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Regular Article |
a Laboratory of Cellular Biology, National Institute of Animal Industry, Kukisaki, Ibaraki 305-0901, Japan
b Department of Beef Cattle Production Technology, Oita Prefectural Livestock Experiment Station, Kujyu, Oita 878-0201, Japan
c Department of Technology, National Livestock Breeding Center, Nishigo, Fukushima 961-8511, Japan
d Tokachi Station, National Livestock Breeding Center, Otofuke, Hokkaido 080-0572, Japan
e Embryo Transplantation Laboratory, Snow Brand Milk Products Co., Ltd., Tomakomai, Hokkaido 059-1365, Japan
f Animal Industry Research Institute, Iwate Prefectural Agriculture Research Center, Takizawa, Iwate 020-0173, Japan
g Department of Animal Production, Hokkaido National Agricultural Experiment Station, Sapporo, Hokkaido 062-8555, Japan
h Embryonic Technology Laboratory, National Institute of Agrobiological Sciences, Kukisaki, Ibaraki 305-8602, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, embryo, female reproductive tract, mammary glands, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is important, however, that livestock animal clones be used not only for practical applications but also for research to elucidate the mechanisms of cloning. Because many questions regarding cloned animals remain unsolved (e.g., male clones that can be produced with difficulty in mice [4] can be produced easily in cattle [8]), comparative research among clones of several species are essential. For breeding schemes in Japan, all bulls and many cows undergo a progeny test, and genetic potentials in bulls and cows are estimated statistically using their progeny's data regarding many traits, such as growth, milk and meat production. Additionally, their lineage is also recorded retroactively to several generations. Because most cloned cattle in Japan were produced from such informative cattle and the identity test between donor cattle and their clones has already started, we will gain favorable knowledge regarding how to solve the questions of cloning in the future. Furthermore, by accumulation of comparative research among clones of several species, mechanisms acting in the process of this promising technique of cloning will gradually be revealed.
Interesting questions have been raised as to how old cloned animals are in terms of genetic age; how long they will be able to live and stand for milk production, reproduction, and so on; and in particular, when to use donor cells derived from old animals. It has been reported in the cloned sheep "Dolly" that the telomere length, which shortens with each cell division, resulting in cellular senescence [17–21], is consistent with the age of the mammary tissue used for nuclear transplantation from the 6-yr-old donor sheep and that the length is significantly shorter than that in age-matched control sheep [22]. This suggests that Dolly may not achieve a full, normal life span. On the other hand, in cases of cloned cattle produced from near-senescent fibroblasts, extension of telomere lengths, in which telomere lengths in cloned cattle were the same as or longer than those in newborn normal calves, has been reported [23]. Tian et al. [24], Wakayama et al. [5], and Betts et al. [25] have also reported normal telomere lengths in clone animals derived from fibroblasts and cumulus cells. Therefore, the question of the discrepancy between Dolly's shortened telomere and others' restored telomeres remains.
The present study was conducted to determine the effects of cloning on telomeres. Focusing our attention on differences in telomere lengths depending on the tissue [26, 27], we had produced cloned cattle from reconstructed embryos with nuclei of epithelial cells from oviduct and mammary gland, skin fibroblasts, and muscle cells [8, 9]. In the present study, we examine variation in telomere lengths in these cloned cattle, and we discuss the effect of cloning on telomere length.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The DNA from leukocytes or cultured cells was prepared with the SDS-phenol-proteinase K extraction methods, and equal amounts of DNA (1 µg) were digested by both the restriction enzyme AluI and HinfI. Samples, 1-kilobase (kb) ladder marker, and 5-kb ladder marker were then loaded on a 1% (w/v) agarose gel (15 x 15 cm). The gels were run by pulse-field electrophoresis to separate long telomeric DNAs [28–30] using a program designed to ensure good separation for size between 1 and 50 kb with the FIGE Mapper electrophoresis system (Bio-Rad, Hercules, CA). The DNA was then transferred to nylon membranes by a standard Southern blotting procedure. DNA was cross-linked to the membrane with 1200 µJ of ultraviolet light.
