Minireview. Telomerase Activity and Telomere Length in Male Germ Cells. Downloaded from Saffet Ozturk 1

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1 BIOLOGY OF REPRODUCTION (2015) 92(2):53, 1 11 Published online before print 7 January DOI /biolreprod Minireview Telomerase Activity and Telomere Length in Male Germ Cells Saffet Ozturk 1 Department of Histology and Embryology, Akdeniz University, School of Medicine, Campus, Antalya, Turkey ABSTRACT Telomeres are located at the outermost ends of all eukaryotic chromosomes and provide for the maintenance of genomic stability and integrity during the life span of organisms. The length of telomeres shortens due to each round of DNA replication, genotoxic insults, and/or reactive oxygen species. To counteract this shortening, certain types of cells, including stem cells, male/female germline cells, granulosa cells, early embryos, and most cancerous cells, express an enzyme known as telomerase, which has the potential of restoring the shortened telomeres. Presence of telomerase activity in the male germ cells ensures maintenance of telomere length at maximum levels during spermatogenesis despite telomere attrition due to DNA replication or other genotoxic factors. In this review, telomerase activity and telomere length in mammalian male germ cells during spermatogenesis are evaluated in detail based on the studies in this field. Also, the relationship between telomerase activity/telomere length and development of male infertility is comprehensively discussed. infertility, male germ cell, spermatogenesis, telomerase activity, telomeres INTRODUCTION Telomeres are evolutionarily conserved tandem repeats of noncoding double-stranded DNA sequences (TTAGGG) n, located at the outermost ends of all eukaryotic chromosomes [1]. The 3 0 end of telomeres includes a short single-stranded guanine-rich tail, which is known as the G-overhang or G-tail [2]. The G-overhang enters into the double-stranded DNA duplex to form telomere loop (T-loop) and displacement loop (D-loop) structures at the end of telomeres. Basically, these exclusive structures protect the G-overhang tail from being sensed as a DNA break, and they interact with specific telomere-associated proteins to stabilize the chromosome termini. Moreover, they play a critical role in the telomere DNA elongation process by serving as primers for telomerase and controlling access of certain factors, such as transcription factors and telomere-associated proteins, to the telomeric sites [3]. For many years, telomeric DNA was accepted as transcriptionally silent sequences; however, several recent 1 Correspondence: Saffet Ozturk, Department of Histology and Embryology, Akdeniz University, School of Medicine, Campus, Antalya 07070, Turkey. sozturk@akdeniz.edu.tr Received: 4 August First decision: 13 September Accepted: 29 December Ó 2015 by the Society for the Study of Reproduction, Inc. eissn: ISSN: studies have revealed that it is actively transcribed into telomere repeat-containing RNAs (TERRAs). The TERRAs are largely composed of UUAGGG repeats and also contain subtelomeric sequences. They localize at the ends of chromosomes in the nuclei, and their lengths range from 100 bp to 9 kb [4, 5]. Although the TERRA molecules are detected at the telomeres of human fetal oocytes at different substages of prophase I [5], their expression pattern, cellular localization, and possible roles in the male germ cells, early embryos, and somatic cells remain to be comprehensively investigated. In addition to DNA sequences of telomeres, telomereassociated proteins, such as TRF1, TRF2, POT1, TIN2, TPP1, RAP1, and TRF-linked proteins, are also key components of telomere structure. The most important functions of these telomere-associated proteins are maintenance of the genomic integrity by forming caps at the chromosome ends and participation in regulation of telomere elongation [6]. The six special telomere-associated proteins TRF1, TRF2, POT1, TIN2, RAP1, and TPP1 are defined as the shelterin complex or the telosome. This particular complex plays an essential role in sensing the stability and length of telomeres [7]. The telomeric repeat-binding factors 1 (TRF1; also known as TERF1) and 2 (TRF2; also known as TERF2) bind to the double-stranded segment of telomeric DNA. The TRF1 protein regulates telomere elongation via inhibiting access of the enzyme telomerase to telomeric sites [8]. As expected, overexpression of the TRF1 protein leads to progressive telomere shortening, and consistently dominant-negative Trf1 mutant causes excessive telomere elongation [9, 10]. Similar to the TRF1 protein, expression levels of the TRF2 protein adversely correlate with telomere lengthening. Furthermore, TRF2 prevents formation of any fusion between chromosome ends and provides for maintenance of the T-loop structure [10, 11]. The dominant-negative mutant allele of Trf2 results in activation of DNA repair system, chromosome instability, and chromosome end-to-end fusion, possibly due to telomere shortening down to critical levels [12]. Taken together, telomeres in combination with telomereassociated proteins contribute to maintenance of genomic integrity in the eukaryotic cells. They also play roles in movement, localization, and anchoring of the chromosomes to the nuclear membrane. Additionally, during cell division, telomeres mediate the pairing of homologous chromosomes, synapsis formation, and homologous recombination during cell division [13, 14]. Notably, detailed information on telomere structure and telomere-associated proteins can be found in three recently published review articles [12, 15, 16]. 1 Article 53

