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Animal Reproduction Institute, Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530005, China
ABSTRACT
Cloning of buffalos (Bubalus bubalis) through nuclear transfer is a potential alternative approach in genetic improvement of buffalos. However, to our knowledge, cloned offspring of buffalos derived from embryonic, fetal, or somatic cells have not yet been reported. Thus, factors affecting the nuclear transfer of buffalo somatic cells were examined, and the possibility of cloning buffalos was explored in the present study. Treatment of buffalo fibroblasts and granulosa cells with aphidicolin plus serum starvation resulted in more cells being arrested at the G0/G1 phase, the proportion of cells with DNA fragmentation being less, and the number of embryos derived from these cells that developed to blastocysts being greater. In addition, a difference was found in the development of embryos reconstructed with fetal fibroblasts from different individuals (P < 0.001). Forty-two blastocysts derived from granulosa cells and fetal fibroblasts were transferred into 21 recipient swamp buffalos, and 4 recipients were confirmed to be pregnant by rectal palpation on Day 60 of gestation. One recipient received two embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two female premature calves. Three recipients maintained pregnancy to term and delivered three female cloned calves after Days 338–349 of gestation. These results indicate that buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to the term of gestation and result in newborn calves.
developmental biology,, early development,, embryo,, pregnancy
Since the first report of live mammals produced by nuclear transfer (NT) of a cultured cell line in 1996 [1], cloned mammals have been produced successfully in sheep [2], cattle [3], mouse [4], goat [5], pig [6], rabbit [7], cat [8], rat [9], horse [10], mule [11], dog [12], and ferret [13] with a variety of somatic cell types as nuclear donors. However, to our knowledge, cloned offspring of buffalos (Bubalus bubalis) derived from embryonic, fetal, or somatic cells have not yet been reported because of their lower fertility, as evidenced by their delayed puberty, silent estrous, long postpartum ovarian inactivity, and lower oocyte recovery number from ovaries in comparison with cattle [14]. Saikhun et al. [15] transferred cloned embryos derived from buffalo fetal fibroblasts into 12 recipients. Three recipients were confirmed to be pregnant on Day 60 after embryo transfer, but no recipient could carry the pregnancy beyond Day 90. Buffalos are important domestic animals that are distributed in the tropical and subtropical regions, providing a high quality of milk, meat, and work power. However, the milk yield of Chinese swamp buffalos is extremely low (normally less than 1000 kg/yr) and is in urgent need of improvement through the propagation of superior germ plasm by modern reproductive technologies. Thus, the cloning of buffalos has a special significance in the genetic improvement of buffalos.
The stage of the donor cell cycle is a major factor contributing to the success of NT in mammals [2, 16]. To facilitate the remodeling and reprogramming of somatic cell nuclei, nonactivated metaphase II oocytes with high levels of maturation-promoting factor are generally employed as recipient cytoplasts for somatic cell NT [17, 18]. Thus, donor cells are usually required to be arrested at the G1 or G0 stage of the cell cycle to avoid chromosomal damage and abnormal ploidy in the resulting embryos. Serum starvation is a commonly used method to arrest cell lines in the G0 phase of the cell cycle [1, 2] but often causes reduced cell survival and increased DNA fragmentation [19], which will, in turn, cause subsequent high embryonic loss after NT [20]. Thus, it is necessary to seek a new method to synchronize the donor cells in the G1 or G0 phase of the cell cycle. Aphidicolin is a reversible inhibitor of mammalian DNA polymerases and can block the cell cycle at the transition from the G1 to S phase [21]. Kues et al. [19] found that aphidicolin treatment (6 µM for 14 h) led to an accumulation of 81.9% porcine fetal fibroblasts at the G1/S transition and that cells returned to the cell cycle rapidly following aphidicolin removal, indicating that aphidicolin was low in toxicity and that its effects were fully reversible. Therefore, treatment with aphidicolin plus serum starvation may be a potential method to synchronize donor cells in the G1 or G0 phase of the cell cycle.
