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Biology of Reproduction 63, 1124-1128 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Telomerase Activity in Bovine Embryos During Early Development1

Jie Xua, and Xiangzhong Yang2,a

a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will 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. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.

developmental biology, IVF/ART, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ends of eukaryotic chromosomes are capped with copies of a hexamer repeat sequence (TTAGGG) and associated proteins [1]. These structures are known as telomeres, and they stabilize the ends of chromosomes during replication [2]. Conventional DNA polymerases replicate DNA only in the 5' to 3' direction and cannot initiate synthesis of a DNA chain de novo [3]. The DNA polymerases that replicate eukaryotic chromosomes use an 8- to 12-base stretch of RNA to prime DNA synthesis. As a consequence, after DNA replication, one end of a linear chromosome will be replicated to the very end, whereas the other end will have a short, 8- to 12-base gap generated by removal of the RNA primer. Because a conventional DNA polymerase cannot fill in this 5' gap, a given DNA end will be incompletely replicated in every other cell division. In yeast, the end of a linear chromosome will shorten by an average of four to six base pairs (bp) per cell division unless telomeres act as substrates for an alternative replication mechanism [1]. In human fibroblast cell culture, telomere length decreases at a rate of 48 ± 21 bp per population doubling [4, 5]. In vivo, Hastie et al. [6] studied human lymphocytes and found that the rate of telomere loss is approximately 33 bp/yr. Many scientists propose the telomere as a "mitotic clock" [4, 7, 8]. Its shortening correlates with the number of cell divisions, and when the telomere length reaches a certain limit, it will signal the cell to undergo replicative senescence, under which the cells stop cell division, become resistant to apoptosis, and alter their normal functions [9].

Telomerase is a ribonucleoprotein [10]. Its RNA subunit acts as a template for the synthesis of telomeric DNA, whereas a protein component catalyzes this process to make up for the inability of polymerase to replicate linear DNA through conventional replication [11]. Mammalian telomerase is developmentally regulated. In humans, telomerase activity is only found in germ cells, immortalized cell lines, and most tumor cells; it is not found in cells of most somatic tissues [12]. Lack of telomerase activity in somatic cells, and the finite length of telomeric DNA limits the number of divisions a cell can undergo before critical telomere shortening signals entry into replicative senescence [1]. Telomerase is active in germ line cells to ensure transmission of full-length chromosomes to progeny [11]. Most tumor and immortal cells have telomerase turned on as a mechanism to counteract telomere loss, thus enabling them to divide infinitely [12, 13].

The challenging question of telomere reprogramming in the cloned animal was raised by Shiels et al. [14], who studied the telomere length of Dolly, the cloned sheep [15], and found that Dolly inherited the shortened telomeres of her mother, the nuclear donor. This study suggested the telomere length of the donor nucleus may not have been completely reprogrammed by cloning, as opposed to what is supposed to happen with fertilization. Therefore, investigating telomere and telomerase reprogramming during early embryo development is of great importance to answer questions about whether reprogramming takes place and, furthermore, how much telomere length can be affected by this process. Due to technical difficulties in determining the telomere length on embryos because of limited cell numbers, we started our study of telomerase reprogramming in the bovine in vitro fertilization (IVF) system. This system could not only serve as a control for further studies on reprogramming in the nuclear transfer system but may also shed light on a more basic concern: how telomere and telomerase are reprogrammed during the fertilization process.

Recent studies in rat germ cells showed that the telomerase activity in early antral and preovulatory follicles was high (comparable to that of transformed 293 cells), whereas its activity in ovulated oocytes was 50-fold lower [16]. Telomerase activity was present at even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis [17]. Researchers also showed that after fertilization, telomerase activity was present in four-cell embryos [16]. Other reports showed that telomerase activity was relatively high in blastocyst-stage embryos of different species, including humans [17] and cattle [18]. The low telomerase activity in ovulated oocytes and the absence of any telomerase activity in spermatozoa raises an interesting question: how and when is telomerase activity acquired in early embryos? Using our IVF bovine embryo model, we demonstrated that telomerase activity is acquired at fertilization and reaches its highest level at the blastocyst stage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection and Maturation of Oocytes

Bovine oocytes used for this research were purchased from BOMED (Madison, WI). Immature oocytes were aspirated from antral follicles (diameter, 2–5 mm) of slaughterhouse ovaries and matured in M199 medium containing serum and hormones using the BOMED standard procedure [19, 20]. These oocytes were placed in sterile cryovials containing pregassed culture medium and shipped to us in a portable incubator at 38.5°C by overnight express. On arrival, oocytes were cultured until 22 h of maturation and were then subjected to IVF.

