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Regular Article |
a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
ABSTRACT
This study examined the telomerase activity in preimplantation bovine embryos derived from either parthenogenetic activation or nuclear transfer. Telomeres are the DNA-protein structures located at the ends of eukaryotic chromosomes. Telomerase is the ribonuclear enzyme that helps to restore telomere length by synthesizing telomeric DNA repeat (5'-TTAGGG-3') from its own RNA template. Without telomerase activity, telomeres shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Previously, we reported the dynamics of telomerase activity in bovine in vitro fertilized (IVF) embryos. In the present study, we examined the telomerase activity in bovine embryos derived either from parthenogenetic activation or somatic cell nuclear transfer (i.e., cloning). Embryos from both sources were harvested at different stages, from zygote to blastocyst. Telomerase activity in embryos derived from parthenogenetic activation and nuclear transfer showed a dynamic profile similar to that of those derived from IVF. Telomerase activity was detected in embryos at all stages examined, with the highest level in the blastocyst stage, regardless of the method of embryo production.
early development, embryo, gamete biology, in vitro fertilization, oocyte development
INTRODUCTION
Telomeres are the DNA-protein structures located at the ends of eukaryotic chromosomes [1]. They function in chromosomal stability and organization and are essential for normal cell division [2]. In eukaryotic cells, the conventional DNA polymerase is unable to replicate lagging strand to the very 5' end [1]. As a consequence, telomeric DNA is progressively lost with each round of cell division [3]. Several teams of scientists have proposed the telomere as a "mitotic clock" for normal cell division [4–6]. Telomere shortening correlates with the number of cell divisions, and when the telomere length reaches a critical limit, it signals the cell to undergo replicative senescence, in which state the cells develop a characteristic large and flat morphology and cease dividing [7]. In human fibroblast cell culture, telomere length decreases at a rate of 48 ± 21 base pairs (bp) per population doubling [4, 8]. Hastie et al. [9] studied human lymphocytes in vivo and found that the rate of telomere loss is approximately 33 bp per year.
Telomerase is a ribonucleoprotein that is normally active in germ cells but not in somatic cells [10]. This enzyme helps to restore telomere length by synthesizing telomeric repeat DNA (5'-TTAGGG-3') from its own RNA template [11]. The lack of telomerase activity in somatic cells and the finite length of telomeric DNA limit the number of divisions a cell can undergo before critical telomere shortening signals entry into replicative senescence [1]. Telomerase is developmentally regulated during embryo development. Recent studies in rat germ cells showed that the telomerase activity in oocytes from early antral and preovulatory follicles was high (comparable to that of transformed 293 cells), whereas its activity in ovulated oocytes was significantly lower [12]. Telomerase activity was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and totally absent in epididymal spermatozoa [13, 14]. Researchers also showed that after fertilization, telomerase activity was present in four-cell-stage embryos [12]. Other reports showed that telomerase activity was relatively high in blastocyst-stage embryos of different species, including humans [13] and cattle [15]. The low telomerase activity found in ovulated oocytes and the absence of any telomerase activity in spermatozoa raises an interesting question: how and when is telomerase activity acquired by early embryos?
In a previous study [16], we examined telomerase activity in bovine in vitro fertilized (IVF) embryos. Telomerase activity was found in all stages of embryos, and this activity gradually increased and reached its highest level by the blastocyst stage. Our results were consistent with those of Betts and King [17], who found that telomerase activity was present during early bovine development from the zygote to the blastocyst stage and that this activity was up-regulated at the morula and blastocyst stages. However, to our knowledge, the dynamics of telomerase activity have not been systematically investigated in parthenogenetically activated (PA) or nuclear transferred (NT) embryos. Parthenogenetic development of an unfertilized oocyte to the blastocyst stage has long been a useful model for studying embryogenic competence without the confounding effects of sperm and fertilization [18]. The PA and IVF systems serve as good developmental controls for the NT studies.
The aim of this study was to investigate the dynamics of bovine telomerase during preimplantation stages in both NT and PA embryos and to directly compare the activity patterns among the IVF, NT, and PA systems.