Hybridization
The telomere-specific oligonucleotide (TTAGGG)3 was end-labeled at 37°C for 15 min using terminal deoxynucleotidyl transferase from the DIG Oligonucleotide 3'-End Labeling Kit (Roche, Mannheim, Germany). The blotted nylon membranes were prehybridized in 40 ml of DIG Easy Hyb (Roche) for 2 h at 37°C, and then were hybridized in 10 ml of DIG Easy Hyb containing 50 pmol of end-labeled, telomere-specific probe for 16 h at 37°C. Membranes were washed 3 times in 50 ml of 0.5x standard saline citrate (SSC; 1x SSC: 0.15 M NaCl plus 0.015 M sodium citrate) for 15 min at 37°C. Chemiluminescence was performed using the DIG Luminescent Detection Kit (Roche) and exposed to x-ray film (RX-U; Fuji Photo Film, Minamiashigara, Japan).
Calculation of Telomere Length
Telomere length was calculated as described by Tian et al. [24] and Norwood and Dimitrov [29]. The chemiluminescence signal was acquired by a Model GS-700 Imaging Densitometer (Bio-Rad) at the highest resolution of 0.01 mm. The acquired 16-bit images were then processed by using the Molecular Analyst software (Bio-Rad). Once the distance calibration was obtained, all distances were converted to telomere lengths, and the dependence of the signal intensity on the telomere length at each point (0.01 mm) was analyzed. Mean terminal restriction fragment (TRF) size was determined as
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Culture of Senescent Cells
To determine a critical telomere length in bovine somatic cells, telomere erosion was examined in culture using a clonal bovine intramuscular preadipocyte (BIP) line [31]. The BIP cells were cultured in Dulbecco modified Eagle medium (Gibco BRL, Grand Island, NY) plus 10% fetal bovine serum and antibiotics at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For each passage, cells were cultured until confluency, were disaggregated by incubation in a 0.02% trypsin and 0.01% EDTA solution, and were allocated to new dishes for further passage. Normally, each passage lasted approximately 3 days. The cell line was maintained routinely until the cells exhibited features of replicative senescence and the number of cells could no longer increase.
Statistical Analysis
The mean TRF analyses in cloned cattle were repeated at least 2 times, and the average is shown in this paper. Because the methods for measuring mean TRF sizes were reported to have an error of approximately 0.3 kb in humans [30] and telomeres in cattle were demonstrated to be 2 times longer than those of humans in this study, when differences between two measurements for the same sample were beyond 0.5 kb, more measurements were carried out until the difference between two measurements was within 0.5 kb. Linear regression of mean TRF sizes in 50 normal Japanese Black cattle against age were analyzed with the least-squares method. Differences among mean TRF sizes in the donor cells, cloned cattle, control cattle, and control sperm were analyzed by Student t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To calibrate the telomere clock in normal cattle, telomere erosion in vivo was analyzed using leukocytes from 50 normal Japanese Black cattle (age, 0–18 yr; Fig. 1). In newborn calves, the mean sizes of the terminal telomere fragments obtained by cutting with restriction enzymes (i.e., mean TRF) were found to be 19.0–21.9 kb (20.43 ± 0.28 kb), whereas in two 18-yr-old animals, which were regarded as aged, the mean TRF sizes were 15.1 and 16.8 kb (Fig. 2A). Then, the analysis yielded a significant linear regression of telomere lengths (LRTL): The mean TRF size regression was -0.23 kb/yr + 20.54 kb (r2 = 0.56, P < 0.01). The mean TRF sizes of bovine spermatozoa were 22.1–23.9 kb (23.0 ± 0.28 kb) (Fig. 2A). Because the mean TRF sizes of spermatozoa in humans are kept longer than those of any tissues beyond generation and then hypothesized as initial lengths of telomere erosion [19], telomere erosion from the beginning of fertilization to birth in cattle was also, thus, revealed to be significantly apparent (P < 0.01) and approximately 2.6 kb. On the other hand, to determine a critical telomere length in bovine somatic cells, telomere erosion in culture was examined using a clonal BIP line that exhibited a fibroblastic appearance and abundant replicative capacity [31]. After 60 passages of culture, BIP cells exhibited features of cellular senescence and almost arrested their proliferation. In such senescent cells, the mean TRF size had also stopped decreasing at 9.7 kb (Fig. 2A).
