In vitro development of reconstructed ibex (Capra ibex) embryosby 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 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, Chinac 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 2006Available online 9 February 2007
bstract
The ibex (Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availabilityf ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat (Capra hircus) cytoplasts toeprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibexmbryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culturend the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cellsnd of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairyoe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets,6% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used ashe control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found betweenreatment groups A and B in embryo cleavage rate, but a significant difference (P < 0.01) was recorded between treatment groups
and B in the blastocyst production rate. There was a significant difference (P < 0.01) between Treatment C and Treatment A/B inmbryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield11%) 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 theitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exoticapra which has the potential of alleviating species on the endangered list.2007 Elsevier B.V. All rights reserved.
The ibex is a member of the genus Capra, a wildmountain goat with large recurved horns. In China, itis only found in the four provinces Xinjiang, Gansu,Nimengu and Qinhai—the total population being less
than 40,000 animals. Although the ibex is not listed inthe endangered categories of the IUCN 2004 Red List forThreatened Species, in 1988 it was listed as a first-classprotected animal by the Chinese government. Today,because of hunting and habitat degradation, the ibex’ssurvival is seriously threatened. They are now few andfacing extinction (China Species Information System,2005).
The loss of biodiversity in animals due to extinc-tion is receiving increasing attention in many countries(Corley-Smith and Brandhorst, 1999; Holt et al., 2004).Advanced reproductive technologies such as the cryop-reservation of gametes/embryos, in vitro fertilization,artificial insemination, embryo transfer and somatic cellnuclear transfer (NT) are being applied in attemptsto conserve and manage endangered species. Due tothe lack of oocytes and surrogate animals, interspeciesomatic cell nuclear transfer (iSCNT) is being exten-sively applied in reconstructed embryo research (Lanzaet al., 2000; Jiang et al., 2005). Investigations have beenaimed at determining whether oocytes from domesticspecies can support embryonic and fetal development,regulated by a somatic nucleus derived from differentspecie (Dominko et al., 1999).
Recently, studies have shown that oocyte cytoplastsfrom cattle and sheep support the reprogramming ofsomatic 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 theaid of iSCNT a live mouflon offspring has been producedusing enucleated domestic sheep (Ovis aries) oocytes ascytoplasts and granulosa cells collected from a mouflon(Ovis orientalis musimon) as donor (Loi et al., 2001).
The objective of the present study was to explorethe feasibility of using the domestic goat (Capra hir-cus) cytoplast to reprogram the nuclei from ibex donorfibroblasts in an effort to produce ibex embryos follow-ing iSCNT.
2. Material and methods
2.1. Preparation of ibex and domestic goat donorfibroblasts
Adult fibroblasts were isolated from skin samplestaken from the ear of a 3-year-old ibex (52 kg BW,Urumqi Zoo, Urumuqi, Xinjiang, China) and a 3-year-old domestic dairy goat (57 kg BW). Primary cultures
were obtained by mincing the dermal tissue into smallcubes of 0.5–1 mm3 using a sterile curved scissors in apetri dish (Becton Dickinson, Lincoln Park, NJ, USA).The culture medium consisted of DMEM/F12 (Gibco,
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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, thetissue cubes were washed by centrifuging in the culturemedium, the supernatant was discarded and fresh cul-ture medium was added to the tubes. This process wasrepeated three times, after which the tissue cubes weretransferred to a filter cap tissue culture flask (25 cm2)(Nalge Nunc, USA) and 3 ml fresh culture medium wasadded. All cultures were incubated at 38.5 ◦C using 5%CO2 in a humidified atmosphere.
After developing fibroblasts were observed (3–5days) around the cubes, half of the culture mediumwas replaced with fresh medium. The medium was thenreplaced every 2 days until a fibroblast cell layer hadbeen established (7–10 days). The confluent cells weretrypsinized (Gibco), counted in a hemocytometer, andre-seeded at a density of 100,000 cells, in a filter captissue culture flask (25 cm2). After two sub-cultures,cultured cells were exposed to a cryoprotectant solu-tion consisting of DMEM/F-12, 20% (v/v) FBS, 10%(v/v) dimethylsulfoxide (DMSO, Sigma) and frozenin aliquots of approximately 1,000,000 cells/ml of cry-oprotectant in CryoTubesTM (Nunc, Fisher ScientificPittsburgh, PA, USA). To prepare the adult fibroblastsas donors for NT, the cells were thawed and cultured.
