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Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts
Cell Biology International 30 (2006) 698e703 www.elsevier.com/locate/cellbi
Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts Md. Abul Hashem a, Dilip P. Bhandari a, Sung Keun Kang a,b, Byeong Chun Lee a,b,*, Hwang Woo Suk a,b,c a
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea b The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, South Korea c School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea Received 5 December 2005; revised 25 February 2006; accepted 26 April 2006
Abstract The present study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. Seven experiments were performed, each with a one-way completely randomized design involving three replicates. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved, and fully confluent stages of Goral cells. In Experiments II and III, the effects of different antioxidants like b-mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 24 h and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ml), were used as in Experiment II. In Experiments VI and VII, the effect of different levels of dimethylsulphoxide (DMSO) at 0%, 0.5%, 1.0% and 2.5% were tested by flow cytometry (FACS). In Experiment I, 68.7% of Goral skin fibroblasts reached the G0/G1 stage (2C DNA content) when subjected to the serum-starved medium, which was more than the cycling (64.9%) and fully confluent groups (61.0%) (P > 0.05). Among the chemically treated group, b-ME, cysteine and DMSO showed better results for synchronization of G0 þ G1 phases than cycling, serum-starved and fully confluent groups. It can thus be concluded that b-ME, cysteine and DMSO at certain concentrations can synchronize the cell cycle effectively, which could have a positive impact on somatic cell nuclear transfer in the goral. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Goral; Cell cycle; Synchronization
1. Introduction Gorals are listed as endangered animals in CITES Appendix I. The grey goral is classified as being at low risk, or nearly threatened, by the 1996 IUCN report (Shackleton, 1997). Gorals derived from the subfamily caprinae have been classified into three tribes, Rupicaprini, Ovibovini and Caprini (Geist, 1987; Gentry, 1992). Within the tribe Rupicaprini, serows and gorals have been assigned to two separate genera (Mead, * Corresponding author. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea. Tel.: þ82 2 880 1269; fax: þ82 2 884 1902. E-mail address: [email protected] (B.C. Lee).
1989; Nowak, 1999), Capricornis and Nemorhaedus, respectively, despite being closely related. The genus Nemorhaedus of Rupicaprini includes three species, N. caudatus (long-tailed goral or Chinese goral), N. goral (Himalayan goral) and N. baileyi (red goral). Nemorhaedus species’ range from northern Pakistan to Northeastern Asia including Korea (Mead, 1989). The longtailed goral (or Chinese goral), N. caudatus, is one of the most endangered mammalian species in Korea (Ministry of Environment of Korea, 2000) and has been designated as a Natural Monument species by the Cultural Properties Administration of the Korean Government. The goral population in South Korea is assumed to be less than 250 (Cultural Properties Administration of Korea, 1999), and their habitats are
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.04.008
Hashem et al. / Cell Biology International 30 (2006) 698e703
restricted and highly fragmented. Therefore, a systematic conservation plan for the revival of this species is urgently needed in South Korea (Min et al., 2004). One should note that some phylogenetic work for Korean gorals has already been done (Min et al., 2004). This phylogenetic study (Min et al., 2004) has shown that Korean and Russian gorals belong to the same clade, and differ from Chinese gorals. Furthermore, they are more genetically distinct than the Chinese goral. Gorals in both countries are highly endangered; probably no more than seven hundred remain in the wild in each country (Cultural Properties Administration of Korea, 1999; Myslenkov and Voloshina, 1989). Advances in Assisted Reproductive Technologies (ARTs) for domestic species have enabled investigators to use similar techniques for the conservation of endangered animals. Examples of ARTs include: embryo recovery/transfer of in vivo produced embryos, artificial insemination, in vitro maturation of oocytes, in vitro fertilization, transfer of in vitro produced embryos, nuclear transfer, and genome resource banking. ARTs have been implemented so as to increase the population of gorals in some zoos. Among ARTs, interspecies somatic cell nuclear transfer (SCNT) has the potential to be a method for maintaining a limited population and conserving goral species. There are four key parameters of nuclear transfer technology that influence the development of cloned embryos: (1) the state and nature of the recipient cytoplast, (2) the cell cycle status of the donor cell, (3) the identity, differentiation state and developmental potency of the donor cell, and, finally, and (4) genetic influences on the successful development of cloned embryos (Eggan and Jaenisch, 2002). One of the most important factors that determine the success of the development of cloned embryos is the cell cycle stage of the cloning donor cells. This is undoubtedly due to differences in DNA content of donor nuclei, which varies according to the phase of the cell cycle. Nuclei transferred to metaphase II recipient cytoplasts before or during activation, when maturation-promoting factor levels are high, must be in the G1 stage in order to maintain correct ploidy of reconstructed embryos at the end of the first cell cycle (Campbell et al., 1996). Most of the experimental findings suggest that the G0/G1 stages of the cell cycle give better results in terms of correct ploidy and development of reconstructed embryos (Boquest et al., 1999; Campbell et al., 1994, 1996; Gomez et al., 2003; Han et al., 2003; Kues et al., 2000; Lee and Piedrahita, 2002; Liu et al., 2004). The highest level of development was observed with the nucleus at the G1 stage fused with cytoplasm at the M stage (Tsunoda and Kato, 1998). This is why synchronization of the cell cycle stages in the G0/G1 phase is one of the key factors determining the success of nuclear transplantation. Serum deprivation, contact inhibition and chemical inhibitors like protease and antioxidant inhibitors are widely used methods for the synchronization of the cell cycle. With a view to improve donor nuclei treatment prior to nuclear transfer, the present study was undertaken to examine the cell cycle characteristics of endangered goral adult skin fibroblasts under a variety of cell cycle-arresting treatments.
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2. Materials and methods 2.1. Establishment of cell lines A skin biopsy was obtained from a 10-year-old female goral (Naemorhedus goral ), housed at the Everland Zoo in Yong-In, Seoul, South Korea. The tissue biopsy was transported to the Laboratory of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University at 40 C in phosphate-buffered saline (PBS; Gibco BRL, Life Technology, NY, USA) supplemented with 0.5% (v/v) penicillinestreptomycin (P/S; SigmaeAldrich, St. Louis, MO, USA). The tissue was washed several times, then cut and minced into small pieces using a sterilized blade, forceps and scissors, in a 60 mm dish. These pieces were digested in 0.25% trypsin EDTA (Gibco) at 39 C in a humidified atmosphere of 5% CO2 for 1e2 h. Digested tissue was vortexed and washed at least 3 or 4 times in PBS by centrifugation at 1500 rpm for 3 min. After a final wash, Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS (v/v) was added to the pellet, and the cell suspension was placed into 60 mm culture dishes at 39 C in 5% CO2 in humidified air and cultured until a confluent monolayer was formed, which took 7e8 days. For maintenance of the cell line, cells were trypsinized for 30 s and passaged into new dishes, and some of them were cryopreserved for further use. Thawed cells were cultured to at least the 3rd passage before being used in this study. Cells were used in the 4th to 7th passage for this experiment.
