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Reproductive Technology |
Department of Animal Science and Center for Regenerative Biology,3 University of Connecticut, Storrs, Connecticut 06269
Department of Animal Science4
and
Department of Veterinary Pathobiology,5 University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Telomeres are special structures at the ends of the eukaryotic chromosomes consisting of repetitive short DNA sequences and their associated proteins [15]. During each cell division, the telomeric DNA in somatic cells of most mammals are shortened by 50–200 base pairs (bp) due to the lack of active telomerase, which elongates telomeric DNA [16, 17]. The shortening of the telomeres with each cell division is believed to cause cellular senescence [18]. Many factors have been found to affect the lengths of telomeres. Telomere lengths are different in animals of different species, individuals of different age within a species, different tissue types within an individual, and different chromosomes in the same cell [19–22].
Little is known about the telomere lengths in pigs from natural reproduction. To date, there has been only one study in pigs in which the telomere lengths of sperm and kidney were compared [22]. An earlier study employing fluorescence in situ hybridization (FISH) reported interstitial telomeric DNA in pig chromosome 6, but provided no measurements of telomere lengths [23]. Despite the success of cloning pigs by numerous groups [3–6, 24–31], the lengths of telomeres in cloned pigs have not been reported. Furthermore, it has been reported that nuclear reprogramming is regulated by a different mechanism in cloned pig embryos than in either cloned bovine or murine embryos [32, 33]. Therefore, information obtained on telomere reprogramming from studies of cloned cattle or mice may not apply in cloned pigs. In the present study, we first established normal profiles of telomere changes with age and tissue type in pigs from natural reproduction. We then examined the telomere lengths of eight cloned pigs and found that they do not differ significantly from those of their age- matched controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To establish the normal telomere length profiles by age and tissue type, samples were collected from major organs of naturally produced pigs at three different ages: fetuses of 70 days of gestation (n = 3), prepubertal pigs of 2.5–5 mo (n = 3), and adults of 16.5–27 mo (n = 6). Organ/tissue samples, including gonad, heart, liver, lung, kidney and skin, were dissected immediately after the animals were killed. All tissue samples were snap frozen in liquid nitrogen and stored at –80°C until analysis. For cloned pigs, skin samples were collected from six live pigs at 4 mo of age and two dead pigs shortly after birth. Four of the cloned pigs (NT1, NT3– NT5) were produced from a fibroblast cell line transduced with the enhanced green fluorescent protein gene (eGFP) [25]. The cell line was established from a 35-day-old crossbred porcine fetus after viral transduction and drug-selection of 13 days for the first round, and then 4 days for each of six additional passages [25]. The other four cloned pigs (NT7–NT10) were generated from a skin fibroblast cell line established from a newborn transgenic pig that expressed eGFP [26]. The cloned pig NT8 died at 3 days of age from a bacterial infection, and NT9, a littermate, died at 7 days of age due to congestive heart failure. As age-matched controls for the clones, skin samples from 4-mo-old pigs of the same breed (n = 6) produced naturally were also collected. To compare telomere lengths of the donor cells with those of the clones, the two donor cell lines used for the generation of the clones were also included in this study. Animal handling and experimentation were in accordance with the National Research Council's publication "Guide for the Care and Use of Laboratory Animals" and approved by Institutional Animal Care and Use Committees at University of Connecticut and University of Missouri-Columbia.
Terminal Restriction Fragment Assay
Genomic DNA from tissue samples and cultured cells was prepared using blood and cell culture DNA kits from Qiagen (Valencia, CA) and was quantified in a spectrophotometer. The terminal restriction fragment (TRF) assay was performed to determine the telomere lengths [16]. Briefly, each DNA sample (2.5 µg) was digested in a mixture of restriction enzymes of RsaI/HinfI (New England Biolabs, Beverly, MA), which do not cut in telomeric DNA but cut frequently in other regions of genomic DNA. The reaction of genomic DNA digestion was carried out at 37°C for 12 h. The digested DNA fragments were then resolved on 1% (w/v) pulsed-field certified agarose gels by pulsed-field electrophoresis with a FIGE Mapper system (Bio-Rad, Hercules, CA) under the following conditions: switch time ramp: 0.1–2.0 sec; forward voltage: 180 V; reverse voltage: 120 V; run time: 16 h. The use of the pulsed-field electrophoresis ensures good separation of the large telomeric DNA fragments, which are normally not well resolved by conventional electrophoresis. The DNA in the agarose was then transferred to nylon membranes (Osmonics, Westborough, MA) after treatment with 0.25 M HCl and 0.4 N NaOH. The nylon membranes were prehybridized in North2South hybridization buffer (Pierce, Rockford, IL) at 55°C for 30 min, and then a biotin-labeled telomere-specific probe (BD Biosciences, San Diego, CA) was added, and then membranes were incubated overnight. Excess probes were removed by washing the blots three times in 2x saline sodium citrate/0.1% SDS at 55°C. The membranes were then subjected to chemiluminescent detection according to the manufacturer's instruction and exposed to x-ray film.