MATERIALS AND METHODS
Collection and Maturation of Oocytes
Bovine oocytes used for this research were purchased from Trans Ova Genetics (Sioux Center, IA). Immature oocytes were aspirated from antral follicles (diameter, 2–5 mm) of slaughterhouse ovaries and matured in M199 medium containing serum and hormones using Trans Ova's standard procedure. These oocytes were placed in sterile cryovials containing pregassed culture medium and shipped to us in a portable incubator (Minitube, Tiefenbach, Germany) at 38.5°C by overnight express. On arrival, oocytes were cultured until 22 h of maturation and then subjected to either PA or NT as described previously [19].
PA of Oocytes
The PA procedure has been described previously [20]. Briefly, after 22 h of maturation, oocytes were stripped of their cumulus. Denuded oocytes with a polar body were selected and subjected to activation. Oocytes were exposed to A23187 (5 µM for 5 min; Sigma, St. Louis, MO), followed by incubation with 6-dimethylaminopurine (6-DMAP) (2.5 mM for 3.5 h; Sigma) in potassium simplex optimized medium (KSOM) containing 0.1% BSA under mineral oil at 39°C in a 5% CO2 atmosphere in humidified air. Following the activation treatment, oocytes were washed in KSOM, then cultured as described above in KSOM plus 0.1% BSA (w/v) for the first 4 days. After that, the medium was switched to KSOM containing 1% BSA for the remaining 3 days required to reach the blastocyst stage. The medium was changed every 2 days during the course of in vitro culture.
Somatic NT
The somatic cell NT (i.e., cloning) procedure has been described previously [21]. Briefly, adult bovine ear-skin fibroblast cells were cultured for several passages and then subjected to serum starvation for 5 days after reaching confluency. Immediately before NT, donor cells were trypsinized, washed by centrifugation (800 x g for 6 min), and resuspended in PBS supplemented with 0.5% fetal bovine serum. Recipient oocyte collection, maturation, and enucleation were conducted at approximately 22 h after maturation. Successful enucleation was confirmed by Hoechst 33342 (B-2261; Sigma) staining. Cells with an approximate diameter of 10–15 µm were selected for NT and inserted into the perivitelline space of the recipient cytoplast using our standard procedure. After transfer, the cell-cytoplast complexes were induced to fuse with two pulses of direct current at 2.5 kV/cm for 10 µsec each by using an Electrocell Manipulator 200 (BTX, San Diego, CA). These electrical pulses also simultaneously induced initial oocyte activation. Fusion was then confirmed by microscopic examination. All fused embryos were further activated by culturing with cycloheximide (10 µg/ml; Sigma) in CR1aa medium for an additional 5 h. Following NT, embryos were washed in KSOM containing 0.1% BSA and then cultured in the same medium under mineral oil at 39°C in the conditions described above.
Embryo Collection and Extraction
Embryos were collected at different stages during in vitro culture (Table 1). At each time point, embryos were collected, washed twice in Ca2+- and Mg2+-free PBS, and then incubated in 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer [22] for 1 h at 4°C at a ratio of one embryo per 5 µl of CHAPS. After incubation, the suspension was briefly centrifuged (12 000 x g for 30 sec), and the supernatant was frozen at -80°C for future analysis. Matured oocytes were denuded of cumulus cells before extraction by CHAPS as described above.