2.2. Treatment of cells In Experiment I, the effects of cyclic, serum-starved (SSd0.5% FBS), and fully confluent stages of goral cells were studied. In Experiments II and III, effects of different antioxidantsdb-Mercaptoethanol (b-ME, 10 mM), cysteine (2 mM) and glutathione (2 mM)dwere examined after cells reached full confluence without serum starvation for 24 and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, protein kinase inhibitor, 2 mM), cycloheximide (protein synthesis inhibitor, 7.5 mg/ml) and cytochalasin B (7.5 mg/ml, cytokinesis inhibitor), were treated as in Experiment II. In Experiments VI and VII, the effects of different levels of dimethylsulphoxide (DMSO, a cytokinesis inhibitor) at 0%, 0.5%, 1.0% and 2.5% on different goral cell cycle stages treated for 24 and 4 h were studied by flow cytometry (FACS). In the case of chemical treatment, the abovementioned treatments were given after 72 h of culture with the cells having 90e100% confluency. In the case of cyclic states, the cells were harvested, fixed and analysed after 24 h of culture. In the case of fully confluent cells, they were harvested, fixed and used for analysis after reaching 90e100% confluency. The aforementioned experiments were each replicated three times. The cell number before plating was normalized by haemocytometer and each treatment had three replication plates with 1 106 cells per plate. Cells from each sample were analysed by FACS on separate occasions. On each occasion, three samples from each treatment were analysed.
2.3. Cell fixation, staining, and cell cycle analysis Cultured cells were harvested using 0.25% Trypsin EDTA (Invitrogen, Carlsbad, CA, USA), and resuspended in DMEM at a concentration of 1 106 cells/tube. The cells were suspended in PBS and centrifuged at 1200 rpm at 4 C for 5 min, the supernatant was decanted and the cells gently re-suspended in PBS. Cells were fixed by adding 0.7 ml cold ethanol (70%) drop-wise in a tube containing 0.3 ml of cell suspension in PBS while vortexing gently. Fixed cells were left at 4 C for 48 h before further analysis. The fixed cells were again centrifuged as above, washed once and re-centrifuged with cold PBS. Centrifuged cells were re-suspended in 0.25 ml PBS containing 5 ml of 10 mg/ml Rnase (Sigma) and incubated at 37 C for 1 h. Incubated cells were stained by adding 10 ml of 1 mg/ml propidium iodide (Sigma). Then, the cells were analysed by FACS using a Becton Dickinson (Rutherford, NJ, USA) flow cytometer at 488 nm. Histogram plots were created using the Cell Quest program (Becton Dickinson, San Jose, CA, USA). The percentages of cells within the various phases of the cell cycle were calculated using Cell
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Quest by gating G0 þ G1, S, and G2 þ M cell populations visualized using a scatterplot of green florescence against red florescence.
Table 2 Effect of different antioxidants on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency
2.4. Statistical analyses
Antioxidants
Percentage of different cell cycle stages (mean SD)
All data were subjected to one-way analysis of variance (ANOVA) and least significant different (LSD) test using general linear models in a statistical analysis system (SAS) program in order to determine differences among experimental groups. When a statistically significant effect was found in each experimental parameter, data were compared by the least squares method. Statistical significance was determined where P was less than 0.05.
G0 þ G1
S
G2/M
b-Mercaptoethanol Cysteine Glutathione
61.1 2.8a 61.2 5.6a 49.9 7.2b
3.3 0.2b 3.3 0.1b 6.5 2.1a
12.2 1.0a 11.6 1.3a 6.3 1.3b
3. Results
phases of cell cycles than 24 h treatments. Synchronization rate was highest in the case of cysteine.
The effect of different cell culture conditions, namely cycling, serum-starved (SS) and fully confluent, on the different cell cycle phases is shown in Table 1. Table 1 shows that there were significant (P < 0.05) differences at S and G2 phases of the cell cycle. All culture conditions were equally effective in G0 þ G1 (P > 0.05). There was no significant difference for G0 þ G1 in the three culture conditions, In the case of S phase, rates of 4.6%, 1.9% and 7.2% were observed for cycling, SS and fully, respectively. However, at G2/M phase, 10.3%, 5.6% and 11.5% were observed for cycling, SS and fully confluent, respectively. 3.2. Experiments II and III The effect of different antioxidants on cells treated for 24 and 4 h after full confluency is shown in Table 2 and in Fig. 1. b-ME and cysteine were equally effective in G0 þ G1, S and G2/M phases but differed significantly (P < 0.05) from results with glutathione. However, the percentage of synchronization of this treatment could not reach a level equal to that of cycling or serum-starved states for G0 þ G1 (Tables 1 and 2). On the contrary, in the 4 h treatment G0 þ G1 values were higher than in the cycling and serumstarved groups (Tables 1 and 3). Irrespective of treatment duration, among the three antioxidants, b-ME and cysteine synchronized the cell cycles more effectively than glutathione (Tables 2 and 3). In the 4 h treatment, cysteine was statistically more effective than glutathione but equally effective as b-ME for G0 þ G1 and S phases of the cell cycles (Table 3). Fig. 1 shows that, irrespective of different antioxidants, 4 h treatments were more effective for the synchronization G0 þ G1 Table 1 Effect of different cell culture condition on the synchronization of cell cycles of adult goral skin fibroblasts
Cycling Serum-starved Fully confluent
Percentage of different cell cycle stages (mean SD) G0 þ G1
S
G2/M
64.9 3.1 68.7 4.7 61.0 1.0
4.6 0.5b 1.9 0.2c 7.2 0.3a
10.3 0.8b 5.6 0.4c 11.5 0.5a
Within columns, values with different superscripts are significantly different (P < 0.05).