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Telomere Lengths in Cloned Cattle Derived from Epithelial Cells of an Old Cow
We produced 5 and 1 cloned cattle by nuclear transfer of cultured oviductal epithelial cells and mammary epithelial cells, respectively, from a 13-yr-old Holstein cow [9] (Table 1). The mean TRF sizes in all 6 newborn cloned calves were significantly smaller (P < 0.01) than those in age-matched controls and surprisingly smaller than those in 18-yr-old control animals (Figs. 1 and 2B). In terms of the age-dependent LRTL, estimated ages taken from leukocytes of cloned calves derived from Holstein oviductal and mammary epithelial cells (as an index of aging not of the whole body but, rather, only of leukocytes) were surprisingly old and approximately 30–35 and 29 yr, respectively (Fig. 3), regardless of the actual bovine lifetime of 15–20 yr at most. However, all of them remained alive and normal at 22–25 mo of age, except that one derived from mammary epithelial cells was slaughtered for deformity. On the other hand, the mean TRF sizes of their donor cells were also revealed to be much shorter than those of leukocytes in age-matched controls in LRTL terms. Telomere erosions from nuclear transfer to birth were revealed to be 1.5–2.7 kb (Table 1) and were the same as or less than telomere erosion from fertilization to birth in normal cattle.
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Telomere Length in Cloned Cattle Derived from Muscle Cells of an Old Bull
The mean TRF sizes of leukocytes from 2 newborn cloned calves that were produced by nuclear transfer of cultured cells derived from the muscle tissue of a 12-yr-old (as of August 1997) Japanese Black bull [8] were within variation among the age-matched control calves (Figs. 1 and 2C). These cloned cattle remained alive and normal at 24–29 mo of age and had calves by artificial insemination using their spermatozoa. The mean TRF sizes of their donor cells, regardless of 6 passages of culture before nuclear transfer, were be much longer than those of leukocytes from cattle at the corresponding age in LRTL terms. Therefore, less telomere erosion from nuclear transfer to birth was revealed in comparison with that from fertilization to birth in normal cattle.
Telomere Length in Cloned Cattle Derived from Cultured Cells of Younger Cattle
We produced 4 cloned cattle by nuclear transfer of cultured oviductal epithelial cells from a 6-yr-old Jersey cow [9]. Like cloned calves derived from epithelial cells of an old Holstein cow, the mean TRF sizes in all 4 newborn cloned calves were significantly smaller (P < 0.01) than those in age-matched controls and surprisingly smaller than those in 18-yr-old control animals (Figs. 1 and 2B). Telomere erosions from nuclear transfer to birth were 0.9–2.0 kb (Table 1) and were less than telomere erosion from fertilization to birth in normal cattle. On the other hand, the mean TRF sizes of leukocytes from 2 cloned cattle that were produced by nuclear transfer of cultured skin cells derived from an ear of a 2-yr-old Japanese Black bull and their donor cells were compared with those of leukocytes obtained from age-matched controls. The mean TRF sizes in the newborn cloned calves were within variation among the age-matched control calves, just as in cloned cattle derived from muscle cells of an old bull (Figs. 1 and 2D). One of the cloned calves was very heavy in body weight (63 kg) compared to the standard body weight for male calves of this breed (20–30 kg) and died shortly after birth because of dystocia at parturition; the other cloned calf was normal in body weight (27 kg) and slaughtered for histopathological research (Table 1). Regardless of a few passages of culture before nuclear transfer, the mean TRF sizes of donor cells from ear skin were to be much smaller than those of leukocytes in the newborn cloned calves. Thus, telomere extension might have occurred from nuclear transfer to birth. The extent of changes in telomere lengths from nuclear transfer to birth was significantly different among their donor cell types of epithelial cells, muscle cells, and fibroblasts (P < 0.01).
