Abstract
Telomerase, an enzyme complex that binds the chromosome ends (telomeres) and maintains telomere length and integrity, is present in germ cells, proliferative granulosa cells, germline stem cells, and neoplastic cells in the ovary, but it is absent in differentiated or aged cells. Activation of telomerase in the ovary underpins both benign and malignant cell proliferation in several compartments, including the germ cells, membrana granulosa, and the ovarian surface epithelium. The difference in telomerase operation between normal and abnormal cell proliferations may lie in the mechanisms of telomerase activation in a deregulated manner. Recent studies have implicated telomerase activity in ovarian cancer as well as oogenesis and fertility. Inhibition of telomerase and the shortening of telomeres are seen in occult ovarian insufficiency. Studies of how telomerase operates and regulates ovary development may provide insight into the development of both germ cells for ovarian reproductive function and neoplastic cells in ovarian cancer. The current review summarizes the roles of telomerase in the development of oocytes and proliferation of granulosa cells during folliculogenesis and in the process of tumorigenesis. It also describes the regulation of telomerase by estrogen in the ovary.
Introduction
Cell proliferation in the ovary is critical to the ovarian functions of producing and releasing germ cells (oocytes) and hormones for reproduction. During the development of oocytes, folliculogenesis is accompanied by significant proliferation of granulosa cells, which is tightly controlled in a temporal and spatial manner. As the overall reproductive lifespan in mammals is predetermined at birth by the number of eggs in each ovary, the ovarian aging manifestations of menopause occur when the finite supply of eggs is depleted. The timing when ovarian aging begins is critical and often reflected by the speed of primordial follicle depletion. The magnitude of the follicular reserve is determined by a discontinuous balance between follicular recruitment, sustainment, and exhaustion (McGee & Hsueh 2000, Adhikari & Liu 2009). In follicular recruitment and sustainment, ovarian cell proliferation plays a primary role, with granulosa cell proliferation, differentiation, and subsequent senescence (aging) and apoptosis (death) to underpin oogenesis and the production of estrogen and progesterone. Recent studies have shown the presence of the cells in adult ovaries with germline stem cell properties of proliferative potential in the postnatal mammalian ovary (Virant-Klun et al. 2009, Zou et al. 2009). Implantation of a cell line established from the germline stem cells of adult mouse ovary into the ovaries of infertile mice reinitiates oogenesis and fertility (Zou et al. 2009). The presence of germline stem cells in adult ovary may reflect suggesting the presence of a potentially renewal mechanism in support of the ovary reproduction, and release of eggs and hormones.
Several lines of evidence indicate that specific genetic mechanisms mediate production by the ovary of germ cells and the hormones needed to support oogenesis. The ovary is surrounded by a single layer of ovarian surface epithelium (OSE) that is a layer of cuboidal cells derived from mesoderm during embryonic development (Auersperg et al. 2001). As a source of ovarian stem cells in humans (Virant-Klun et al. 2009) and mice (Lee et al. 2008), OSE undergoes hyperplasia in response to estrogen treatment to potentially lead to tumorigenesis (Laviolette et al. 2010). Estrogen accelerates tumorigenesis initiated by the simian virus 40 (SV40) T antigens, resulting in an early onset and reduced overall survival time (Laviolette et al. 2010). In the follicular compartment, estrogen is released by and acts on folliculogenesis. In contrast, recent studies showed that phosphatase and tensin homolog (PTEN) that binds and dephosphorylates the phosphatidylinositol (3,4,5)-trisphosphate has a chief function in suppressing folliculogenesis and ovulation (Fan et al. 2008, Marx 2008, Reddy et al. 2008, Adhikari & Liu 2009). Lacking PTEN in oocytes causes the entire primordial follicle pool to become activated, followed by depletion of all primordial follicles in young adult mice (Reddy et al. 2008, Gleicher et al. 2009), whereas inactivating PTEN in the granulosa cells results in not only enhanced ovulation but also persistent non-steroidogenic luteal structures in the adult mouse ovary (Fan et al. 2008). To date, however, neither the downstream signaling or effector of PTEN, nor the opposing positive regulatory pathway in follicular activation is known.
One of the major genetic mechanisms determining cell proliferative capacity is the maintenance of telomeres. Work from several independent laboratories has shown that forced expression of the telomerase reverse transcriptase (TERT) gene to maintain telomeres in cultured OSE cells results in increased proliferative potential of the cells, causing them to resemble stem cells (Davies et al. 2003, Kusakari et al. 2003, Alvero et al. 2004, Maeda et al. 2005, Li et al. 2007, Yang et al. 2007a, 2007b). This fundamental effect of telomere remodeling on OSE cells demonstrates proof of principle that the regulation of telomere remodeling is closely linked to OSE cell proliferative capacity. However, substantial in vivo evidence also shows that telomere remodeling is regulated by various factors, including hormones, growth factors, and cytokines, under diverse conditions. Intriguingly, during folliculogenesis, ovarian aging, and neoplastic development, the pattern of TERT-induced telomerase activity is dependent on cell type, time, and location. This review presents the current understanding of the fundamental roles of telomerase and telomere maintenance in ovarian cell proliferation.