3.3. Experiments IV and V The effect of different protease inhibitors during treatment for 24 and 4 h on the cell cycle phases is shown in Tables 4 and 5 and in Fig. 2. Tables 4 and 5 show that none of the antioxidants could synchronize the cell cycle as G0 þ G1 phases as effectively as cyclic and SS. Among these three chemicals, cycloheximide showed better results than 6-DMAP and cytochalasin B for G0 þ G1 and G2/M at 24 h of treatment (P < 0.05). In G0 þ G1 phases, cycloheximide and cytochalasin B were equally effective and significantly (P < 0.05) different from 6-DMAP. On the other hand, in S phase, 6DMAP and cytochalasin B were equally effective but significantly (P < 0.05) different from cycloheximide (Table 5). Fig. 2 shows that in the case of 24 h and 4 h treatments, cycloheximide shows better results for the synchronization of G0 þ G1 phases than cytochalasin B and 6-DMAP. 3.4. Experiments VI and VII The effect of different levels of DMSO treatment for 24 and 4 h is shown in Table 6 and in Fig. 3. With increasing levels of DMSO, a statistically significant difference (P < 0.05) was observed in all stages except the M phase of the cell cycle for the 24 h treatment. Maximum synchronization at G0 þ G1 stage was 74.8% and 75.2% in 0.5% and 1% of DMSO, respectively (Table 6). However, at the highest level of 2.5% DMSO, G0 þ G1 frequency decreased very sharply Percentages of Go+G1 phases
3.1. Experiment I
Cell culture conditions
Within columns, values with different superscripts are significantly different (P < 0.05).
90 80 70 60 50 40 30 20 10 0
24h 4h
beta mercaptoethanol
Cysteine
Glutathione
Different antioxidants Fig. 1. Effect of different antioxidants, after 24 h and 4 h treatment, on the synchronization of goral skin fibroblasts at G0 þ G1 phases of the cell cycle.
Hashem et al. / Cell Biology International 30 (2006) 698e703 Table 3 Effect of different antioxidants on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency Percentage of different cell cycle stages (mean SD) G0 þ G1 b-Mercaptoethanol Cysteine Glutathione
S ab
67.2 4.5 75.9 5.3a 62.9 3.2b
G2/M b
2.6 0.1 2.9 0.3ab 4.9 2.0a
9.0 3.7 9.5 1.1 10.2 1.1
(6.7%). The S and G2/M levels were higher in control (0.0%) and 0.5% level of DMSO and with increasing levels of DMSO, S and G2/M levels also decreased. For the G0 þ G1 phases of cell cycle control, 0.5% and 1% DMSO were equally effective but significantly (P < 0.05) different from 2.5% DMSO (Table 6). In contrast, for the 4 h treatment, 0.5% and 1.0% DMSO were equally effective at G0 þ G1 phases and were significantly (P < 0.05) better than 2.5% DMSO (Table 7). Fig. 3 shows that at 0.5% level of DMSO, synchronization rate of cells at G0 þ G1 phases was almost similar in both 24 and 4 h treatments, whereas 1.0% level of DMSO was better after 4 h treatment. In contrast, 2.5% level of DMSO was found detrimental after 24 h treatment. 4. Discussion Synchronization between the cell cycle of the karyoplast and the cytoplast is very important when trying to increase the efficiency of somatic cell nuclear transfer technology (SCNT). The most critical aspect of cell cycle coordination is maintenance of normal ploidy in the reconstructed nuclear transfer (NT) embryo (Campbell et al., 1996). For SCNT, using pre-activated cytoplasts as recipients results in poorer development compared with non-activated metaphase II cytoplasts (Heyman et al., 2002; Wakayama and Yanagimachi, 2001). Non-activated cytoplasts are high in activity of maturationpromoting factor (MPF), a complex of cyclin B and cyclindependent kinase 1 (CDK 1 or p34cdc2). When an interphase donor nucleus is introduced into a high MPF milieu, it undergoes nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). For donor cells in S-phase, this results in developmental failure because of fragmentation
Table 4 Effect of different protease inhibitors on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency
6-DMAP Cycloheximide Cytochalasin B
Percentage of different cell cycle stages (mean SD) G0 þ G1
S b
3.9 2.9 60.1 3.7a 10.8 5.3b
0.9 0.7 2.6 0.2 1.8 0.5
Protease inhibitors 6-DMAP Cycloheximide Cytochalasin B
Within columns, values with different superscripts are significantly different (P < 0.05).
Protease inhibitors
Table 5 Effect of different protease inhibitors on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency
G2/M b
0.8 0.8 7.0 0.6a 1.3 0.5b
Within columns, values with different superscripts are significantly different (P < 0.05).
Percentage of different cell cycle stages (mean SD) G0 þ G1
S b
37.3 10.0 58.7 3.2a 56.2 5.9a
G2/M b
1.9 0.6 3.5 0.3a 2.5 0.1b
10.3 0.7 12.5 3.6 11.3 1.7
Within columns, values with different superscripts are significantly different (P < 0.05).
of partially replicated DNA and incorrect ploidy (Collas et al., 1992). Among the donor cell cycle stages which are compatible with maintaining ploidy, there are two principal scenarios, depending on DNA content: G0/G1-phase donor nuclei and G2/ M-phase donor nuclei. For G0/G1-phase donor nuclei, the donor chromatin has not yet replicated (2C), and chromosomes are not ready to segregate. Both quiescent G0- and proliferating G1-phase somatic donors have been successfully used to produce cloned offspring (Gibbons et al., 2002; Kasinathan et al., 2001; Wells et al., 2001). In contrast, the cell cycle stage of transferred nuclei influences the morphology of condensed chromosomes: G1-stage prematurely condensed chromosomes exhibit single chromatids, S-phase PCC can be seen as patches of chromatin, and G2-phase prematurely condensed chromosomes have two chromatids (Fulka et al., 1998). Therefore, following activation, MPF levels decline, chromatin decondenses and a nuclear envelope is formed. All nuclei that have undergone nuclear envelope breakdown will then undergo DNA synthesis. Hence donor nuclei must be in G0 or G1 when transferred to metaphase II recipient oocytes with high levels of MPF in order to condense chromosomes normally and maintain correct ploidy of reconstructed embryos at the end of the first cell cycle (Han et al., 2003). To obtain somatic cells in the G1 stage of the cell cycle, several methods have been employed (Gibbons et al., 2002; Gomez et al., 2003; Han et al., 2003; Hayes et al., 2005; Kubota et al., 2000; Kues et al., 2000; Liu et al., 2004; Zou et al., 2002).