In the present study, aphidicolin plus serum starvation was first employed to synchronize the cell cycle of buffalo fetal fibroblasts and ovarian granulosa cells, and then the effects of donor cell individuals on the development of NT embryos were also examined. In addition, a noninvasive enucleation method (the Spindle View System; SpindleView, Woburn, MA) was employed to prepare cytoplasts, and an efficient fusion method (platinum needle electrode) was applied to fuse the donor cell and cytoplast. Finally, three cloned buffalos from fetal fibroblasts and adult ovarian granulosa cells were produced successfully.
All chemicals used in this study were purchased from Sigma (St. Louis, MO), with the exception of TCM 199 powder, which was purchased from Gibco BRL (Paisley, Scotland, U.K.), and fetal calf serum (FCS) and Dulbecco modified Eagle medium (DMEM), which were purchased from Hyclone (Logan, UT). The in vitro maturation medium was TCM 199 supplemented with 26.2 mM NaHCO3, 5 mM Hepes, 5% estrous cow serum (ECS, self-preparation), 2% bovine follicular fluid (collected without regard to the stage of the reproductive cycle), and FSH at 0.1 µg/ml. The embryo culture medium (CM) was TCM 199 supplemented with 3% ECS. The basic micromanipulation medium was TCM 199 supplemented with 5 mM NaHCO3, 5 mM Hepes, and 5% ECS. All of the media were supplemented with penicillin G at 60 µg/ml and streptomycin sulfate at 100 µg/ml and then sterilized by passing through a 0.20-µm filter and stored at 4°C for up to 4 wk.
Preparation of Recipient Cytoplasts
Buffalo oocytes from Chinese swamp buffalo ovaries taken at slaughter were matured in a 30-mm glass dish containing 1.5 ml of maturation medium for 20–22 h under a humidified 5% CO2 in air at 38.5°C. After in vitro maturation, the surrounding cumulus cells were removed by manual pipetting in the presence of hyaluronidase at 2 mg/ml, and oocytes with an extruded first polar body were selected for enucleation. The selected oocytes were placed into the manipulation medium drop supplemented with cytochalasin B at 5 µg/ml and 0.1 M sucrose for 10 min before micromanipulation. The first polar body and metaphase II plate were removed by aspiration with a 25-µm-inner-diameter beveled pipette under the Nikon TE300 inverted microscope equipped with a Narishige micromanipulator (Tokyo, Japan) and the Spindle View System. Exposing all the removed cytoplasm to the Spindle View System and checking for the presence of the removed metaphase plate confirmed successful enucleation (Fig. 1).
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Preparation of Donor Karyoplasts
Tissues from the skin of three Chinese swamp buffalo female fetuses (about 3 mo) were enzymatically digested with 0.25% trypsin and 0.05% EDTA for 30 min and then with 0.2% collagenase for 45 min. The disaggregated cells were washed three times in DMEM supplemented with 10% FCS by centrifugation at 500 x g for 5 min and then placed in culture in a 60-mm tissue culture dish under a humidified 5% CO2 in air atmosphere at 38.5°C. After 7–10 days of culture, confluent fibroblast monolayers were obtained and then routinely passaged with an enzymatic solution (0.25% trypsin and 0.05% EDTA) for 7 min. Small aliquots of progressively growing cell lines that were established were frozen in DMEM supplemented with 10% FCS and 10% dimethyl sulfoxide (DMSO) for further study.
Granulosa cells from 2- to 6-mm-diameter follicles of one aged Chinese swamp buffalo (about 15 yr old) ovary taken at slaughter were washed three times in DMEM supplemented with 10% FCS by centrifugation at 500 x g for 5 min and then placed in culture in a 60-mm tissue culture dish under a humidified 5% CO2 in air atmosphere at 38.5°C for 4–5 days. The confluent granulosa cell monolayers were trypsinized with a solution of 0.25% trypsin and 0.05% EDTA and then frozen in DMEM supplemented with 10% FCS and 10% DMSO for further study.