2 OZTURK THE ENZYME TELOMERASE SYNTHESIZES THE TELO- MERIC REPEATS A portion of telomeres in a replicating cell inevitably shortens at the end of each DNA replication process due to the inability of DNA replication machinery to replenish this lost portion by using a conventional DNA polymerase enzyme [17 19]. Therefore, telomeric DNA progressively diminishes from 50 to 1500 bp as a result of each cell division [20]. In addition to DNA replication, genetic predisposition, lifestyle factors, psychological stress, exogenous and endogenous genotoxic insults, and long-term exposure to reactive oxygen species (ROS) may deplete telomeres in both dividing and nondividing cells [21 24]. The main solution for cells that have critically short telomeres is to elongate the telomeres by using the enzyme telomerase or the alternative lengthening of telomeres (ALT) mechanism. Telomerase is uniquely specialized to synthesize telomeric DNA sequences de novo by adding tandem arrays of TTAGGG repeats onto 3 0 ends of chromosomes during genomic DNA replication [25, 26]. The enzyme telomerase is composed of a ribonucleoprotein complex that, in its active form, consists of two principal subunits called the telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) [25, 27]. In addition to the two main subunits, this complex also includes an associated protein subunit known as dyskerin encoded by the DKC1 gene [25]. The TERT uses the TERC subunit as an RNA template complementary to the 6-nt telomeric sequences in order to synthesize telomeric repeats [28, 29]. As expected, the TERT subunit is localized in the cell nucleus, and it does not displace its subcellular localization during cell-cycle progression [30]. The TERC subunit is a single-stranded RNA and is transcribed at higher levels in the telomerase-positive cells than in the telomerase-negative cells. Similarly, it resides in the cell nucleus [31, 32]. Telomerase is exclusively expressed in highly proliferative cells, including germline cells, granulosa cells, early embryos, stem cells, activated lymphocytes, hematopoietic cells, basal epidermal cells, immortal cells, and certain types of cancer cell populations [33 36]. Although it is diminished in most cells during embryonic development [37 39], a large number of mouse somatic cells show telomerase expression at low levels [40]. Measuring protein and mrna levels of the TERT gene in most cases is a reliable biomarker to determine the telomerase activity [41]. Although telomerase expression is controlled both transcriptionally and translationally, the TERT mrna profiles correlate closely with the telomerase activity [31, 42]. However, the TERC level does not seem to be a reasonable marker of telomerase activity because it is ubiquitously transcribed in most cell types, even in telomerase-negative cells [41]. Apart from telomere lengthening by the enzyme telomerase, the ALT process is also available for telomere extension in several somatic and germ cells [43 45]. Briefly, telomere elongation by the ALT mechanism is carried out by two consecutive steps: telomere sister chromatid exchange (T-SCE) and DNA replication. Whereas T-SCE provides for telomeric exchange between nonhomologous chromosomes, DNA replication machinery extends the short telomeric strands by using long telomeric sequences as a template [46]. Although the presence of this mechanism has been identified in particular somatic cells [47, 48] and early embryos [43], its molecular details are not known with any clarity in male germ cells. TELOMERE LENGTH AND TELOMERASE ACTIVITY IN TESTICULAR TISSUES Briefly, spermatogenesis, which occurs within seminiferous tubules of the testes of the sexually mature male, can be divided into three main phases: proliferative (spermatogonial), meiotic, and spermiogenesis [49, 50]. In the proliferative phase, spermatogonial cells located adjacent to the seminiferous tubules undergo several rounds of mitotic divisions and then differentiate into primary spermatocytes. Subsequently, primary spermatocytes enter the first meiotic division and form secondary spermatocytes after completing the first meiotic division in the meiotic stage. Next, the secondary spermatocytes experience a second meiotic division, giving rise to haploid round spermatids [51 53]. During spermiogenesis, round spermatids undergo morphological changes, such as acrosome formation, nuclear condensation, and flagella development, and finally become spermatozoa [49]. The maturation of the spermatids into spermatozoa includes 16 steps in mice. Round spermatids are created at steps 1 7, and elongating spermatids are formed during steps During steps 13 16, elongated spermatids are developed, and finally, mature spermatozoa are generated [54]. To summarize, differentiation of spermatogonial cells into spermatozoa includes many consecutive mitotic and two meiotic divisions. Also, many intracellular and intercellular interactions must occur during this process to complete spermatogenesis correctly [55, 56]. Because spermatogenic cell types in rodent testes are located in distinct compartments of the seminiferous tubules, they can be easily characterized. Therefore, most of the investigations on the spatial and temporal telomerase expression and telomere length in the spermatogenic cells have been performed in rodent testes. Greenberg et al. [57] and Martin- Rivera et al. [30] found that mature mouse testes expressed both the TERT protein and mrna and therefore exhibited telomerase activity. Additionally, the presence of telomerase activity in a subset of somatic tissues, such as thymus, spleen, intestine, and liver, was identified in both studies. Although mature mouse testes had telomerase activity, it was absent in the immature mouse testes at the ages of 2 and 4 wk [58]. As expected, telomerase activity was also detected at the later developmental stages of the mouse testes at the ages of 6, 8, 10, 13, and 16 wk [58]. These findings are partially contradicted by Yamamoto et al. [59], who found telomerase activity not only in the mature mouse testes at the age of 10 wk, but also in the immature postnatal mouse testes at the ages of 17 and 22 days. When the Tert mrna expression was analyzed in the developing postnatal mouse testes at the ages of 6, 17, 29, and 46 days as well as 5 mo, the highest level was found in the 6- day-old testes. Although low levels of the Tert mrna were observed in the testes from 17-, 29-, and 46-day-old mice, a slightly increased Tert mrna expression was detected in the 5-mo-old adult mouse testis [60]. Additionally, Weise and Gunes [60] demonstrated that early postnatal mouse testes at the ages of 6, 17, and 29 days possessed telomerase activity, which is similar to the finding by Yamamoto et al. [59], but in sharp contrast to the previous report [58]. As mentioned, Prowse and Greider [58] found an absence of telomerase activity in the immature mouse testes. This discrepancy between former [58] and later [59, 60] studies may originate from the use of different mouse strains and/or methodological difference for measuring telomerase activity (Table 1). In parallel to the telomerase activity, the terminal restriction fragment (TRF) length analysis has documented that the telomeres of mature mouse testes (age, 12 wk), including all 2 Article 53