24h
70
Percentage of Go+G1 phases
Antioxidants
701
4h
60 50 40 30 20 10 0
6-DMAP
Cycloheximide
Cytochalasin B
Different protease inhibitors Fig. 2. Effect of different protease inhibitors, after 24 h and 4 h treatment, on the synchronization of goral skin fibroblasts at G0 þ G1 phases.
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Table 6 Effect of different levels of dimethylsulphoxide (DMSO) on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency Level of DMSO (%)
Percentage of different cell cycle stages (mean SD) G0 þ G1
S
G2/M
0% 0.5% 1.0% 2.5%
66.0 4.2b 74.8 0.2a 75.2 0.8a 6.7 0.5c
4.2 0.5a 1.4 0.1b 0.8 0.1c 0.1 0.02d
11.7 0.9a 5.4 0.4b 4.0 0.3c 0.1 0.0d
Within columns, values with different superscripts are significantly different (P < 0.05).
Percentage of Go+G1 Phases
In this study, the different chemicals and non-chemical effects on adult goral skin fibroblast cell were analysed using FACS to determine the relative proportions of cells in different phases of the cell cycle, particularly G0/G1. We observed that cycling, SS and fully confluent conditions were equally effective for synchronization of cells at G0 þ G1 stages (P > 0.05). Within the chemical treatment group, b-ME, cysteine and DMSO had better results over the cycling, SS and fully confluent group (Tables 2e7). Our results varied with the findings of other authors. Among them, Liu et al. (2004) reported that chemical synchronization of the cell cycle stage of rabbit foetal fibroblasts treated with aphidicolin (0.1 mg/ml, 6 h) and demicoline (0.5 mg/ml, 10 h) to G0/G1 (25e50%) was less effective than serum deprivation. Gibbons et al. (2002) found that treating adult bovine granulosa cells with cycling dependent kinase 2 inhibitor, roscovitine, synchronized them at G0/ G1 (82.4%) phase of the cell cycle, which is higher than serum-starved cells (76.7%). A higher percentage of African wildcat and domestic cat nuclei were in the G0/G1 phase after cells were serum-starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%) or after roscovitine (56% vs. 84%) treatments, respectively (Gomez et al., 2003). Kues et al. (2000) found 60.6e73.3% in G0/G1 stages (2C DNA content) in serum-supplemented medium, 77.9e 80.2% in serum deprived medium, and 81.9% of cells at the 90
24h
80
4h
70 60 50 40 30 20 10 0
0.5% level
1.0% level
2.5% level
Level of DMSO Fig. 3. Effect of different levels of DMSO, after 24 h and 4 h treatments, on the synchronization of goral skin fibroblasts at G0 þ G1 phases.
Table 7 Effect of different levels of Dimethylsulphoxide (DMSO) on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency Level of DMSO (%)
Synchronization rate (%) of different cell cycle stages (mean SD) G0 þ G1
0.5% 1.0% 2.5%
a
75.4 1.5 79.5 5.7a 65.0 0.7b
S
G2/M
3.0 1.4 2.3 0.6 6.4 2.7
7.1 1.6a 8.3 1.5a 3.0 1.2b
Within columns, values with different superscripts are significantly different (P < 0.05).
G1/S transition in aphidicolin treatment in pigs. There are reports of differences in different stages of cells in goat foetal fibroblast (Zou et al., 2002), adult fibroblast cells of cattle (Hayes et al., 2005), panda fibroblast cells (Han et al., 2003), and adult cattle fibroblasts (Kubota et al., 2000). These variations may be due to different species, different chemicals used to synchronize cell cycle stages, as well as experimental protocols. For example, from the findings of Gomez et al. (2003) in African wild cats and domestic cats had different values for G0/G1 stages in case of cycling, SS and chemical treated groups. In summary, the studies reported here have demonstrated that b-ME, cysteine and DMSO had better results compared with cycling, serum-starved and fully confluent groups for the synchronization of goral skin fibroblast cells at G0 þ G1 stages. This finding could have a positive effect on somatic cell nuclear transfer in this endangered species. Acknowledgements This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00). References Boquest AC, Day BN, Prather RS. Flow cytometric analysis of cultured porcine fetal fibroblast cells. Biol Reprod 1999;60:1013e9. Campbell KH, Loi P, Cappai P, Wilmut I. Improved development to blastocyst of bovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol Reprod 1994;50:1385e93. Campbell KHS, Loi P, Otaegui PJ, Wilmut I. Cell cycle coordination in embryo cloning by nuclear transfer. Rev Reprod 1996;1:40e6. Collas P, Balise JJ, Robl JM. Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol Reprod 1992; 46:492e500. Cultural Properties Administration of Korea. A report for distribution and ecological studies of Korean natural Monuments, goral and musk deer. 1999. Eggan K, Jaenisch R. Nuclear reprogramming: biological and technological constraints. In: Cibelli J, Lanza RP, Campbell KHS, West MD, editors. Principles of cloning. Amsterdam: Academic Press, An imprint of Elsevier Science; 2002. p. 85e108. Fulka JJ, First NL, Moor RM. Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod 1998;4:41e9. Geist V. On the evolution of the Caprinae. In: Soma H, editor. The biology and management of Capricornis and related mountain antelopes. London: Croon Helm; 1987. p. 3e40.