After three to six passages, either confluent fibroblasts or granulosa cells were treated by culture in DMEM plus 10% FCS for 72 h, serum starvation (culture in DMEM plus 0.5% FCS for 72 h), or a combination of aphidicolin and serum starvation (culture in aphidicolin at 0.1 µg/ml for 24 h and then in DMEM plus 0.5% FCS for 48 h) before being used as donor nuclei or for analysis of the cell cycle.
Cell Cycle Analysis by Flow Cytometry
Cell cycle analysis was performed as previously described [22] with a little modification. Briefly, cells to be submitted for flow cytometry sorting were trypsinized, washed, and resuspended in 1 ml of Dulbecco PBS. Cells were fixed in 75% ethanol at 4°C for at least 24 h and then washed twice with PBS. The cell pellets were stained with propidium iodide at 50 µg/ml for 30 min at 37°C. The cell cycle stages of each cell population (1 x 107 cells/ml) were analyzed on a FACS vantage flow cytometer (Becton Dickinson, San Jose, CA), and the percentages of the cells at the G0/G1, S, and G2/M phases of the cell cycle were calculated.
Trypsinized donor cells were transferred into the perivitelline space of enucleated oocytes with a 25-µm micropipette. The couplet was transferred to a droplet of 100 µl of fusion medium (0.28 M mannitol, 0.1 mM CaCl2, 0.1 mM MgSO4, 5 mM Hepes, and 0.1% BSA) overlaid with mineral oil and then placed on the micromanipulator with two platinum needle electrodes (0.2 mm apart). The fusion was induced by the application of an alternating current pulse of 2 V for 1 sec and then three direct current pulses of 1 kV/cm for 15 µsec with an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, CA). Couplets were then washed in CM and incubated in this medium for 30 min at 38.5°C. The fusion of couplets was checked at 200x magnification under an inverted microscope. Three hours after the fusion, the activation of fused embryos was induced by exposure to 5 µM ionomycin in CM for 5 min and subsequent incubation in 2 mM 6-dimethylaminopurine for 3 h at 38.5°C and 5% CO2 in air.
In Vitro Culture of Reconstructed Embryos
After activation, reconstructed embryos were placed in coculture with granulosa cell monolayers in a 30-µl droplet of CM overlaid with mineral oil under a humidified atmosphere of 5% CO2 in air at 38.5°C. The granulosa cell monolayers were established 48–72 h before the introduction of embryos. After the introduction of embryos, half of the medium was replaced with fresh medium every 24 h. After 2 days of coculture, the cleavage of reconstructed embryos was checked, and the number of developed blastocysts was recorded within 8 days of coculture.
Day 6 cloned blastocysts derived from either granulosa cells or fetal fibroblasts that had been treated with aphidicolin plus serum starvation were transferred nonsurgically into the uterine horn ipsilateral to the ovary containing a palpable corpus luteum of recipient Chinese swamp buffalos at Day 6 of the native estrous cycle. Each recipient received two embryos, and the pregnancy status was determined 60 days after embryo transfer by rectal palpation.
All live animals were raised in the Experimental Animal Farm of Guangxi University under good feeding conditions. All procedures described within were reviewed and approved by the Experimental Animal Care and Use Committee of Guangxi University and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
To identify whether the cloned buffalo calves derived from donor cells, a microsatellite analysis of genomic DNA from the various samples was performed with 20 microsatellite markers (ILSTS038, ILSTS097, ILSTS008, ILSTS028, CSRM60, CSSM047, CSSM019, BM1818, CSSM036, ILSTS020, ILSTS030, CSSM032, ILSTS029, ILSTS061, CSSM66, ILSTS019, BM1824, CSSM022, CSSM033, and CSSM041). The total genomic DNA from three cloned claves (CB1, CB2, and CB3) and three foster mothers (RB1, RB2, and RB3) was obtained from their ear skin. The DNA from donor cells (DB2, DB1, and DB3) was prepared from frozen ovary tissue and fetal fibroblast cell lines. The PCR primers for microsatellite markers labeled with fluorescent dyes (6 FAM, 9 HEX, and 5 TET) were synthesized by Beijing AUGCT Biotechnology. The PCR analysis was carried out for 35 cycles, and products were separated by 2% agarose gels. All multiplexing and loci evaluations were performed on an ABI 3730XL automated sequencer by GeneMapper v. 3.5 software (Applied Biosystems, Foster City, CA).