IVF of Bovine Oocytes

The IVF procedure has been described in detail elsewhere [21]. Briefly, frozen semen was thawed in a 37°C water bath. The sperm were washed twice by centrifugation (500 x g, 15 min) with washing medium, which was based on Brackett defined medium [21, 22], and then capacitated with heparin and coincubated for 6 h with cumulus-enclosed oocytes in Brackett fertilization medium [21]. The cumulus cells were then removed by pipetting with fire-polished glass pipettes of different diameters. The oocytes were washed and cultured in 100-µl droplets of KSOM containing 0.1% BSA under mineral oil at 39°C in an atmosphere of 5% CO2 in humidified air for the first 4 days. After 4 days of incubation, the medium was changed to KSOM containing 1% BSA for the remaining 3 days required to reach the blastocyst stage. Medium was changed every 2 days during the course of in vitro culture. The average blastocyst rate using this system in our lab is approximately 50% (n = 272, BL = 148). Retrospective samples of embryos at the morula and blastocyst stages were taken to determine cell count following epifluorescent staining by Hoechst 33342 (B-2261; Sigma, St. Louis, MO).

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 PBS, then incubated in 0.5% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) buffer [12] for 1 h at 4°C at a ratio of one embryo per 5 µl of CHAPS. After incubation, the buffer was briefly centrifuged (12000 x g, 30 sec), and the supernatant was frozen at -80°C for future analysis. Matured oocytes were denuded before being extracted by CHAPS in the same way as described earlier to treat embryos. Protein concentration assay was carried out using the Bicinchoninic Acid Protein Assay Kit (B-9643; Sigma, St. Louis, MO). A relatively constant protein concentration (0.257 ± 0.025 µg/µl) was found in the extracts among all stages that were assayed. Two-microliter lysis extracts (~0.50 µg, 0.4 embryo equivalent) were chosen for further Telomeric Repeat Amplification Protocol (TRAP) assay, as recommended by Kim et al [12].


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  		TABLE 1. Timing of oocyte and embryo collection

Telomerase Activity Assay

Telomerase activity of embryo extracts was analyzed using TRAP, as adapted from the method described by Kim et al. [12]. 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 BSA, 0.5 U Taq DNA polymerase, and 0.1 µg 32P-labeled substrate for telomerase and upstream primer for PCR [TS] primer [5'-AATCCG TCGAGC AGAGTT-3']). The tubes were incubated at room temperature for 60 min. Then, 2 µl of 100 ng/µl downstream primer for PCR (CX) downstream primer (5'-[CCCTTA]3 CCCTAA-3') was added to the reaction tubes, and 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 1.5 min. The PCR products were analyzed on 12% nondenaturing acrylamide gels, which were electrophoresed at 500 V for 80 min. Dried gels were exposed to x-ray films at -20°C. 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 represent the telomerase activity of the corresponding embryo extract. Relative activity was calculated by the formula

where I is XXX (the intensity of telomeric bands).

Statistical Analysis

The relative telomerase activity of different-stage embryos was subjected to a one-way ANOVA using the General Linear Model (GLM) of the Statistical Analysis System (SAS; Statistical Analysis System Institute, Inc., Cary, NC). A significance level of 0.05 was used unless otherwise stated. The relationship of relative telomerase activity and dilution factor of positive cell extract was subjected to regression analysis using the GLM of the SAS. The relationship of relative telomerase activity and embryo developmental stage was also subjected to regression analysis using the GLM of the SAS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Before the telomerase assay on embryos, a dose-response curve using telomerase-positive, 293-cell extract (45227K; Pharmingen, San Diego, CA) was established by using dilution factors of 1, 10, 100, and 1000. A representing gel of telomerase activities at different dilutions is shown in Figure 1. Relative quantities of telomerase activities are graphed in Figure 2. The figures clearly show a dose response resulting from progressive dilutions. A linear relationship was found between the relative telomerase activity and dilution factor (r = 0.952, P < 0.05). These data demonstrate that the TRAP procedure is a reasonable method for quantification of telomerase activity.