3 TELOMERASE AND TELOMERE LENGTH IN MALE GERM CELLS TABLE 1. Schematic representation of telomerase activity levels in the mammalian testes and male germ cells. Testis a Spermatogenic cells a,b Species Immature Mature SG SC RS EgS ES Sperm Method c Parameter d Reference Mouse ND þþþþ NA NA NA NA NA NA TRAP T. activity [58] þ? þ? þ? þ? þ? þ? þ? ND TRAP T. activity [59] þþþþ þþþþ NA NA NA NA NA NA TRAP T. activity [60] NA NA þþ þ/ þ/ þþþþ þ/ NA In situ TRAP T. activity [61] NA NA þþ þþþþ ND þþþþ þþþ ND IHC TERT [61] þþþþ þþþ NA NA NA NA NA NA RT-PCR Tert [60] Rat þþþþ þþ þþþþ þþ þþþ NA NA ND TRAP T. activity [68] NA NA þþþþ þþ þþþ NA NA ND TRAP T. activity [71] NA NA NA þþþ þþþþ NA NA ND TRAP T. activity [74] Human þþ þþþþ NA NA NA NA NA ND TRAP T. activity [39] NA NA þþþþ NA NA NA NA NA TRAP T. activity [70] þþ þþ þ þþþ NA NA NA ND ISH TERC [69] a The lowest and the highest telomerase activity are denoted by þ and þþþþ, respectively. Very weak telomerase activity is denoted by þ/. NA, not analyzed; ND, not detected;?, no quantitative comparison among spermatogenic cells and testicular tissues. b SG, spermatogonia, SC, spermatocytes; RS, round spermatids; EgS, elongating spermatids; ES, elongated spermatids. c TRAP, telomeric repeat amplification protocol; IHC, immunohistochemistry; ISH, in situ hybridization; RT-PCR, reverse transcription-polymerase chain reaction. d T. activity, telomerase activity; TERT and Tert, telomerase reverse transcriptase; TERC, telomerase RNA component. spermatogenic cell types, were longer than those in sexually immature testes (age, 4 wk), which did not have elongated spermatids and spermatozoa [61]. The presence of telomerase activity in either immature or mature mouse testes probably contributes to increased telomeres in the mature mouse testes. It is noteworthy that although the timing of spermatozoa production in mouse testes can slightly change depending on the mouse strains, sperm generation and sexual maturity generally begin at approximately Day 35 of postnatal development in mice [50, 58, 62]. Also, participation of the spermatogenic cell types during postnatal testicular development is being based on the developmental stages [63, 64]. Immunohistochemical analysis on the mature mouse testis sections showed that the TERT protein was expressed in the spermatogonia, early spermatocytes, and elongating spermatids at stage X of the seminiferous epithelial cycle, whereas the TERT signal was not detected in the pachytene spermatocytes and round spermatids [61]. However, no TERT expression was found in certain elongating spermatids at different steps of the seminiferous epithelial cycle, which indicated a mosaic TERT expression in the elongating spermatids [61, 65]. Fluorescence in situ hybridization (FISH) analysis of testis sections revealed that the elongated spermatids had longer telomere length than any of other spermatogenic cell types [61] (Table 2). The most interesting aspect of this finding is that although the elongated spermatids have longer telomeres, they do not undergo any DNA replication process, which facilitates telomere elongation by the enzyme telomerase and/or ALT mechanism. Therefore, telomere lengthening in these cells seems to be carried out by a distinct mechanism, which has not yet been identified. However, a limited part of the sperm nuclear genome is packaged with histone, which may facilitate telomeric elongation [66, 67]. Importantly, because the nuclear genome in the spermatids and spermatozoa is strictly packed with protamine, it is possible that the length of telomeres in these cells could not be measured correctly due to lack of easy access of the specific DNA probes to the telomeric regions. In contrast to the findings of some studies on the postnatal mouse testes, telomerase activity in the rat testes was detectable at birth, and it progressively increased during early testicular development from 1 to 4 wk of age. However, it gradually decreased in 5- to 10-wk-old postnatal rat testes [68]. Conversely, a mature human testis from a 37-yr-old man possessed predominantly higher telomerase activity than a fetal testis. Furthermore, telomerase activity was detected at low levels in the newborn human testes [39]. In the fetal and TABLE 2. The length of telomeres in the immature and mature testes and in the spermatogenic cells during spermatogenesis. Testis a Spermatogenic cells a,b Species Immature Mature SG SC RS EgS ES Sperm Method c Reference Mouse þþ þþþþ NA NA NA NA NA NA TRF [61] NA NA þ þþ þ NA NA NA TRF [61] NA NA þþ þþþþ* þþ NA NA NA Q-FISH [75] Rat NA NA TRF-S. [71] Human NA NA NA NA NA NA NA 10.5 TRF [55] NA NA NA NA NA NA NA TRF [81] NA NA NA NA NA NA NA TRF-FIGE [82] NA NA NA NA NA NA NA TRF [83] NA NA NA NA NA NA NA Q-FISH [84] NA NA þþ þþþþ þþ þþ þþ þ Q-FISH [88] a Telomere lengths in kilobases (kb) are presented as the mean 6 SD or SEM. The lowest and the highest relative telomere length are denoted by þ and þþþþ, respectively. NA, not analyzed. b An asterisk indicates that the data for primary spermatocytes (spermatocytes I) were used. SG, spermatogonia; SC, spermatocytes; RS, round spermatids; EgS, elongating spermatids; ES, elongated spermatids. c TRF, telomere restriction fragment; S., Southern blot; TRF-FIGE, terminal restriction fragment-field inversion gel electrophoresis; Q-FISH, quantitative fluorescent in situ hybridization. 3 Article 53

4 OZTURK postpubertal human testes, spatial and temporal expression of the TERC subunit was also analyzed. Although the TERC subunit was observed in the gonocytes and Sertoli cell precursors of fetal human testes, it was expressed in the spermatogonia and in the primary and secondary spermatocytes, but not in the spermatids and spermatozoa, of postpubertal human testis. It is noteworthy that the primary spermatocytes had considerably higher TERC levels than other spermatogenic cells [69]. In a recently published work, high level telomerase activity in the human spermatogonial stem cells was additionally reported based on the increased expression profile of the TERT subunit [70]. In parallel with telomerase activity, the human TERT promoter activity in the htertp-lacz transgenic mice at 6, 17, 29, and 46 days and at 5 mo of age was gradually enhanced with aging except in the 46-day-old testis, and it peaked in the 5-mo-old adult mouse testis [60]. In line with the finding of telomerase activity, the TERC gene expression was detected in the embryonic, fetal, and adult human testicular tissues as well as in the spermatogenic cells [69]. Taken together, those studies on the postnatal mouse [30, 57, 58], rat [68], and human [39] testes revealed a striking difference in the telomerase activity between immature and mature testes of these species. New studies are required to understand the possible underlying reasons for this discrepancy in the telomerase activity between mouse and rat testes, which