Within each experiment, the difference between treatments in frequencies of oocytes undergoing cleavage and developing to the blastocyst stage was analyzed by the chi-square test. The cell cycle distribution of donor cells was analyzed by a one-way ANOVA (GraphPad software, v. 2.02; GraphPad, San Diego, CA). Probability values less than 0.05 were considered significant.
Effects of Culture Methods on the Cell Cycle Distribution of Donor Cells
To seek an efficient treatment for synchronizing more donor cells at the G0/G1 phase, buffalo fetal fibroblasts and granulosa cells were treated by confluent culture (10% FCS for 72 h), serum starvation (0.5% FCS for 72 h), or aphidicolin plus serum starvation (aphidicolin at 0.1 µg/ml for 24 h and then 0.5%). As shown in Tables 1 and 2, significantly more fetal fibroblasts or granulosa cells treated by aphidicolin plus serum starvation were synchronized at the G0/G1 phase (87.9%–89.0%) than the cells treated by serum starvation (76.6%–87.9%, P < 0.007) and confluent culture (63%–66%, P < 0.0004). In addition, the DNA fragmentation proportion of cells treated with aphidicolin plus serum starvation (2.6%–2.8%) was significantly less than that of serum starvation only (8.3%–9.1%, P < 0.0002), although it was significantly higher than that of normal serum supplementation (1.1%–1.4%, P < 0.05).
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Development of NT Embryos from Donor Cells Treated with Different Methods
To compare the NT efficiency of donor cells treated with different methods, either fibroblasts or granulosa cells that had been treated with confluent culture, serum starvation, or aphidicolin plus serum starvation were transferred into the enucleated buffalo oocytes. As illustrated in Tables 3 and 4, the cleavage rate (70%–74%) and blastocyst yield (21%–22%) of reconstructed embryos from fetal fibroblasts and granulosa cells treated with aphidicolin plus serum starvation were significantly higher than those from fetal fibroblasts and granulosa cells treated with either confluent culture in 10% FCS supplementation medium or serum starvation only (P < 0.02).
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Effects of Donor Cell Individuals on the Development of NT Embryos
To examine the donor cell individual contributions to the development of NT embryos, three fibroblast cell lines from three fetuses were employed as the NT donor cells. Significantly more NT embryos from the fetal fibroblasts of individual 011010 developed to blastocysts (23.6%, P < 0.001) than the NT embryos from the fetal fibroblasts of either individual 030323 (8.8%) or individual 030410 (10.8%), although there was no significant difference in the cleavage rate of NT embryos among the three fetal fibroblast cell lines (Table 5).
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Pregnancy and Calf Birth Following Embryo Transfer
To examine the developmental competence of reconstructed embryos from different donor cells, a total of 42 blastocysts derived from granulosa cells and fetal fibroblasts that had been treated with aphidicolin plus serum starvation were transferred into 21 recipient buffalos (Table 6). Four recipients were confirmed to be pregnant by rectal palpation at Day 60 of gestation. One pregnant buffalo received two NT embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two premature female calves (9 and 17 kg) (Fig. 2A). One pregnant buffalo underwent a cesarean section on Day 343 of gestation (19 November 2004) and delivered a female calf (CB1) from fetal fibroblasts (Fig. 2B). Unfortunately, the calf survived for only 20 min after birth, which may have been due to the amniotic fluid from the respiration tract of the calf not having been expelled immediately, because all of the organs appeared to be normal after dissection. The two remaining pregnant buffalos delivered a female calf (CB2) derived from granulosa cells (Fig. 2C) on Day 349 of gestation (17 March 2005) and a female calf (CB3) derived from fetal fibroblasts (Fig. 2E) on Day 338 of gestation (25 October 2005). The birth weight of the three calves (29, 23, and 23 kg) was similar to the normal buffalo calf (21–39 kg), and none of them showed the large calf syndrome as described in cloned calves [23, 24]. The cloned buffalo calf derived from ovarian granulosa cells began to appear hyperpyrexic and to display an ‘adult' appearance with rough hairs progressively on Day 5 after birth (Fig. 2D). The calf was cured on Day 8 with ampicillin, showed hyperpyrexia again on Day 12, and died on Day 14. Cloned calf CB3, derived from fetal fibroblasts, grew well and is still alive (Fig. 2F).
