Telomerase Activity in Female and Male Rat Germ Cells ... - CiteSeerX

BIOLOGY OF REPRODUCTION 56, 1120-1125 (1997)

Telomerase Activity in Female and Male Rat Germ Cells Undergoing Meiosis and in Early Embryos Karen M. Eisenhauer,3 Rachel M. Gerstein, 4 Choy-Pik Chiu,5 Marco Conti,3 and Aaron J.W. Hsueh 2 3, Department of Gynecology and Obstetrics3 and Department of Genetics, 4 Stanford University Medical Center, Stanford, California 94305 Geron Corporation,' Menlo Park, California 94025 ABSTRACT Telomerase isa ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity inthe following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in 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. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality. INTRODUCTION Telomeres are specialized structures at the ends of eukaryotic chromosomes and are composed of conserved sequences of DNA repeats [1]. Telomeric DNA is not completely synthesized by conventional DNA polymerase, and in the absence of the enzyme telomerase, a ribonucleoprotein, chromosomes lose 50-200 base pairs per mitotic division [2, 3]. Cellular aging is characterized by a decrease in telomere length, and this has been implicated as a mitotic clock that signals cells to stop division when telomeres reach a critically short length [4]. Conversely, maintenance of telomere length appears necessary for cellular immortality [5, 6]. Initially identified in ciliates, telomerase activity has also been detected in Xenopus [7], mouse [8], and human [9, Accepted December 9, 1996. Received July 23, 1996. 'Supported in part by grants CA42509 to Leonard A. Herzenberg and A134762 to Leonore A. Herzenberg. R.M.G. is a recipient of a Postdoctoral Fellowship from the Irvington Foundation for Immunological Research. 2Correspondence: Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, CA 94305-5317. FAX: (415) 725-7102; e-mail: aaron.hsueh @forsythe.stanford.edu

10] tissues. In humans, it was originally reported that telomerase is repressed in all normal somatic tissues but active in transformed cells, implicating a role for telomerase in tumorigenesis [10]. Recently, it has been demonstrated that low levels of telomerase activity are also present in a subset of hematopoietic cells [11-13]. Since telomeres in these cells continue to shorten, the biological significance of this finding has not been determined. The telomere hypothesis suggests that telomerase activity is high in embryonic cells and that it decreases in somatic tissues during development and differentiation. Telomerase is expected to remain active in germline cells to ensure the transmission of full-length chromosomes to progeny. Consistent with this hypothesis, telomerase activity has been detected in extracts of human ovaries and testes [10]. Little is known about telomerase activity in specific ovarian cell types, however. Telomere length has not been measured in oocytes because of difficulty in obtaining sufficient amounts of DNA for analysis. Telomerase activity has been detected in Xenopus oocytes throughout oogenesis [7], but amphibian oogenesis differs from that of mammals in that a subpopulation of oogonia undergo mitotic divisions each breeding season. In mammals, the process of folliculogenesis does not involve germ cell division since the entire pool of primordial follicles is formed before birth or soon after birth. During reproductive life, only meiotic progression is found in oocytes. Changes in telomerase activity in the mammalian oocyte during folliculogenesis have not been previously investigated. The presence of telomerase activity in testes was anticipated from the long telomeres in sperm and the longer telomere length in sperm of older men [14]. Similarly, mouse testes telomere lengths are 1-3 kilobase pairs longer than those in somatic tissues [8]. The observation that mouse testicular telomerase activity cannot be detected until 42 days of age suggests that telomerase activity is low until advanced stages of spermatogenesis [8]; but telomerase activity has not been analyzed in male germ cells during spermatogenesis, and the specific cell types expressing telomerase activity in the testes are unknown. The objective of the present study was to determine whether selected rat female and male germ cells have telomerase activity and to study potential changes in activity in these cells during their differentiation and after fertilization. We examined levels of telomerase activity in oocytes at two different stages of folliculogenesis and after ovulation, in male germ cells at different stages of spermatogenesis, and in early embryos. Since the distribution of telomerase may differ between species [8, 10] and very little is known about telomerase activity in the rat, we also performed a survey of telomerase activity in several rat somatic tissues.