belong to the same order (Rodentia). In contrast to having higher telomerase activity in the immature rat testes than in mature testes [68], the immature (age, 9 days) rat testes had significantly lower average telomere length than the mature (age, 70 days) testes ( vs kb, respectively) [71] (Table 2). This suggests an inverse correlation between telomerase activity and telomere length in the immature and mature rat testes [68, 71]. Probably, this results from the increased telomerase activity during early postnatal testicular development, which plays a role in the length of telomeres at the later stages of development. TELOMERE LENGTH AND TELOMERASE ACTIVITY IN MALE GERM CELLS The immature rodent testes at early postnatal development include type A spermatogonia, and during postnatal development, other spermatogenic cells, such as spermatocytes, round spermatids, elongating/elongated spermatids, and sperm cells, emerge based on the developmental timetable [72, 73]. Telomerase activity and telomere length were analyzed in the individual spermatogenic cell types isolated from seminiferous tubules of immature and mature rat testes [71]. Those researchers found that telomerase activity was at the highest level in the type A spermatogonia, isolated from immature rat testes, compared to the pachytene spermatocytes, round spermatids, and spermatozoa obtained from mature rat testes or epididymis. While pachytene spermatocytes and round spermatids had appreciable telomerase activity, no telomerase activity was present in the epididymal spermatozoa [71], which was reported in other studies as well [68, 74]. These results suggest that the presence of higher telomerase activity in the type A spermatogonia likely provides immature testes with higher telomerase activity than that of mature testes. Similar findings were also obtained in the isolated mouse spermatogenic cells, where telomerase activity was detected in the primary spermatocyte, secondary spermatocyte, and spermatid fractions and was absent in the spermatozoa or Sertoli cell fractions [59] (Table 1). To understand whether telomerase activity in the spermatogenic cells correlates with telomere length, telomeres have been measured in isolated rat spermatogenic cells [71]. The mean telomere length of the type A spermatogonia isolated from 7-day-old immature rat testes was kb, whereas the pachytene spermatocytes obtained from 70-day-old mature rat testes had a telomere length of kb, which was significantly higher than that in the type A spermatogonia. On the other hand, the length of telomeres in the round spermatids from mature testes was kb, almost similar to the length measured in the type A spermatogonia. Strikingly, the epididymal spermatozoa from mature rat possessed the highest telomere length ( kb) compared to other spermatogenic cell types [71]. Similarly, in the mouse, Reig-Viader et al. [75] found that primary spermatocytes (spermatocytes I) had the longest telomere length compared to other germ cell populations: spermatogonia, secondary spermatocytes (spermatocytes II), and round spermatids [75] (Table 2). As a result, telomere length gradually increases during spermatogenesis from spermatogonia up to spermatozoa, though a small decline exists in the round spermatids. Further telomere lengthening from round spermatids to spermatozoa is an interesting finding of these works [61, 71] because transcriptional activity and DNA replication are terminated at the midspermiogenesis stage of spermatogenesis. Telomere extension in the mitotic cells is largely carried out during S- phase of the cell cycle, at which time telomerase recruits to a subset of telomeres to elongate the telomeres [76, 77]. Although both spermatogonial cells and primary spermatocytes undergo DNA replication [61], no DNA replication process occurs in the later stages of spermatogenesis, such as in the spermatids and spermatozoa [78, 79]. DNA replication is required for extending telomeres with the enzyme telomerase or the ALT mechanism. Therefore, telomere lengthening in the spermatids and/or spermatozoa seems to be carried out independent from these two basic mechanisms. Another telomere extension mechanism or mechanisms may be working in these cells, and this requires further investigation. It is important to note that higher telomerase activity in the type A spermatogonia may contribute to telomere lengthening in the resulting spermatocytes and perhaps in the subsequent spermatogenic cells. Consistent with the calculated telomere lengths, telomeric signal intensity of each spermatogenic cell manifested remarkable differences during spermatogenesis in mice [61]. The signal intensity originating from telomeres began to diminish in the pachytene spermatocytes, and it reached the lowest levels in the round spermatids (until the end of step 7 of spermiogenesis). Surprisingly, telomeric signals started to increase in the elongating spermatids and approached the highest levels in the elongated spermatids. In addition to representing telomeric signal intensity, the quantitative FISH (Q-FISH) also gave the approximate number of telomeric repeats, which reflects the relative telomere length. In parallel, the elongated spermatids had longer telomeric repeats than that of spermatocytes and round spermatids [61] (Table 2). Probably, the increase of telomerase activity in the elongating spermatids resulted in extended telomeres in the elongated spermatids, which showed no telomerase activity [61, 65]. These findings suggest another possibility for the increased telomeres in the postmeiotic male germ cells namely, that the telomere elongation and telomeric reorganization in the postmeiotic spermatids may be carried out before protaminehistone exchange in the nucleosomes. 4 Article 53