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Genetic Analysis of Cloned Calves
To identify whether the cloned buffalo calves derived from donor cells, a microsatellite analysis of genomic DNA from the various samples was performed with 20 microsatellite markers. Comparison of the pattern of alleles in the cloned buffalo calves with their donor cell populations and surrogate mothers indicated that two cloned calves (CB1 and CB3) derived from fetal fibroblasts and that one cloned calf (CB2) derived from granulosa cells, as there was 100% identity to donor cells (DB1, DB2, and DB3) and a significant difference from the surrogate mothers (RB1, RB2, and RB3) at all 20 microsatellite markers (Table 7).
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The present study shows that the development of buffalo NT embryos may depend on the donor cell genetic characteristics and culture treatment, and buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to gestation and result in newborn buffalo calves.
To set up an efficient NT procedure, one of the most important steps is to remove the nucleus of recipient oocytes completely. The enucleation of oocytes prior to NT is of crucial importance 1) to avoid aneuploidy with its detrimental effects on later development, 2) to eliminate any genetic contribution of the recipient cytoplasm, and 3) to exclude the possibility of parthenogenetic activation and embryo development without the participation of the newly introduced nucleus. Although enucleation has been accomplished successfully in a range of species by labeling the oocyte DNA with Hoechst 33342 [25], exposure to ultraviolet light for 30 sec causes a loss in membrane integrity, decreases methionine incorporation, alters protein synthesis patterns in bovine oocytes [25], and decreases viability in rabbit oocytes [26]. Enucleation under the Spindle View System (Pol-Scope image) is a reliable, noninvasive new method, which has been proved to be efficient in the enucleation of mammalian oocytes [27]. Thus, we removed the nucleus of recipient oocytes under the Spindle View System to prepare cytoplasts for subsequent NT (Fig. 1).
Apart from the recipient cytoplast, one of the important factors contributing to the success of somatic cell NT is appropriate cell cycle coordination. The cell cycle-stage synchrony between the donor nucleus and the recipient oocyte is critical to ensure the development of NT embryos [28], although donor nuclei from nonquiescent or cycling cells could be successfully used to clone cattle as well [29]. Serum starvation is a commonly used method to arrest cell lines in the G0 phase of the cell cycle [1, 2] but often causes reduced cell survival and increased DNA fragmentation [19], which will result in subsequent high embryonic loss after NT [20]. Except for serum starvation, several reversible chemical inhibitors are also effective in blocking the cell cycle at various positions. Aphidicolin is a reversible inhibitor of mammalian DNA polymerases and can block the cell cycle at the transition from the G1 to S phase [21]. It has been shown that rodent and human fibroblasts can be synchronized in a reversible manner at the G1/S transition with 6 µM aphidicolin [30]. Thus, we synchronized donor cells at the G0/G1 phase by a combination of aphidicolin and serum starvation treatment and found that significantly more fibroblasts or granulosa cells were synchronized at the G0/G1 phase than cells treated by serum starvation and confluent culture in normal serum supplementation. In addition, the DNA fragmentation proportion of cells treated with aphidicolin plus serum starvation was significantly less than that of serum starvation. Results of the subsequent NT experiment also show that the cleavage rate and blastocyst yield of reconstructed embryos from donor cells treated by aphidicolin and serum starvation were significantly higher than those of donor cells treated with either confluent culture in 10% FCS supplementation medium or serum starvation only. The DNA synthesis of donor cells may be inhibited by treatment with aphidicolin and sustained by following serum starvation in a physiological status. Thus, treatment of donor cells with aphidicolin plus serum starvation can reduce the DNA damage caused by direct serum starvation and is an appropriate procedure for donor cell cycle synchronization in buffalo somatic cell NT.