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Telomerase Activity Assay
Telomerase activity of embryo extracts was analyzed using TRAPeze kit (Intergen Company, Pruchase, NY). The Telomeric Repeats Amplification Protocol (TRAP) was adapted from that described by Kim et al. [22]. Briefly, 2 µl of lysate from different-stage embryos (0.4 embryo equivalent) was added to a polymerase chain reaction (PCR) tube containing 23 µl of reaction buffer (20 mM Tris-HCl [pH 8.3], 63 mM KCl, 1 mM EGTA, 1.5 mM MgCl2, 0.005% Tween 20, 0.1 mg/ml of BSA, 0.5 U Taq DNA polymerase, and 0.1 µg of 32P-labeled TS primer [5'- AATCCG TCGAGC AGAGTT-3']). The tubes were incubated at room temperature for 60 min, then 2 µl of 100 ng/µl of CX downstream primer (5'-[CCCTTA]3 CCCTAA-3') were added to the reaction. All reaction tubes were heated at 94°C for 2.5 min to end the telomeric elongation reaction. Samples were then subjected to 31 PCR cycles of 94°C for 30 sec, 50°C for 30 sec, and 70°C for 90 sec. The PCR products were analyzed on 12% nondenaturing acrylamide gels that were electrophoresed at 500 V for 80 min. Dried gels were exposed to x-ray film (Fujifilm, Tokyo, Japan) at -20°C, and the films were scanned and analyzed by Quantity One 4.01 (Bio-Rad, Hercules, CA). The collective intensity of ladder-like telomeric bands was used to quantitate the telomerase activity of the corresponding embryo extract. Telomerase-positive human 293 cell extract and distilled water were used as positive and negative controls in this study, respectively. Because telomerase is an RNase-sensitive enzyme, RNase-treated 293 cell extract was also used as a negative control. A quantitation control template R8 was used as well.
Statistical Analysis
The relative telomerase activity of different-stage embryos was subjected to a one-way analysis of variance using the General Linear Model (GLM) of the SAS program (SAS Institute, Cary, NC). A significance level of 0.05 was used unless otherwise stated. The relationship of relative telomerase activity and the dilution factor of positive cell extracts was subjected to regression analysis using the GLM of the SAS program, as was the relationship of relative telomerase activity and embryo developmental stage.
RESULTS
Before the telomerase assay, a dose-response curve using a telomerase-positive, 293-cell extract (45227K; Pharmingen, San Diego, CA) was established by using dilution factors of 1, 10, 100, and 1000 [16]. A linear relationship was found between the relative telomerase activity and the dilution factor (r = 0.952, P < 0.05). These results demonstrate that the TRAP procedure is a reasonable method for quantification of telomerase activity.
Embryo samples were taken to determine embryonic cell counts in early embryos at different stages. A summary of these data are presented in Table 1. Average cell counts per embryo were 43.5 ± 1.5 (n = 58) for morula (Day 5) and 93.8 ± 5.6 (n = 48) for blastocyst (Day 7) in PA embryos and 35.1 ± 1.1 (n = 51) for morula (Day 5) and 81.0 ± 3.7 (n = 46) for blastocyst (Day 7) in NT embryos. Previously, cell counts and protein contents were determined in our IVF system [16], and these were used as reference values for the present study.
For telomerase-activity assays, matured oocytes and embryos at various stages (from zygote to blastocyst; Table 1) were used. The experiments were replicated three times for both PA and NT groups (n = 104 and 98, respectively). For each replication, each sample was assayed at least twice, and an average value was taken. Relative telomerase activities in PA and NT embryos at different stages are shown in Figures 1 and 2, respectively.
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In the PA system (Fig. 1), telomerase activity was not significantly different among stages from oocyte to morula. However, we saw a trend involving an increase from the oocyte to zygote stage, a decrease at the eight-cell stage, and then an increase again at the morula stage. A significant increase was found at the blastocyst stage (P < 0.05).
In the NT system (Fig. 2), telomerase activity decreased from the oocyte to the cleaved stage (r = 0.966, P < 0.05). From the cleaved stage onward, the activity increased gradually to the blastocyst stage (r = 0.978, P < 0.05), during which the highest telomerase activity was detected (P < 0.05).
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A direct comparison of telomerase activity among zygote- and blastocyst-stage embryos of the IVF, PA, and NT systems, along with matured oocytes, is shown in Figures 3 (representative gel) and 4 (quantitative data). Higher telomerase activity was found in the blastocysts from all three systems (P < 0.05); however, telomerase activity in blastocysts from the PA system appeared to be higher than that in those from the IVF (P < 0.05) or NT systems (P < 0.05).