Telomere and telomerase: a mechanism for cell regeneration
Telomeres, the specialized structures at the ends of all eukaryotic chromosomes, are formed from a unique DNA nucleotide sequence and specific coat proteins. Telomeres from different species assume various tertiary and quaternary structures. The telomeric DNA sequence in humans comprises extended arrays of a few thousand tandem repeats of TTAGGG complementary double strands. However, telomeres end with a single-stranded 3′-end overhang of ∼150 bp. This 3′ single-stranded overhang is protected from being sensed as DNA breaks by protein complexes or within the double-stranded duplex of the telomere (de Lange 2005, Zhao et al. 2008, Lue 2009; Fig. 1). Entry of the telomere 3′ overhang into the duplex results in a displacement loop and a larger telomere loop at each end of the chromosome (Griffith et al. 1999). The specialized configuration of telomeric DNA has a critical role in protecting chromosomes. During each round of telomeric DNA replication, telomeres are shortened due to the end replication problem (Olovnikov 1996). With each round of chromosome replication, 100–200 bp of telomeric DNA are lost; so, telomeres shorten from 10–15 to 2–5 kb in a lifespan of human cells. When telomeres shorten to a critical length, the cell exits the cell cycle, which is characteristic of cell senescence (Counter et al. 1992, Shay 1999, Blackburn 2001, Wong & Collins 2003). Thus, the initial size of telomeres and the extent of telomere shortening during cell divisions define a cell's replicative lifespan.
Simplified cartoon structures of linear and circular telomeres at the ends of chromosomes. Double-stranded telomere DNAs are bound by telomeric repeat-binding factor 1 (TRF1, also known as TERF1) and TRF2 (TERF2). The 3′ single-stranded overhang of telomere is capped by telomerase complex (circled; which is composed by TERT and TERC). The long telomere hides its 3′ single-stranded overhang in the telomere double-stranded DNA duplex, resulting in a local displacement loop (D-loop) as indicated and a large telomere loop (T-loop). Symbol keys are indicated for TRF1, TRF2, TERT, and TERC.
Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0008
The telomere end replication problem is evolutionarily solved by the enzyme complex telomerase (Szostak & Blackburn 1982, Greider & Blackburn 1985). Telomerase binds, protects, and positively regulates telomere length (Fig. 1). Telomerase is conserved from yeast to higher eukaryotes to synthesize telomeres by RT and to maintain telomeres for continuous renewal and proliferation, particularly of highly proliferative cells, such as germ lines and stem cells in mammals (Shay 1999, Blackburn 2001). As a ribonucleoprotein complex, telomerase contains the catalytic subunit TERT, the telomerase RNA components (TERC), and binding proteins (Cohen et al. 2007, Venteicher et al. 2009). Murine TERT comprises 1122 amino acid residues encoded by a gene on chromosome 13 (D13Mit8; Greenberg et al. 1998). Murine TERC comprises 430 nucleotides (Blasco et al. 1995). Reconstitution of telomerase demonstrates that TERT is the rate-limiting factor capable of lengthening telomeric DNA when expressed in cells (Bodnar et al. 1998). Expression of TERT enables telomerase to function in telomere lengthening and length maintenance; conversely, deficiency of either TERT or TERC abolishes telomerase activity and results in short telomeres (Fig. 2).
Model of the mechanisms by which estrogen stimulates telomerase activity, telomere lengthening, and tumorigenesis in the ovary. (A) Estrogen (E2) is produced by granulosa cells (GC) under the action of GNRH and FSH, and the autocrine actions of estrogen. Estrogen stimulation of telomerase (TE) activity underlies GC proliferation. (B) Oversupply of estrogen induces high telomerase activity, lengthening of telomeres, and GC tumor formation. (C) Spillover of estrogen to the epithelial compartment causes telomerase activation, telomere lengthening, and ovarian carcinoma.
Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0008
Telomerase is required for oocyte development
Telomerase is present in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes, and at markedly decreased levels during oocyte maturation (Eisenhauer et al. 1997, Betts & King 1999). Knock out in mice of TERT (Leri et al. 2003, Wong et al. 2003) or TERC (Blasco et al. 1997, Hande et al. 1999) eliminates telomerase activity, compromises telomere maintenance, and results in defects of cell developments in multiple tissues. In later generations of TERC-deficient mice, highly proliferative tissues such as those of the reproductive organs, immune system, hematopoietic system, and the germ line show compromised proliferative capacity (Blasco et al. 1995, 1997, Lee et al. 1998). Infertility is a major phenotype of late generations of telomerase-deficient mice (Lee et al. 1998, Herrera et al. 1999). A significant decline in litter size occurred by the fourth generation, when telomeres are markedly shortened, and no progeny was produced from eight different intercrosses of sixth-generation mice (Lee et al. 1998, Herrera et al. 1999). The uterine horns of infertile mice lacking TERC are significantly shorter in length and diameter, and the ovarian tissues are considerably smaller (Lee et al. 1998). Histological analysis confirmed the reduced size of the TERC-deficient ovary, and also revealed reduced myometrial thickness with increased atrophy of the smooth muscle cells in the uterine horns (Lee et al. 1998). Intercrosses between wild-type male and TERC-deficient female mice produced an average of 3.9±2.9 fertilized eggs per TERC-deficient female compared to 9.8±3.8 per female in wild-type litters (Lee et al. 1998). The atrophic uterus and compromised ovulation are indicative of decreased steroid hormone production by granulosa cells and decreased oocyte development in telomerase-deficient mice.
