Telomere biology in mammalian germ cells and during development

Developmental Biology 274 (2004) 15 – 30 www.elsevier.com/locate/ydbio

Review

Telomere biology in mammalian germ cells and during development Sofie Bekaerta, Hanane Derradjia,b, Sarah Baatoutb,* a

Laboratory for Biochemistry and Molecular Cytology, Department for Molecular Biotechnology, FLTBW-Ghent University, Belgium b Laboratory of Radiobiology, Belgian Nuclear Research Centre, SCK!CEN, B-2400 Mol, Belgium Received for publication 8 July 2003, revised 18 June 2004, accepted 21 June 2004 Available online 5 August 2004

Abstract The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on. D 2004 Elsevier Inc. All rights reserved. Keywords: Telomere length; Telomerase; Oocyte; Spermatozoa; Embryo; Fetus; Human; Mouse; Aging disorders; Malformations

Introduction Telomere structure: specialized nucleoprotein complex Telomere length erosion has been postulated as a causal determinant for biological aging (reviewed in Campisi, 2001). Telomeres are specialized nucleoprotein complexes at the termini of linear chromosomes that, in vertebrates, are composed of hundreds of TTAGGG tandem repeats (reviewed in Blackburn, 1991) that together with specific * Corresponding author. Laboratory of Radiobiology, Belgian Nuclear Research Centre, SCK!CEN, Boeretang 200, B-2400 Mol, Belgium. Fax: +32 14 31 47 93. E-mail address: [email protected] (S. Baatout). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.06.023

proteins cap the chromosome ends and prevent them from being recognized as unnatural breaks (Fig. 1A). The current model of the telomeric complex within mitotic cells is that of a molecule of higher order, established by the invasion and protection of the single-stranded G-rich 3V overhang between the double-stranded telomeric strand (D-loop), forming a particular T-loop structure (Fig. 1B) (Griffith et al., 1999). This formation of a complex telomeric cap is shown to be mediated by a large number of telomerebinding proteins that are summarized in Table 1 (reviewed in Shay, 1999). The number of telomeric repeats within an organism is not fixed, but changes dynamically. A fraction of the terminal telomere sequences is lost during each round of DNA replication, the so-called bend replicationQ problem

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Fig. 1. The telomeric protective cap. (A) Extended focus (confocal) image of an embryonic mitotic murine cell (8-cell stage) after in situ hybridization with FITC-labeled telomere-specific PNA probes (green) and DNA counterstaining with propidium iodide (red). (B) Mammalian telomeres are suggested to form a protective cap by an interaction of the telomeric tract with a certain number of telomere-binding proteins like the double-stranded binding TRF1 and 2 and associating factors, and single-stranded binding proteins like POT1, resulting in a higher-order complex (t-loop) in which the single-stranded G-rich overhang is protected by invasion within the double-stranded telomere (displacement loop). Telomere-binding proteins are also shown to recruit DNA repair proteins like the RAD50-MRE11-NBS1-complex, DNA-dependent kinases, and Ku proteins (see also Table 1). Telomeres can become dysfunctional by critical shortening, direct DNA damage, or deficient binding of telomere-specific proteins. As a consequence of dysfunctional telomeres, the cell is signaled into replicative senescence via p53/pRB checkpoint pathways, which can give rise to aging phenotypes. In the absence of p53, telomere dysfunction may lead to programmed cell death. When critically short telomeres are not recognized by the cells’ checkpoint machinery, cells survive with concomitant genomic instability that might lead to tumorigenesis.

(Olovnikov, 1973). Conventional DNA polymerases replicate DNA only in the 5V to 3V direction and cannot initiate synthesis of a DNA chain de novo (Watson, 1972), a 8- to 12-base stretch of RNA provides the necessary 3V-OH end-to-prime DNA synthesis. During discontinuous lagging strand synthesis the most distal primer cannot be replaced. As a consequence, telomeres shorten with every round of cell division. Such reduction in telomere length plays an important role in replicative senescence, which induces an irreversible cell cycle arrest in G0/G1 activated via p53/p21/p19 and p16/Rb pathways (Fig. 1B). In human fibroblast cell culture, telomere length decreases at a rate of 50–100 bp per population doubling (Allsopp et al., 1992; Harley et al., 1990). Hastie et al. (1990) studied human lymphocytes in vivo and found that the rate of telomere loss is approximately 33 bp/year. The telomere attrition rate is not a constant function of cell division but is also negatively influenced by environmental factors like oxidative stress (von Zglinicki et al., 2000). Both reliable reference values

and determinants of telomere length within representative populations are still undefined. Telomere length is species, individual, tissue, and chromosome specific Telomeric sequences do not contain protein-encoding genes but are crucial for the preservation of genome integrity. Telomere length is species specific: in budding yeast the average length of telomeric tracts is stabilized at 300 bp (Walmsley and Petes, 1985), mice have ultralong telomeres (20–150 kb) that are highly polymorphic between inbreds (Kipling and Cooke, 1990; Starling et al., 1990) and in human cells, average telomere length measures between 5 and 15 kb (Cross et al., 1989). Studies performed on data sets from human monozygous twins have revealed that telomere length is, to a large extent, genetically determined (Graakjaer et al., 2003; Slagboom et al., 1994). Data on telomere length setting during gestational life in utero are scarce. Mean telomere length assessment in

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Table 1 Telomere-binding proteins and interacting factors Telomere-binding factor, human

Postulated function(s)