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Liu CT, Yu KC, Ju JC. Cell cycle stage analysis of rabbit foetal fibroblasts and cumulus cells. Reprod Domest Anim 2004;39:385e90. Mead JI. Nemorhaedus goral. Mamm Spp 1989;335:1e5. Ministry of Environment of Korea. Illustrated magazine of endangered, threatened and protective species of Korea. 2000; p. 12. Min MS, Okumura H, Jo DJ, An JH, Kim KS, Kim CB, et al. Molecular phylogenetic status of the Korean goral and Japanese serow based on partial sequences of the mitochondrial cytochrome b gene. Molecules Cells 2004; 17:365e72. Myslenkov AI, Voloshina IV. Ecology and behaviour of the Amur goral. Moscow: Nauka; 1989. Nowak RM. Walker’s mammals of the world. 6th ed. Baltimore: Johns Hopkins University Press; 1999. p. 1209e12. Shackleton DM, the IUCN/SSC Caprinae Specialist Group, editors. Wild sheep and goats and their relatives. Status survey and action plan for Caprinae. Switzerland and Cambridge, UK: IUCN; 1997. Tsunoda Y, Kato Y. Full-term development after transfer of nuclei from 4-cell and compacted morula stage embryos to enucleated oocytes in the mouse. J Exp Zool 1998;278:250e4. Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001;122:49e60. Wells D, Oliver J, Miller A, Forsyth J, Berg M, Cockrem K, et al. Bovine cells selected in G1 of the cell cycle are totipotent following somatic cell nuclear transfer. In: Proceedings of the 32nd Annual Conference on Soc Reprod Biol 2001 [abstract 25]. Zou X, Wang Y, Cheng Y, Yang Y, Ju H, Tang H, et al. Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle. Mol Reprod Dev 2002;61:164e72.
Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts Md. Abul Hashem a, Dilip P. Bhandari a, Sung Keun Kang a,b, Byeong Chun Lee a,b,*, Hwang Woo Suk a,b,c a
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea b The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, South Korea c School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea Received 5 December 2005; revised 25 February 2006; accepted 26 April 2006
Abstract The present study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. Seven experiments were performed, each with a one-way completely randomized design involving three replicates. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved, and fully confluent stages of Goral cells. In Experiments II and III, the effects of different antioxidants like b-mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 24 h and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ml), were used as in Experiment II. In Experiments VI and VII, the effect of different levels of dimethylsulphoxide (DMSO) at 0%, 0.5%, 1.0% and 2.5% were tested by flow cytometry (FACS). In Experiment I, 68.7% of Goral skin fibroblasts reached the G0/G1 stage (2C DNA content) when subjected to the serum-starved medium, which was more than the cycling (64.9%) and fully confluent groups (61.0%) (P > 0.05). Among the chemically treated group, b-ME, cysteine and DMSO showed better results for synchronization of G0 þ G1 phases than cycling, serum-starved and fully confluent groups. It can thus be concluded that b-ME, cysteine and DMSO at certain concentrations can synchronize the cell cycle effectively, which could have a positive impact on somatic cell nuclear transfer in the goral. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Goral; Cell cycle; Synchronization
1. Introduction Gorals are listed as endangered animals in CITES Appendix I. The grey goral is classified as being at low risk, or nearly threatened, by the 1996 IUCN report (Shackleton, 1997). Gorals derived from the subfamily caprinae have been classified into three tribes, Rupicaprini, Ovibovini and Caprini (Geist, 1987; Gentry, 1992). Within the tribe Rupicaprini, serows and gorals have been assigned to two separate genera (Mead, * Corresponding author. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea. Tel.: þ82 2 880 1269; fax: þ82 2 884 1902. E-mail address: [email protected] (B.C. Lee).
1989; Nowak, 1999), Capricornis and Nemorhaedus, respectively, despite being closely related. The genus Nemorhaedus of Rupicaprini includes three species, N. caudatus (long-tailed goral or Chinese goral), N. goral (Himalayan goral) and N. baileyi (red goral). Nemorhaedus species’ range from northern Pakistan to Northeastern Asia including Korea (Mead, 1989). The longtailed goral (or Chinese goral), N. caudatus, is one of the most endangered mammalian species in Korea (Ministry of Environment of Korea, 2000) and has been designated as a Natural Monument species by the Cultural Properties Administration of the Korean Government. The goral population in South Korea is assumed to be less than 250 (Cultural Properties Administration of Korea, 1999), and their habitats are
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.04.008
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restricted and highly fragmented. Therefore, a systematic conservation plan for the revival of this species is urgently needed in South Korea (Min et al., 2004). One should note that some phylogenetic work for Korean gorals has already been done (Min et al., 2004). This phylogenetic study (Min et al., 2004) has shown that Korean and Russian gorals belong to the same clade, and differ from Chinese gorals. Furthermore, they are more genetically distinct than the Chinese goral. Gorals in both countries are highly endangered; probably no more than seven hundred remain in the wild in each country (Cultural Properties Administration of Korea, 1999; Myslenkov and Voloshina, 1989). Advances in Assisted Reproductive Technologies (ARTs) for domestic species have enabled investigators to use similar techniques for the conservation of endangered animals. Examples of ARTs include: embryo recovery/transfer of in vivo produced embryos, artificial insemination, in vitro maturation of oocytes, in vitro fertilization, transfer of in vitro produced embryos, nuclear transfer, and genome resource banking. ARTs have been implemented so as to increase the population of gorals in some zoos. Among ARTs, interspecies somatic cell nuclear transfer (SCNT) has the potential to be a method for maintaining a limited population and conserving goral species. There are four key parameters of nuclear transfer technology that influence the development of cloned embryos: (1) the state and nature of the recipient cytoplast, (2) the cell cycle status of the donor cell, (3) the identity, differentiation state and developmental potency of the donor cell, and, finally, and (4) genetic influences on the successful development of cloned embryos (Eggan and Jaenisch, 2002). One of the most important factors that determine the success of the development of cloned embryos is the cell cycle stage of the cloning donor cells. This is undoubtedly due to differences in DNA content of donor nuclei, which varies according to the phase of the cell cycle. Nuclei transferred to metaphase II recipient cytoplasts before or during activation, when maturation-promoting factor levels are high, must be in the G1 stage in order to maintain correct ploidy of reconstructed embryos at the end of the first cell cycle (Campbell et al., 1996). Most of the experimental findings suggest that the G0/G1 stages of the cell cycle give better results in terms of correct ploidy and development of reconstructed embryos (Boquest et al., 1999; Campbell et al., 1994, 1996; Gomez et al., 2003; Han et al., 2003; Kues et al., 2000; Lee and Piedrahita, 2002; Liu et al., 2004). The highest level of development was observed with the nucleus at the G1 stage fused with cytoplasm at the M stage (Tsunoda and Kato, 1998). This is why synchronization of the cell cycle stages in the G0/G1 phase is one of the key factors determining the success of nuclear transplantation. Serum deprivation, contact inhibition and chemical inhibitors like protease and antioxidant inhibitors are widely used methods for the synchronization of the cell cycle. With a view to improve donor nuclei treatment prior to nuclear transfer, the present study was undertaken to examine the cell cycle characteristics of endangered goral adult skin fibroblasts under a variety of cell cycle-arresting treatments.
