Lv b , Tingting Liu b , Zhiqiang Shuai b , Hong Gao b , Tao Cai b , Xu Cao b , Hanqing Wang a,∗ a
c
Key Laboratory for Nature Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Road 342#, Lanzhou 730000, China b Xinjiang Goldcattle Bio. Inc., Xiaoyun Road 15#, Economic & Technology Development Zone, Urumqi 830026, Xinjiang, China Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing South Road 40#, Urumqi 830011, Xinjiang, China d Graduate School of the Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100039, China Received 11 July 2006; received in revised form 20 December 2006; accepted 21 December 2006 Available online 9 February 2007
Abstract The ibex (Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat (Capra hircus) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference (P < 0.01) was recorded between treatment groups A and B in the blastocyst production rate. There was a significant difference (P < 0.01) between Treatment C and Treatment A/B in embryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield (11%) of iSCNT embryos derived from female ibex fibroblasts (Treatment A) were lower than those of nuclear transfer embryos (Treatment B) and parthenogenetic development embryos (Treatment C). The domestic goat (C. hircus) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list. © 2007 Elsevier B.V. All rights reserved. Keywords: Ibex; Interspecies; Nuclear transfer; Embryo
1. Introduction
∗ Corresponding author. Tel.: +86 991 3750767; fax: +86 991 3716067. E-mail address: [email protected] (H. Wang).
0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.12.009
The ibex is a member of the genus Capra, a wild mountain goat with large recurved horns. In China, it is only found in the four provinces Xinjiang, Gansu, Nimengu and Qinhai—the total population being less
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than 40,000 animals. Although the ibex is not listed in the endangered categories of the IUCN 2004 Red List for Threatened Species, in 1988 it was listed as a first-class protected animal by the Chinese government. Today, because of hunting and habitat degradation, the ibex’s survival is seriously threatened. They are now few and facing extinction (China Species Information System, 2005). The loss of biodiversity in animals due to extinction is receiving increasing attention in many countries (Corley-Smith and Brandhorst, 1999; Holt et al., 2004). Advanced reproductive technologies such as the cryopreservation of gametes/embryos, in vitro fertilization, artificial insemination, embryo transfer and somatic cell nuclear transfer (NT) are being applied in attempts to conserve and manage endangered species. Due to the lack of oocytes and surrogate animals, interspecie somatic cell nuclear transfer (iSCNT) is being extensively applied in reconstructed embryo research (Lanza et al., 2000; Jiang et al., 2005). Investigations have been aimed at determining whether oocytes from domestic species can support embryonic and fetal development, regulated by a somatic nucleus derived from different specie (Dominko et al., 1999). Recently, studies have shown that oocyte cytoplasts from cattle and sheep support the reprogramming of somatic cell nuclei from other species (Dominko et al., 1999; White et al., 1999; Bui et al., 2002; Li et al., 2002; Lee et al., 2003; Sansinena et al., 2003, 2005). With the aid of iSCNT a live mouflon offspring has been produced using enucleated domestic sheep (Ovis aries) oocytes as cytoplasts and granulosa cells collected from a mouflon (Ovis orientalis musimon) as donor (Loi et al., 2001). The objective of the present study was to explore the feasibility of using the domestic goat (Capra hircus) cytoplast to reprogram the nuclei from ibex donor fibroblasts in an effort to produce ibex embryos following iSCNT. 2. Material and methods 2.1. Preparation of ibex and domestic goat donor fibroblasts Adult fibroblasts were isolated from skin samples taken from the ear of a 3-year-old ibex (52 kg BW, Urumqi Zoo, Urumuqi, Xinjiang, China) and a 3-yearold domestic dairy goat (57 kg BW). Primary cultures were obtained by mincing the dermal tissue into small cubes of 0.5–1 mm3 using a sterile curved scissors in a petri dish (Becton Dickinson, Lincoln Park, NJ, USA). The culture medium consisted of DMEM/F12 (Gibco,