Telomere-binding site

hTRF1

Telomere length regulation, functional telomere structure

x

hTRF2

Telomere end-protection, protection single strand overhang, telomere length regulation

x

hKu70/86, DNA-PKc (DNA-PK) complex

DNA damage repair, regulation of telomere length and structure, telomere nuclear localization Telomere length regulation via ribosylation of TRF1

x TRF1, TRF2 interacting

Double strand

Tankyrase (TRF1-interacting, ankyrin-related ADP-ribose polymerase) TIN2 hRAP1 hnRNP A1 (UP1)

Mediates architectural role of TRF1 Telomere length regulation Telomere length regulation

hPot1

Telomere length regulation

hTERT

catalytic subunit of telomerase

Bibliography Single strand Bilaud et al. (1997), Chong et al. (1995), Iwano et al. (2004), Smith and de Lange (1997), van Steensel and de Lange, 1997, Zhong et al. (1992) Bilaud et al. (1997), Broccoli et al. (1997), Griffith et al. (1999), Smogorzewska et al. (2000), Song et al. (2000), van Steensel et al. (1998), Zhu et al. (2000b) Bianchi et al. (1999), Hsu et al. (2000)

TRF1 interacting

Sbodio et al. (2002), Smith and de Lange (1999, 2000), Smith et al. (1998)

TRF1 interacting TRF2 interacting

Kim et al. (1999, 2003) Li et al. (2000) Burd and Dreyfuss (1994), Ishikawa et al. (1993), LaBranche et al. (1998), Riva et al. (1986) Baumann and Cech, 2001, Loayza and de Lange (2003) Nakamura et al. (1997)

x

x

The list of known proteins that specifically bind the telomere is rapidly increasing. Two main TBP groups are distinguished based on their specificity for telomeric single or double strands.

intercrossed interfertile mice with distinct average telomere lengths revealed a bimodal distribution (Zhu et al., 1998). Subsequent backcrosses resulted in telomere length equilibration toward the longer telomere length, suggesting a dominant and trans-acting telomere elongation mechanism, mapped to chromosome 2 in mouse. In humans, an X-linked telomere length inheritance was recently proposed (Nawrot et al., 2004). At birth, average telomere length is not significantly different between male and female newborns (Okuda et al., 2002), thus sex differences in telomere lengths observed later in life (average telomere lengthwoman N average telomere lengthman) would reflect differences in biological aging set by environmental determinants. A marked synchrony exists in average telomere length between different tissues of human fetuses at different gestational ages (between 15 and 19 weeks) and newborns, telomere length is similar in white blood cells (WBC), skin, and umbilical cord artery cells from individual neonatal donors but differs extensively between newborn donors (Okuda et al., 2002; Youngren et al., 1998). This synchrony is lost during extrauterine life, most probably as a result of different proliferative programs and presence or absence of telomerase between organs and tissues (Wright et

al., 1996). The same conclusion was drawn from experiments in mouse (Prowse and Greider, 1995). At the cellular level, a chromosome-specific telomere length setting is displayed, which is largely conserved in different tissues from an individual organism (Martens et al., 1998; Zijlmans et al., 1997). Indeed, from cytometric analysis of telomere length using a telomere-specific fluorescently labeled PNA probe and digital imaging analysis, it is known that telomere length is heterogeneous among different chromosomes (Lansdorp et al., 1996; Martens et al., 1998). Recent experiments both by ourselves and others via independent approaches have revealed telomere length polymorphisms between homologous chromosomes and surprisingly also between sister chromatids (Baird et al., 2003; Bekaert et al., 2002; Londono-Vallejo et al., 2001; Rubelj and Vondracek, 1999) that explains to a large extent the telomere length heterogeneity, that is, broad and fuzzy appearance of telomere signals on Southern blots, as observed in human cells. Recent cytometric telomere length analysis of both monozygotic and dizygotic twins revealed that this chromosome-specific telomere length setting is inherited and maintained during normal embryonic, fetal, and postnatal somatic cell development (Graak-

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jaer et al., 2003). This underscores the importance of initial (genetic) telomere length setting in utero in addition to environmental and epigenetic effects later in life. Yet, the (genetic) determinants for telomere length polymorphisms between individuals, tissues, and chromosomes during development in the in utero environment are largely unknown. Telomere length as biomarker for human aging: initial telomere length setting and aging in vivo Although a direct correlation between replicative senescence and organismal aging does not exist, an interesting and challenging question is whether it will be possible in the near future to make predictions about the predisposition to certain aging disorders later in life from telomere length of an oocyte or a blastomere from a preimplanted embryo. Recent studies have evidenced intriguing relationships between telomere length and aging disorders like vascular aging (reviewed in Aviv et al., 2003). The same holds true for fetal and postnatal growth in relation to the etiology of disease. Growth restriction in utero followed by a catch-up growth period postnatally was associated with shorter life span and shorter kidney telomeres in male rats (Jennings et al., 1999). If not causally involved in human life span determination, telomere length certainly has to be considered as a valuable biomarker indicating or predicting on the aging phenotype (or aging disorders) later in life. This hypothesis is only on the verge of being investigated at this moment. Besides longitudinal epidemiological studies, basic insight into the senescence signaling within individual cells must help elucidate this presumed link between telomeric molecular clock (mean telomere length or individual telomere length?) and early growth with the etiology of aging diseases. Via a novel PCR-based methodology for allele-specific, single telomere length analysis (STELA), bimodal distributions of telomeres were detected in normal fibroblasts, which originate from inter-allelic differences. Baird et al. (2003) suggested that these inter-allelic telomere length differences pinpoint toward a stochastic telomere inheritance in the germline. This implies that a single telomere (like 17 or Xp/q) can only function as a mitotic clock within a single cell in a stochastic way depending on the maternal and paternal telomeric length inherited and further preserved throughout development. Revealing the complete set of (genetic and environmental) determinants of telomere length and telomere length variability in utero will be crucial for the complete understanding of development and even more so to further unravel the in vivo aging phenotype.