2.2. Preparation of recipient cytoplasts
Fresh domestic goat oocytes were collected froman abattoir (Urumqi, Xinjiang, China) and oocytessurrounded by at least three layers of granulosa cellswere selected for in vitro maturation (IVM) culture.Maturation medium (TCM-199, Gibco) was enrichedwith 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/mlP/S. IVM was carried out in a humidified atmosphereof 5% CO2 at 39 ◦C for 24 h. After 21–22 h of in vitromaturation, the cumulus–oocyte complexes (COCs)from domestic goats were treated for 3–5 min in TCMcontaining 0.3–0.5 mg/ml hyaluronidase (Sigma),followed by gentle pipetting to remove the cumuluscells. Mature, metaphase-II oocytes were selected,based on the visualization of a first polar body, andrandomly assigned to three treatment groups. TreatmentA consisted of enucleation and reconstruction withadult ibex fibroblasts (Fig. 1), Treatment C was a
control for pathogenic division, while Treatment B wasa comparison of the homologous NT.
After incubation with 10 �g/ml Hoechst 33342 for15 min at 38.5 ◦C for staining the metaphase plate, the
L. Wang et al. / Small Ruminant Re
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Fig. 1. Adult ibex fibroblasts used in the experiment.
ocytes in M2 medium (Sigma) containing 7.5 �g/mlytochalasin B (Sigma) at 35 ◦C were manipulated usingn inverted microscope (Nikon) fitted with a Narishigeicromanipulation station. The metaphase plate was
isualized under a short UV light exposure (3–5 s),nd the metaphase spindle and polar body with a smallmount of surrounding cytoplasm was removed byegative pressure applied to a beveled 15 �m diametericropipette (Fig. 2). Once enucleated, cytoplasts were
ept in modified TCM (TCM-199 supplemented with0% FBS and 100 IU/ml P/S) and allowed to recoverrom the cytochalasin B treatment for 30 min in thencubator.
.3. Nuclear transfer
In Treatments A and B the donor cells from ibexnd domestic adult dairy goat were third generation
ig. 2. Goat oocytes derived from abattoir enucleated during the exper-ment.
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fibroblasts. Donor cells were starved (0.5% FBS) for3–5 days to induce quiescence, prior to NT procedures(Campbell et al., 1996a,b). Reconstruction was per-formed in groups of 20 cytoplasts in a 100 �l dropletof M2 medium containing 7.5 �g/ml cytochalasin B. Asingle adult ibex fibroblast (Treatment A) or a singleadult dairy goat fibroblast (Treatment B) was insertedinto the perivitelline space of an enucleated oocyte.Fusion of karyoplast–cytoplast couplets was carried outin a Voltain Model EP-1 Cell fusion/Activation Sys-tem (Cryologic, Australia). Couplets were manuallyaligned between the electrodes of a 0.5 mm gap fusionchamber overlaid with fusion buffer (0.28 M mannitol,0.5 mM MgSO4·7H2O, 0.1 mM CaCl2, 0.5 mM HEPESand 1 mg/ml of BSA (Fraction V, Sigma)). The coupletswere then electrofused by two consecutive DC pulses(1.4 kV/cm for 20 �s).
Couplets were microscopically evaluated for fusionafter 0.5 h. The fused couplets were collected. Theoocytes that were randomly allocated to the partheno-genetic activation treatment group (Treatment C) wereplaced in the fusion chamber and pulsed twice(1.4 kV/cm for 20 �s) to initiate activation. The embryosfrom the treatment groups A and B were reprogrammedin IVM medium in a humidified atmosphere of 5% CO2at 38.5 ◦C for 4 h.
2.4. Chemical activation of reconstructed embryos
After the 4 h reprogramming of the embryos for Treat-ments A and B in IVM medium, chemical activation ofall treatment groups was performed by incubation in IVCmedium (Choi et al., 2002) of 7% (v/v) ethanol for 7 minat room temperature (IVC medium: SOF medium sup-plemented with 2% (v/v) basal medium Eagle (BME)essential amino acids (Sigma), 1% (v/v) minimum essen-tial medium (MEM) nonessential amino acids, 1 mMglutamine, 6 mg/ml BSA (fatty acids free) and 0.5 mg/mlmyoinositol), followed by 2.5–4 h of culture in IVCmedium containing 2 mM 6-dimethylaminoputine (6-DMAP).