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TELOMERASE IN RAT GERM CELLS MATERIALS AND METHODS Cell Collection Oocytes. Sprague-Dawley rats (Simenson Laboratories, Gilroy, CA) (n = 12) were implanted with diethylstilbestrol capsules at 24 days of age to induce the growth of a cohort of early antral follicles. Rats were killed at 27 days of age, and oocytes were removed from follicles by puncture of the follicle wall. Oocytes from preovulatory follicles were obtained by treating rats (n = 19) with 10 IU eCG at 25 days of age. Rats were killed 2 days later, and the largest (> 500 pim) follicles were dissected using watchmaker forceps. Oocytes collected from follicles were denuded by treatment with 0.1% collagenase-dispase (Boehringer-Mannheim, Indianapolis, IN ) in 0.2 mM EDTA for 20 min at 37°C, followed by pipetting through small-bore pipettes. For collection of ovulated oocytes, immature rats (n = 24) were treated with 10 IU eCG followed by a single injection of 1 ig hCG. Oocytes were collected from the oviduct 16-18 h later and were denuded with 1 mg/ml hyaluronidase for 15 min at 22°C. All oocytes were pipetted through at least three changes of media to remove contaminating somatic cells and then snap-frozen and stored at -70°C. Male germ cells. Testes were collected from adult male Sprague-Dawley rats (n = 6). Pachytene spermatocyte and round spermatids were isolated using velocity sedimentation [15]. As previously described, the pachytene spermatocyte preparations contain 85-90% pachytene spermatocytes, and the round spermatid preparation contains 86% round spermatids [16]. Spermatozoa were isolated from caput and cauda epididymis [17]. Early embryos. Adult female rats (n = 6) were mated, and embryos were collected from the oviducts 72 h after mating. Tissue Preparation Somatic tissues and ovaries were collected from female rats at 27 days of age (n = 2), since this corresponded to the approximate age at which oocytes were collected. Testes were collected from male rats at 27 days of age (n = 2). Tissues were snap-frozen and then homogenized in lysis buffer as previously described [10]. Protein concentrations of the lysates were determined using the Bradford method (Bradford assay; Bio-Rad, Hercules, CA), and aliquots of lysate equivalent to 5 ig total protein were used in the telomeric repeat amplification protocol assay.

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ilar to those developed for the TRAP-eze kit (Oncor, Gaithesburg, MD). Preparation of cell lysates. Cells were lysed as previously described [10]. For oocytes and embryos, a concentration of 5 oocytes/lil lysis buffer was used, whereas for all other cell types, 106 cells/50 l lysis buffer were used. In order to ensure lysis of spermatozoa, these cells were also subjected to three cycles of freeze/thaw and/or sonication. These additional steps did not affect telomerase activity in the 293 cells (data not shown). RNase pretreatment Aliquots of cell lysates were incubated with RNase A (200 ig/ml) for 10 min at 23°C as a negative control for the TRAP assay as previously described [10], since telomerase is a ribonucleoprotein that is inactivated by RNase. Formation of telomerase products. The assay was carried out in 25 il of reaction mixture with 0.1 g TS oligonucleotide (5'-AATCCGTCGAGCAGAGTT-3') in 20 mM Tris-HCl (pH 8.3), 1.5 mM MgC12, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, and 50 LiM of each dNTP (TRAP buffer) in a 0.5-ml HotStart 50 tube (Molecular Bioproducts, San Diego, CA). After 30 min of incubation at room temperature, tubes were heated at 94C for 3 min to stop the telomerase reaction. PCR amplification of telomerase products. A mixture of 0.1 g of labeled RP, 1 U Taq DNA polymerase, and 3.3 l TRAP buffer was added to each tube, and PCR was carried out for 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec. Analysis of TRAP products. PCR products were resolved on 10% denaturing polyacrylamide gels at 1700 V for 3.5 h and then exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA). Enzymatic activity was expressed in arbitrary units as total counts in the RNase-sensitive reaction products, determined using ImageQuant software (Molecular Dynamics). Dilutions of lysates containing the equivalent of 15-70 oocytes, 2.5-5 embryos, and 100025 000 pachytene spermatocytes, round spermatids, or spermatozoa were assayed in duplicate for the purposes of quantitation. At these concentrations, these cell lysates exhibited dose dependency, i.e., there was a clear relationship between cell number and the amount of TRAP product generated. The TRAP products generated by the lysates were compared to those of a 293 cell standard curve that was assayed and run in parallel in each gel. A minimum of two separate assays using two dilutions of each cell lysate were used to determine the mean percentage of activity of each sample. The relative telomerase activity in each extract is expressed as a percentage of the activity obtained with 293 cell extracts on a per cell basis.