Telomere biology during development Telomere length in somatic cells originates from telomere length setting in the germline. In normal human somatic cells, telomere shortening is considered to act as a tumor

suppression mechanism that limits proliferation capacity and irreversibly induces replicative senescence when a critical telomere length is reached. However, in germline cells like in most tumor cells and immortalized cell lines (Autexier and Greider, 1996; Shay and Bacchetti, 1997), telomere erosion during cell division is counterbalanced by the activation of telomerase, a reverse transcriptase that counters telomere attrition via synthesis of telomeric tandem repeats onto the ends of chromosomes (Greider and Blackburn, 1985; Morin, 1989; reviewed in Greider, 1990; Nugent and Lundblad, 1998) (Fig. 2). Early in development, telomerase activity compensates for telomere length erosion from consecutive rounds of proliferation during tissue growth and differentiation, thus establishing a dynamic equilibrium and ensuring a normal replicative potential of somatic cells later during extrauterine life. Although regulation of telomerase activity is supposed to be crucially important for telomere length setting during development and even though fetal telomere length is highly synchronized, no direct correlation could yet be found

Fig. 2. Telomere length equilibrium is maintained within germline cells. In these cells, telomere erosion is counterbalanced by telomerase-mediated addition of telomeric sequences. The telomerase holoenzyme complex is composed of a catalytic protein subunit (hTERT in humans, mTERT in mouse), an RNA molecule (hTR in human, mTR in mouse) that is used as template for the addition of new telomeric repeats, and several associated proteins. Introduction of the telomerase catalytic subunit into primary nonimmortal human cells that have no detectable telomerase activity is sufficient to restore enzymatic activity, to elongate and maintain telomeres, and in some cases to allow immortal growth (Bodnar et al., 1998). The halflife of the telomerase enzyme is approximately 24 h in resting cells, but repression of the enzyme can be induced much faster in, for example, terminally differentiating cells, and a correlation has been reported between inactivation, differentiation, and increased telomere erosion (Ulaner et al., 2001). The regulation of telomerase is suggested to be mediated at different levels both at the transcriptional and posttranscriptional (alternative splicing) level and during assembly of the active holoenzyme complex. The template-RNA component of telomerase TR is shown to be constitutively expressed whereas the catalytic component is rate limiting.

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between telomere length setting and telomerase activity (Youngren et al., 1998). Additional factors might be determinant for telomere length during development. Understanding all players that contribute to telomere length variability set during intrauterine life will be important. In the following sections, we will summarize the current knowledge on telomere biology during the different phases of normal and abnormal development. Importance of telomeres in meiosis From an evolutionary point of view, meiosis is of crucial importance to eukaryotic organisms, providing variation as a result of recombination and chromosomal rearrangements. From cytological studies, it is known that telomeres play an important role in the functional nuclear re-organization occurring at meiosis that facilitates homologue recognition and recombination (Cooper et al., 1998; Dernburg et al., 1995; Nimmo et al., 1998; Rockmill and Roeder, 1998). In yeast, telomere defects induce meiotic asynapsis and incorrect chromosome segregation. In Schizosaccharomyces pombe, the interaction occurring during the prophase stage of meiosis (during which the nucleus has a horsetail shape) between the telomere and the spindle pole bodies (which are the microtubule organizing centers analogous to the centrosome material in higher eukaryotes and are known to participate in mitosis, meiosis, and nuclear movements) aids recognition and recombination of homologous chromosomes. Furthermore, genetic mutations in yeast that alter telomere biology induce meiotic asynapsis (failure of homologous chromosomes to pair during meiosis), incorrect chromosome segregation, and defects in the production or release of spores (sporulation) (Cooper et al., 1998; Nimmo et al., 1998). Specific structural telomere-binding proteins, like Taz1 in the fission yeast S. pombe that shares sequence homology with the telomere-binding protein TRF1 in humans, have been shown to be critical for homologue alignment and pairing. Other proteins, like Ndj1p in Saccharomyces cerevisiae, are shown to have a meiosis-specific telomerebinding function affecting meiotic recombination and chromosome segregation (Conrad et al., 1997; Rockmill and Roeder, 1998). In Caenorhabditis elegans, telomere shortening leads to a failure in the segregation of meiotic chromosomes (Ahmed and Hodgkin, 2000). In mammals, consequences of telomere dysfunction in meiosis might lead to apoptosis. In mammals, however, defects associated with telomere dysfunction presumably manifest themselves before defects in chromosome pairing in meiosis (synapsis) do so. Even though telomeres demonstrate particular joining during meiosis (Dernburg et al., 1995; Scherthan et al., 1994), the consequences of telomere dysfunction or telomerase deficiency in meiosis are still not fully understood. During the meiotic