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  		FIG. 1. TRAP gel picture of positive 293-cell extract (45227k). Telomerase-positive 293-cell extract was progressively diluted by factor of 1 (no dilution), 10, 100, and 1 000. The TRAP assay was then carried out to measure the activity. The PCR products were analyzed on 12% nondenaturing, 1.0-mm-thick acrylamide gels, which were electrophoresed at 500 V for 80 min. The gels were dried under a vacuum at 70°C for 2 h and then at room temperature for an additional 30 min. Dried gels were exposed to x-ray films at -20°C. Lane 1: 293-cell extract; lane 2: 10x diluted 293-cell extract; lane 3: 100x diluted 293-cell extract; lane 4: 1000x diluted 293-cell extract



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  		FIG. 2. Histogram for the relative telomerase activity of different dilutions of positive 293-cell extract. Gel film (Fig. 1) was scanned and analyzed by Quantity One 4.01. The cumulative intensity of ladder-like telomeric bands was used to represent the telomerase activity of the corresponding embryo extract. Relative activity was calculated by the formula

where I is XXX. A linear relationship is found between relative telomerase activity and dilution factor of the cell extract (r = 0.952)

Mature oocytes and embryos at various stages (from zygote to blastocyst, Table 1) were assayed for telomerase activity. The experiment was replicated four times, with a total of 314 oocytes and embryos. For each replication, each sample was assayed at least twice, and an average value was taken. Relative telomerase activities in embryos at different stages are shown in Figure 3. Low telomerase activity was found in the mature oocytes with a significant increase of activity (P < 0.05) found after IVF. Regressional analysis indicates that between the zygote and eight-cell stage, a gradual decline of telomerase activity takes place (Fig. 3, r = 0.959, P < 0.05). From the eight-cell to blastocyst stage, telomerase activity gradually increases (Fig. 3, r = 0.997, P < 0.05) and reaches a significantly higher level by the blastocyst stage (P < 0.05).



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  		FIG. 3. Relative telomerase activity (RTA) in early bovine IVF embryos at different stages expressed on a per-embryo or per-cell basis. The formula of calculating relative activity is

where I is XXX, or

A) Per-embryo basis. Low telomerase activity was found in the mature oocytes. A significant increase of telomerase activity was found after IVF (P < 0.05). Between the zygote to eight-cell stage, a gradual decline of telomerase activity was observed (r = 0.959). From the eight-cell to blastocyst stages, telomerase activity gradually increased (r = 0.997) and reached a significantly higher level at the blastocyst stage (P < 0.05). Different letters represent statistically significant differences (P < 0.05). B) Per-cell basis. A progressive decrease of telomerase activity from the zygote to blastocyst stages was found (r = 0.753, P < 0.05)

To determine whether the enzymatic activity changes were due to cell number or protein content increases, retrospective embryo samples were taken to determine embryonic cell counts and total protein contents in early embryos at different stages. Average cell counts per embryo were 28 ± 0.9 (n = 80) for morula-stage (Day 5 IVF) and 108 ± 3.6 (n = 68) for blastocyst-stage (Day 7) embryos (not shown in Table 1). Total protein content was 0.25, 0.27, 0.30, 0.26, and 0.20 µg/µl for oocyte, zygote, cleaved, morula-stage, and blastocyst-stage embryos, respectively (Table 2). No difference (P > 0.05) was found in total protein content during early embryo development among the embryos stages examined, suggesting that the higher telomerase activity in blastocysts was due to enzymatic activity but not an increase in total protein. However, when the telomerase activity was expressed on a per-cell basis, a progressive decrease in activity was found (Fig. 3, r = 0.753, P < 0.05).