Grand Island, NY, USA), with 20% fetal bovine serum (FBS, Gbico) and 100 IU/ml penicillin-streptomycin (P/S, SIGMA, St. Louis, MO, USA). Prior to culture, the tissue cubes were washed by centrifuging in the culture medium, the supernatant was discarded and fresh culture medium was added to the tubes. This process was repeated three times, after which the tissue cubes were transferred to a filter cap tissue culture flask (25 cm2 ) (Nalge Nunc, USA) and 3 ml fresh culture medium was added. All cultures were incubated at 38.5 ◦ C using 5% CO2 in a humidified atmosphere. After developing fibroblasts were observed (3–5 days) around the cubes, half of the culture medium was replaced with fresh medium. The medium was then replaced every 2 days until a fibroblast cell layer had been established (7–10 days). The confluent cells were trypsinized (Gibco), counted in a hemocytometer, and re-seeded at a density of 100,000 cells, in a filter cap tissue culture flask (25 cm2 ). After two sub-cultures, cultured cells were exposed to a cryoprotectant solution consisting of DMEM/F-12, 20% (v/v) FBS, 10% (v/v) dimethylsulfoxide (DMSO, Sigma) and frozen in aliquots of approximately 1,000,000 cells/ml of cryoprotectant in CryoTubesTM (Nunc, Fisher Scientific Pittsburgh, PA, USA). To prepare the adult fibroblasts as donors for NT, the cells were thawed and cultured. 2.2. Preparation of recipient cytoplasts Fresh domestic goat oocytes were collected from an abattoir (Urumqi, Xinjiang, China) and oocytes surrounded by at least three layers of granulosa cells were selected for in vitro maturation (IVM) culture. Maturation medium (TCM-199, Gibco) was enriched with 10% FBS, 5 g/ml FSH (Sigma), 5 g/ml LH (Sigma), 1 g/ml E2 (Sigma), 0.3 mM sodium pyruvate (Sigma), 100 M cysteamine (Sigma) and 100 IU/ml P/S. IVM was carried out in a humidified atmosphere of 5% CO2 at 39 ◦ C for 24 h. After 21–22 h of in vitro maturation, the cumulus–oocyte complexes (COCs) from domestic goats were treated for 3–5 min in TCM containing 0.3–0.5 mg/ml hyaluronidase (Sigma), followed by gentle pipetting to remove the cumulus cells. Mature, metaphase-II oocytes were selected, based on the visualization of a first polar body, and randomly assigned to three treatment groups. Treatment A consisted of enucleation and reconstruction with adult ibex fibroblasts (Fig. 1), Treatment C was a control for pathogenic division, while Treatment B was a comparison of the homologous NT. After incubation with 10 g/ml Hoechst 33342 for 15 min at 38.5 ◦ C for staining the metaphase plate, the
L. Wang et al. / Small Ruminant Research 73 (2007) 135–141
Fig. 1. Adult ibex fibroblasts used in the experiment.
oocytes in M2 medium (Sigma) containing 7.5 g/ml cytochalasin B (Sigma) at 35 ◦ C were manipulated using an inverted microscope (Nikon) fitted with a Narishige micromanipulation station. The metaphase plate was visualized under a short UV light exposure (3–5 s), and the metaphase spindle and polar body with a small amount of surrounding cytoplasm was removed by negative pressure applied to a beveled 15 m diameter micropipette (Fig. 2). Once enucleated, cytoplasts were kept in modified TCM (TCM-199 supplemented with 10% FBS and 100 IU/ml P/S) and allowed to recover from the cytochalasin B treatment for 30 min in the incubator. 2.3. Nuclear transfer In Treatments A and B the donor cells from ibex and domestic adult dairy goat were third generation
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fibroblasts. Donor cells were starved (0.5% FBS) for 3–5 days to induce quiescence, prior to NT procedures (Campbell et al., 1996a,b). Reconstruction was performed in groups of 20 cytoplasts in a 100 l droplet of M2 medium containing 7.5 g/ml cytochalasin B. A single adult ibex fibroblast (Treatment A) or a single adult dairy goat fibroblast (Treatment B) was inserted into the perivitelline space of an enucleated oocyte. Fusion of karyoplast–cytoplast couplets was carried out in a Voltain Model EP-1 Cell fusion/Activation System (Cryologic, Australia). Couplets were manually aligned between the electrodes of a 0.5 mm gap fusion chamber overlaid with fusion buffer (0.28 M mannitol, 0.5 mM MgSO4 ·7H2 O, 0.1 mM CaCl2 , 0.5 mM HEPES and 1 mg/ml of BSA (Fraction V, Sigma)). The couplets were then electrofused by two consecutive DC pulses (1.4 kV/cm for 20 s). Couplets were microscopically evaluated for fusion after 0.5 h. The fused couplets were collected. The oocytes that were randomly allocated to the parthenogenetic activation treatment group (Treatment C) were placed in the fusion chamber and pulsed twice (1.4 kV/cm for 20 s) to initiate activation. The embryos from the treatment groups A and B were reprogrammed in IVM medium in a humidified atmosphere of 5% CO2 at 38.5 ◦ C for 4 h. 2.4. Chemical activation of reconstructed embryos After the 4 h reprogramming of the embryos for Treatments A and B in IVM medium, chemical activation of all treatment groups was performed by incubation in IVC medium (Choi et al., 2002) of 7% (v/v) ethanol for 7 min at room temperature (IVC medium: SOF medium supplemented with 2% (v/v) basal medium Eagle (BME) essential amino acids (Sigma), 1% (v/v) minimum essential medium (MEM) nonessential amino acids, 1 mM glutamine, 6 mg/ml BSA (fatty acids free) and 0.5 mg/ml myoinositol), followed by 2.5–4 h of culture in IVC medium containing 2 mM 6-dimethylaminoputine (6DMAP). 2.5. In vitro culture of reconstructed embryos
Fig. 2. Goat oocytes derived from abattoir enucleated during the experiment.