699
2. Materials and methods 2.1. Establishment of cell lines A skin biopsy was obtained from a 10-year-old female goral (Naemorhedus goral ), housed at the Everland Zoo in Yong-In, Seoul, South Korea. The tissue biopsy was transported to the Laboratory of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University at 40 C in phosphate-buffered saline (PBS; Gibco BRL, Life Technology, NY, USA) supplemented with 0.5% (v/v) penicillinestreptomycin (P/S; SigmaeAldrich, St. Louis, MO, USA). The tissue was washed several times, then cut and minced into small pieces using a sterilized blade, forceps and scissors, in a 60 mm dish. These pieces were digested in 0.25% trypsin EDTA (Gibco) at 39 C in a humidified atmosphere of 5% CO2 for 1e2 h. Digested tissue was vortexed and washed at least 3 or 4 times in PBS by centrifugation at 1500 rpm for 3 min. After a final wash, Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS (v/v) was added to the pellet, and the cell suspension was placed into 60 mm culture dishes at 39 C in 5% CO2 in humidified air and cultured until a confluent monolayer was formed, which took 7e8 days. For maintenance of the cell line, cells were trypsinized for 30 s and passaged into new dishes, and some of them were cryopreserved for further use. Thawed cells were cultured to at least the 3rd passage before being used in this study. Cells were used in the 4th to 7th passage for this experiment.
2.2. Treatment of cells In Experiment I, the effects of cyclic, serum-starved (SSd0.5% FBS), and fully confluent stages of goral cells were studied. In Experiments II and III, effects of different antioxidantsdb-Mercaptoethanol (b-ME, 10 mM), cysteine (2 mM) and glutathione (2 mM)dwere examined after cells reached full confluence without serum starvation for 24 and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, protein kinase inhibitor, 2 mM), cycloheximide (protein synthesis inhibitor, 7.5 mg/ml) and cytochalasin B (7.5 mg/ml, cytokinesis inhibitor), were treated as in Experiment II. In Experiments VI and VII, the effects of different levels of dimethylsulphoxide (DMSO, a cytokinesis inhibitor) at 0%, 0.5%, 1.0% and 2.5% on different goral cell cycle stages treated for 24 and 4 h were studied by flow cytometry (FACS). In the case of chemical treatment, the abovementioned treatments were given after 72 h of culture with the cells having 90e100% confluency. In the case of cyclic states, the cells were harvested, fixed and analysed after 24 h of culture. In the case of fully confluent cells, they were harvested, fixed and used for analysis after reaching 90e100% confluency. The aforementioned experiments were each replicated three times. The cell number before plating was normalized by haemocytometer and each treatment had three replication plates with 1 106 cells per plate. Cells from each sample were analysed by FACS on separate occasions. On each occasion, three samples from each treatment were analysed.
2.3. Cell fixation, staining, and cell cycle analysis Cultured cells were harvested using 0.25% Trypsin EDTA (Invitrogen, Carlsbad, CA, USA), and resuspended in DMEM at a concentration of 1 106 cells/tube. The cells were suspended in PBS and centrifuged at 1200 rpm at 4 C for 5 min, the supernatant was decanted and the cells gently re-suspended in PBS. Cells were fixed by adding 0.7 ml cold ethanol (70%) drop-wise in a tube containing 0.3 ml of cell suspension in PBS while vortexing gently. Fixed cells were left at 4 C for 48 h before further analysis. The fixed cells were again centrifuged as above, washed once and re-centrifuged with cold PBS. Centrifuged cells were re-suspended in 0.25 ml PBS containing 5 ml of 10 mg/ml Rnase (Sigma) and incubated at 37 C for 1 h. Incubated cells were stained by adding 10 ml of 1 mg/ml propidium iodide (Sigma). Then, the cells were analysed by FACS using a Becton Dickinson (Rutherford, NJ, USA) flow cytometer at 488 nm. Histogram plots were created using the Cell Quest program (Becton Dickinson, San Jose, CA, USA). The percentages of cells within the various phases of the cell cycle were calculated using Cell
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Quest by gating G0 þ G1, S, and G2 þ M cell populations visualized using a scatterplot of green florescence against red florescence.
Table 2 Effect of different antioxidants on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency
2.4. Statistical analyses
Antioxidants
Percentage of different cell cycle stages (mean SD)
All data were subjected to one-way analysis of variance (ANOVA) and least significant different (LSD) test using general linear models in a statistical analysis system (SAS) program in order to determine differences among experimental groups. When a statistically significant effect was found in each experimental parameter, data were compared by the least squares method. Statistical significance was determined where P was less than 0.05.
G0 þ G1
S
G2/M
b-Mercaptoethanol Cysteine Glutathione
61.1 2.8a 61.2 5.6a 49.9 7.2b
3.3 0.2b 3.3 0.1b 6.5 2.1a
12.2 1.0a 11.6 1.3a 6.3 1.3b
3. Results
phases of cell cycles than 24 h treatments. Synchronization rate was highest in the case of cysteine.
The effect of different cell culture conditions, namely cycling, serum-starved (SS) and fully confluent, on the different cell cycle phases is shown in Table 1. Table 1 shows that there were significant (P < 0.05) differences at S and G2 phases of the cell cycle. All culture conditions were equally effective in G0 þ G1 (P > 0.05). There was no significant difference for G0 þ G1 in the three culture conditions, In the case of S phase, rates of 4.6%, 1.9% and 7.2% were observed for cycling, SS and fully, respectively. However, at G2/M phase, 10.3%, 5.6% and 11.5% were observed for cycling, SS and fully confluent, respectively. 3.2. Experiments II and III The effect of different antioxidants on cells treated for 24 and 4 h after full confluency is shown in Table 2 and in Fig. 1. b-ME and cysteine were equally effective in G0 þ G1, S and G2/M phases but differed significantly (P < 0.05) from results with glutathione. However, the percentage of synchronization of this treatment could not reach a level equal to that of cycling or serum-starved states for G0 þ G1 (Tables 1 and 2). On the contrary, in the 4 h treatment G0 þ G1 values were higher than in the cycling and serumstarved groups (Tables 1 and 3). Irrespective of treatment duration, among the three antioxidants, b-ME and cysteine synchronized the cell cycles more effectively than glutathione (Tables 2 and 3). In the 4 h treatment, cysteine was statistically more effective than glutathione but equally effective as b-ME for G0 þ G1 and S phases of the cell cycles (Table 3). Fig. 1 shows that, irrespective of different antioxidants, 4 h treatments were more effective for the synchronization G0 þ G1 Table 1 Effect of different cell culture condition on the synchronization of cell cycles of adult goral skin fibroblasts
Cycling Serum-starved Fully confluent
Percentage of different cell cycle stages (mean SD) G0 þ G1
S
G2/M
64.9 3.1 68.7 4.7 61.0 1.0
4.6 0.5b 1.9 0.2c 7.2 0.3a
10.3 0.8b 5.6 0.4c 11.5 0.5a
Within columns, values with different superscripts are significantly different (P < 0.05).