Telomere Length in Embryonic Cell-Cloned Cattle
We investigated telomere lengths in embryonic cell-cloned cattle that had been produced by nuclear transfer of the nuclei of 28- to 49-cell stage blastomeres to enucleated oocytes. All of 6 embryonic cell-cloned cattle we tested had significantly longer telomeres than age-matched controls (P < 0.01), and their offspring calves, which were obtained by artificial insemination of normal spermatozoa to female embryonic cell-cloned cattle, also had telomere lengths somewhere in between those of age-matched control calves and embryonic cell-cloned cattle (Fig. 2E and Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The number of samples in the present study was limited and the conditions for comparison were inequitable, but the most likely explanation for the remarkable variation among telomere lengths in our cloned cattle was that it reflected the telomere length variation of their donor cells and the extent of telomere length change induced by cloning. First, the variation among donor cells may be caused by cell type-specific factors in vivo, e.g., frequencies of cell division in each tissue and intracellular environments such as a concentration of superoxide that is detrimental to DNA helices, and so on. It was suggested that the telomere erosion rate of the oviductal and mammary epithelial cells in vivo may be much more rapid than that in leukocytes, and that the extremely shortened telomere lengths of resultant donor cells may lead to the remarkably short telomere lengths of the cloned calves. In contrast, it was suggested that telomere lengths of the muscle cells could be retained even in the old donor bull, because the donor cells were possibly derived from muscle satellite cells that play a role in restoring injured muscle tissue and are inactive for cell division except when performing that function. Therefore, telomere lengths of the muscle cells might lead to a longer mean TRF of leukocytes in the cloned calves than that in the donor bull. Second, it was also suggested that the effect of aging in vivo of donor cattle might contribute to the telomere length variation of donor cells, because the difference between telomere lengths in oviductal epithelial cells from 2 cows in the present study was reflected by the different ages of the cows. Furthermore, the difference might result in telomere length variation among cloned cattle that is significantly regressive (r2 = 0.840, P < 0.01). The possibility that telomere length variation of donor cells was contributed to by the genetic background of the donor cattle was not excluded, because variation among telomere lengths in the leukocytes of control cattle was not so small. On the other hand, telomere length variation among donor cells might not be induced by culture so much, because the donor cells were used after only a few passages.
Not only initial telomere lengths in donor cells but also the extent of telomere length change induced after cloning might lead to a remarkable variation among telomere lengths in cloned cattle. In all previous reports except the case of Dolly [22], it was demonstrated that shortened telomeres in donor fibroblasts and cumulus cells both extended and restored from nuclear transfer to birth; therefore, normal telomere lengths in cloned calves were obtained [23–25]. Betts et al. [25] pointed out that the rebuilding of shorter telomere lengths in donor cells could be due to telomerase activities, which are detected as early as the blastocyst stage in reconstructed embryos. The present study showed that some cloned cattle had longer telomere lengths than those of their donor cells, and that others had telomere lengths just a bit shorter than those of their donor cells. Additionally, the extent of telomere length changes from nuclear transfer to birth were significantly different among donor cell types (P < 0.01). It is of interest that less telomere erosion or telomere extension from nuclear transfer to birth was observed in most cloned cattle in comparison with telomere erosion from fertilization to birth in control cattle (2.6 kb). Thus, it was suggested that cloning does not necessarily restore the telomere clock but, rather, may commonly trigger an elongation of telomere lengths, probably more or less according to cell types. Not only in cattle but also in humans, telomere lengths were reported to decrease from 22 kb in spermatozoa to 10 kb in newborn-baby leukocytes [17]. Hence, it is most probable that telomeres also decrease from fertilization to birth in sheep. If so, then Dolly's telomere length, which was the same as that of the donor mammary epithelial cell, might elongate transiently after cloning, resulting in compensation for the loss during embryo-fetus development.