The activated embryos were cultured in 500 l IVC medium, overlaid with 500 l mineral oil (Sigma, embryo tested) at 38.5 ◦ C and a 5% CO2 , 5% O2 and 90% N2 humidified atmosphere. Embryos were evaluated for cleavage after 3 days of culture and the medium was replaced every 2 days. After 2 days of culture the cleavage embryos were transferred and cultured in SOF medium supplemented with 2% (v/v) BME essential
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Fig. 3. Interspecies NT ibex blastocysts.
amino acids, 1% (v/v) MEM non-essential amino acids, 1 mM glutamine, 6 mg/ml BSA (fatty acids free) and 5% FBS. Blastocysts from all three treatment groups (Fig. 3) were selected after 7 days in culture (fusion = day 0) and the data were statistically analyzed. 2.6. Statistical analysis The proportion of oocytes undergoing cleavage (cleavage rate after 3 days of culture) and developing to blastocysts after 7 days of culture in each treatment group were compared using Chi-square analysis. Probability values <0.05 were considered as being statistically significant (SAS Institute Inc., Cary, NC, 1992). 3. Results The development of interspecies embryos reconstructed with female ibex fibroblasts (Treatment A), NT embryos reconstructed with domestic adult dairy goat fibroblasts (Treatment B), and the parthenogenet-
ically activated oocytes (Treatment C) are summarized in Table 1. The percentage of oocytes cleaved and developing to blastocysts in Treatment C (control) was significantly higher than those of embryos reconstructed in Treatments A and B (P < 0.01). Out of a total of 190 parthenogenetically activated oocytes, 90% of the embryos cleaved within 72 h and 43% reached the blastocyst stage by 168 h of IVC. In Treatment A, 832 oocytes were enucleated of which 95% were successfully reconstructed and 86% subsequently fused following two electrical pulses. Of the fused couplets, 68% of the embryos cleaved within 72 h and 11% reached the blastocyst stage by 168 h of IVC. In Treantment B, 205 oocytes were enucleated into cytoplasts, 96% of which were reconstructed and 81% subsequently fused following two electrical pulses. Of the fused couplets, 76% of the embryos cleaved within 72 h and 31% reached the blastocyst stage by 168 h of IVC. No significant difference between treatment groups A and B was recorded regarding embryo cleavage rate (68% versus 76%), but there was a significant difference (P < 0.01) in blastocyst production rate (11% versus 31%). 4. Discussion Interspecies nuclear transfer is a powerful tool for the protection of endangered species from extinction, and some exciting successes have been attained (Dominko et al., 1999; Lanza et al., 2000; Loi et al., 2001). However, a number of problems associated with this technology still remain and its efficiency until now is very low—less than 1% of the cloned embryos develop into viable offspring (Han et al., 2003). Most of the reasons for this extremely high failure rate are unknown. In nuclear transfer experiments, many factors may affect the results—the donor cell being one of the most important. Suitable donor cells must not only have healthy cell membranes, but also good compatibility with the recipient oocytes. Some reports have shown that donor cells with high cell passage numbers result in lower
Table 1 Development of interspecies NT embryos reconstructed with female adult ibex fibroblasts, NT embryos reconstructed with domestic adult dairy goat fibroblasts and the parthenogenic controls Treatment group (fibroblasts)
No. of MII1 (oocytes)
No. (%) (enucleated)
No. (%) (reconstructed)
No. (%) (fused)
No. (%) (cleaved)
No. (%) (blastocysts)
A (ibex fibroblasts) B (goat fibroblasts) C (parthenogenic)
861 205 190
832 (97) 193 (94) –
790 (95) 185(96) –
678 (86) 150 (81) 190 (100)
461 (68)aA 114 (76)aA 171 (90)B
76 (11)A 46 (31)B 82 (43)C
Values with different superscripts (a and b) differ significantly (P < 0.05). Values with different superscripts (A–C) differ significantly (P < 0.01). 1 MII = metaphase-II spindle.