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  		TABLE 2. Protein content in different stage embryos


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results confirm the previous observations in other species [16] that telomerase activity is relatively low in matured oocytes. Furthermore, we demonstrated for the first time, to our knowledge, that telomerase activity increases after IVF and then decreases gradually until the eight-cell stage. In addition, we found that from the eight-cell stage onward, telomerase activity increases progressively with advancing embryo stage and reaches its highest level at the blastocyst stage. We demonstrated that these telomerase changes were due to increased enzymatic activity, because a relatively constant protein concentration was found in early embryo extracts among the different stages examined. This was not surprising, because total cytoplasmic volume does not change significantly from the oocyte to blastocyst stages.

Current hypotheses of cellular aging and immortalization postulate that telomerase is active in germ line cells to ensure the transmission of full-length chromosomes to progeny [11]. Although telomerase activity clearly is present and functions to maintain telomere length in germ cell lines [6, 12], little work has been done to establish how transmission of full-length chromosomes is accomplished during the embryo development process. This question becomes more remarkable because before fertilization, both the sperm and the ovulated oocyte have little or undetectable telomerase activity [17, 23].

Telomerase activity was expected in embryos, as reported previously [16, 17, 24]. The telomere hypothesis suggests that telomerase is active in embryo cells, and that this activity decreases during differentiation in a tissue- and a cell-specific manner [16]. Our findings demonstrate that telomerase activity is present in all stages of bovine IVF embryos that were assayed, which is in agreement with a recent report on bovine embryos by Betts and King [24]. Those authors also found that telomerase activity was detected during early development from the zygote to blastocyst stages, and that its activity was up-regulated in morula- and blastocyst-stage embryos. The results obtained from the present study are also consistent with those observations established in other species. In rats, telomerase activity is detected in four-cell-stage embryos [16], and in humans, telomerase activity is detected in blastocyst-stage embryos [17].

Telomerase activity could not be detected in individual human oocytes [17]; however, the developmental stage of the assayed oocytes was not specified in this study. Lower levels of activity in mature (ovulated) than in preovulatory or early antral follicular oocytes were reported in rats [16]. For the male germ cells, low-level telomerase activity was detectable in spermatocytes and spermatids, but telomerase activity could not be detected in spermatozoa from either caput or cauda epididymis [23, 25]. Lack of telomerase activity was also found in ejaculated human spermatozoa [17]. The present study showed that telomerase activity of oocytes significantly increased after fertilization. We propose that the fertilization process, particularly the fertilization-induced calcium oscillations and their downstream effects, are the likely reason for this significant increase of telomerase activity from the mature oocyte to the zygote. In mammalian cells, intracellular calcium oscillations affect gene activation through both amplitude and frequency modulation [26]. Ovulated oocytes have the potential to decode the calcium signal and to resume meiosis and, thus, further development [27, 28]. Moreover, intracellular calcium [29] and mitogen-activated protein (MAP) kinase [30] play regulatory roles in the telomerase activity of human epidermal stem cells and human solid tumor cells, respectively. More work needs to be done to elucidate these regulatory mechanisms in early embryo development. Candidate regulators likely include calcium, metaphase-promoting factors, MAP kinase, small G proteins, as well as other factors.

In our study, we also found that telomerase activity decreased gradually from the zygote stage to the eight-cell stage. After the eight-cell stage, the activity increased again, and it reached its highest level at the blastocyst stage. Interestingly, this changing pattern of telomerase activity coincides with the maternal-zygotic transition. Barnes and First [31] studied the embryonic transcription of in vitro-cultured (IVC) bovine embryos, and they suggested that embryonic transcription was initiated sometime between the four- to six-cell stage of development in IVF/IVC embryos. This was indicated by the presence of {alpha}-amanitin-sensitive protein synthesis at 36 to 48 h after insemination. Similarly, other studies using bovine embryos collected surgically also have indicated that transcription begins only when the embryo reaches the eight-cell stage of development [32]. It is reasonable to speculate that telomerase activity follows a similar pattern of maternal-zygotic transition during the early developmental stages of the IVF bovine embryos used in our study. We hypothesize that the oocytes have inactive forms of telomerase before fertilization. The fertilization process, possibly through calcium oscillations or other pathways, either activates these inactive telomerase proteins or initiates the translation of maternal stock mRNAs. The activity would then gradually decrease, because embryonic transcription does not occur until approximately the eight-cell stage but degradation of the maternal proteins, including telomerase, does occur. Once embryonic transcription starts, new telomerase proteins will be synthesized, and this could account for the increase of activity after the eight-cell stage. Similar to the results recently reported by Betts and King [24], we noticed that blastocysts have the highest level of telomerase activity. However, Betts and King [24] found that the telomerase activity of blastocyst-stage embryos is approximately 40-fold greater than that of eight-cell-stage embryos. Our findings indicate that the activity in blastocyst-stage embryos is only 1.5-fold greater than that of eight-cell-stage embryos, although significant differences are found between these two stages. This discrepancy possibly results from the different assay kits and/or quantification methods used in the two studies. A 36-bp internal control was employed by Betts and King; no such control was used in the present study.