5 TELOMERASE AND TELOMERE LENGTH IN MALE GERM CELLS TELOMERE LENGTH AND TELOMERASE ACTIVITY IN HUMAN SPERM CELLS Characterization of telomerase activity and telomere length in men has been largely conducted on sperm cells, which can be easily obtained compared to other spermatogenic cells. Similar to the findings for mouse, rat [68, 71, 74], and pig [80] sperm cells, no telomerase activity has been reported in human sperm cells [58, 81]. However, human sperm cells have predominantly higher telomere length than somatic cells [55, 56], and similar results have been found for pig and cow sperm cells [55, 80]. The length of telomeres in spermatozoa does not display prominent changes during their maturation process, which occurs at different regions of epididymis. This reveals that they acquire their final telomere length in seminiferous tubules [80]. As expected, the ejaculated and epididymal sperm cells exhibit no telomerase activity as if it is not detected in the spermatozoa within seminiferous tubules [39, 80]. The average telomere length of the sperm cells recruited from normozoospermic men was and kb as a result of resolution of the PCR products with terminal restriction fragment-field inversion gel electrophoresis after digestion with MnlI/RsaI or with HinfI/RsaI restriction enzymes, respectively [82]. Similarly, the mean telomere length of the sperm cells taken from healthy donors has been measured as kb [83], 10.5 kb [55], and kb [81] (Table 2). In contrast, Turner and Hartshorne [84] measured it as kb in the sperm cells collected from 50 unselected men. Additionally, those authors did not observe any correlation between certain semen parameters (sperm concentration, sperm morphology, and progressive sperm motility) and average sperm telomere length. The huge variations in the mean telomere lengths of the human sperm samples among the studies mentioned above probably arise from use of distinct methods for measuring telomeres. The former studies [55, 81, 82] employed the TRF method to determine the telomeric lengths, which tends to measure telomeres as 3 4 kb greater than their actual length [85, 86]. Conversely, the latter work by Turner and Hartshorne [84] utilized the Q-FISH method that specially measures telomeric regions. Because the TRF needs an isolated genomic DNA, some cells, such as immature germ cells and/or testicular somatic cells, in the semen sample may contaminate the sperm cell fraction, which might cause less accurate measurements. Various sperm preparation methods may be an additional factor. Santiso et al. [87], for example, used the swim-up technique in selecting sperm [87], and as is well known, the swim-up technique helps separate the sperm cells that have longer telomeres and lower fragmented DNA [87]. Individual differences in the donor populations may also lead to measuring distinct telomere lengths among healthy donors, and in addition to differently measured telomere lengths among men, sperm samples from the same individual have also exhibited distinct telomere lengths [82, 87]. Having a different telomere length in each individual sperm cell presumably results from the presence of a variable telomerase activity in the spermatogenic cells at the early stage of spermatogenesis and/ or exposure of these cells to oxidative stress at different levels during spermatogenesis. A newly published study by Jørgensen et al. [88] also characterized the telomere lengths in the spermatogenic cell types of human testes at the ages of between 31 and 40 yr. Those authors found that the primary spermatocytes had the highest telomere length compared to other spermatogenic cells. Although spermatogonial cells and spermatids possessed similar telomere lengths, spermatozoa had slightly lower length than these cells [88] (Table 2). As a result, telomeres are generally the longest in mammalian spermatozoa compared to other spermatogenic cells. Conversely, telomerase activity gradually decreases from spermatogonia to spermatozoa (Fig. 1). Because telomeres are capable of anchoring to nuclear membrane, the long telomeres in the differentiated spermatozoa are considered to function in the nuclear organization, where dramatic morphological changes occur during spermiogenesis [89]. Since the genomic DNA replication occurs during male pronucleus formation following fertilization, a recently published study examined telomere lengths [84]. Those researchers found that the length of telomeres in the male pronuclei during development from 1 to 16 h after fertilization did not display any predominant changes. However, it should be noted that the male pronuclei had significantly shorter telomere length than the female pronuclei (6.16 vs kb) [84]. PATERNAL AGE CORRELATES WITH TELOMERE LENGTH IN THE SPERM CELLS The length of telomeres progressively erodes with age in most proliferative somatic tissues, which results in a decreased number of cell divisions and eventually replicative senescence [83, 90]. Similarly, telomere length in the oocytes declines with female aging [91]. However, sperm cells are capable of elongating their telomeres during paternal aging. Consistent with that, sperm cells from older men have longer telomeres with respect to sperm cells from younger men [56, 82, 83]. A similar significant positive correlation between telomere length and paternal age at the time of conception has been found in the offspring of 18- to 19-yr-old high school students [92]. The annual rate of telomere lengthening in the sperm cells during paternal aging has been reported differently by a variety of studies: 70 bp [56], 135 bp [82], and 17 bp [93]. This difference may arise from the use of distinct methods for measuring telomere length and from heterogeneity of the populations tested in these works. Inconsistent with the telomere lengthening during paternal aging, the sperm cells from men aged 35 yr or older had significantly longer average telomere length than those from men younger than 35 yr (6.69 vs kb) [84]. This relationship was also observed across multiple generations; for example, grandchildren of an older grandfather possessed much longer telomere length at birth than their fathers [94]. Of note, no remarkable difference in the telomere lengthening based on the paternal aging was found between male and female offspring at birth [93]. However, advanced paternal age causes telomere lengthening at a size of half to more than double the annual attrition, and it results in increased telomere length in the offspring [83, 93, 95]. The precise mechanism for the telomere lengthening in the sperm cells from aged men has not been clearly identified. A recent study aimed to elucidate the possible reason for this increase in the spermatozoa of older men [88]. For this purpose, telomeres in the spermatogenic cells from old and young men were measured by the Q-FISH method [88]. This study disclosed no significant difference in their telomere length profiles of the spermatogenic cells. This indicates that no wider window of telomere lengthening process existed in the spermatogenic cells of the older men [88]. The increase of telomere length in the sperm cells during paternal aging may have several reasons. First, only a subset of sperm cells that have higher telomere length may be selected in the aged men [83]. The surveillance mechanism specifically recognizing the sperm cells with longer telomeres has not been clearly identified. Oxidative stress seems to be one ingredient playing a role in this selection process [96]. Second, increased 5 Article 53