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fusion rates and lower blastocyst development rates in the reconstructed embryos (Wilmut et al., 1997; Kato et al., 1998; Hill et al., 2000; Roh et al., 2000; Bhuiyan et al., 2004). The possible explanation is that longterm in vitro culture alters the donor cell metabolism and unbalanced regulation of imprinted genes may be induced, thus affecting nuclear remodeling (Walker et al., 1996). In fact, the donor cells of most successfully cloned animals come from early-passage G0/G1 fibroblasts (Cibelli et al., 1998; Baguisi et al., 1999; Wells et al., 1999; Onishi et al., 2000). Contrary opinions have also been expressed (Wakayama et al., 1999; Kubota et al., 2000). The key factor being whether the differentiated nucleus of donor cells from different species can be reprogrammed successfully. Only the complete reprogramming of the nucleus can smoothly conduct the development of reconstructed embryos in vitro and in vivo, particularly when the species of donor karyoplasts and recipient cytoplasts are far apart in their taxonomic classification (Dominko et al., 1999; Lanza et al., 2000). In this study, the donor cells for the iSCNT and the NT were third generation fibroblasts. The iSCNT embryos derived from the ibex fibroblasts could develop to the blastocyst stage, but both cleavage rate and blastocyst yield of iSCNT embryos (68 and 11%) derived from the ibex fibroblasts and NT embryos (76 and 31%) derived from the domestic goat fibroblasts were significantly lower than those of parthenogenetic developed embryos (90 and 43%). This indicates incompatibilities between the new components synthesized by the donor nucleus and the components left over in the recipient cytoplasm (Dominko et al., 1999). In this case, the donor cells may be insufficiently reprogrammed in recipient oocytes (Lu et al., 2005). Other possible reasons are that when enucleation was performed by labeling the oocyte DNA with Hoechst 33342 (Smith, 1993). Cell membrane integrity was damaged and exposure to UV light decreased the viability of the reconstructed embryo (Yang et al., 1990; Smith, 1993). In interspecies cloned animals, the recipient oocyte is another important factor, since the dominant distribution of mtDNA is from recipient oocytes (Steinborn et al., 2002; Takeda et al., 2003). To some extent, the reprogramming of the donor cell nucleus is determined by the cell cycle stage of the recipient cytoplasm (Dominko et al., 1999). Studies have indicated that following electrofusion, if the reconstructed embryos with a MII cytoplast and G0/G1 somatic nucleus are reprogrammed in IVM medium for an extended period of time, benefits later development (Wakayama et al., 1998; Dominko et al., 1999). Similar results were obtained
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in this study. In normal embryos, the earliest stages of embryogenesis are regulated by maternally inherited gene products stored within the oocyte cytoplasm. The progress in the development of embryogenesis is finally determined by whether or not the embryonic genes are activated at species-specific developmental stages (Telford et al., 1990). The same events occur in IVC of iSCNT embryos. Thus whether the maternal-toembryonic transition occurs smoothly in the recipient cytoplasm is determined by whether the differentiated nucleus of donor cells from different species can be reprogrammed successfully or not. In comparison with the NT embryos derived from domestic goat fibroblasts, the iSCNT embryos derived from ibex fibroblasts had a much lower blastocyst rate (11% versus 31%). The ibex fibroblasts were less efficient than the domestic goat fibroblasts in nuclear transfer embryo development. One reason may be the difference in donor cell types as donor cells may differ in their ability to reprogram recipient oocytes (Saikhun et al., 2002). The more distantly related the species, the more difficult is the maternal-to-embryonic transition (Wells et al., 1999). The nuclear–cytoplasmic interaction may be perturbed, leading to difficulty in activating the genes of the donor cell (Gurdon, 1986). Interestingly, interspecies NT studies have made important contributions in iSCNT embryo development and endangered animal clones and interspecies NT has been reported live offspring in the gaur (Lanza et al., 2000) and the mouflon (Loi et al., 2001). In the present study, iSCNT embryos derived from ibex fibroblasts reached the blastocyst stage of development and a blastocyst development rate of 11% was attained. This demonstrated that the domestic goat cytoplast supported mitotic cleavage of the ibex karyoplast and was capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra. Acknowledgements The authors would like to thank the Urumqi Zoo, Xinjiang, China for providing the adult Siberian ibex ear tissue samples. This study was financially supported by Xinjiang Goldcattle Bio. Inc. References Baguisi, A., Behboodi, E., Melican, D.T., Pollock, J.S., Destrempes, M.M., Cammuso, C., Williams, J.L., Nims, S.D., Porter, C.A., Midura, P., Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G.,