High levels of telomerase activity are present in germline stem cells of ovarian origin (Virant-Klun et al. 2009, Zou et al. 2009), which have the capacity to differentiate into oocytes on implantation into the mouse ovary and confer fertility on infertile ovaries (Zou et al. 2009). Consistently, human ovarian germline stem cells with high telomerase activity (scraped from 21 post-menopausal women) proliferate in vitro to produce oocyte-, blastocyst-, and embryo-like structures (Virant-Klun et al. 2009). The human embryonic stem cell lines H9 and hES-NCL1 have been used for differentiation to primordial germ cells (Tilgner et al. 2008). Similarly, mouse embryonic stem cells have been shown to form ovarian follicles and oocyte-like cells (Qing et al. 2007, Virant-Klun et al. 2009). A stem cell-like line with high telomerase activity has been established from preantral follicle cultures from 35 mouse blastocysts from 193 parthenotes (Lee et al. 2008). Mouse embryonic bodies cultured in testis-conditioned medium developed into ovarian structures containing putative oocytes that expressed oocyte-specific markers such as FIGLA (Fig-α) and ZP3, and were surrounded by one to two layers of flattened cells without a visible zona pellucida (Lacham-Kaplan et al. 2006). Collectively, these studies demonstrate that telomerase activity is important in the processes of oocyte development and parthenogenesis.
In determining the effects of telomerase deficiency on germ cell development, researchers have investigated the cleavage and pre-implantation development of embryos derived from in vivo and IVF of sperm with oocytes with or without TERC (Liu et al. 2002). Fertilization of oocytes containing short telomeres by TERC wild-type sperm leads to aberrant cleavage and development of the embryos. More than 50% of the fertilized eggs developed only one pronucleus, and had a high incidence of cytofragmentation (Liu et al. 2002). These data suggest that short telomeres in the oocytes contribute to anomalous fertilization of gametes and abnormal cleavage of embryos. The defects of short telomeres from oocytes appear not to be reset sufficiently following fertilization. So despite significant telomere lengthening during early cleavage development, the data showing that telomere repair is insufficient in the absence of telomerase argue in favor of a significant role for telomerase in the reprogramming of telomere length after fertilization. Under normal conditions, oocytes have shorter telomeres than somatic cells; so telomere reprogramming for lengthening must take place following fertilization. Telomeres are reprogrammed by a recombination-based mechanism during early cleavage cycles, and from the blastocyst stage onwards, telomerase maintains the telomere length established by this alternative mechanism (Liu et al. 2007). Telomerase activity is present at low levels in ovulated oocytes, significantly increased in four-cell rat embryos following fertilization, bovine zygotes, and two- to five-cell embryos, but decreased in six- to eight-cell embryos, morulae, and blastocysts (Eisenhauer et al. 1997, Betts & King 1999, Xu & Yang 2000). Thus, following fertilization, the dramatic telomere remodeling involves both telomerase activity and telomere homologous recombination to enable embryonic development.
Role of telomerase in granulosa cell proliferation during folliculogenesis
Granulosa cells share many characteristics with other epithelia, including their origin as epithelial stem cells. In developing ovarian follicles, where intense cell division is crucial to sustain the estrous cycle and drive oocyte development, the formation and regression of granulosa cells determines the development of germ cells and also the timing and the amount of hormones secretion. Studies have consistently shown that high telomerase activity is present in the smallest preantral follicles, and telomerase activity declines gradually during the transition from small- to medium-sized follicles (Lavranos et al. 1999). In situ hybridization and in situ assay of telomerase activity demonstrate the association of high levels of telomerase activity with the highly proliferative granulosa cells in growing follicles (Lavranos et al. 1999, Yamagata et al. 2002), suggesting that the high proliferative activity of granulosa cells depends on telomerase activity to maintain telomeres. Following ovulation, the follicle develops into the corpus luteum with the granulosa cells to exit the cell cycle undergoing differentiation to luteal cells and apoptosis. Recently, luteinizing granulosa cells isolated from the ovarian follicles of infertile patients have been shown to survive in cultures for a long period of time, and a subset of the differentiated granulosa cells shows in vitro differentiation into other cell types such as neurons, chondrocytes, and osteoblasts (Kossowska-Tomaszczuk et al. 2009). In the large follicles undergoing atretic changes, however, telomerase activity is significantly less, and the inhibition of telomerase is prevented by estrogen administration in rats, suggesting that telomerase withdrawal plays an integral role in granulosa cell apoptosis and follicular atresia induced by estrogen withdrawal (Yamagata et al. 2002).
Analysis (by immunohistochemistry and fluorescence in situ hybridization) of the distribution of TERT during folliculogenesis and its correlation with telomeres in porcine ovarian primary and preantral follicles showed that TERT is expressed in granulosa cells with a typical nuclear location, and that its expression correlates with the size of telomeres. Long telomeres were seen from preantral to antral follicles (Russo et al. 2006). Telomerase activity was detected in two distinct populations of porcine granulosa cells: relatively undifferentiated granulosa cells isolated from small (1–2 mm) antral follicles and differentiated cells with advanced functionality obtained from large (5–7 mm) antral follicles (Tomanek et al. 2008). However, epidermal growth factor stimulated telomerase activity to a greater extent in the granulosa cells isolated from small follicles (Tomanek et al. 2008), suggesting that telomerase activity and telomere lengthening are needed for granulosa cell proliferation, particularly in small, rapidly growing follicles. A cross-sectional study on 54 women ≤37 years of age undergoing IVF with classified occult ovarian insufficiency investigated telomerase activity and telomere length in the granulosa cells acquired at the time of oocyte retrieval (Butts et al. 2009). Lack of granulosa cell telomerase activity and short telomeres were found, suggesting that aberrant telomere homeostasis is associated with occult ovarian insufficiency in young women (Butts et al. 2009).
Thus, abnormalities in telomere length and telomerase activity in human granulosa cells may serve as molecular markers for occult ovarian insufficiency (Butts et al. 2009). Compromised telomerase maintenance of telomeres in granulosa cells might play a causal role in infertility due to occult ovarian insufficiency. The age-related decline in ovarian follicle number has been attributed to estrogen deficiency, but the cause of failed maintenance of telomeres in occult ovarian insufficiency in young women is poorly understood.