The development of cloned embryos differs among donor cell lines, even if they derived from the same tissue or organ [31]. Similarly, results obtained by Miyoshi et al. [32] from NT with four primary cell lines of adult bovine somatic cells indicate that the primary donor cell culture affects in vitro blastocyst development, initial pregnancy rates, and the percentage of live births. Powell et al. [33] evaluated the NT efficiency of bovine fetal fibroblasts harvested from six Jersey fetuses and concluded that a significant component in determining somatic cell NT success is the source of the nuclear donor cells. In the present study, a significant difference in the blastocyst formation of cloned embryos was also found among fetal fibroblasts from three buffalo fetuses. Thus, the reprogramming of the donor nucleus in the cytoplasts may be dependent on their genetic characteristics, and the selection of donor cells may be important to ensure NT efficiency.
The gestation length for the cloned pregnancies (average, 343 days; range, 338–349 days) in the present study seems to be longer than that for normal breeding in swamp buffalos (average, 330 days; range, 320–340 days) [34]. A similar result was observed by Kubota et al. [35] in cloned cattle, in which the gestation periods for the cloned pregnancies were 9 days longer than the average gestation period for the breed. The prolongation of gestation length for the cloned pregnancies may be due to the delayed maturation of the cloned fetal adrenal gland.
The cloned buffalo calf derived from the ovarian granulosa cells of one aged buffalo in the present study displayed an ‘adult' appearance with rough hairs and died 14 days after birth, indicating a donor aging effect on the survivability of cloned offspring. Although a 21-yr-old Brahman bull and a 17-yr-old Japanese Black Beef bull were cloned successfully [35, 36] and even two clones derived from a 12-yr-old Japanese Black sire that had become infertile have been proved to have normal fertility [37], the aging characteristics were observed in many male clones derived from a bull aged 10 yr [31], and a significant cardiopulmonary pathology and juvenile-onset diabetes at birth were also associated with the male clone from a 21-yr-old Brahman bull [36]. Thus, donor cell age may have an influence on the viability of clones.
The success of this NT procedure in buffalos may be because of modifications in the following three aspects. First, a noninvasive enucleation method was employed with the Spindle View System, which can ensure the removal of oocyte nuclei completely by removing a small amount of cytoplasm and then maintaining the correct chromosome ploidy of the reconstructed embryos that may be very important to their subsequent development. Second, aphidicolin plus serum starvation was employed to synchronize the donor cell cycle at the G0/G1 phase without resulting in damage to the DNA in comparison with serum treatment alone, which may favor the development of reconstructed embryos. A similar result was reported in the NT of bovine somatic cells treated by roscovitine with a high fetal and calf survival after embryo transfer [38]. Third, a new method (platinum needle electrode) has been applied to fuse the donor cell and cytoplast, in which a high fusion rate can be achieved in a relative low intensity of electronic field (1 kV/cm, 15 µsec), and then the viability of reconstructed embryos can be maintained maximally after electronic fusion.
The successful development of NT in buffalos will speed the genetic improvement of buffalo herds and also allow us to prepare transgenic cloning buffalos. However, only three cloned swamp buffalos were obtained, and the efficiency is still low. Thus, more research should be done in the future.
ACKNOWLEDGMENTS
We are grateful to Prof. Ning Li and Dr. Yinhua Huang of China Agricultural University for their assistance in the microsatellite analysis of buffalo clones.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by China High Technology Development Program (2002AA206651) and China Natural Science Foundation (30460090).
Correspondence: 2FAX: 86 771 323 9202; e-mail: ardsshi{at}gxu.edu.cn
Received: 25 January 2007.
First decision: 16 February 2007.
Accepted: 27 April 2007.
REFERENCES
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