Telomeric Repeat Amplification Protocol (TRAP) Assay Telomerase activity was determined using the TRAP assay [10] with the modifications described below. In the present study, the sensitivity of the TRAP assay was increased by lengthening the time for formation of telomerase products from 10 to 30 min and increasing the number of polymerase chain reaction (PCR) cycles from 27 to 30. In addition, a modified CX primer (RP) was used, which was designed to reduce dimerization with TS and result in an improved signal-to-noise ratio. This primer has been used for the TRAP assay in recent studies [18-20]. The RP primer was 5' end-labeled with [y-32 P]ATP and T4 polynucleotide kinase (Pharmacia, Piscataway, NJ). These modifications allowed detection of telomerase activity in one 293 cell. The reagents and primers used in this study were sim-

RESULTS Quantitative Analysis of Telomerase Activity Using the TRAP Assay To provide a standard to quantitate telomerase activity, varying amounts of cell lysates (corresponding to 1-100 cells) from an immortalized human embryonic kidney cell line (293) were assayed, and the total incorporation of labeled primer into PCR products was determined (Fig. 1A). The formation of TRAP products is proportional to cell number in the range of 1-100 cells (Fig. 1B). These quantities of the 293 cells (1-100 cells/TRAP reaction) were run in duplicate in each assay along with the germ cell and embryo lysates.

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FIG. 2. Telomerase activity in ovulated oocytes and pachytene spermatocytes. Aliquots of 293 cells, oocytes, and pachytene spermatocytes were lysed and incubated for 10 min at 21°C with or without RNase. Untreated (-) and RNase-treated (+) lysates were then assayed for telomerase activity as described in Materials and Methods. Lanes 1-2, 1000 293 cells; lanes 3-4, 10 ovulated oocytes (OC); lanes 5-6, 10 000 pachytene spermatocytes (PS). Refer to Figure 1 for an explanation of the assay.

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FIG. 1. Quantitation of TRAP products obtained with 293 cell lysates. Aliquots of cell lysates were incubated with the TS oligonucleotide in the presence of dNTPs, and elongated products were amplified by PCR, resolved on polyacrylamide gels, and visualized after exposure to Phosphorlmager screens. Telomerase elongation of the TS primer yields a characteristic sixnucleotide repeat ladder. A) Lane 1, negative control (NC), reaction carried out in the absence of cell lysate; lanes 2-5, reactions with extracts from 1, 10, 30, or 100 cells; lane 6, lysate from 100 cells pretreated with RNase A. B) Relationship between TRAP product signals and number of 293 cells in the assay.

Activity in Gonadal Cells and Early Embryos The specificity of the TRAP assay for telomerase activity in cell lysates is tested by pretreating lysates with RNase, which destroys the RNA component of telomerase and thus inactivates the enzyme. Telomerase activity in ovulated oocytes (Fig. 2; OC) and in oocytes from early antral and preovulatory follicles (data not shown) was RNase sensitive. The following activity levels are expressed on a per cell basis as the percentage relative to 293 cells assayed in parallel. The levels of telomerase activity in oocytes from early antral and preovulatory follicles were 83% and 159%, respectively (Fig. 3A), while the level in ovulated oocytes was 2% (Fig. 3A). Similarly, RNase-sensitive telomerase activity was found in pachytene spermatocytes (Fig. 2; PS) and round spermatids (data not shown). Quantitation revealed pachytene spermatocyte activity to be 0.31%, while activity of round spermatids was 0.55% (Fig. 3B; RS). In contrast, no telomerase activity was detected in sperm from either the caput or cauda epididymis. After fertilization, activity was present in early embryos, averaging 7% (Fig. 3A; EE). Telomerase Activity in Rat Somatic and Gonadal Tissues Some telomerase activity was present in all tissue surveyed at 27 days of age. High activity was present in the

TELOMERASE IN RAT GERM CELLS

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thymus, ovaries, and testes (Fig. 4); lower activity was detected in liver, brain, and spleen, and the lowest level was found in the kidney.