Regulation of telomerase activity by estrogen
The ovary is both a major source of estrogen and an important target organ of the hormone. By autocrine mechanisms, estrogen exerts profound effects on the ovary (Weihua et al. 2003). Withdrawal of estrogen induces structural atrophy and dysfunction of the ovaries. Conversely, prolonged exposure to estrogen leads to the risk of certain types of tumors, such as ovarian cancer (Lacey et al. 2002). Estrogen acts by binding to estrogen receptors (ESR) that dimerize and bind to estrogen response elements (ERE) in the promoters of estrogen target genes to regulate gene transcription (Bjornstrom & Sjoberg 2005). The estrogen–ESR complex also transactivates gene expression through interactions with other DNA-binding elements (Huang et al. 2004). Estrogen acts mainly through ESR1 (ERα) and ESR2 (ERβ; Couse et al. 2000), two members of the nuclear receptor superfamily. Both ESR forms are present in granulosa cells of the rodent ovary, with ESR2 suggested to be the predominant form (Palter et al. 2001). While ESR2 is essential for granulosa cell proliferation, ESR1 has specific roles in ovulation, corpus luteum formation, and interstitial glandular cell development (Couse et al. 2000, Dupont et al. 2000). In addition, both ESR1 and ESR2 are implicated in regulating TERT gene transcription in cancer and healthy differentiated cells (Kondoh et al. 2007, Grasselli et al. 2008).
Estrogen activates TERT transcription by a direct action on the gene in ESR-positive human cancer cell lines (Kyo et al. 1999, Misiti et al. 2000, Ling et al. 2006). Moreover, in human OSE cells, a putative ERE identified in the TERT gene promoter binds ESR1 (Misiti et al. 2000). In vivo DNA footprinting showed an ESR-dependent remodeling of the TERT gene promoter in OSE cells (Misiti et al. 2000). Estrogen and ESR1, but not ESR2, markedly stimulate TERT gene promoter activity in an ERE-dependent manner; the estrogen-induced telomerase activity occurs within 3 h of treatment of OSE cells (Misiti et al. 2000).
Although both ESR1 and ESR2 are present in murine ovary, the mouse TERT promoter does not appear to possess a canonical ERE. Estrogen stimulates the expression of transcription factor MYC (c-myc) in rat granulosa cells in vivo (Piontkewitz et al. 1997). The MYC gene is likely to be a direct target of ESR, as stimulation of MYC gene transcription occurs within 1 h of estrogen exposure (Santos et al. 1988). Estrogen stimulation of telomerase activity is dependent on the integrity of the MYC gene in human choriocarcinoma cells (Sarkar et al. 2006). These data suggest that estrogen positively regulates TERT gene transcription by both a direct action on the TERT gene promoter and an indirect mechanism involving the MYC gene. In ovarian cancers, MYC mediates aurora-A kinase-induced telomerase activity (Yang et al. 2004), and the actions of MYC on the TERT gene are further controlled by the tumor suppressor BACR1 (Zhou & Liu 2003) and the transforming growth factor-β family signaling transducer SMAD3 (Li & Liu 2007, Cassar et al. 2009, Liu et al. 2010).
Evidence suggests that prolonged exposure of the ovary to high levels of estrogen is associated with the development of ovarian tumors. Estrogen replacement therapy may also be involved in promoting ovarian cancer (Lacey et al. 2002). The mitogenic effects of estrogen have been studied in various ESR-positive ovarian cancer cell lines, and anti-estrogens, such as tamoxifen and glyceollins, suppress ovarian cancer cell growth in vitro and in animal models. Moreover, estrogen induces ovarian papillary neoplasms in several species, including dogs (Jabara 1962), rabbits (Bai et al. 2000), and pigs (Silva et al. 1998). Expression of ESR even at low levels has been associated with an earlier-stage, higher tumor differentiation and better prognosis (Geisler et al. 1996, Munstedt et al. 2000). These in vitro and in vivo studies support the notion that estrogen plays a significant part in ovarian tumorigenesis by regulating the genes underlying tumor cell immortalization and transformation. Further studies are required to investigate the mechanism by which estrogen induces tumorigenesis. During multi-step ovarian carcinogenesis, many alterations occur in the interactive network of estrogen-responsive genes, including the proto-oncogene MYC, anti-apoptotic protein BCL2, and TERT (Chow et al. 1996). These estrogen-regulated genes may play important roles during ovarian tumorigenesis, such as aberrant epithelial cell proliferation, immortalization, transformation, invasion, and metastasis (Li et al. 2008; Fig. 1).
Reactivation of telomerase in OSE and the role in ovarian regeneration and oncogenesis
The replicative lifespan is limited in cells lacking telomerase activity by replication-associated telomere shortening. Therefore, in the context of ovarian regeneration, telomerase activation might extend the lifespan of replicating OSE and other ovarian progenitor cells. On the other hand, high levels of telomerase activity are a hallmark of cancer, including ovarian epithelial carcinoma. Accumulating data indicate that telomerase activation is an early event in ovarian carcinogenesis. At present, it is unclear whether telomerase activation preserves the non-malignant phenotype and replicative longevity of ovarian reproductive cells or constitutes an early alteration needed for subsequent unlimited proliferation and malignant transformation.