2.5. In vitro culture of reconstructed embryos
The activated embryos were cultured in 500 �l IVCmedium, overlaid with 500 �l mineral oil (Sigma,embryo tested) at 38.5 ◦C and a 5% CO2, 5% O2 and90% N humidified atmosphere. Embryos were evalu-
2ated for cleavage after 3 days of culture and the mediumwas replaced every 2 days. After 2 days of culture thecleavage embryos were transferred and cultured in SOFmedium supplemented with 2% (v/v) BME essential
138 L. Wang et al. / Small Ruminant Re
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) andthe data were statistically analyzed.
2.6. Statistical analysis
The proportion of oocytes undergoing cleavage(cleavage rate after 3 days of culture) and developingto blastocysts after 7 days of culture in each treatmentgroup were compared using Chi-square analysis. Proba-bility values <0.05 were considered as being statisticallysignificant (SAS Institute Inc., Cary, NC, 1992).
3. Results
The development of interspecies embryos recon-structed with female ibex fibroblasts (Treatment A),NT embryos reconstructed with domestic adult dairygoat fibroblasts (Treatment B), and the parthenogenet-
Table 1Development of interspecies NT embryos reconstructed with female adult ibgoat fibroblasts and the parthenogenic controls
Values with different superscripts (a and b) differ significantly (P < 0.05). Val1 MII = metaphase-II spindle.
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ically activated oocytes (Treatment C) are summarizedin Table 1. The percentage of oocytes cleaved anddeveloping to blastocysts in Treatment C (control) wassignificantly higher than those of embryos reconstructedin Treatments A and B (P < 0.01). Out of a total of190 parthenogenetically activated oocytes, 90% of theembryos cleaved within 72 h and 43% reached the blas-tocyst stage by 168 h of IVC. In Treatment A, 832oocytes were enucleated of which 95% were successfullyreconstructed and 86% subsequently fused followingtwo electrical pulses. Of the fused couplets, 68% ofthe embryos cleaved within 72 h and 11% reached theblastocyst stage by 168 h of IVC. In Treantment B, 205oocytes were enucleated into cytoplasts, 96% of whichwere reconstructed and 81% subsequently fused follow-ing two electrical pulses. Of the fused couplets, 76% ofthe embryos cleaved within 72 h and 31% reached theblastocyst stage by 168 h of IVC. No significant differ-ence between treatment groups A and B was recordedregarding embryo cleavage rate (68% versus 76%), butthere was a significant difference (P < 0.01) in blastocystproduction rate (11% versus 31%).
4. Discussion
Interspecies nuclear transfer is a powerful tool for theprotection of endangered species from extinction, andsome exciting successes have been attained (Dominko etal., 1999; Lanza et al., 2000; Loi et al., 2001). However,a number of problems associated with this technol-ogy still remain and its efficiency until now is verylow—less than 1% of the cloned embryos develop intoviable offspring (Han et al., 2003). Most of the rea-sons for this extremely high failure rate are unknown.In nuclear transfer experiments, many factors may affectthe results—the donor cell being one of the most impor-
tant. Suitable donor cells must not only have healthycell membranes, but also good compatibility with therecipient oocytes. Some reports have shown that donorcells with high cell passage numbers result in lower
ex fibroblasts, NT embryos reconstructed with domestic adult dairy
%)nstructed)
No. (%)(fused)
No. (%)(cleaved)
No. (%)(blastocysts)
95) 678 (86) 461 (68)aA 76 (11)A
6) 150 (81) 114 (76)aA 46 (31)B
190 (100) 171 (90)B 82 (43)C
ues with different superscripts (A–C) differ significantly (P < 0.01).