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In vitro development of reconstructed ibex (Capra ibex) embryos by nuclear transfer using goat (Capra hircus) oocytes Liang Wang a,c,d , Tao Peng b , Hai Zhu b , Zili Lv b , Tingting Liu b , Zhiqiang Shuai b , Hong Gao b , Tao Cai b , Xu Cao b , Hanqing Wang a,∗ a
c
Key Laboratory for Nature Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Road 342#, Lanzhou 730000, China b Xinjiang Goldcattle Bio. Inc., Xiaoyun Road 15#, Economic & Technology Development Zone, Urumqi 830026, Xinjiang, China Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing South Road 40#, Urumqi 830011, Xinjiang, China d Graduate School of the Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100039, China Received 11 July 2006; received in revised form 20 December 2006; accepted 21 December 2006 Available online 9 February 2007
Abstract The ibex (Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat (Capra hircus) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference (P < 0.01) was recorded between treatment groups A and B in the blastocyst production rate. There was a significant difference (P < 0.01) between Treatment C and Treatment A/B in embryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield (11%) of iSCNT embryos derived from female ibex fibroblasts (Treatment A) were lower than those of nuclear transfer embryos (Treatment B) and parthenogenetic development embryos (Treatment C). The domestic goat (C. hircus) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list. © 2007 Elsevier B.V. All rights reserved. Keywords: Ibex; Interspecies; Nuclear transfer; Embryo
1. Introduction
∗ Corresponding author. Tel.: +86 991 3750767; fax: +86 991 3716067. E-mail address: [email protected] (H. Wang).
0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.12.009
The ibex is a member of the genus Capra, a wild mountain goat with large recurved horns. In China, it is only found in the four provinces Xinjiang, Gansu, Nimengu and Qinhai—the total population being less
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than 40,000 animals. Although the ibex is not listed in the endangered categories of the IUCN 2004 Red List for Threatened Species, in 1988 it was listed as a first-class protected animal by the Chinese government. Today, because of hunting and habitat degradation, the ibex’s survival is seriously threatened. They are now few and facing extinction (China Species Information System, 2005). The loss of biodiversity in animals due to extinction is receiving increasing attention in many countries (Corley-Smith and Brandhorst, 1999; Holt et al., 2004). Advanced reproductive technologies such as the cryopreservation of gametes/embryos, in vitro fertilization, artificial insemination, embryo transfer and somatic cell nuclear transfer (NT) are being applied in attempts to conserve and manage endangered species. Due to the lack of oocytes and surrogate animals, interspecie somatic cell nuclear transfer (iSCNT) is being extensively applied in reconstructed embryo research (Lanza et al., 2000; Jiang et al., 2005). Investigations have been aimed at determining whether oocytes from domestic species can support embryonic and fetal development, regulated by a somatic nucleus derived from different specie (Dominko et al., 1999). Recently, studies have shown that oocyte cytoplasts from cattle and sheep support the reprogramming of somatic cell nuclei from other species (Dominko et al., 1999; White et al., 1999; Bui et al., 2002; Li et al., 2002; Lee et al., 2003; Sansinena et al., 2003, 2005). With the aid of iSCNT a live mouflon offspring has been produced using enucleated domestic sheep (Ovis aries) oocytes as cytoplasts and granulosa cells collected from a mouflon (Ovis orientalis musimon) as donor (Loi et al., 2001). The objective of the present study was to explore the feasibility of using the domestic goat (Capra hircus) cytoplast to reprogram the nuclei from ibex donor fibroblasts in an effort to produce ibex embryos following iSCNT. 2. Material and methods 2.1. Preparation of ibex and domestic goat donor fibroblasts Adult fibroblasts were isolated from skin samples taken from the ear of a 3-year-old ibex (52 kg BW, Urumqi Zoo, Urumuqi, Xinjiang, China) and a 3-yearold domestic dairy goat (57 kg BW). Primary cultures were obtained by mincing the dermal tissue into small cubes of 0.5–1 mm3 using a sterile curved scissors in a petri dish (Becton Dickinson, Lincoln Park, NJ, USA). The culture medium consisted of DMEM/F12 (Gibco,