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DISCUSSION
The discovery that a whole animal can be cloned from a differentiated somatic cell of an adult individual [23] demonstrates that genes inactivated during tissue differentiation can be completely reactivated via cloning. Using differential display methodology, De Sousa et al. [24] compared blastocyst stage-specific mRNA expression of bovine embryos produced by IVF and NT of fetal fibroblasts versus in vivo embryo controls. They demonstrated that after NT, gene expression in fetal fibroblast cells is reprogrammed to mimic that of preimplantation embryo development. This suggests that the NT process induced the dramatic changes in gene transcription.
The complicated question of telomere reprogramming in the cloned animal was raised by Shiels et al. [25], who studied the telomere length of Dolly, the cloned sheep [23], and found that Dolly inherited the shortened telomeres of her mother, the nuclear donor. This study suggested that the length of telomeres in the donor nucleus may not have been completely reprogrammed by cloning, as opposed to what is supposed to happen with fertilization. However, Lanza et al. [26] recently demonstrated that the telomere length in their six cloned calves derived from populations of senescent donor somatic cells was extended beyond that of newborn and age-matched control animals. Our group [27] also found that the telomere length of 10 cloned calves derived from cultured fibroblast cells is indistinguishable from that of their age-matched controls. The discrepancies found in these various groups may come from species differences and/or different NT techniques or donor cell types [26]. Our data from this study on comparing telomerase activity in early embryo development after various procedures suggest that telomere lengths of cloned animals could be restored via the increased telomerase activity in cloned embryos, similar to what is supposed to occur after fertilization.
Current hypotheses of cellular aging and immortalization postulate that telomerase is active in germ line cells to ensure transmission of full-length chromosomes to progeny [11]. Although telomerase activity clearly is present and functions to maintain telomere length in germ cell lines [9, 22], little work has been done to establish how full-length chromosome transmission is accomplished during the embryo development process, especially in NT and PA embryos.
Our present work provides, to our knowledge for the first time, data on stage-specific telomerase activity during early embryo development following PA and NT. For both types of embryos, we demonstrated that telomerase activity was highest at the blastocyst stage (Figs. 1 and 2). Additionally, we found that in the NT system, up-regulation of telomerase activity began as early as the morula stage (Fig. 2), which is similar to our findings for the IVF system, whereas in the PA system, such up-regulation was not observed until the blastocyst stage (Fig. 1).
Telomerase activity was expected in embryos and has been reported previously [12, 13, 17]. The telomere hypothesis suggests that telomerase is active in embryo cells, and that this activity decreases during differentiation in a tissue- and cell-specific manner [12]. Our previous findings [16] demonstrated that telomerase activity is present at all stages of the bovine IVF embryos that were assayed, which is in agreement with the report of Betts and King [17] on bovine embryos. They found that telomerase activity was present during early development from the zygote to blastocyst stage, and that its activity was up-regulated in the morula and blastocyst stages. Recently, Lanza et al. [26] and Tian et al. [27] independently reported high telomerase activity in bovine blastocyst-stage embryos derived from NT. Our results from this experiment extend the previous findings, in that the telomerase activity increases at the blastocyst stage in cloned embryos in parallel with activity in PA and IVF embryos. The results of our study are also consistent with observations in other species. In rats, telomerase activity has been detected in four-cell-stage embryos [12], and in humans, telomerase activity has been detected in blastocysts [13].