Increasing telomerase activity by overexpressing TERT alone (Alvero et al. 2004), TERT plus SV40 large T antigen (Davies et al. 2003, Kusakari et al. 2003), or TERT plus human papillomavirus-16 antigens (Maeda et al. 2005) extended the proliferative lifespan of human OSE cells toward immortality. These studies independently showed the requirement for inactivation of the p53 and RB1 (Rb) tumor suppressor mechanisms in TERT-induced cell immortalization. To establish whether a single disruption of either the p53 or RB1 pathway would promote telomerase-induced human OSE cell immortalization, Liu et al. used RNAi technology to knock down either RB1 or p53 in combination with ectopic expression of the TERT in human OSE cells. Significantly, knockdown of either RB1 (Yang et al. 2007a) or p53 (Yang et al. 2007b) in the presence of TERT is sufficient to immortalize human OES cells. It was later shown that human OSE cells could be immortalized by TERT expression without functional disruption of RB1 and p53 (Li et al. 2007). Thus, while telomerase activity is indispensable in the immortalization step of human OSE, p53 and RB1 are not, at least under certain conditions. Furthermore, TERT-immortalized human OSE cells have lengthened telomeres, are diploid without chromosomal instability, and maintain epithelial characters without tumorigenicity (Kusakari et al. 2003, Yang et al. 2007a, 2007b, Sasaki et al. 2009).
The importance of telomerase in tumorigenesis has been fully recognized (Shay & Bacchetti 1997, Shay & Wright 2005). Telomerase activity and dysfunctional telomeres have been demonstrated in human ovarian carcinoma (Counter et al. 1994, Baykal et al. 2004). Telomerase is not present in normal OSE and premalignant lesions, but is up-regulated in 90–97% of ovarian carcinomas (Gorham et al. 1997, Yokoyama et al. 1998, Baykal et al. 2004, Bayne & Liu 2005, Bayne et al. 2007, Shay & Wright 2007). By in situ hybridization, 90% (28 of 31) of ovarian carcinomas expressed TERT. However, TERT gene expression has also been found in benign epithelial tissues, so it is unlikely to be a useful marker for differentiating between benign and malignant tumors (Baykal et al. 2004).
Ovarian cancer is highly lethal. Based on the pattern of epithelial differentiation and morphological criteria, there are four major subtypes of ovarian epithelial cancer: serous, mucinous, endometrioid, and clear cell. Serous tumors are most common, representing about 50% of all ovarian cancers derived from epithelial cells. Endometrioid adenocarcinomas account for 20–25% of ovarian cancer, and clear cell and mucinous adenocarcinomas account for <10% of ovarian cancer (Soslow 2008). Aggressive ovarian tumors result from the uncontrolled proliferation of ovarian stem cells (Bapat et al. 2005, Szotek et al. 2006), and epithelial ovarian cancer is thought to arise from OSE (Young et al. 2004). Consistently, TERT- and SV40 large T antigen-immortalized human OSE cells undergo anchorage-independent growth in vitro and tumorigenesis in mice upon forced expression of either oncogene ERBB2 (c-erbB-2) or mutant HRAS (Ha-Ras; Kusakari et al. 2003). Recently, oncogenic transformation of human OSE cells has been investigated with defined cellular oncogenes (Sasaki et al. 2009). Non-viral transduction of primary human OSE cells with human genes encoding mutant cyclin-dependent kinase 4, cyclinD1, and TERT establishes normal diploid OSE cells with extended proliferation without chromosomal instability (Sasaki et al. 2009). Inactivation of p53 plus oncogenic transduction of the KRAS oncogene did not lead to tumor formation, but the additional transduction of AKT, or combined transduction of MYC with BCL2, resulted in tumor formation (Sasaki et al. 2009). The findings of Sasaki et al. (2009) demonstrate that the presence of telomerase, together with tumor suppressor p53, proto-oncogenes RAS and MYC, and the anti-apoptotic pro-survival BCL2, causes the development of ovarian epithelial cancer in vitro.
The future
The discoveries from various studies indicating that telomerase has an indispensable role in ovarian germ cell development highlight a regulatory mechanism through which telomerase determines different cell proliferative potentials by regulating ovarian telomere homeostasis. The regulation of telomerase activity may thus provide a means to modulate germ cell development in the ovary. The regulation of telomerase activity by estrogen implies a pathway through which telomere length and cell proliferative potential can be modulated to support the various functions of the ovary. The regulation of the telomerase–telomere axis by estrogen may also reflect how granulosa cells facilitate germ cell development and how aging occurs when estrogen is withdrawn. Further studies are required to determine a potential intermediate role of telomerase activity in estrogen deficiency-induced ovarian aging. Elucidation of a temporal and spatial mechanism of estrogen regulation of telomerase activity (and the roles of other hormones in telomere remodeling) would provide a basis for the design of interventions to treat ovarian aging and infertility.
However, it is clear that telomerase is a double-edged sword in germ cell development and neoplastic immortalization. The ability to control telomerase activity in OSE could allow the prevention and treatment of ovarian cancers derived from these cells. Multiple signaling molecules and pathways could be involved in telomerase activation during ovarian cancer cell immortalization. They warrant investigation of their relationships to OSE cell immortalization and transformation as seen in ovarian cancer.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from the National Health and Medical Research Council of Australia and Cancer Council of Victoria, Australia.
References
Adhikari D & Liu K 2009 Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocrine Reviews 30 438–464.
Alvero AB, Fishman DA, Qumsiyeh MB, Garg M, Kacinski BM & Sapi E 2004 Telomerase prolongs the lifespan of normal human ovarian surface epithelial cells without inducing neoplastic phenotype. Journal of the Society for Gynecologic Investigation 11 553–561.
Auersperg N, Wong AST, Choi K-C, Kang SK & Leung PCK 2001 Ovarian surface epithelium: biology, endocrinology, and pathology. Endocrine Reviews 22 255–288.
Bai W, Oliveros-Saunders B, Wang Q, Acevedo-Duncan ME & Nicosia SV 2000 Estrogen stimulation of ovarian surface epithelial cell proliferation. In Vitro Cellular & Developmental Biology. Animal 36 657–666.