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FIG. 3. Telomerase activity in germ cells and early embryos. Cell lysates corresponding to 5-70 oocytes, 2.5-5 early embryos, and 1 x 103-2.5 X 104 pachytene spermatocytes, round spermatids, or spermatozoa were assayed. Activity is expressed on a per cell basis as the percentage relative to 293 cells assayed in parallel. Data are the mean - SEM of at least 6 determinations from 2 or 3 separate assays. A) Activity in oocytes and early embryos. EA, Oocytes from early antral follicles; PO, oocytes from preovulatory follicles; OV, ovulated oocytes; EE, early embryos. B)Activity in spermatogenic cells. PS, pachytene spermatocytes; RS, round spermatids; Caput, spermatozoa from caput epididymis; Cauda, spermatozoa from cauda epididymis. N.D., Not detectable. Refer to Figure 1 for an explanation of the assay.

This study demonstrates that telomerase activity is present in female and male rat germ cells but cannot be detected in spermatozoa. Our data indicate that telomerase activity was present in all three types of oocytes examined (preantral, preovulatory, and ovulated). Levels of telomerase activity were high in the oocytes from early antral and preovulatory follicles. These oocytes are fully grown and are arrested in prophase of meiosis I. After the LH surge and ovulation, oocytes proceed through meiosis I, resulting in the ovulation of a 2C oocyte. The reason lower levels of telomerase activity were observed in ovulated oocytes than in preovulatory oocytes is not known but could reflect the biochemical changes occurring during germinal vesicle breakdown. During this process, the rates of protein and RNA synthesis decline dramatically, with RNA synthesis decreasing to barely detectable levels after breakdown of the nuclear envelope and chromosome condensation [21]. Indeed, as much as one half of the polyadenylated RNA accumulated during oocyte growth is either degraded or deadenylated during meiotic maturation [21]. In a recent study, telomerase activity could not be detected in individual human oocytes [22], but the developmental stage of the oocytes assayed was not determined. We detected lower levels of activity in mature (ovulated) than in preovulatory or early antral oocytes; this activity might have been difficult to detect at a single-cell level. Further studies are necessary to determine whether there are species differences in the distribution of oocyte telomerase activity. Telomerase activity was also found in male germ cells, as was previously hypothesized [10]. Our results demonstrate that telomerase activity is present at two stages of spermatogenesis: pachytene spermatocytes and round spermatids. The former is one of the longest steps of spermatoFIG. 4. Telomerase activity in tissues from 27-day-old rats. Somatic tissues and ovary were collected from a 27-day-old female rat; testis was from a 27-day-old male rat. Tissues were lysed and then assayed as described in Materials and Methods. Aliquots of lysate equivalent to 5 ug protein were assayed either with (+) or without (-) RNase pretreatment. Tissues assayed were thymus (Th), liver (L), brain (B), kidney (K), spleen (S), ovary (0), and testis (T). Similar results were obtained in two separate experiments. Refer to Figure 1 for an explanation of the assay.

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genesis, when synapsis along homologous chromosomes is complete and recombination occurs. At the round spermatid stage, meiosis I and II have been completed, and the haploid spermatid undergoes the process of terminal differentiation. Since the cell preparations that were used contained a small percentage of intermediate and elongated spermatids, we cannot exclude the possibility that these cells may also have contributed to the telomerase activity in the cell preparations. In contrast to observations in spermatocytes and spermatids, telomerase activity could not be detected in spermatozoa from either caput or cauda epididymis. This is not surprising, since it is known that transcription and translation do not occur in spermatozoa and that DNA polymerase is also not active [23]. This is consistent with the lack of telomerase activity in ejaculated human spermatozoa [22]. In Xenopus spermatozoa, however, low levels of telomerase activity have been observed [7]. The finding of telomerase activity in both female and male meiotic germ cells suggests that in addition to its previously hypothesized functions in germ cells, telomerase may also be important during meiosis. Evidence from a variety of organisms suggests that telomeres associate with each other and with the nuclear membrane during early stages of meiosis, and that these interactions may be important for homologous chromosome pairing [24] and recombination [25]. Further studies are necessary to determine whether telomerase plays a critical role in maintaining full-length chromosomes during meiotic progression and gamete differentiation in germ cells. The present study demonstrates the presence of telomerase activity in 4-cell embryos. Telomerase activity was expected in embryos, since the telomere hypothesis suggests that telomerase is active in embryonic cells and that this activity decreases during differentiation in a tissue- and cell-specific manner. In humans, telomerase activity is present in blastocyst cells and in most somatic tissues at 1620 wk of development, but not in neonatal somatic tissues [22]. In mice, telomere lengths of various tissues are similar, and longest, in the neonatal period and become heterogeneous during development [8]. It has recently been demonstrated that induction of cellular differentiation results in a pronounced down-regulation of telomerase activity, providing a direct link between telomerase activity and terminal differentiation [26, 27]. We have detected telomerase activity in several somatic tissues (thymus, liver, brain, kidney, spleen) in 27-day-old rats. It has recently been reported that telomerase activity is also present in colon and liver tissue from adult rats [28]. These results are similar to those reported for the mouse, where activity has been detected in kidney, liver, and spleen [8, 29]. This is in contrast to the restricted telomerase activity found in human germline and hematopoietic cells [10-12]. The significance of these differences is unknown. It has been hypothesized that long-lived species may have evolved more stringent control mechanisms than short-lived species to limit cellular proliferation, since the regulation of proliferation is thought to play a role in protection against carcinogenesis [30]. Although the rat has some telomerase activity in many tissues, we found the highest levels in the gonads and thymus. While the role of telomerase in these tissues remains to be determined, the high level of activity in these tissues is correlated with extensive replicative potential. The high level of telomerase activity in the thymus (also observed in mouse thymus; R. Gerstein, unpublished results) may en-