Grand Island, NY, USA), with 20% fetal bovine serum (FBS, Gbico) and 100 IU/ml penicillin-streptomycin (P/S, SIGMA, St. Louis, MO, USA). Prior to culture, the tissue cubes were washed by centrifuging in the culture medium, the supernatant was discarded and fresh culture medium was added to the tubes. This process was repeated three times, after which the tissue cubes were transferred to a filter cap tissue culture flask (25 cm2 ) (Nalge Nunc, USA) and 3 ml fresh culture medium was added. All cultures were incubated at 38.5 ◦ C using 5% CO2 in a humidified atmosphere. After developing fibroblasts were observed (3–5 days) around the cubes, half of the culture medium was replaced with fresh medium. The medium was then replaced every 2 days until a fibroblast cell layer had been established (7–10 days). The confluent cells were trypsinized (Gibco), counted in a hemocytometer, and re-seeded at a density of 100,000 cells, in a filter cap tissue culture flask (25 cm2 ). After two sub-cultures, cultured cells were exposed to a cryoprotectant solution consisting of DMEM/F-12, 20% (v/v) FBS, 10% (v/v) dimethylsulfoxide (DMSO, Sigma) and frozen in aliquots of approximately 1,000,000 cells/ml of cryoprotectant in CryoTubesTM (Nunc, Fisher Scientific Pittsburgh, PA, USA). To prepare the adult fibroblasts as donors for NT, the cells were thawed and cultured. 2.2. Preparation of recipient cytoplasts Fresh domestic goat oocytes were collected from an abattoir (Urumqi, Xinjiang, China) and oocytes surrounded by at least three layers of granulosa cells were selected for in vitro maturation (IVM) culture. Maturation medium (TCM-199, Gibco) was enriched with 10% FBS, 5 g/ml FSH (Sigma), 5 g/ml LH (Sigma), 1 g/ml E2 (Sigma), 0.3 mM sodium pyruvate (Sigma), 100 M cysteamine (Sigma) and 100 IU/ml P/S. IVM was carried out in a humidified atmosphere of 5% CO2 at 39 ◦ C for 24 h. After 21–22 h of in vitro maturation, the cumulus–oocyte complexes (COCs) from domestic goats were treated for 3–5 min in TCM containing 0.3–0.5 mg/ml hyaluronidase (Sigma), followed by gentle pipetting to remove the cumulus cells. Mature, metaphase-II oocytes were selected, based on the visualization of a first polar body, and randomly assigned to three treatment groups. Treatment A consisted of enucleation and reconstruction with adult ibex fibroblasts (Fig. 1), Treatment C was a control for pathogenic division, while Treatment B was a comparison of the homologous NT. After incubation with 10 g/ml Hoechst 33342 for 15 min at 38.5 ◦ C for staining the metaphase plate, the
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Fig. 1. Adult ibex fibroblasts used in the experiment.
oocytes in M2 medium (Sigma) containing 7.5 g/ml cytochalasin B (Sigma) at 35 ◦ C were manipulated using an inverted microscope (Nikon) fitted with a Narishige micromanipulation station. The metaphase plate was visualized under a short UV light exposure (3–5 s), and the metaphase spindle and polar body with a small amount of surrounding cytoplasm was removed by negative pressure applied to a beveled 15 m diameter micropipette (Fig. 2). Once enucleated, cytoplasts were kept in modified TCM (TCM-199 supplemented with 10% FBS and 100 IU/ml P/S) and allowed to recover from the cytochalasin B treatment for 30 min in the incubator. 2.3. Nuclear transfer In Treatments A and B the donor cells from ibex and domestic adult dairy goat were third generation
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fibroblasts. Donor cells were starved (0.5% FBS) for 3–5 days to induce quiescence, prior to NT procedures (Campbell et al., 1996a,b). Reconstruction was performed in groups of 20 cytoplasts in a 100 l droplet of M2 medium containing 7.5 g/ml cytochalasin B. A single adult ibex fibroblast (Treatment A) or a single adult dairy goat fibroblast (Treatment B) was inserted into the perivitelline space of an enucleated oocyte. Fusion of karyoplast–cytoplast couplets was carried out in a Voltain Model EP-1 Cell fusion/Activation System (Cryologic, Australia). Couplets were manually aligned between the electrodes of a 0.5 mm gap fusion chamber overlaid with fusion buffer (0.28 M mannitol, 0.5 mM MgSO4 ·7H2 O, 0.1 mM CaCl2 , 0.5 mM HEPES and 1 mg/ml of BSA (Fraction V, Sigma)). The couplets were then electrofused by two consecutive DC pulses (1.4 kV/cm for 20 s). Couplets were microscopically evaluated for fusion after 0.5 h. The fused couplets were collected. The oocytes that were randomly allocated to the parthenogenetic activation treatment group (Treatment C) were placed in the fusion chamber and pulsed twice (1.4 kV/cm for 20 s) to initiate activation. The embryos from the treatment groups A and B were reprogrammed in IVM medium in a humidified atmosphere of 5% CO2 at 38.5 ◦ C for 4 h. 2.4. Chemical activation of reconstructed embryos After the 4 h reprogramming of the embryos for Treatments A and B in IVM medium, chemical activation of all treatment groups was performed by incubation in IVC medium (Choi et al., 2002) of 7% (v/v) ethanol for 7 min at room temperature (IVC medium: SOF medium supplemented with 2% (v/v) basal medium Eagle (BME) essential amino acids (Sigma), 1% (v/v) minimum essential medium (MEM) nonessential amino acids, 1 mM glutamine, 6 mg/ml BSA (fatty acids free) and 0.5 mg/ml myoinositol), followed by 2.5–4 h of culture in IVC medium containing 2 mM 6-dimethylaminoputine (6DMAP). 2.5. In vitro culture of reconstructed embryos
Fig. 2. Goat oocytes derived from abattoir enucleated during the experiment.