Bapat SA, Mali AM, Koppikar CB & Kurrey NK 2005 Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Research 65 3025–3029.
Baykal A, Thompson JA, Xu XC, Hahn WC, Deavers MT, Malpica A, Gershenson DM, Silva EG & Liu J 2004 In situ human telomerase reverse transcriptase expression pattern in normal and neoplastic ovarian tissues. Oncology Reports 11 297–302.
Bayne S & Liu JP 2005 Hormones and growth factors regulate telomerase activity in ageing and cancer. Molecular and Cellular Endocrinology 240 11–22.
Bayne S, Jones ME, Li H & Liu JP 2007 Potential roles for estrogen regulation of telomerase activity in aging. Annals of the New York Academy of Sciences 1114 48–55.
Betts DH & King WA 1999 Telomerase activity and telomere detection during early bovine development. Developmental Genetics 25 397–403.
Bjornstrom L & Sjoberg M 2005 Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Molecular Endocrinology 19 833–842.
Blackburn EH 2001 Switching and signaling at the telomere. Cell 106 661–673.
Blasco MA, Funk W, Villeponteau B & Greider CW 1995 Functional characterization and developmental regulation of mouse telomerase RNA. Science 269 1267–1270.
Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA & Greider CW 1997 Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91 25–34.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C-P, Morin GB, Harley CB, Shay JW, Lichtsteiner S & Wright WE 1998 Extension of life-span by introduction of telomerase into normal human cells. Science 279 349–352.
Butts S, Riethman H, Ratcliffe S, Shaunik A, Coutifaris C & Barnhart K 2009 Correlation of telomere length and telomerase activity with occult ovarian insufficiency. Journal of Clinical Endocrinology and Metabolism 94 4835–4843.
Cassar L, Nicholls C, Pinto AR, Li H & Liu JP 2009 Bone morphogenetic protein-7 induces telomerase inhibition, telomere shortening, breast cancer cell senescence, and death via Smad3. FASEB Journal 23 1880–1892.
Chow SN, Chien CH & Chen CT 1996 Molecular biology of human ovarian cancer. International Surgery 81 152–157.
Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ & Reddel RR 2007 Protein composition of catalytically active human telomerase from immortal cells. Science 315 1850–1853.
Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB & Bacchetti S 1992 Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO Journal 11 1921–1929.
Counter CM, Hirte HW, Bacchetti S & Harley CB 1994 Telomerase activity in human ovarian carcinoma. PNAS 91 2900–2904.
Couse JF, Curtis Hewitt S & Korach KS 2000 Receptor null mice reveal contrasting roles for estrogen receptor α and β in reproductive tissues. Journal of Steroid Biochemistry and Molecular Biology 74 287–296.
Davies BR, Steele IA, Edmondson RJ, Zwolinski SA, Saretzki G, von Zglinicki T & O'Hare MJ 2003 Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen. Experimental Cell Research 288 390–402.
Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P & Mark M 2000 Effect of single and compound knockouts of estrogen receptors alpha (ERα) and beta (ERβ) on mouse reproductive phenotypes. Development 127 4277–4291.
Eisenhauer KM, Gerstein RM, Chiu CP, Conti M & Hsueh AJ 1997 Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos. Biology of Reproduction 56 1120–1125.
Fan H-Y, Liu Z, Cahill N & Richards JS 2008 Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells. Molecular Endocrinology 22 2128–2140.
Geisler JP, Wiemann MC, Miller GA & Geisler HE 1996 Estrogen and progesterone receptor status as prognostic indicators in patients with optimally cytoreduced stage IIIc serous cystadenocarcinoma of the ovary. Gynecologic Oncology 60 424–427.
Gleicher N, Weghofer A, Oktay K & Barad D 2009 Do etiologies of premature ovarian aging (POA) mimic those of premature ovarian failure (POF)? Human Reproduction 24 2395–2400.
Gorham H, Yoshida K, Sugino T, Marsh G, Manek S, Charnock M, Tarin D & Goodison S 1997 Telomerase activity in human gynaecological malignancies. Journal of Clinical Pathology 50 501–504.
Grasselli A, Nanni S, Colussi C, Aiello A, Benvenuti V, Ragone G, Moretti F, Sacchi A, Bacchetti S & Gaetano C et al. 2008 Estrogen receptor-α and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase. Circulation Research 103 34–42.
Greenberg RA, Allsopp RC, Chin L, Morin GB & DePinho RA 1998 Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation. Oncogene 16 1723–1730.
Greider CW & Blackburn EH 1985 Identification of a specific telomere terminal transferase enzyme with two kinds of primer specificity. Cell 51 405–413.
Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H & de Lange T 1999 Mammalian telomeres end in a large duplex loop. Cell 97 503–514.
Hande MP, Samper E, Lansdorp P & Blasco MA 1999 Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. Journal of Cell Biology 144 589–601.
Herrera E, Samper E & Blasco MA 1999 Telomere shortening in mTR−/− embryos is associated with failure to close the neural tube. EMBO Journal 18 1172–1181.
Huang J, Li X, Yi P, Hilf R, Bambara RA & Muyan M 2004 Targeting estrogen responsive elements (EREs): design of potent transactivators for ERE-containing genes. Molecular and Cellular Endocrinology 218 65–78.
Jabara AG 1962 Induction of canine ovarian tumours by diethylstilboestrol and progesterone. Australian Journal of Experimental Biology and Medical Science 40 139–152.