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prophase of the first meiotic division, telomeres cluster near the nuclear membrane in a conformation known as the dboucquetT stage. At pachytene, the boucquet organization is disrupted and dispersed to enable homologous telomere pairing at the nuclear periphery. This phenomenon is universal during meiosis and has also been observed in human spermatocytes and is known to be involved in the ataxia telangiectasia mutant (ATM) (Pandita et al., 1999; Rasmussen and Holm, 1978). It was suggested that the structure of sperm telomeres would play a role in the initiation of sperm nucleus reorganization that occurs during male pronucleus development after fertilization (Zalenskaya et al., 2000). Furthermore, severe telomere shortening, and as a consequence telomere dysfunction and chromosomal instability, in late-generation mTR / mice triggers cell death in the testes (Chin et al., 1999). Dysfunctional telomeres: DNA repair and apoptosis factors implicated in telomere biology in germ cells Telomere shortening can lead to dysfunctional telomeres, chromosomal instability, and apoptosis both in somatic and germ cells. Apoptosis is a normal series of events in a cell, which leads to its programmed death and is a crucial mechanism during development, aging, as well as under various pathological conditions. Experiments have revealed that proteins implicated in DNA repair and apoptosis are also located at the telomeric cap and play an indispensable role in the control of telomere length regulation and functionality (reviewed in Gasser, 2000). In particular, a link has been made between the chromosomal ends and Ku70/Ku86 proteins. Ku70 and Ku86 are members of the DNA-PKc complex that is involved in DNA double-stranded break repair, nonhomologous recombination, and modulation of transcription (Fig. 1B). In mTR / mice, apoptosis occurs specifically in cells lacking detectable Ku70/Ku86 proteins. Ku70 is also known to bind telomeres in vivo and is thought to play a role in normal telomere function (Hemann et al., 2001; Hsu et al., 2000; Laroche et al., 1998). In germ cells, the absence of Ku70 or Ku86 and the resulting decrease in nonhomologous recombination prevent chromosome fusion as a mechanism to repair dysfunctional telomeres. In the absence of chromosome fusions, dysfunctional telomeres may be recognized as a DNA double-strand break and this would induce apoptosis (Espejel et al., 2002). Apoptosis occurring in cells with dysfunctional telomeres could be due directly to telomere dysfunction or indirectly as a consequence of subsequent chromosome breaks and genomic instability. This kind of tissue-specific surveillance of DNA breaks has also been shown to exist in mouse neural development (Gao et al., 1998). In the absence of nonhomologous recombination proteins (including Ku70), unrepaired double-strand breaks specifically elicit neuronal apoptosis (see also neural tube defects).

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mTR / mice exhibit a high proportion of apoptotic germ cells. Surprisingly, no chromosome fusions could be identified in germ cells. Therefore, chromosome fusion has been proposed as a mechanism by which somatic cells specifically restrain the damage caused by dysfunctional telomeres. A dysfunctional telomere may be recognized by the cell as a DNA break. The removal of a DNA break by chromosome fusion may permit the cell to move forward in the cell cycle. In the germline, however, the transmission of any cytogenetic abnormalities could have tremendous developmental effects on the next generation. Hence, a mechanism of telomere surveillance within germ cells might be present that preserves the germline from chromosomal abnormalities (Hemann et al., 2001). If the telomere length setting in the germline was not equilibrated, this might have dramatic implications, even during evolution. Stindl (2004) speculated that gradual telomere attrition over many thousands of generations could lead to weakening and even result in the extinction of a specific species. Telomerase and apoptosis during development. Zhu et al. (2000a) suggested recently that the catalytic subunit of telomerase (mTERT) might protect embryonic cells against apoptosis. Indeed, the decrease of the level and function of mTERT in embryonic mouse neurons in culture significantly augmented their sensitivity to apoptosis as seen by the higher levels of oxidative stress and mitochondrial dysfunction. In contrast, overexpression of the catalytical subunit in pheochromocytoma cells (benign tumor cells of the adrenal medulla or the sympathetic nervous system) resulted in diminished sensitivity to apoptosis (Zhu et al., 2000a). Thus, the catalytic subunit of telomerase might protect embryonic cells against apoptosis. Interestingly, similarities between the dynamics of telomerase activity and the dynamics of apoptosis have also been shown in early bovine embryos. Indeed, apoptosis could not be detected from the two- to eight-cell stage, but the percentage of dead cells increased from the 8-cell stage onwards: 50–79% of morulae or early blastocysts exhibited apoptosis and 100% of expanded blastocysts had at least one apoptotic cell, meaning that apoptosis is a physiological process that starts early during development. Telomere biology in reproductive cells Telomere length in spermatozoa in human, porcine, and bovine organisms is substantially longer compared to normal somatic cells (Allsopp et al., 1992; Cooke and Smith, 1986; de Lange et al., 1990; Kozik et al., 1998). Germline cells are the most likely targets for initial telomere length setting, before fertilization and onset of early developmental programs. Their main task is to preserve the inheritance of intact, full-length chromosomes to the progeny. Yet only limited data have been published concerning telomere biology in the germline.

Reproduction is highly dependent upon germ-cell expansion capacity and thus telomerase activity to preserve the dynamic telomere length equilibrium. Telomerase activity was initially demonstrated in Xenopus oocytes and embryos (Mantell and Greider, 1994). In humans, telomerase activity was found in fetal, newborn, and adult ovaries and testes (Fujisawa et al., 1998), but not in mature spermatozoa or oocytes (Wright et al., 1996). More recent evaluation of telomerase activity employing a more sensitive telomerase assay enabling quantitative single-cell analysis has revealed telomerase, albeit at low levels, in mature oocytes (Wright et al., 2001). Female germ cells: telomerase activity decreases in function of oocyte maturation stages. In rats, a high telomerase activity has been observed in oocytes from follicles at the early antral and preovulatory stages. It then declines at the 1mm stage and even further at the 3-mm stage (Eisenhauer et al., 1997). In ovulated oocytes, the telomerase activity is found to be 50-fold lower (Eisenhauer et al., 1997; Lavranos et al., 1999) (Figs. 2 and 3). Brenner et al. (1999) found differences in human telomerase catalytic subunit mRNA expression in individual human oocytes at germinal vesicle (GV), metaphase of 1st meiosis (MI) or metaphase of 2nd meiosis (MII) stages, day3 embryos, and blastocysts (Fig. 3), thus demonstrating that telomerase was actively transcribed in the oocyte. In lategeneration mTR / mice, a reduced number of oocytes was counted during ovulation without significant decreases in plugging capacity (Lee et al., 1998). The absence of telomerase activity in some abnormal human oocytes has been linked to shortened telomeres and is therefore associated with reproductive senescence or chromosomal abnormalities like aneuploidy or translocations and could be used as a marker for the health status of the oocyte and the future embryo. If telomerase contained in the oocyte cytoplasm is necessary for the maintenance of chromosomal stability and telomere length, inoculation of telomerase or some oocyte cytoplasm should be able to restart or interfere with the btelomeric clockQ in abnormal human oocytes (Cohen et al., 1998). Male germ cells: telomere biology during maturation. Nuclear architecture changes importantly during spermatogenesis in mammals and the telomere domains play a leading role in this reorganization (Ward and Zalensky, 1996; Zalensky et al., 1995). In spermatogonia, telomeres are randomly scattered throughout nucleoplasm, but in spermatocytes, all telomeres relocalize to the nuclear membrane and form specific dimeric and tetrameric associations (Zalensky et al., 1997). Telomere dimers and higher-order associations have also been demonstrated in sperm of five other mammals (Zalensky et al., 1997). An interesting question is whether these relocalizations and interactions with the nuclear membrane are telomere length dependent.