Pigs have been reported to have interstitial telomeric DNA on chromosome 6 [23], which is not affected by cell division or nuclear reprogramming. This interstitial telomeric DNA is potentially detectable by our TRF assay, and could, therefore, confound the results. For this reason, we carried out experiments to determine whether this interstitial telomeric DNA was in fact included in the telomeres located at the ends of chromosomes and measured by TRF assay. Briefly, exonuclease BAL-31 (New England Biolabs), which digests the DNA from both ends of the chromosomes, was used to treat genomic DNA samples before RsaI/HinfI digestion [34]. A time course of BAL-31 treatment was developed in which 5 µg of genomic DNA was incubated for 15, 30, 60, 120, and 300 min in the presence of BAL-31 (40 U/ml) at 30°C followed by inactivation in 20 mM EGTA at 65°C for 10 min and then ethanol precipitation. The BAL-31 digested DNA was then subjected to the TRF assays as described above. It was found that, with the increasing time of digestion, the telomeric DNA measured by the TRF assay became progressively shorter. No signal could be detected after 120 min (the minimum effective treatment duration for eliminating the telomeric DNA at the chromosome ends) of BAL-31 treatment (Fig. 1A), while the majority of the genomic DNA fragments were still 12–50 kilobases (kb) in length before RsaI/HinfI treatment (data not shown). This demonstrated a complete removal of chromosome-end telomeric DNA by this treatment and failure of TRF assay to detect the interstitial telomeric DNA sequence. We carried out this BAL- 31 treatment to all DNA samples used in this study and were unable to detect any signals of the interstitial telomeric DNA (data not shown). This clearly demonstrated that the results of the TRF assays in this study only pertain to the lengths of telomere located at chromosome ends.
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Calculation of Telomere Lengths
The x-ray film images were scanned and processed using the Quantity One software (Bio-Rad). Each telomere signal smear was evenly divided into at least 30 grids and the mean telomere size was determined according to the formula: L =
(ODi·Li)/(ODi), where ODi and Li are the signal intensity and telomere length, respectively, at position i within the lane.
Statistical Analysis
The TRF assay was repeated at least three times for each sample, and the averages were reported. Differences among groups were analyzed by student t-test and GLM procedure using SAS software (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A representative TRF assay on transgenic cloned pigs is shown in Figure 1B and the mean telomere lengths, in different tissue types, of naturally produced pigs of different ages are summarized in Figure 2. In general, the pig telomeres ranged from 9 to more than 23 kb. At the fetal stage, telomere lengths averaged 21.2 kb and varied from 20.7 to 21.7 kb among six major organs. As expected, telomeres were significantly shortened in both the prepubertal and adult age groups. For all the tissue types examined, telomere shortening of approximately 4 kb was observed from fetus to prepuberty, and further shortening of telomeres by about 1.5 kb occurred from prepuberty to adulthood. When telomeres from different tissues were compared within a given age group, the results for the fetal pigs showed their lengths were relatively uniform and no significant differences were found among different tissues (P > 0.05). At both the prepubertal and adult stages, however, significant differences in telomere lengths were found among different tissues (P < 0.05). Particularly, during the prepubertal stage, telomere lengths of skin and gonad (18.0 ± 0.4 and 18.2 ± 0.6 kb, respectively) were approximately 2 kb longer than those of liver, lung, and heart (16.3 ± 0.4, 16.1 ± 0.6, and 15.8 ± 0.4 kb, respectively). Similarly, in adult pigs, telomeres in skin and gonad (16.1 ± 0.8 and 16.6 ± 0.9 kb, respectively) were longer than those in other tissues, with the shortest telomeres found in the liver (14.0 ± 1.0 kb).