Kondoh K, Tsuji N, Asanuma K, Kobayashi D & Watanabe N 2007 Inhibition of estrogen receptor β-mediated human telomerase reverse transcriptase gene transcription via the suppression of mitogen-activated protein kinase signaling plays an important role in 15-deoxy-Delta(12,14)-prostaglandin J(2)-induced apoptosis in cancer cells. Experimental Cell Research 313 3486–3496.
Kossowska-Tomaszczuk K, De Geyter C, De Geyter M, Martin I, Holzgreve W, Scherberich A & Zhang H 2009 The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells 27 210–219.
Kusakari T, Kariya M, Mandai M, Tsuruta Y, Hamid AA, Fukuhara K, Nanbu K, Takakura K & Fujii S 2003 C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro. British Journal of Cancer 89 2293–2298.
Kyo S, Takakura M, Kanaya T, Zhuo W, Fujimoto K, Nishio Y, Orimo A & Inoue M 1999 Estrogen activates telomerase. Cancer Research 59 5917–5921.
Lacey JV Jr, Mink PJ, Lubin JH, Sherman ME, Troisi R, Hartge P, Schatzkin A & Schairer C 2002 Menopausal hormone replacement therapy and risk of ovarian cancer. Journal of the American Medical Association 288 334–341.
Lacham-Kaplan O, Chy H & Trounson A 2006 Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 24 266–273.
de Lange T 2005 Shelterin: the protein complex that shapes and safeguards human telomeres. Genes and Development 19 2100–2110.
Laviolette LA, Garson K, Macdonald EA, Senterman MK, Courville K, Crane CA & Vanderhyden BC 2010 17β-Estradiol accelerates tumor onset and decreases survival in a transgenic mouse model of ovarian cancer. Endocrinology 151 929–938.
Lavranos TC, Mathis JM, Latham SE, Kalionis B, Shay JW & Rodgers RJ 1999 Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles. Biology of Reproduction 61 358–366.
Lee HW, Blasco MA, Gottlieb GJ, Horner JW II, Greider CW & DePinho RA 1998 Essential role of mouse telomerase in highly proliferative organs. Nature 392 569–574.
Lee ST, Choi MH, Lee EJ, Gong SP, Jang M, Park SH, Jee H, Kim DY, Han JY & Lim JM 2008 Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis. Fertility and Sterility 90 1910–1920.
Leri A, Franco S, Zacheo A, Barlucchi L, Chimenti S, Limana F, Nadal-Ginard B, Kajstura J, Anversa P & Blasco MA 2003 Ablation of telomerase and telomere loss leads to cardiac dilatation and heart failure associated with p53 upregulation. EMBO Journal 22 131–139.
Li H & Liu JP 2007 Mechanisms of action of TGF-β in cancer: evidence for Smad3 as a repressor of the hTERT gene. Annals of the New York Academy of Sciences 1114 56–68.
Li NF, Broad S, Lu YJ, Yang JS, Watson R, Hagemann T, Wilbanks G, Jacobs I, Balkwill F & Dafou D et al. 2007 Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression. Cell Proliferation 40 780–794.
Li YF, Hu W, Fu SQ, Li JD, Liu JH & Kavanagh JJ 2008 Aromatase inhibitors in ovarian cancer: is there a role? International Journal of Gynecological Cancer 18 600–614.
Ling S, Zhou L, Li H, Dai A, Liu JP, Komesaroff PA & Sudhir K 2006 Effects of 17β-estradiol on growth and apoptosis in human vascular endothelial cells: influence of mechanical strain and tumor necrosis factor-α. Steroids 71 799–808.
Liu L, Blasco M, Trimarchi J & Keefe D 2002 An essential role for functional telomeres in mouse germ cells during fertilization and early development. Developmental Biology 249 74–84.
Liu L, Bailey SM, Okuka M, Munoz P, Li C, Zhou L, Wu C, Czerwiec E, Sandler L & Seyfang A et al. 2007 Telomere lengthening early in development. Nature Cell Biology 9 1436–1441.
Liu JP, Nicholls C, Chen SM, Li H & Tao ZZ 2010 Strategies of treating cancer by cytokine regulation of chromosome end remodelling. Clinical and Experimental Pharmacology and Physiology 37 88–92.
Lue NF 2009 Closing the feedback loop: how cells “count” telomere-bound proteins. Molecular Cell 33 413–414.
Maeda T, Tashiro H, Katabuchi H, Begum M, Ohtake H, Kiyono T & Okamura H 2005 Establishment of an immortalised human ovarian surface epithelial cell line without chromosomal instability. British Journal of Cancer 93 116–123.
Marx J 2008 Developmental biology. Aging of the ovary linked to PTEN pathway. Science 319 558–559.
McGee EA & Hsueh AJW 2000 Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews 21 200–214.
Misiti S, Nanni S, Fontemaggi G, Cong YS, Wen J, Hirte HW, Piaggio G, Sacchi A, Pontecorvi A & Bacchetti S et al. 2000 Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells. Molecular and Cellular Biology 20 3764–3771.
Munstedt K, Steen J, Knauf AG, Buch T, von Georgi R & Franke FE 2000 Steroid hormone receptors and long term survival in invasive ovarian cancer. Cancer 89 1783–1791.
Olovnikov AM 1996 Telomeres, telomerase, and aging: origin of the theory. Experimental Gerontology 31 443–448.
Palter SF, Tavares AB, Hourvitz A, Veldhuis JD & Adashi EY 2001 Are estrogens of import to primate/human ovarian folliculogenesis? Endocrine Reviews 22 389–424.
Piontkewitz Y, Sundfeldt K & Hedin L 1997 The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo. Journal of Endocrinology 152 395–406.
Qing T, Shi Y, Qin H, Ye X, Wei W, Liu H, Ding M & Deng H 2007 Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 75 902–911.
Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, Shen Y, Du C, Tang W, Hamalainen T & Peng SL et al. 2008 Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science 319 611–613.
Russo V, Berardinelli P, Martelli A, Di Giacinto O, Nardinocchi D, Fantasia D & Barboni B 2006 Expression of telomerase reverse transcriptase subunit (TERT) and telomere sizing in pig ovarian follicles. Journal of Histochemistry and Cytochemistry 54 443–455.
Santos GF, Scott GK, Lee WM, Liu E & Benz C 1988 Estrogen-induced post-transcriptional modulation of c-myc proto-oncogene expression in human breast cancer cells. Journal of Biological Chemistry 263 9565–9568.
Sarkar P, Shiizaki K, Yonemoto J & Sone H 2006 Activation of telomerase in BeWo cells by estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin in co-operation with c-Myc. International Journal of Oncology 28 43–51.
Sasaki R, Narisawa-Saito M, Yugawa T, Fujita M, Tashiro H, Katabuchi H & Kiyono T 2009 Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes. Carcinogenesis 30 423–431.
Shay JW 1999 Toward identifying a cellular determinant of telomerase repression [editorial; comment]. Journal of the National Cancer Institute 91 4–6.
Shay JW & Bacchetti S 1997 A survey of telomerase activity in human cancer. European Journal of Cancer 33 787–791.
Shay JW & Wright WE 2005 Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 26 867–874.
Shay JW & Wright WE 2007 Hallmarks of telomeres in ageing research. Journal of Pathology 211 114–123.
Silva EG, Tornos C, Deavers M, Kaisman K, Gray K & Gershenson D 1998 Induction of epithelial neoplasms in the ovaries of guinea pigs by estrogenic stimulation. Gynecologic Oncology 71 240–246.
Soslow RA 2008 Histologic subtypes of ovarian carcinoma: an overview. International Journal of Gynecological Pathology 27 161–174.
Szostak JW & Blackburn EH 1982 Cloning yeast telomeres on linear plasmid vectors. Cell 29 245–255.
Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R, Dombkowski D, Preffer F, Maclaughlin DT & Donahoe PK 2006 Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian inhibiting substance responsiveness. PNAS 103 11154–11159.
Tilgner K, Atkinson SP, Golebiewska A, Stojkovic M, Lako M & Armstrong L 2008 Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 26 3075–3085.
Tomanek M, Chronowska E, Kott T & Czernekova V 2008 Telomerase activity in pig granulosa cells proliferating and differentiating in vitro. Animal Reproduction Science 104 284–298.
Venteicher AS, Abreu EB, Meng Z, McCann KE, Terns RM, Veenstra TD, Terns MP & Artandi SE 2009 A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 323 644–648.
Virant-Klun I, Rozman P, Cvjeticanin B, Vrtacnik-Bokal E, Novakovic S, Rulicke T, Dovc P & Meden-Vrtovec H 2009 Parthenogenetic embryo-like structures in the human ovarian surface epithelium cell culture in postmenopausal women with no naturally present follicles and oocytes. Stem Cells and Development 18 137–150.
Weihua Z, Andersson S, Cheng G, Simpson ER, Warner M & Gustafsson JA 2003 Update on estrogen signaling. FEBS Letters 546 17–24.
Wong JM & Collins K 2003 Telomere maintenance and disease. Lancet 362 983–988.
Wong KK, Maser RS, Bachoo RM, Menon J, Carrasco DR, Gu Y, Alt FW & DePinho RA 2003 Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing. Nature 421 643–648.
Xu J & Yang X 2000 Telomerase activity in bovine embryos during early development. Biology of Reproduction 63 1124–1128.
Yamagata Y, Nakamura Y, Umayahara K, Harada A, Takayama H, Sugino N & Kato H 2002 Changes in telomerase activity in experimentally induced atretic follicles of immature rats. Endocrine Journal 49 589–595.
Yang H, Ou CC, Feldman RI, Nicosia SV, Kruk PA & Cheng JQ 2004 Aurora-A kinase regulates telomerase activity through c-Myc in human ovarian and breast epithelial cells. Cancer Research 64 463–467.
Yang G, Rosen DG, Colacino JA, Mercado-Uribe I & Liu J 2007a Disruption of the retinoblastoma pathway by small interfering RNA and ectopic expression of the catalytic subunit of telomerase lead to immortalization of human ovarian surface epithelial cells. Oncogene 26 1492–1498.
Yang G, Rosen DG, Mercado-Uribe I, Colacino JA, Mills GB, Bast RC Jr, Zhou C & Liu J 2007b Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells. Carcinogenesis 28 174–182.
Yokoyama Y, Takahashi Y, Shinohara A, Lian Z & Tamaya T 1998 Telomerase activity in the female reproductive tract and neoplasms. Gynecologic Oncology 68 145–149.
Young TW, Mei FC, Yang G, Thompson-Lanza JA, Liu J & Cheng X 2004 Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry. Cancer Research 64 4577–4584.
Zhao Y, Hoshiyama H, Shay JW & Wright WE 2008 Quantitative telomeric overhang determination using a double-strand specific nuclease. Nucleic Acids Research 36 e14.
Zhou C & Liu J 2003 Inhibition of human telomerase reverse transcriptase gene expression by BRCA1 in human ovarian cancer cells. Biochemical and Biophysical Research Communications 303 130–136.
Zou K, Yuan Z, Yang Z, Luo H, Sun K, Zhou L, Xiang J, Shi L, Yu Q & Zhang Y et al. 2009 Production of offspring from a germline stem cell line derived from neonatal ovaries. Nature Cell Biology 11 631–636.