A decrease in telomerase activity was found in cleavage-stage embryos from both PA and NT systems as well as in IVF embryos. This coincides with the maternal-zygotic transition model established by Barnes and First [28], who suggested that embryonic transcription of in vitro-cultured bovine embryos was initiated sometime between the four- to six-cell-stage of development. Similarly, other studies [29] using bovine embryos collected surgically have also indicated that transcription begins only when the embryo reaches the eight-cell stage. It is reasonable to speculate that telomerase activity follows a pattern similar to that of the maternal-zygotic transition during early developmental stages in the PA, NT, and IVF systems. The maternal telomerase activity gradually decreases until approximately the eight-cell-stage, during which embryonic transcription starts. Once embryonic transcription initiates, new telomerase proteins are synthesized, and these could account for the increased activity detected during later stages. Furthermore, De Sousa et al. [24] also reported that reprogramming occurred after NT by comparing the gene expression pattern of normal, in vivo-derived embryos with that of NT embryos and nuclear donor cells in bovines using a differential display PCR technique. They reported that donor cell mRNA expression is significantly modified to become embryo specific in nuclear reconstructed blastocysts; in other words, the donor genes were successfully reprogrammed after NT. Their finding supports our observations that telomerase activity in cloned embryos followed a similar pattern to that in the PA and IVF embryos, suggesting successful reprogramming of telomerase after NT.
To determine whether the changes in enzymatic activity resulted from protein content increases, a protein concentration assay was carried out during our previous study [16] on IVF-derived embryos. A relatively constant protein concentration (0.257 ± 0.025 µg/µl) was found in the extracts among all stages that were assayed. This was not surprising, because total cytoplasmic volume does not change significantly from the oocyte to the blastocyst stage in both IVF and PA embryos. However, we do suspect a cytoplasmic decrease in the NT system, because a portion of the cytoplasm was removed during the enucleation process. Nevertheless, the volume of cloned embryos at various stages should be similar, because the same enucleation procedure was applied. As we noticed, the telomerase activity of cloned blastocyst was, indeed, lower than that in PA and IVF blastocysts (Figs. 3 and 4). This may come from a loss of cytoplasm in the enucleation step or, possibly, from the variance between embryos. Krussel et al. [30] showed in humans that even with the single blastomeres of preimplantation embryos, great differences in expression levels for various genes could be found. We also noticed that a significantly higher telomerase activity was found in the PA blastocysts than in those derived from either IVF or NT (Fig. 4). The reasons for this observation are not known, but the nonmaternal genes from sperm or somatic cells may have actually suppressed the telomerase gene expression and, hence, reduced the telomerase activity. In a study by Chian et al. [31], bovine oocyte activation by sperm and PA induced different cytoplasmic responses for protein synthesis.
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A retrospective cell counting on early embryos derived from PA and NT was conducted in this study. Interestingly, if we plot telomerase activity on a per-cell instead of a per-embryo basis (figure not shown), we find that the activity actually decreased gradually from the zygote to the blastocyst stage in PA (r = 0.69, P < 0.05) and NT (r = 0.69, P < 0.05) embryos, which is consistent with the result from our previous IVF study [16]. The biological significance of these patterns is not clear. More studies are needed to examine the underlying mechanisms.
We draw the following conclusions from our results: Telomerase activity in bovine embryos derived from PA and NT follows a transition pattern similar to that in embryos derived from IVF. This suggests successful reprogramming of telomerase in embryos after activation or cloning treatments. The activity is present throughout the early developmental stages, and the highest level is detected at the blastocyst stage in PA, NT, and IVF embryos. The level of telomerase activity per cell, however, decreases during early embryonic development in the PA and NT systems. Further investigations are required to examine how and when telomere restoration occurs during embryo development after NT.
ACKNOWLEDGMENTS
Ovine FSH and LH used throughout our research were kindly provided by the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease, the National Institute of Child Health and Human Development, and the U.S. Department of Agriculture. The authors wish to thank S. Jiang and S. Jones for sample collections, X. Tian for discussion on developing the telomerase assays, and M. Julian for critical reading of this manuscript.
FOOTNOTES
First decision: 5 September 2000.
1 Supported in part by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under agreement 96-35203-3268 and by a grant from Connecticut Innovations, Inc. This paper is a scientific contribution (number 1975) of the Storrs Agricultural Experiment Station of the University of Connecticut. 
2 Correspondence: X. Jerry Yang, Department of Animal Science, University of Connecticut, 1390 Storrs Rd. U-4163, Storrs, CT 06269-4163. FAX: 860 486 0534; jyang{at}canr.uconn.edu
Accepted: October 10, 2000.
Received: August 10, 2000.
REFERENCES
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