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Telomere Lengths in Cloned Pigs
The telomere lengths of all cloned pigs and their donor cells are summarized in Table 1. The telomere lengths of the eGFP-transduced fetal fibroblast cell line and the eGFP- expressing newborn fibroblast cell line averaged 18.5 and 19.1 kb, respectively. The telomeres in the skin cells of the newborn transgenic pig, cells of which were used to establish the newborn cell line, were 20.1 kb. Because the cells were passaged six times, cells in culture shortened their telomeres approximately 100 bp per cell doubling. We do not have tissues to examine the fetus used to generate the fetal cell line. However, the 18.5-kb telomeres of this cell line were nearly 2 kb shorter than control fetuses, suggesting loss of telomeres during drug selection.
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Similar to pigs of natural reproduction, the telomeres of cloned pigs also ranged from 9 to greater than 23 kb. At 4 mo of age, the skin telomeres of cloned pigs were not significantly different from those of their age-matched controls (18.7 ± 2.2 kb versus 18.0 ± 1.4 kb, P > 0.05; Fig. 3). For the two cloned pigs that died at 3 and 7 days of age, NT8 and NT9, the skin telomere lengths were 24.7 and 21.9 kb, respectively. These lengths were not significantly different from those of fetuses at 70 days of gestation (23.3 ± 2.0 kb versus 21.7 ± 0.6 kb).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, Southern blotting was used to measure telomere lengths in pigs. As mentioned earlier, an interstitial telomeric sequence is present in porcine chromosome 6 [23]. This interstitial telomeric sequence alone, however, was undetectable using the standard TRF assay, likely due to its small proportion in the total telomeric DNA in the cells. Although quantitative FISH (Q-FISH) [35] can detect the lengths of the interstitial telomere, it is more effective when a small number of cells, such as a preimplantation embryo, are characterized. Additionally, the use of Q-FISH can add another variable to telomere measurements in our study because Q-FISH requires the establishment of cell cultures from the cloned animals and culturing cells will shorten telomere lengths, confounding the measurement of the vast majority of the telomeric DNA located at the ends of chromosomes. Therefore, Southern blotting is the method of choice for our study.
We found that telomere lengths are inversely related to age. In pigs produced naturally, telomeres shortened on average 3.5 kb during the first 4–5 mo after birth. This telomere loss is primarily due to body growth from cell divisions. It has been reported that, in humans, telomeres shorten by 30–70 bp each year and approximately 1.7 kb of telomeres are lost from a fetus to an adult of 24 yr of age [16, 36]. It is obvious that the telomeres in pigs shorten at a much faster rate than those of humans [37, 38]. This is likely because humans reach their adult size in approximately 18 yr, while it only takes pigs several months. This faster growth rate in pigs is thus correlated with a faster loss of telomeres than in humans. From prepubertal to adult pigs, telomere shortening was much reduced, only about 1.5 kb were lost from 4 to 5 mo to up to 26 mo of age. This may be due to the fact that prepubertal pigs have already reached more than approximately half of their adult body size. The reduced rate of telomere loss results directly from a reduced growth rate in addition to cell division for the replacement of dead cells. This observation also agrees with the finding in humans, where only about 0.7 kb of telomere reduction was seen from 24 yr of age to 91 yr of age [16].
In fetal pigs from natural reproduction, we observed a synchrony of telomere lengths in tissues and tissue-specific telomere shortening in vivo during postnatal development. Telomere length synchrony among different tissues had previously been found in human fetuses [39] and newborn mice [19]. Similar to humans and mice, the telomere synchrony in pig tissues was lost during postfetal life, resulting in large variation in telomere lengths in different tissues/ organs. These observations can be explained by the fact that telomere lengths of somatic cells are proportional to their proliferation rates in the absence of active telomerase, as is the case in most mammalian species. Internal organs such as the liver, for example, grow to a much bigger size than many other smaller organs and likely undergo more rounds of cell division. Although mouse somatic tissues have active telomerase, telomeres also vary in different mouse tissues, suggesting tissue-specific telomerase activities [20].