The activated embryos were cultured in 500 l IVC medium, overlaid with 500 l mineral oil (Sigma, embryo tested) at 38.5 ◦ C and a 5% CO2 , 5% O2 and 90% N2 humidified atmosphere. Embryos were evaluated for cleavage after 3 days of culture and the medium was replaced every 2 days. After 2 days of culture the cleavage embryos were transferred and cultured in SOF medium supplemented with 2% (v/v) BME essential
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Fig. 3. Interspecies NT ibex blastocysts.
amino acids, 1% (v/v) MEM non-essential amino acids, 1 mM glutamine, 6 mg/ml BSA (fatty acids free) and 5% FBS. Blastocysts from all three treatment groups (Fig. 3) were selected after 7 days in culture (fusion = day 0) and the data were statistically analyzed. 2.6. Statistical analysis The proportion of oocytes undergoing cleavage (cleavage rate after 3 days of culture) and developing to blastocysts after 7 days of culture in each treatment group were compared using Chi-square analysis. Probability values <0.05 were considered as being statistically significant (SAS Institute Inc., Cary, NC, 1992). 3. Results The development of interspecies embryos reconstructed with female ibex fibroblasts (Treatment A), NT embryos reconstructed with domestic adult dairy goat fibroblasts (Treatment B), and the parthenogenet-
ically activated oocytes (Treatment C) are summarized in Table 1. The percentage of oocytes cleaved and developing to blastocysts in Treatment C (control) was significantly higher than those of embryos reconstructed in Treatments A and B (P < 0.01). Out of a total of 190 parthenogenetically activated oocytes, 90% of the embryos cleaved within 72 h and 43% reached the blastocyst stage by 168 h of IVC. In Treatment A, 832 oocytes were enucleated of which 95% were successfully reconstructed and 86% subsequently fused following two electrical pulses. Of the fused couplets, 68% of the embryos cleaved within 72 h and 11% reached the blastocyst stage by 168 h of IVC. In Treantment B, 205 oocytes were enucleated into cytoplasts, 96% of which were reconstructed and 81% subsequently fused following two electrical pulses. Of the fused couplets, 76% of the embryos cleaved within 72 h and 31% reached the blastocyst stage by 168 h of IVC. No significant difference between treatment groups A and B was recorded regarding embryo cleavage rate (68% versus 76%), but there was a significant difference (P < 0.01) in blastocyst production rate (11% versus 31%). 4. Discussion Interspecies nuclear transfer is a powerful tool for the protection of endangered species from extinction, and some exciting successes have been attained (Dominko et al., 1999; Lanza et al., 2000; Loi et al., 2001). However, a number of problems associated with this technology still remain and its efficiency until now is very low—less than 1% of the cloned embryos develop into viable offspring (Han et al., 2003). Most of the reasons for this extremely high failure rate are unknown. In nuclear transfer experiments, many factors may affect the results—the donor cell being one of the most important. Suitable donor cells must not only have healthy cell membranes, but also good compatibility with the recipient oocytes. Some reports have shown that donor cells with high cell passage numbers result in lower
Table 1 Development of interspecies NT embryos reconstructed with female adult ibex fibroblasts, NT embryos reconstructed with domestic adult dairy goat fibroblasts and the parthenogenic controls Treatment group (fibroblasts)
No. of MII1 (oocytes)
No. (%) (enucleated)
No. (%) (reconstructed)
No. (%) (fused)
No. (%) (cleaved)
No. (%) (blastocysts)
A (ibex fibroblasts) B (goat fibroblasts) C (parthenogenic)
861 205 190
832 (97) 193 (94) –
790 (95) 185(96) –
678 (86) 150 (81) 190 (100)
461 (68)aA 114 (76)aA 171 (90)B
76 (11)A 46 (31)B 82 (43)C
Values with different superscripts (a and b) differ significantly (P < 0.05). Values with different superscripts (A–C) differ significantly (P < 0.01). 1 MII = metaphase-II spindle.