3.3. Experiments IV and V The effect of different protease inhibitors during treatment for 24 and 4 h on the cell cycle phases is shown in Tables 4 and 5 and in Fig. 2. Tables 4 and 5 show that none of the antioxidants could synchronize the cell cycle as G0 þ G1 phases as effectively as cyclic and SS. Among these three chemicals, cycloheximide showed better results than 6-DMAP and cytochalasin B for G0 þ G1 and G2/M at 24 h of treatment (P < 0.05). In G0 þ G1 phases, cycloheximide and cytochalasin B were equally effective and significantly (P < 0.05) different from 6-DMAP. On the other hand, in S phase, 6DMAP and cytochalasin B were equally effective but significantly (P < 0.05) different from cycloheximide (Table 5). Fig. 2 shows that in the case of 24 h and 4 h treatments, cycloheximide shows better results for the synchronization of G0 þ G1 phases than cytochalasin B and 6-DMAP. 3.4. Experiments VI and VII The effect of different levels of DMSO treatment for 24 and 4 h is shown in Table 6 and in Fig. 3. With increasing levels of DMSO, a statistically significant difference (P < 0.05) was observed in all stages except the M phase of the cell cycle for the 24 h treatment. Maximum synchronization at G0 þ G1 stage was 74.8% and 75.2% in 0.5% and 1% of DMSO, respectively (Table 6). However, at the highest level of 2.5% DMSO, G0 þ G1 frequency decreased very sharply Percentages of Go+G1 phases
3.1. Experiment I
Cell culture conditions
Within columns, values with different superscripts are significantly different (P < 0.05).
90 80 70 60 50 40 30 20 10 0
24h 4h
beta mercaptoethanol
Cysteine
Glutathione
Different antioxidants Fig. 1. Effect of different antioxidants, after 24 h and 4 h treatment, on the synchronization of goral skin fibroblasts at G0 þ G1 phases of the cell cycle.
Hashem et al. / Cell Biology International 30 (2006) 698e703 Table 3 Effect of different antioxidants on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency Percentage of different cell cycle stages (mean SD) G0 þ G1 b-Mercaptoethanol Cysteine Glutathione
S ab
67.2 4.5 75.9 5.3a 62.9 3.2b
G2/M b
2.6 0.1 2.9 0.3ab 4.9 2.0a
9.0 3.7 9.5 1.1 10.2 1.1
(6.7%). The S and G2/M levels were higher in control (0.0%) and 0.5% level of DMSO and with increasing levels of DMSO, S and G2/M levels also decreased. For the G0 þ G1 phases of cell cycle control, 0.5% and 1% DMSO were equally effective but significantly (P < 0.05) different from 2.5% DMSO (Table 6). In contrast, for the 4 h treatment, 0.5% and 1.0% DMSO were equally effective at G0 þ G1 phases and were significantly (P < 0.05) better than 2.5% DMSO (Table 7). Fig. 3 shows that at 0.5% level of DMSO, synchronization rate of cells at G0 þ G1 phases was almost similar in both 24 and 4 h treatments, whereas 1.0% level of DMSO was better after 4 h treatment. In contrast, 2.5% level of DMSO was found detrimental after 24 h treatment. 4. Discussion Synchronization between the cell cycle of the karyoplast and the cytoplast is very important when trying to increase the efficiency of somatic cell nuclear transfer technology (SCNT). The most critical aspect of cell cycle coordination is maintenance of normal ploidy in the reconstructed nuclear transfer (NT) embryo (Campbell et al., 1996). For SCNT, using pre-activated cytoplasts as recipients results in poorer development compared with non-activated metaphase II cytoplasts (Heyman et al., 2002; Wakayama and Yanagimachi, 2001). Non-activated cytoplasts are high in activity of maturationpromoting factor (MPF), a complex of cyclin B and cyclindependent kinase 1 (CDK 1 or p34cdc2). When an interphase donor nucleus is introduced into a high MPF milieu, it undergoes nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). For donor cells in S-phase, this results in developmental failure because of fragmentation
Table 4 Effect of different protease inhibitors on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency
6-DMAP Cycloheximide Cytochalasin B
Percentage of different cell cycle stages (mean SD) G0 þ G1
S b
3.9 2.9 60.1 3.7a 10.8 5.3b
0.9 0.7 2.6 0.2 1.8 0.5
Protease inhibitors 6-DMAP Cycloheximide Cytochalasin B
Within columns, values with different superscripts are significantly different (P < 0.05).
Protease inhibitors
Table 5 Effect of different protease inhibitors on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency
G2/M b
0.8 0.8 7.0 0.6a 1.3 0.5b
Within columns, values with different superscripts are significantly different (P < 0.05).
Percentage of different cell cycle stages (mean SD) G0 þ G1
S b
37.3 10.0 58.7 3.2a 56.2 5.9a
G2/M b
1.9 0.6 3.5 0.3a 2.5 0.1b
10.3 0.7 12.5 3.6 11.3 1.7
Within columns, values with different superscripts are significantly different (P < 0.05).
of partially replicated DNA and incorrect ploidy (Collas et al., 1992). Among the donor cell cycle stages which are compatible with maintaining ploidy, there are two principal scenarios, depending on DNA content: G0/G1-phase donor nuclei and G2/ M-phase donor nuclei. For G0/G1-phase donor nuclei, the donor chromatin has not yet replicated (2C), and chromosomes are not ready to segregate. Both quiescent G0- and proliferating G1-phase somatic donors have been successfully used to produce cloned offspring (Gibbons et al., 2002; Kasinathan et al., 2001; Wells et al., 2001). In contrast, the cell cycle stage of transferred nuclei influences the morphology of condensed chromosomes: G1-stage prematurely condensed chromosomes exhibit single chromatids, S-phase PCC can be seen as patches of chromatin, and G2-phase prematurely condensed chromosomes have two chromatids (Fulka et al., 1998). Therefore, following activation, MPF levels decline, chromatin decondenses and a nuclear envelope is formed. All nuclei that have undergone nuclear envelope breakdown will then undergo DNA synthesis. Hence donor nuclei must be in G0 or G1 when transferred to metaphase II recipient oocytes with high levels of MPF in order to condense chromosomes normally and maintain correct ploidy of reconstructed embryos at the end of the first cell cycle (Han et al., 2003). To obtain somatic cells in the G1 stage of the cell cycle, several methods have been employed (Gibbons et al., 2002; Gomez et al., 2003; Han et al., 2003; Hayes et al., 2005; Kubota et al., 2000; Kues et al., 2000; Liu et al., 2004; Zou et al., 2002).