The exact explanation for the difference among donor cell types in the extent of telomere length elongation from nuclear transfer to birth is unclear at this moment. In humans, it was reported that telomerase activities are not detected in most somatic cells except for a part of the stem cells and fetal cells [32–34], and that telomerase activities even in telomerase-positive somatic cells are lost quickly by culture and particularly during passage [35]. In cattle, similar results were obtained [25], and telomerase activities in oviduct, mammary, muscle, and ear skin tissue were very weak or not detected in our study (data not shown). Thus, it was suggested that the difference among donor cell types in telomere length elongation was not caused by telomerase activities taken from donor cells. It may, instead, be partially explained by a possibility that donor cell type-specific sensitivities to telomerase, based on telomere-binding proteins [36, 37], lead to the difference in the extent of telomere elongation in reconstructed embryos. For example, telomere-binding protein TRF1, which inhibits telomerase activities, was expressed differentially according to the tissue type [37].
To confirm whether telomere elongation is only specific to somatic cell-cloned cattle, we investigated telomere lengths in embryonic cell-cloned cattle that had been produced by nuclear transfer of blastomeres to enucleated oocytes. They are regarded as being homogeneous to normal cattle, because totipotent cells in germline were used as their donor cells. All of 6 embryonic cell-cloned cattle and their offspring calves that we tested had significantly (P < 0.01) longer telomeres than the age-matched controls. These results might indicate that elongation of telomeres did not result from cloning with "somatic" cells but, rather, was induced by the technique of nuclear transfer itself.
To determine a critical telomere length in bovine somatic cells, telomere erosion was examined in culture using a clonal BIP cell line, in which the mean TRF size had also stopped decreasing at 9.7 kb. This critical length is longer than that from human fibroblast [20] and leukocytes [38] but is shorter than the telomere lengths of sterile mice derived from successive crossing of mice lacking telomerase RNA and exhibiting decreased proliferation in highly proliferative organs [39, 40]. Because the mean TRF sizes of leukocytes in old cattle were much longer than the critical length found in the BIP line, it may be difficult to relate telomere erosion to senescence in normal cattle. Therefore, it was indicated that cloned calves derived from epithelial cells could live regardless of the ages estimated from telomeres in leukocytes, which were surprisingly old at approximately 24–33 yr. However, it is possible that cloned cattle produced from oviductal and mammary epithelial cells may lose function sooner in tissues with cells regulated at a high rate of telomere erosion and, consequently, die young because of their remarkably short telomeres.
It is often said that we must take care of the decreased genetic variation in the livestock population and the resultant increased risk of inbreeding depression and genetic disease induced by the overdiffusion of cloning. Likewise, it may be important to be careful of telomere lengths, not only for individual cloned animals but also for whole species. If cloned animals and their offspring that had short telomeres were selectively reproduced for their high performance and disseminated without due care, the whole species might become short-lived and, in some cases, face extinction. When applying cloning techniques in the livestock industry, we must check telomere lengths and take them into account, for the sake of longevity, when making mating plans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Funded by the 21st Century Green Frontier, Clone Project of the Ministry of Agriculture, Forestry & Fisheries of Japan. This work was supported by the Department of Animal Breeding and Reproduction in National Institute of Livestock and Grassland Sciences. 
2 Correspondence and current address: Norikazu Miyashita, Animal Cell Biology Laboratory, National Institute of Agrobiological Sciences, Kukisaki, Ibaraki 305-8602, Japan. FAX: 81 298 38 8689; nmiya{at}affrc.go.jp
3 Current address: Laboratory of Functional Morphology, Tohoku University, Aoba, Sendai 981-8555, Japan
Accepted: December 20, 2001.
Received: August 22, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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