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fusion rates and lower blastocyst development rates in the reconstructed embryos (Wilmut et al., 1997; Kato et al., 1998; Hill et al., 2000; Roh et al., 2000; Bhuiyan et al., 2004). The possible explanation is that longterm in vitro culture alters the donor cell metabolism and unbalanced regulation of imprinted genes may be induced, thus affecting nuclear remodeling (Walker et al., 1996). In fact, the donor cells of most successfully cloned animals come from early-passage G0/G1 fibroblasts (Cibelli et al., 1998; Baguisi et al., 1999; Wells et al., 1999; Onishi et al., 2000). Contrary opinions have also been expressed (Wakayama et al., 1999; Kubota et al., 2000). The key factor being whether the differentiated nucleus of donor cells from different species can be reprogrammed successfully. Only the complete reprogramming of the nucleus can smoothly conduct the development of reconstructed embryos in vitro and in vivo, particularly when the species of donor karyoplasts and recipient cytoplasts are far apart in their taxonomic classification (Dominko et al., 1999; Lanza et al., 2000). In this study, the donor cells for the iSCNT and the NT were third generation fibroblasts. The iSCNT embryos derived from the ibex fibroblasts could develop to the blastocyst stage, but both cleavage rate and blastocyst yield of iSCNT embryos (68 and 11%) derived from the ibex fibroblasts and NT embryos (76 and 31%) derived from the domestic goat fibroblasts were significantly lower than those of parthenogenetic developed embryos (90 and 43%). This indicates incompatibilities between the new components synthesized by the donor nucleus and the components left over in the recipient cytoplasm (Dominko et al., 1999). In this case, the donor cells may be insufficiently reprogrammed in recipient oocytes (Lu et al., 2005). Other possible reasons are that when enucleation was performed by labeling the oocyte DNA with Hoechst 33342 (Smith, 1993). Cell membrane integrity was damaged and exposure to UV light decreased the viability of the reconstructed embryo (Yang et al., 1990; Smith, 1993). In interspecies cloned animals, the recipient oocyte is another important factor, since the dominant distribution of mtDNA is from recipient oocytes (Steinborn et al., 2002; Takeda et al., 2003). To some extent, the reprogramming of the donor cell nucleus is determined by the cell cycle stage of the recipient cytoplasm (Dominko et al., 1999). Studies have indicated that following electrofusion, if the reconstructed embryos with a MII cytoplast and G0/G1 somatic nucleus are reprogrammed in IVM medium for an extended period of time, benefits later development (Wakayama et al., 1998; Dominko et al., 1999). Similar results were obtained
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in this study. In normal embryos, the earliest stages of embryogenesis are regulated by maternally inherited gene products stored within the oocyte cytoplasm. The progress in the development of embryogenesis is finally determined by whether or not the embryonic genes are activated at species-specific developmental stages (Telford et al., 1990). The same events occur in IVC of iSCNT embryos. Thus whether the maternal-toembryonic transition occurs smoothly in the recipient cytoplasm is determined by whether the differentiated nucleus of donor cells from different species can be reprogrammed successfully or not. In comparison with the NT embryos derived from domestic goat fibroblasts, the iSCNT embryos derived from ibex fibroblasts had a much lower blastocyst rate (11% versus 31%). The ibex fibroblasts were less efficient than the domestic goat fibroblasts in nuclear transfer embryo development. One reason may be the difference in donor cell types as donor cells may differ in their ability to reprogram recipient oocytes (Saikhun et al., 2002). The more distantly related the species, the more difficult is the maternal-to-embryonic transition (Wells et al., 1999). The nuclear–cytoplasmic interaction may be perturbed, leading to difficulty in activating the genes of the donor cell (Gurdon, 1986). Interestingly, interspecies NT studies have made important contributions in iSCNT embryo development and endangered animal clones and interspecies NT has been reported live offspring in the gaur (Lanza et al., 2000) and the mouflon (Loi et al., 2001). In the present study, iSCNT embryos derived from ibex fibroblasts reached the blastocyst stage of development and a blastocyst development rate of 11% was attained. This demonstrated that the domestic goat cytoplast supported mitotic cleavage of the ibex karyoplast and was capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra. Acknowledgements The authors would like to thank the Urumqi Zoo, Xinjiang, China for providing the adult Siberian ibex ear tissue samples. This study was financially supported by Xinjiang Goldcattle Bio. Inc. References Baguisi, A., Behboodi, E., Melican, D.T., Pollock, J.S., Destrempes, M.M., Cammuso, C., Williams, J.L., Nims, S.D., Porter, C.A., Midura, P., Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G.,
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