sure that the clonal progeny of progenitor T cells have the capacity for numerous cell divisions. In conclusion, high levels of telomerase activity were observed in oocytes, and lower levels of activity were present in spermatogenic cells. These findings suggest that in addition to its well-known role during mitosis, telomerase may play an important role in lengthening telomeres during meiotic divisions. Since oocyte telomerase may be important for maintaining chromosomal integrity, the extremely sensitive TRAP assay may be useful for studying the effects of hormonal treatments, chemotherapeutic agents, and environmental toxins on oocyte quality. REFERENCES 1. Blackburn EH. Structure and function of telomeres. Nature 1991; 350: 569-573. 2. Greider CW, Blackburn EH. Identification of specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985; 43:405413. 3. Greider CW. Blackburn EH. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 1987; 51:887-898. 4. Harley CB, Vaziri H, Counter CM, Allsopp RC. The telomere hypothesis of cellular aging. Exp Gerontol 1992; 27:375-382. 5. Counter CM, Avillon AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992; 11:1921-1929. 6. Counter CM, Hirte HW, Bacchetti S, Harley CB. Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci USA 1994; 91: 2900-2904. 7. Mantell LL, Greider CW. Telomerase activity in germline and embryonic cells of Xenopus. EMBO J 1994; 13:3211-3217. 8. Prowse KR, Greider CW. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 1995; 92:4818-4822. 9. Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989; 59: 521-529. 10. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266:2011-2015. 11. Broccoli D, Young JW, DeLange T Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci USA 1995; 92: 9082-9086. 12. Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S. Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995; 85:2315-2320. 13. Chiu C-P, Dragowska W, Kim NW, Vaziri H, Yui J, Thomas TE, Harley CB, Lansdorp PM. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells 1996; 14:239-248. 14. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990; 346:866-868. 15. Bellve A, Cavicchia J, Millette C, O'Brien D, Bhatnagar Y, Dym M. Spermatogenic cells of the prepubertal mouse: isolation and morphological characterization. J Cell Biol 1977; 74:68-85. 16. Conti M, Adamo S, Geremia R, Monesi V. Developmental changes of cyclic adenosine monophosphate-dependent protein kinase activity during spermatogenesis in the mouse. Biol Reprod 1983; 28:860-869. 17. Horowitz JA, Toeg H, Orr GA. Characterization and localization of cAMP-dependent protein kinases in rat epididymal sperm. J Biol Chem 1984; 259:832-838. 18. Chiu C-P, Dragowska W, Kim NW, Vaziri H, Yui J, Thomasm TE, Harley CB, Lansdorp PM. Differential expression of activity in hematopoietic progenitors from adult human bone marrow. Stem Cells 1996; 14:239-248. 19. Morrison SJ, Prowse KR, Ho P, Weissman IL. Telomerase in hematopoietic cells is associated with self-renewal potential. Immunity 1996; 5:1-20. 20. Avillion AA, Piatyszek MA, Gupta J, Shay JW, Baccheti S, Greider

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