6 OZTURK FIG. 1. Schematic diagram of telomere length and telomerase activity dynamics during male germ differentiation from spermatogonia to spermatozoa. In general, the length of telomeres increases from spermatogonia to spermatozoa, but telomerase activity inversely decreases in the same direction. Small fluctuations related to telomere length and telomerase activity exist in the spermatogenic cells based on the mammalian species, and no telomerase activity is detected in the sperm cells. Note that the relative telomere length and telomerase activity gradually decrease from green to red. SG, spermatogonia; SC, spermatocytes; RS, round spermatids; EgS, elongating spermatids; ES, elongated spermatids. telomerase activity in the spermatogonial stem cells leads to longer telomeres, and sperm cells in the aged men originating from these cells may possess longer telomere length [88]. Third, as spermatogonial cells undergo less proliferation in elderly men, they experience less telomere erosion [88]. This may contribute to the sperm cells of the aged men having longer telomeres. Fourth, changing levels of the hormones and/or other intracellular factors regulating telomere length and telomerase activity in the male germ cells may result in extended telomeres or selection of certain sperm cells that have longer telomeres in the older men. Finally, in the aged men, there may be an ALT mechanism that operates more actively in the spermatogenic cells. The possible advantages of telomere elongation in the sperm cells from an older father are to compensate for the short telomeres derived from oocytes of aged women so that the life span of the older male s offspring is extended. NUCLEAR LOCALIZATION OF TELOMERES DURING SPERMATOGENESIS Recent studies have also revealed that the position of telomeres in the nucleus of spermatogenic cells manifests dynamic changes during spermatogenesis. Telomeres disperse all around of the nuclei in the spermatogonia and leptotene spermatocytes [61]. Then, telomeres transiently cluster in one pole of the nucleus in the early zygotene spermatocytes, and in the late zygotene spermatocytes, they spread to peripheral parts of the nucleus. This distribution enables chromosomes to attach to the nuclear envelope, which thus contributes to homologous pairing and correct segregation [61, 97]. Peripheral dispersions of the telomeres endure during the first meiotic division in the pachytene spermatocytes [61, 98]. Following that, they separate from the peripheral regions in the secondary spermatocytes and accumulate mainly in the center of the nucleus, around the nucleolus, in the round spermatids [61, 99]. In the elongating spermatids, telomeres spread in the nucleus again as the nucleolus fades [61]. REGULATION OF TELOMERASE ACTIVITY IN MALE GERM CELLS In general, regulation of telomerase activity is initially carried out on the transcriptional level of the TERT gene, which encodes the catalytic subunit of telomerase [100]. Certain transcription factors, such as v-myc avian myelocytomatosis viral oncogene homolog (c-myc), mitotic arrest deficient 1 (MAD1), estrogen receptor (ER), and other factors, especially regulate the TERT mrna expression [101]. MAD1 along with paired amphipathic helix protein, msin3, represses transcription of the TERT gene so that repression of the telomerase activity in the differentiated somatic cells may be carried out this way [101, 102]. On the other hand, both estrogen and c-myc increase the transcriptional expression of the TERT gene, which results in increased telomerase activity [ ]. Analogously, administration of 17b-estradiol to the Michigan Cancer Foundation-7 (MCF-7) cell cultures led to up-regulated telomerase activity. Notably, the MCF-7 cells, a breast cancer cell line, are known to express ER [104]. The promoter region of the TERT gene includes an estrogen-responsive element where ER specifically binds. Additionally, the E-boxes in the TERT promoter are bound by the c-myc, the expression of which is induced following estrogen treatment [104]. As expected, estrogen deficiency created by targeted disruption of aromatase in mice caused loss of telomerase activity in the adrenal gland and resulted in compromised cell proliferation and shortened telomeres [106]. It is important to note that fluctuation of the endogenous estrogen levels during reproductive ages may directly affect telomerase activity and the length of telomeres in ER-expressing cells [107]. This may play a direct role in the decrease of telomere length in the oocytes during maternal aging. The regulation of telomerase activity in male germ cells is not completely clarified, but some regulators, such as Kit ligand (KITL; also known as stem cell factor) and estrogen, are involved. The KITL increases production of both TERT protein and TERC levels in the spermatogonial cells, and thus telomerase activity is promoted [108]. Although primordial germ cells have telomerase activity at low levels, their telomerase activity has been enhanced upon KITL induction. Additionally, the PI3K/AKT pathway also mediates activation of the KIT receptor in the mouse spermatogonial cells [109, 110]. Consistently, activation of the protein kinase AKT, one of the PI3K downstream targets, leads to an increase in human telomerase activity through phosphorylation of the TERT subunit [111]. In turn, the increased telomerase activity stimulates germ cell proliferation [108]. Notably, no significant relationship existed between telomerase activity and parameters such as sperm count, follicle-stimulating hormone, luteinizing hormone, and testosterone levels [105]. THE RELATIONSHIP BETWEEN TELOMERE LENGTH/ TELOMERASE ACTIVITY AND MALE INFERTILITY To evaluate the importance of telomerase activity on the reproductive system, Terc knockout (Terc / ) mouse models 6 Article 53