In the present study, we found that telomeres were longer in gonads than in most other types of tissue examined, except for the skin. Additionally, the decrease of telomere lengths in gonads was also less dramatic than that in other tissues. These observations may have been caused by the presence of large numbers of germ cells in this organ. It has been reported that germ cells have active telomerase activity and have longer telomeres than other tissues [40]. The slow rate of telomere shortening in gonads may reflect the decrease in the somatic portions of this organ, with the germ cells maintaining the original length of their telomeres and therefore, overall less telomere shortening was observed because the entire organs were used for analysis.
Interestingly, the telomeres in the skin cells of pigs were also longer than in other tissues analyzed. Although the same observation has also been reported for humans [41] when skin was compared to blood leukocytes and synovial tissues, it is unclear why this is the case. Because skin cells do not contain active telomerase, the likely explanation for this is that skin cells do not divide as much as the cells of other organs during postnatal growth.
In the present study, we found that the transduced cell line from a 35-day-old fetus had shorter telomeres than the newborn cell line. This may be due to the fact that the transduced fetal cell line was subjected to long periods of culture for a total of 37 days. This is equivalent to at least 25–30 doublings in ideal culture conditions. In contrast, the newborn cell line was only passaged six times, for approximately 12 cell doublings [25]. Despite differences between these donor cells, clones generated from these two cell lines did not differ in telomere length when compared with age- matched controls from natural reproduction. This suggests that telomeres were reprogrammed during the nuclear transfer process. This observation is consistent with those in cloned cattle and sheep [11, 12, 14, 42]. The telomere restoration in these cloned animals may have resulted from active telomerase activity in cloned embryos as reported in the bovine [11, 12].
One of the cloned piglets generated from the transduced fetal cell line (NT3), however, had telomeres of 15.6 kb at 4 mo of age, which is approximately 2.4 kb shorter than her age-matched controls. Because skin samples at birth were unavailable, it is impossible to conclude whether this pig had accelerated shortening of telomeres after birth or the embryo had insufficient telomere restoration during nuclear reprogramming. Previously, Miyashita et al. [13] found remarkable differences in telomere length among cloned cattle derived from different cell types, suggesting that not all cloned embryos can reverse the telomere attrition caused by aging of the donor animals and culture of donor cells. The failure of nuclear transfer to fully restore telomere lengths may be the case in this particular cloned pig. This is because 4-mo-old piglets of natural reproduction lose at least 3.5 kb of telomere from late fetal stage to prepuberty. If this cloned piglet (NT3) also had lost 3.5 kb by 4 mo of age, then this pig's telomeres would be estimated to have been approximately 19 kb at late gestation. Furthermore, the telomere lengths in pigs are similar to those in cattle, which have an estimated loss of 2.6 kb from fertilization to birth [13]. Using this figure as an estimate for the loss in pigs, the cloned piglet NT3 would, at embryo transfer, have had telomeres approximately 21.7 kb. This would indicate that some telomere reprogramming by the cloned embryo (Table 1) had taken place, which restored telomere lengths by 3.2 kb from the original 18.5 kb in the donor cells.
In summary, we found that telomere lengths in pigs from natural reproduction decrease with age and that different tissues have different telomere lengths after birth. Telomeres of cloned pigs are similar to those in age-matched controls from natural reproduction, indicating telomere reprogramming occurs during the nuclear transfer process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: X. Cindy Tian, 1392 Storrs Road, Storrs, CT 06269- 4243. FAX: 860 486 8809, xtian{at}canr.uconn.edu
Received: 25 August 2003.
First decision: 23 September 2003.
Accepted: 22 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002 295:1089-10921,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002 20:251-255[CrossRef][Medline]1,3-Galactosyltransferase-knockout cloned pigs expressing human 1,2-fucosylosyltransferase. Biol Reprod 2003 69:437-445This article has been cited by other articles:
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  H. Niemann, X C. Tian, W A. King, and R. S F Lee Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning Reproduction, February 1, 2008; 135(2): 151 - 163. [Abstract] [Full Text] [PDF]
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 V. J. Hall, P. Stojkovic, and M. Stojkovic Using Therapeutic Cloning to Fight Human Disease: A Conundrum or Reality? Stem Cells, July 1, 2006; 24(7): 1628 - 1637. [Abstract] [Full Text] [PDF]
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 R. S. Prather, P. Sutovsky, and J. A. Green Nuclear Remodeling and Reprogramming in Transgenic Pig Production Experimental Biology and Medicine, December 1, 2004; 229(11): 1120 - 1126. [Abstract] [Full Text] [PDF]
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