24h
70
Percentage of Go+G1 phases
Antioxidants
701
4h
60 50 40 30 20 10 0
6-DMAP
Cycloheximide
Cytochalasin B
Different protease inhibitors Fig. 2. Effect of different protease inhibitors, after 24 h and 4 h treatment, on the synchronization of goral skin fibroblasts at G0 þ G1 phases.
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Table 6 Effect of different levels of dimethylsulphoxide (DMSO) on the synchronization of cell cycles of adult goral skin fibroblasts treated for 24 h after full confluency Level of DMSO (%)
Percentage of different cell cycle stages (mean SD) G0 þ G1
S
G2/M
0% 0.5% 1.0% 2.5%
66.0 4.2b 74.8 0.2a 75.2 0.8a 6.7 0.5c
4.2 0.5a 1.4 0.1b 0.8 0.1c 0.1 0.02d
11.7 0.9a 5.4 0.4b 4.0 0.3c 0.1 0.0d
Within columns, values with different superscripts are significantly different (P < 0.05).
Percentage of Go+G1 Phases
In this study, the different chemicals and non-chemical effects on adult goral skin fibroblast cell were analysed using FACS to determine the relative proportions of cells in different phases of the cell cycle, particularly G0/G1. We observed that cycling, SS and fully confluent conditions were equally effective for synchronization of cells at G0 þ G1 stages (P > 0.05). Within the chemical treatment group, b-ME, cysteine and DMSO had better results over the cycling, SS and fully confluent group (Tables 2e7). Our results varied with the findings of other authors. Among them, Liu et al. (2004) reported that chemical synchronization of the cell cycle stage of rabbit foetal fibroblasts treated with aphidicolin (0.1 mg/ml, 6 h) and demicoline (0.5 mg/ml, 10 h) to G0/G1 (25e50%) was less effective than serum deprivation. Gibbons et al. (2002) found that treating adult bovine granulosa cells with cycling dependent kinase 2 inhibitor, roscovitine, synchronized them at G0/ G1 (82.4%) phase of the cell cycle, which is higher than serum-starved cells (76.7%). A higher percentage of African wildcat and domestic cat nuclei were in the G0/G1 phase after cells were serum-starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%) or after roscovitine (56% vs. 84%) treatments, respectively (Gomez et al., 2003). Kues et al. (2000) found 60.6e73.3% in G0/G1 stages (2C DNA content) in serum-supplemented medium, 77.9e 80.2% in serum deprived medium, and 81.9% of cells at the 90
24h
80
4h
70 60 50 40 30 20 10 0
0.5% level
1.0% level
2.5% level
Level of DMSO Fig. 3. Effect of different levels of DMSO, after 24 h and 4 h treatments, on the synchronization of goral skin fibroblasts at G0 þ G1 phases.
Table 7 Effect of different levels of Dimethylsulphoxide (DMSO) on the synchronization of cell cycles of adult goral skin fibroblasts treated for 4 h after full confluency Level of DMSO (%)
Synchronization rate (%) of different cell cycle stages (mean SD) G0 þ G1
0.5% 1.0% 2.5%
a
75.4 1.5 79.5 5.7a 65.0 0.7b
S
G2/M
3.0 1.4 2.3 0.6 6.4 2.7
7.1 1.6a 8.3 1.5a 3.0 1.2b
Within columns, values with different superscripts are significantly different (P < 0.05).
G1/S transition in aphidicolin treatment in pigs. There are reports of differences in different stages of cells in goat foetal fibroblast (Zou et al., 2002), adult fibroblast cells of cattle (Hayes et al., 2005), panda fibroblast cells (Han et al., 2003), and adult cattle fibroblasts (Kubota et al., 2000). These variations may be due to different species, different chemicals used to synchronize cell cycle stages, as well as experimental protocols. For example, from the findings of Gomez et al. (2003) in African wild cats and domestic cats had different values for G0/G1 stages in case of cycling, SS and chemical treated groups. In summary, the studies reported here have demonstrated that b-ME, cysteine and DMSO had better results compared with cycling, serum-starved and fully confluent groups for the synchronization of goral skin fibroblast cells at G0 þ G1 stages. This finding could have a positive effect on somatic cell nuclear transfer in this endangered species. Acknowledgements This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00). References Boquest AC, Day BN, Prather RS. Flow cytometric analysis of cultured porcine fetal fibroblast cells. Biol Reprod 1999;60:1013e9. Campbell KH, Loi P, Cappai P, Wilmut I. Improved development to blastocyst of bovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol Reprod 1994;50:1385e93. Campbell KHS, Loi P, Otaegui PJ, Wilmut I. Cell cycle coordination in embryo cloning by nuclear transfer. Rev Reprod 1996;1:40e6. Collas P, Balise JJ, Robl JM. Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol Reprod 1992; 46:492e500. Cultural Properties Administration of Korea. A report for distribution and ecological studies of Korean natural Monuments, goral and musk deer. 1999. Eggan K, Jaenisch R. Nuclear reprogramming: biological and technological constraints. In: Cibelli J, Lanza RP, Campbell KHS, West MD, editors. Principles of cloning. Amsterdam: Academic Press, An imprint of Elsevier Science; 2002. p. 85e108. Fulka JJ, First NL, Moor RM. Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod 1998;4:41e9. Geist V. On the evolution of the Caprinae. In: Soma H, editor. The biology and management of Capricornis and related mountain antelopes. London: Croon Helm; 1987. p. 3e40.
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