Telomerase synthesizes the TTAGGG repeats using an RNA template that contains a partially redundant sequence complementary to TTAGGG. Telomerase extends the primer until reaching the end of the template sequence. The primer is then unwound from the template and either dissociated from the nonprocessive telomerase or held by the processive telomerase for repriming at the beginning of the template [33]. Telomerase processivity differs from species to species. Human telomerase extends a primer in a processive manner, producing the characteristic six-bp ladder pattern when analyzed by polyacrylamide gel electrophoresis [34]. Mouse telomerase produces one or two repeat additions under standard assay conditions, which is consistent with a mostly nonprocessive activity [35]. Telomerase processivity is also modulated by several other factors, including dNTP (mix of dATP, dTTP, dGTP, dCTP) concentration, especially dGTP, temperature, and concentration of K+ [34]. We noted a positive linear relationship between the activity and processivity of telomerase in our assay system; as shown in Figure 1, the processivity decreases with the increasing of the dilution factor. We thus attribute the low-processive telomeric band pattern from the bovine embryo extracts to the following three factors: 1) species difference, 2) nonoptimized reaction condition, and 3) low activity.

Together, we draw the following conclusions from our results: The telomerase activity is low in mature bovine oocytes. Telomerase activity is present throughout the early developmental stages in bovine embryos. Fertilization is the likely trigger for the increased telomerase activity at the zygote stage. In addition, a stage-dependent, gradual decrease of telomerase activity was found between the zygote to the eight-cell stages, whereas a gradual increase of telomerase activity was observed from the eight-cell stage onward, coinciding with maternal-zygotic transition in this species. The highest level of telomerase activity was detected at the blastocyst stage. The level of telomerase activity per cell, however, decreased during early embryonic development. The biological significance of these observations on telomere reprogramming is not clear. More studies are needed to examine the underlying mechanisms responsible for these observations.


   ACKNOWLEDGMENTS
 
Ovine FSH 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 for sample collections, X. Tian for discussion on developing the telomerase assays, and M. Julian for critical reading of this manuscript.


   FOOTNOTES
 
First decision: 21 February 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 (1933) of the Storrs Agricultural Experiment Station of the University of Connecticut. Back

2 Correspondence: Xiangzhong Yang, Department of Animal Science, University of Connecticut, 3636 Horsebarn Rd., Ext. 40, Storrs, CT 06269. FAX: 860 486 4375; jyang{at}canr.uconn.edu Back

Accepted: May 19, 2000.

Received: January 21, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. Schaetzlein, A. Lucas-Hahn, E. Lemme, W. A. Kues, M. Dorsch, M. P. Manns, H. Niemann, and K. L. Rudolph
Telomere length is reset during early mammalian embryogenesis
PNAS, May 25, 2004; 101(21): 8034 - 8038.
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 J. Exp. Med.Home page
R. C. Allsopp, S. Cheshier, and I. L. Weissman
Telomerase Activation and Rejuvenation of Telomere Length in Stimulated T Cells Derived from Serially Transplanted Hematopoietic Stem Cells
J. Exp. Med., December 2, 2002; 196(11): 1427 - 1433.
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 Mol Hum ReprodHome page
D. L. Wright, E. L. Jones, J. F. Mayer, S. Oehninger, W. E. Gibbons, and S. E. Lanzendorf
Characterization of telomerase activity in the human oocyte and preimplantation embryo
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 Biol. Reprod.Home page
J. Xu and X. Yang
Telomerase Activity in Early Bovine Embryos Derived from Parthenogenetic Activation and Nuclear Transfer
Biol Reprod, March 1, 2001; 64(3): 770 - 774.
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