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Fig. 3. Summary of telomerase activity in human and rodent oocytes (at different stages of maturation) and early embryos (from fertilization to the blastocyst stage).

Telomerase activity has been monitored in detail in the male germline. During embryogenesis, the human telomerase expression starts before the differentiation of sex and cord. From the end of fetal development up to the prepubertal period, a high expression of human telomerase can be observed in the majority of the tubular cells of the testis, the majority of which are immature Sertoli cells, whereas no expression has been found in gonocytes (immature nondividing germ cells). For information, the Sertoli cells separate meiotic spermatocytes and spermatids (in the adluminal compartment) from the spermatogonia and early spermatocytes (in the basal compartment). They play not only an important supportive role for the seminiferous epithelium but also additionally supply germ cells with growth factors and nutrients throughout their development. In contrast, in the adult, the highest level of telomerase expression has been discovered in the testis, in particular, the seminiferous tubules consisting predominantly of germinative cells (Yashima et al., 1998a,b). This level is equivalent to that expressed by many epithelial tumors. The expression has been found to be higher in primary spermatocytes than in spermatogonia, their precursors (Figs. 4 and 5). Furthermore, the expression was less important in secondary spermatocytes, which are actually postmeiotic cells, whereas no expression could be detected in nondividing spermatids as well as spermatozoa. In

hyalinized seminiferous tubules composed of a majority of Sertoli cells, the expression remained high even at the eighth decade of life. In actively dividing spermatocytes, the high level of expression can easily be understood by the constant production of spermatozoa, starting from puberty. The level of telomerase activity in pachytene spermatocytes and round spermatids is lower than in ovulated oocytes. Lack of telomerase activity has been found in spermatozoa from two areas of the epididymis: the caput and the cauda. The epididymis is the single convoluted tubule extending from the vas efferentia to the vas deferens; it plays a role in the transport, concentration, maturation, and storage of spermatozoa, and secretion of energy substrates and materials coating spermatozoa, and is divided into three anatomical areas called the caput (head), corpus (body), and cauda (tail) (Wright et al., 1996) (Figs. 4 and 5). No telomerase activity has been detected in ejaculated human spermatozoa (Eisenhauer et al., 1997). Telomerase activity increases at fertilization and telomerase is dispensable at early cleavage Liu et al. (2002) have observed aberrant fertilization and first mitotic divisions in TR / mice. They found that fertilization of mTR / eggs with either wild-type or mTR / sperm and fertilization of TR / sperm with

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Fig. 4. Schema of telomerase activity at the different stages of maturation during human spermatogenesis.

wild-type eggs all manifested similar low rates of cleavage and development to morula and blastocysts, suggesting that only a few gametes from TR / mice are capable of

fertilization and early development, nonetheless of whether they were hetero- or homozygous for telomerase deletions. That the homozygous mTR / embryos could develop to

Fig. 5. Schematic overview of the telomerase activity and hTR expression during different stages of human fetal development and in the adult. Telomerase activity (measured as the catalytic activity in extraction) and hTR expression during fetal and neonatal development (after Ulaner et al., 1998, 2001; Wright et al., 1996, 2001; Yashima et al., 1998a,b). In humans, telomerase activity is detected from the blastocyst stage until 20 weeks of gestation in most embryonic tissues (Wright et al., 1996). During fetal development, tissue- and developmental phase-dependent regulation generates different levels of telomerase activity. Telomerase activity disappears more rapidly during fetal development in heart, brain, and kidney as compared to liver, lung, testes, and spleen (Ulaner and Giudice, 1997). Whereas hTR expression is measured in the kidney up to 21 weeks postgestation, no telomerase activity is measured from 17 weeks onwards due to inactivation hTERT via alternative splicing (Ulaner et al., 1998). Whereas telomerase is absent in most adult tissues, some exceptions exist with expression of telomerase at different levels, closely linked to dividing cells. Telomerase activity has been detected in lymphocyte stem and progenitor cells, and in peripheral blood lymphocytes upon proliferation induction (Counter et al., 1995; Hiyama et al., 1995). High expression was found in primary spermatocytes and Sertoli cells within the testis, intermediate expression in lymphoid germinal centers, and weak expression was detected in regenerative epithelia (Yashima et al., 1998a,b).