7 TELOMERASE AND TELOMERE LENGTH IN MALE GERM CELLS have been created. Although no significant abnormality was found in the early generations, the third-generation Terc / mice had a two- to threefold lower sperm count in their cauda epididymis compared to wild-type mice [112]. Furthermore, the motility of sperm from third-generation Terc / male mice was at minimal levels in comparison with that of the sperm cells from wild-type mice. However, at the fourth generation, the Terc / male mice did not have any viable mature sperm cells, and only spermatids existed in their small testes [112]. As a result, telomeric shortening at the late generations due to absence of telomerase activity in the spermatogenic cells of the Terc / male mice caused chromosome segregation error, spermatogenic cell depletion, reduced sperm count, impaired fertility, and increased apoptotic index [97, 113, 114]. Because spermatogenic cells with critically shortened telomeres are largely eliminated by the apoptotic pathway, this most likely led to increasing the apoptotic index in the Terc / male germ cells of the later generations [114, 115]. Note that the apoptotic pathway is a regular process during germ cell development to remove distorted germ cells, which are not capable of producing normal sperm cells [116]. The association between male infertility and telomerase expression/activity in the mouse [59] and human [105, 117] testicular tissues has also been investigated. In the mouse, Yamamoto et al. [59] did not discover any significant relationship between healthy testes and cryptorchid testes arrested at primary spermatocyte or round spermatid stage by using telomeric repeat amplification protocol and sensitive quantitative telomerase assay methods, both of which were for measuring telomerase activity. However, as expected, mouse testes with Sertoli cell-only syndrome (SCOS) did not exhibit telomerase activity, probably due to lack of spermatogenic cell types [59] (Table 3). Consistent with the data on the pathological mouse testis samples, telomerase activity levels in the human testicular biopsies from patients diagnosed as infertile due to maturation arrest, obstructive azoospermia, or oligozoospermia were not different from each other [105]. Additionally, no significant discrepancy was found in the telomerase activity between early maturation arrest (arrested at spermatocytes) and late maturation arrest (arrested at round spermatids) testes. In contrast, both early and late maturation arrest testes had significantly higher telomerase activity than the testes from patients with SCOS. Because spermatogenic cells were the main sources of telomerase activity in the seminiferous tubules, lack of spermatogenic cells in the SCOS testis resulted in loss of telomerase activity, as expected [105] (Table 3). Similarly, Schrader et al. [118] did not find either telomerase activity or TERT expression in the human testes with SCOS; however, all of the SCOS testes expressed the TERC. Those authors also found that human maturation-arrested testes with evidence of spermatocytes and early spermatids had telomerase activity in 8 of 9 cases and TERT mrna expression in all nine patients, but the TERT expression was positive in only 5 of 9 patients. As expected, all 12 patients with obstructive azoospermia with normal spermatogenesis possessed telomerase activity and expressed both the TERT and TERC [118] (Table 3). The study found no considerable positive correlation between telomerase activity and the TERT expression, which may arise from differences in the sensitivity of the methods used for measuring the two parameters or difference in the TERT mrna expression pattern. Another study by Schrader et al. [119] revealed that expressional levels of the TERT mrna could be used as a parameter to predict the presence of germ cells in the testes of patients with nonobstructive azoospermia. Testes of patients with nonobstructive azoospermia and full spermatogenesis had markedly higher TERT mrna levels than those of testes with maturation arrested at the primary or secondary spermatocyte stage. However, the patients with SCOS exhibited substantially reduced TERT mrna expression profiles, as expected [119]. In a different work, the same group investigated whether detection of the TERT mrna expression or telomerase activity could be used as a molecular diagnostic tool for subclassification of spermatogenesis disorders, such as maturation arrest, SCOS, and obstructive azoospermia [120] (Table 3). Collectively, these findings were promising in the sense that expression levels of the TERT mrna could be used in distinguishing three subgroups: full spermatogenesis, maturation arrest, and SCOS; however, telomerase activity analysis was not found to be as robust as the TERT mrna analysis. Among these three groups, the TERT mrna expression was the highest in the testes with obstructive azoospermia and full spermatogenesis compared to maturation arrest or SCOS testis. TABLE 3. Telomerase activity, TERT mrna expression, and the length of telomeres in the normal and pathological human testis or sperm cells and mouse testis. Testicular pathology a,b Material EMA LMA SCOS OA NOA IDI OS Control a,c Method d Parameter e Reference Human testis þþþþ þþþþ ND þþþþ NA NA þþþþ NA TRAP-ELISA T. activity [105] þ? þ? ND þ? NA NA NA NA TRAP T. activity [118] NA NA þ NA NA NA NA NA SQTA T. activity [122] þþ þþþ þ/ NA NA NA NA þþþþ qrt-pcr TERT [117] þ? þ? ND þ? NA NA NA NA RT-PCR TERT [118] þþ NA þ/ NA þþþþ NA NA NA RT-PCR TERT [119] þþ NA þ/- þþþþ NA NA NA NA RT-PCR TERT [120] Human sperm NA NA NA NA NA NA Q-PCR T. length [92] NA NA NA NA NA NA Q-PCR T. length [113] Mouse testis þþ þþ ND NA NA NA NA þþ TRAP-SQTA T. activity [59] a The lowest and the highest telomerase activity are denoted by þ and þþþþ, respectively. Very weak telomerase activity is denoted by þ/. NA, not analyzed; ND, not detected;?, no quantitative comparison among analyzed testicular tissues. b EMA, early maturation arrest; LMA, late maturation arrest; SCOS, Sertoli cell-only syndrome; OA, obstructive azoospermia; NOA, nonobstructive azoospermia; IDI, idiopathic infertile; OS, oligozoospermia. c Normal spermatogenesis. d TRAP-ELISA, telomeric repeat amplification protocol-enzyme-linked immunosorbent assay; SQTA, sensitive quantitative telomerase assay; TRAP, telomeric repeat amplification protocol; RT-PCR, reverse transcription-polymerase chain reaction; qrt-pcr, quantitative real-time polymerase chain reaction; Q-PCR, quantitative polymerase chain reaction. e T. activity, telomerase activity; T. length, telomere length. TERT, telomerase reverse transcriptase. 7 Article 53