nant Re
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usion rates and lower blastocyst development rates inhe reconstructed embryos (Wilmut et al., 1997; Kato etl., 1998; Hill et al., 2000; Roh et al., 2000; Bhuiyant al., 2004). The possible explanation is that long-erm in vitro culture alters the donor cell metabolismnd unbalanced regulation of imprinted genes may benduced, thus affecting nuclear remodeling (Walker etl., 1996). In fact, the donor cells of most successfullyloned animals come from early-passage G0/G1 fibrob-asts (Cibelli et al., 1998; Baguisi et al., 1999; Wells etl., 1999; Onishi et al., 2000). Contrary opinions havelso been expressed (Wakayama et al., 1999; Kubotat al., 2000). The key factor being whether the differ-ntiated nucleus of donor cells from different speciesan be reprogrammed successfully. Only the completeeprogramming of the nucleus can smoothly conducthe development of reconstructed embryos in vitro andn vivo, particularly when the species of donor kary-plasts and recipient cytoplasts are far apart in theiraxonomic classification (Dominko et al., 1999; Lanzat al., 2000).
In this study, the donor cells for the iSCNT and the NTere third generation fibroblasts. The iSCNT embryoserived from the ibex fibroblasts could develop to thelastocyst stage, but both cleavage rate and blastocystield of iSCNT embryos (68 and 11%) derived from thebex fibroblasts and NT embryos (76 and 31%) derivedrom the domestic goat fibroblasts were significantlyower than those of parthenogenetic developed embryos90 and 43%). This indicates incompatibilities betweenhe new components synthesized by the donor nucleusnd the components left over in the recipient cytoplasmDominko et al., 1999). In this case, the donor cells maye insufficiently reprogrammed in recipient oocytes (Lut al., 2005). Other possible reasons are that when enu-leation was performed by labeling the oocyte DNA withoechst 33342 (Smith, 1993). Cell membrane integrityas damaged and exposure to UV light decreased theiability of the reconstructed embryo (Yang et al., 1990;mith, 1993).
In interspecies cloned animals, the recipient oocytes another important factor, since the dominant distri-ution of mtDNA is from recipient oocytes (Steinbornt al., 2002; Takeda et al., 2003). To some extent,he reprogramming of the donor cell nucleus is deter-
ined by the cell cycle stage of the recipient cytoplasmDominko et al., 1999). Studies have indicated that fol-owing electrofusion, if the reconstructed embryos with
MII cytoplast and G0/G1 somatic nucleus are repro-
rammed in IVM medium for an extended period ofime, benefits later development (Wakayama et al., 1998;ominko et al., 1999). Similar results were obtained
search 73 (2007) 135–141 139
in this study. In normal embryos, the earliest stagesof embryogenesis are regulated by maternally inher-ited gene products stored within the oocyte cytoplasm.The progress in the development of embryogenesis isfinally determined by whether or not the embryonicgenes are activated at species-specific developmentalstages (Telford et al., 1990). The same events occur inIVC of iSCNT embryos. Thus whether the maternal-to-embryonic transition occurs smoothly in the recipientcytoplasm is determined by whether the differentiatednucleus of donor cells from different species can bereprogrammed successfully or not.
In comparison with the NT embryos derived fromdomestic goat fibroblasts, the iSCNT embryos derivedfrom ibex fibroblasts had a much lower blastocystrate (11% versus 31%). The ibex fibroblasts were lessefficient than the domestic goat fibroblasts in nucleartransfer embryo development. One reason may be thedifference in donor cell types as donor cells may differin their ability to reprogram recipient oocytes (Saikhunet al., 2002). The more distantly related the species, themore difficult is the maternal-to-embryonic transition(Wells et al., 1999). The nuclear–cytoplasmic interac-tion may be perturbed, leading to difficulty in activatingthe genes of the donor cell (Gurdon, 1986).
Interestingly, interspecies NT studies have madeimportant contributions in iSCNT embryo developmentand endangered animal clones and interspecies NT hasbeen reported live offspring in the gaur (Lanza et al.,2000) and the mouflon (Loi et al., 2001).
In the present study, iSCNT embryos derived fromibex fibroblasts reached the blastocyst stage of devel-opment and a blastocyst development rate of 11%was attained. This demonstrated that the domesticgoat cytoplast supported mitotic cleavage of the ibexkaryoplast and was capable of reprogramming thenucleus to achieve a blastocyst stage embryo in exoticCapra.
Acknowledgements
The authors would like to thank the Urumqi Zoo, Xin-jiang, China for providing the adult Siberian ibex eartissue samples. This study was financially supported byXinjiang Goldcattle Bio. Inc.
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