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morula and blastocysts suggests that telomerase is dispensable for the early cleavage stage of embryo development. Furthermore, fertilized eggs from TR / exhibited a high incidence of apoptosis, as evidenced by both cytofragmentation and nuclear DNA fragmentation. Cytofragmentation also corresponded with the development of only one pronucleus (the female one) and might be attributable to telomere dysfunction that results in meiotic defects. Telomerase activity of oocytes increases considerably after fertilization (Fig. 3) (Dolmetsch et al., 1997). One hypothesis is that fertilization causes a calcium level fluctuation that, as an indirect and downstream effect, increases telomerase activity from the mature oocyte stage to the zygote stage. It is well known that, in mammals, intracellular calcium fluctuations induce changes in gene activation by modifying both the amplitude and the frequency (Dolmetsch et al., 1997). A calcium signal causes ovulated oocytes to complete meiosis and to begin embryonic development (Dupont, 1998; Ozil, 1998). Furthermore, the mitogen-activated protein (MAP) kinase and the intracellular calcium (both involved in fertilization) take part in the regulation of the telomerase activity of human solid tumor cells and epidermal stem cells, respectively (Bickenbach et al., 1998; Seimiya et al., 1999). Nevertheless, regulatory mechanisms during the early embryo development remain to be elucidated, and candidates are calcium, MAP kinase, metaphase-promoting factor, small G proteins, and other proteins. Lack of telomerase activity is linked to infertility mTR / mice are reported to be initially fertile. However, successive generations of these mice lead to a steady decrease in fertility, observed as decreased litter size, testis size, and number of primary spermatocytes, finally resulting in sterility (Lee et al., 1998; Liu et al., 2002). A testicular atrophy associated with germ cell depletion accompanies infertility in mTR / males derived from the 6th generation through interbreeding, meaning that litters from the seventh generation are extremely rare (Rudolph et al., 1999). However, the reproductive systems of those young males appear to be structurally and functionally normal up to the third mTR / generation (Lee et al., 1998). In the following generations, a severe decrease in fertility, due to apoptosis in the germ cells, has been reported (Lee et al., 1998). In the early embryo, telomerase activity decreases up to the 8-cell stage and increases again at the blastocyst stage Eisenhauer et al. (1997) showed that telomerase activity was present in four-cell embryos whereas Xu and Yang (2000) showed that it increased after in vitro fertilization and decreased gradually until the eight-cell

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stage (Fig. 3). They also found that from the eight-cell stage onwards, telomerase activity increases gradually, becoming relatively high in blastocysts of certain species like humans and bovines (Betts and King, 1999; Eisenhauer et al., 1997; Xu and Yang, 2000). Activation of inactive telomerase proteins or an initiation of the translation of maternal stock mRNAs occurs just after fertilization (maybe via calcium fluctuations or other pathways). From fertilization up to the 8-cell stage, the telomerase activity would then progressively diminish because embryonic transcription only starts around the eight-cell stage in contrast to degradation of maternal proteins (and telomerase as well). As soon as embryonic transcription begins, a new telomerase protein is synthesized, and this could explain the increase in activity between the eight-cell and the blastocyst stages. Embryonic transcription (indicated by the presence of a-amanitin-sensitive protein synthesis) has been examined by Barnes and First (1991) in bovine embryos fertilized in vitro and starts sometime between the four- and six-cell stage of embryonic development (corresponding to 36–48 h after insemination). Another study performed on bovine embryos collected and cultured after fertilization showed that transcription only started once the embryo reached the eight-cell stage of embryonic development (Kopecny et al., 1989). The basal or absent telomerase activity in human oocytes contrasts with studies in Xenopus eggs where telomerase activity was shown to be high (Mantell and Greider, 1994; Wright et al., 1996, 2001). However, this difference might be due to the maternal products and their storage in mammals in comparison with the frog. Indeed, in mammals, zygotic transcription starts after the first division whereas in Xenopus, maternal products are preponderant throughout the first 12 embryonic divisions (Newport and Kirschner, 1982). Therefore, telomerase is required during the first embryonic divisions, it must have a maternal origin in frogs, whereas, in mammals, it could be generated following zygotic transcription. Telomerase activity during development: different levels of regulation We have described how telomerase must be activated within germ cells to guarantee the transmission of fulllength chromosomes to the offspring (Dahse et al., 1997; Hastie et al., 1990). However, it is not yet clear how this transmission is achieved during embryonic development. Whether telomerase per se plays a direct biological role in mammalian development is not sure, but its counterbalancing function against telomere shortening is shown to be vital for organ and tissue homeostasis and cell viability. Transient telomerase activity, especially during development, is crucially important. Telomerase activation must enable telomere length setting and maintenance, yet