8 OZTURK As expected, the SCOS testes had TERT mrna expression at minimal levels [120]. On the other hand, Giannakis et al. [121] found that testicular telomerase activity levels could be employed as a marker for determining the presence of testicular spermatozoa in the azoospermic men with varicoceles pre- and postvaricocelectomy [121]. Interestingly, some studies mentioned above showed that certain subpopulations of the testes diagnosed as SCOS demonstrated telomerase activity at low levels. Probably, this seems to originate from foci of primary spermatocytes or round spermatids/spermatozoa in the rare seminiferous tubules of testes with SCOS [122]. In parallel with that, human testes with SCOS had the lowest levels of TERT mrna expression compared to the testes with normal spermatogenesis and with premeiotic or postmeiotic spermatogenic arrest. Additionally, those authors found that the TERT mrna expression progressively increased from premeiotic arrest testis to postmeiotic arrest testis and reached the highest levels in the normal human testis [117] (Table 3). In a recently published work, no relationship between male infertility and sperm telomere length was determined [84]. Because telomere length is reset during early embryo development following fertilization [123], short telomeres in sperm cells can be elongated before they adversely affect pregnancy progression. However, Thilagavathi et al. [113] found a correlation between the two parameters. In that investigation, the length of telomeres in the sperm cells obtained from control and idiopathic infertile men was analyzed by using quantitative PCR. The relative mean telomere length (T [telomere assay]/s [single copy gene assay]) of the sperm from idiopathic infertile men was significantly lower than that in the control group ( vs ) (Table 3). However, no correlation was observed between telomere length and certain sperm parameters, such as sperm count, sperm forward motility, sperm morphology, ROS levels in the semen, and DNA fragmentation index [113]. In a different work, the same group showed that telomere length in the peripheral blood leukocytes collected from both women and men has been associated with idiopathic recurrent pregnancy loss compared to controls [124]. On the other hand, Ferlin et al. [92] observed a significant positive correlation between sperm telomere length and total sperm count: Sperm from oligozoospermic (total sperm count,,39 million/ejaculate) men had significantly lower sperm telomere length than normozoospermic (total sperm count, 39 million/ejaculate) men ( vs ) [92] (Table 3). A paper by Reig-Viader et al. [125] revealed that the nuclear distribution and expression levels of TERRA molecules in spermatocytes exhibited significant changes in infertile patients compared with control individuals. Additionally, reduced meiotic recombination and decreased TERT levels at telomeres were also observed in the infertile patients; however, no alteration was reported in the telomerase-terra association and the length of telomeres in the spermatocytes of affected patients [125]. This study has provided new insight into the relationship between telomere biology and male infertility development by addressing the molecular biological roles of TERRA molecules in the meiotic process in spermatocytes [126]. Some studies have also investigated the relationship between genetic variants on the telomere/telomerase-associated genes and male infertility. Yan et al. [127] examined the single nucleotide polymorphisms (SNPs) in the TERT and telomeraseassociated protein 1 (TEP1) genes. Some SNPs in those genes were found to be linked with increasing risk of male infertility. Additionally, although no loss of telomeric material was visible, the increased rate of associations between certain chromosome telomeres in the majority blood cells was found to be associated with azoospermia in men [128]. Overall, few studies have examined the relationship between male infertility and telomere length/telomerase activity, and no correlation between the two parameters has been clearly found [84, 105]. Therefore, further studies are needed in larger groups to understand whether a significant correlation exists between development of male infertility and telomere length/telomerase activity. Also, analyzing telomeres and telomerase activity of each spermatogenic cell type obtained from testes of healthy and infertile men should give us more reliable data to determine the effect of changed telomere lengths and telomerase activity on male infertility. CONCLUSIONS AND FUTURE DIRECTIONS In conclusion, telomeres in the mammalian male germ cells progressively increase in length from spermatogonia to spermatozoa during spermatogenesis. However, telomerase activity is gradually down-regulated during germ cell differentiation from spermatogonia to spermatozoa, and no telomerase activity occurs in the spermatozoa. Although telomere length and telomerase activity in the male germ cells during spermatogenesis have been largely characterized in rodents and partially in humans, the regulatory mechanisms that act on the telomere elongation and telomerase expression need further studies. Characterization of these mechanisms and more studies on this subject would help us understand the importance of telomere length and telomerase activity for proper progression of spermatogenesis. Also, few studies have attempted to identify the relationship between telomerase activity/telomere length and male infertility. Further studies with larger homogenous infertile patient groups using the most suitable techniques in this field are required to clearly address this relationship. To date, many studies have been performed to characterize telomerase expression and telomere length in male germ cells of certain mammalian species, but only a limited number of studies on human germ cells have appeared. Because prominent differences in telomere length and telomerase expression parameters exist between human and other mammalian species based on available studies, more research is required on human male germ cells, such as sperm cells readily obtained for intracytoplasmic sperm injection or in vitro fertilization and testicular biopsy samples taken for detecting the underlying causes of male infertility. Using these samples, following ethics permissions, the telomere length and telomerase activity levels can be accurately determined in the spermatogenic cells from spermatogonia to sperm cells during spermatogenesis in men. One of the major challenges in this field is the difficulty of finding healthy human testicular tissues with normal spermatogenesis. In general, testicular biopsy samples taken by orchiectomy due to suspicion of testicular tumor or other reasons are commonly used as a control. However, these testis samples may not reflect healthy individuals because of the risk for developing tumor-like tissue that may adversely influence spermatogenic cells. Another challenge involves different techniques, samples, and species strains being used to detect telomerase expression and to measure telomere length, which produce different results in similarly designed studies. Therefore, similar techniques and sampling methods along with identical strains should be used in future works. 8 Article 53