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telomerase must be inactivated well before the telomeres become too long to impair cell viability. The telomerase activity within a given cell is regulated at different levels. The level of hTERT expression is rate limiting for telomerase. Differences in hTR accumulation result in differences in telomerase activity only when hTERT expression levels are substantial. The patterns of TERT expression are regulated by factorinduced transcription combined with posttranscriptional regulation via alternative splicing. The hTERT promoter region is rich in transcription factor binding sites and the oncoprotein c-myc, Sp1, estrogen, and progesterone have been shown to upregulate hTERT (Kyo et al., 2000; Wu et al., 1999). Alternative mRNA splicing of the human telomerase catalytic subunit (hTCS) may play a regulatory role in telomerase activity of oocytes and early embryos. Three different PCR product sizes (457, 421 and 275 bp) of the hTCS have been described. Interestingly, normal human oocytes and early embryos only exhibit the 457-bp PCR product, which is identical to the first described hTCS gene, whereas some damaged oocytes, spermatozoa, or early embryos possess the three different splicing variants of hTCS (Brenner et al., 1999; Ulaner et al., 1998). These three hTCS PCR products have also been observed in some normal tissues, fetal tissues, tumors, and cell lines. The importance of the splicing variants of hTCS in the regulation of telomerase activity in damaged embryos, and whether they induce the production of proteins with different biochemical functions, is not yet understood. Additionally, whether the splicing patterns of hTCS can serve as a potential marker of health status and survival of embryos has yet to be proved. Another level of regulation of telomerase activity consists out of the holoenzyme complex assembly in combination with its telomere-binding capacity. Inaccurate holoenzyme assembly results in nonfunctional telomerase enzymes, and posttranslational modifications such as phosphorylations are shown to disable telomere-binding capacity. Recent studies demonstrate that mutations in genes encoding two telomerase holoenzyme components reduce the maximal level of telomerase activation and dramatically compromise the proliferative renewal of hematopoietic and epithelial tissues (Mitchell et al., 1999; Vulliamy et al., 2001). These findings reveal a much greater requirement for telomerase in the processes of human growth and development than previously suspected. Telomerase activity during development varies in function of the organs Only limited information is available about the expression of telomerase during development and differentiation (Fig. 6). Telomerase regulation appears to be tissue dependent: in heart, for example, a decrease in telomerase

activity is accompanied by inhibition of hTERT mRNA (chromatin-mediated) expression, in kidney on the other hand, different hTERT splice variants appear dependent on the level of telomerase activity (Ulaner et al., 1998). In 16week-old human fetuses (when the embryonic period and organogenesis are finished), telomerase is expressed in many tissue-specific stem cells. Analogously, in mice, the RNA component of telomerase is present in all the newborn tissues tested but is downregulated together with mTERT directly after birth, whereas in humans, telomerase is expressed in fetal as well as newborn and adult testes and ovaries (Blasco et al., 1995, 1997; Wright et al., 1996). The majority of human somatic tissues at 16–20 weeks of development (apart from the brain tissue) expressed telomerase activity at a very high level (Wright et al., 1996). However, because whole tissues were examined in this study, no distinction could be made between the different cells present in those tissues, and only part of a very complex process was examined. A complete understanding of the mechanism is therefore still lacking. The detailed characterization of telomerase activity during the latter stage of gestation is prevented by the unavailability of human fetal material between 18 weeks and birth. High proliferative demand within newborns exhausts the telomere reserve in some tissues at a higher rate than in adult life (Frenck et al., 1998; Rufer et al., 1999). Greater telomerase activation has been measured in extracts of peripheral blood mononuclear cells during the first years of life (Hiyama et al., 1995). In the fetal kidney, expression is low or absent in temporary tissues such as the adrenal cortex and the mesonephros. However, even if the differentiation into specialized kidney cells is, in the majority of cases, accompanied by a decrease or even a complete loss of expression, maturation of immature epithelial cells was associated with retained expression and, in case of the metanephros, with increased expression. Therefore, telomere maintenance by telomerase would play a primordial role during embryonic development. It is proposed that during human embryonic development, telomerase would be present and active and then, just after birth, would be downregulated (Wright et al., 1996). So far, during in situ hybridization experiments in human embryos, the highest levels of hTR were detected in the central nervous system, in particular in undifferentiated neuroepithelium, with reduced but detectable levels in other epithelial tissues (Yashima et al., 1998a). Maturation of primitive neuroepithelial cells into nondividing central nervous tissue is linked to a lack of detectable expression of telomerase. Whereas the factors responsible for these differences in hTR accumulation are unknown, recent experiments show the existence of a complex interrelationship of hTR expression with human embryonic development, cell differentiation, and cell division (Ulaner et al., 2001; Yashima et al., 1998b).

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Fig. 6. Description of some neural tube defects in which telomerase could be involved.

Disturbed telomere regulation is linked to abnormal development Aging disorders. Disturbed telomere length regulation in prenatal development may account for various diseases such as progeria-like Hutchinson–Gilford progeria (a laminopathy originating from a single heterozygous splicing mutation in the LMNA gene that encodes for lamina/C proteins) as well as Werner (mutation in WRN helicase gene), Bloom (mutation of BLM helicase), and Down (trisomy 21) syndromes. These syndromes are characterized by accelerated premature aging in vivo and reduced proliferative capacity in vitro, which is shown by an increase in telomere attrition rates in cultured progeria donor cells. Increased telomere attrition rates are also apparent in vivo in blood lymphocytes isolated from Down syndrome donors (Dorland et al., 1998; Vaziri et al., 1993). In contrast, normal telomere lengths are found in vivo in Bloom syndrome cells (Yankiwski et al., 2000). Furthermore, an example of the direct impact of telomerase deficiency or insufficiency in the occurrence of certain pathologies is the dyskeratosis congenita (DKC) disorder. DKC is characterized by mucosal patchiness, reticulate skin pigmentation, erosion of regenerative tissues like skin and nail, resulting finally in bone marrow failure,

and ultimately death. It was demonstrated recently that lymphocytes and fibroblasts isolated from DKC patients contain a defect in hTR expression level and stability, leading to lower levels of telomerase activity, short telomeres leading to genomic instability with catastrophic impact on normal development (Lee et al., 1998; Mitchell et al., 1999). Neural tube defects are linked to abnormal telomerase activity. The coordinating function of the central nervous system is primordial for normal functioning of an entire human organism. And more specifically, biological timing during subsequent developmental programs is registered particularly by this control unit within the organism. Neural tube defects, including spina bifida, anencephaly, and congenital hydrocephalus, are among the major causes of infant mortality (Fig. 6). The closure of the neural tube includes cell proliferation, migration, as well as apoptosis, and only a few genes have so far been shown to be involved in this complex developmental process (reviewed in Harris and Juriloff, 1997). Therefore, mouse models of neural tube defects are essential to build up the molecular understanding of this process. Telomerase is actively present in the brain during embryonic development and TERT was shown to be

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involved in neuronal differentiation and viability (Greenberg et al., 1999). Regulation of developmental death and survival in neurons is presumably determined by, respectively, suppression of telomerase activity and TERT expression, which was found to induce p53-dependent apoptosis and overexpression of TERT, and presumably protects against neuronal apoptosis, for example, in experimental models of neurodegenerative diseases (Zhu et al., 2000a; reviewed in Mattson et al., 2001). Herrera et al. have shown that mice mTR / exhibited defects in neural tube closure. Those defects were shown to be associated with telomere loss, augmented chromosomal instability, and apoptosis (Herrera et al., 1999). The defects of the neural tube closure were observed in four different areas of the nervous system (forebrain, midbrain (mesencephalon), cervical, and caudal regions), which correspond to the four places where the neural tube fuses (Sakai, 1989). Some malformed mTR / embryos also showed a bilateral dissymmetry while the majority had normal symmetry. This would mean that the neural tube formation would be one of the processes most sensitive to a lack of telomerase, that is, telomere length equilibrium, during development. Furthermore, in those mTR / mice, telomeres have been reported to shorten approximately 5 kb per generation. This was due mainly to the absence of telomerase activity and was associated with an increase in aneuploidy and end-to-end chromosomal fusions (Blasco et al., 1997; Hande et al., 1999). Therefore, a possible correlation between frequency of malformed mTR / embryos and the decrease of telomere length per mTR / mouse generation has been hypothesized. The neural tube defect in mTR / embryos would be a consequence of both telomere shortening and telomere loss from chromosome ends. Indeed, other mouse models in which neural tube malformations have been reported have been shown to exhibit chromosomal abnormalities, such as trisomy 12 and trisomy 14 strains, with neural tube defects in 100% or 50% of the embryos, respectively (Putz and Morriss-Kay, 1981). These nondividing brain cells and neurons are particularly interesting to exclusively study the impact of environmental determinants on telomere attrition rates. Increasing (biological) age is a major risk factor in the development of Alzheimer’s disease, and biochemical changes such as an increase in cellular oxidative stress may be effectors of synaptic dysfunction and neuronal degeneration. Fu et al. (1999, 2000) and Zhu et al. (2000a) demonstrated, via independent experiments, that ectopic overexpression of telomerase in neural cells has a protective, anti-apoptotic effect. Inhibition of the catalytic subunit of telomerase on the other hand increases the probability of neuronal cell death by oxidative and apoptotic damage. The impact on telomere length within these neurons is not known. The understanding of biological timekeeping and, more specifically, biological time setting is of major importance in light of recent discoveries about the presence and preservation of neural stem cell populations (subventricular zone and denate gyrus

of the hippocampus) and the suggestion to use embryonic stem cell therapy in neurodegenerative disorder therapy. What happens during cloning? The question of telomere reprogramming in cloned animals was initially proposed by Shiels et al. (1999), who measured the telomere length of Dolly, the cloned sheep (Wilmut et al., 1997), and discovered that Dolly’s shortened telomeres were inherited from her mother, the nuclear donor indicating that no telomere length reequilibration occurred during cloning in contrast to normal reproduction. According to Shiels et al., in contrast to what is believed to happen during fertilization, the telomere length of the donor nucleus might not be fully reprogrammed by cloning. Consequently, examining and studying telomere biology and reprogramming during early development are of considerable importance for understanding how reprogramming occurs and how telomere length can be altered by this phenomenon. The cloning of livestock from adult nuclei raises the question what the effect of telomere length (re)equilibrium is in the cloned stock. Taken into consideration that telomerase is activated by the blastocyst stage, it is probable that the reprogramming of the adult nucleus (irrespective of the age of the adult donor and of its initial telomere length) is associated with the reexpression of telomerase and the resetting of telomere length up to a normal level seen in blastocysts (Betts and King, 1999; Kubota et al., 2000). This was established by comparing telomere lengths from cloned fetuses (1523 kb) and adult donor cells (14–18 kb), which showed that telomere length in cloned fetuses appeared to be longer than in adult donor cells with no difference observed between the age-matched controls (Shiels et al., 1999; Wilmut et al., 1997). No telomere shortening or premature aging was observed in cloned mice (Wakayama et al., 2000). In cloned cattle, even telomere extension beyond the length of age-matched control animals was measured (Lanza et al., 2000). These and future cloning experiments will provide further insights into the aging phenotype in vivo, that is, from the single-cell level to the organism level.

Conclusion The control mechanism of telomere biology in general and telomere length determination in particular during human development is complex and not yet fully understood. It is shown to be cell, tissue, and organ specific, and appears to be closely related to cell maturation, differentiation, proliferation, and apoptosis. Future studies must focus on the regulatory mechanisms by which both telomere length and telomerase activity are regulated during development. If telomeres serve as molecular clocks in human

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beings even at the organismal level, understanding the determinants that contribute to heterogeneity of telomere length during intrauterine life will help to unravel human developmental biology and biological aging.

Acknowledgments The authors would like to thank Prof. Dr. P. Van Oostveldt, Dr. Paul Jacquet, and Prof. Dr. Max Mergeay for continuous support. Dr. ir. Sofie Bekaert is funded by a 2- to 4-year Research Project (01109502) from the Special Research Fund, Ghent University and ir. Hanane Derradji is supported by a doctoral grant from SCK!CEN/Ghent University. This work is currently supported by a research contract from the European Union under the Nuclear Euratom Programme (FIS5-2002-00029).

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