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Regular Article |
a Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia 20007
ABSTRACT
Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.
spermatogenesis, testes
INTRODUCTION
Telomeres, located at the ends of eukaryotic chromosomes, consist of evolutionarily conserved repetitive noncoding DNA sequences that function as stabilizing elements for the chromosomes during divisions [1, 2]. During each replication cycle of the cell, a portion of the telomere that is overlaid with the primers for DNA polymerase is not replicated [3, 4]. Therefore, the length of the telomeres shortens progressively with each cell division. As a result, telomeres are shorter in senescent cells than in younger cells [5, 6]. The male germ-line cells appear to be an exception to this process of telomere shortening after successive divisions [7–9]. Male germ cell differentiation is a complex process beginning with the proliferation of diploid type A spermatogonial stem cells and terminating with morphologically distinct haploid spermatozoa [10]. The type A spermatogonia successively divide mitotically to form intermediate type spermatogonia, type B spermatogonia, and preleptotene spermatocytes. Preleptotene spermatocytes through the stages of leptotene, zygotene, pachytene, and diplotene undergo the first meiotic division to yield the secondary spermatocytes. The secondary spermatocytes undergo the reductional second meiotic division and give rise to haploid round spermatids that elongate and differentiate into spermatozoa. Despite both mitotic and meiotic divisions of the proliferating and differentiating germ cells, spermatozoa, the terminally differentiated male gametes, exhibit a very long telomere length [9, 11]. It could be hypothesized that type A spermatogonia have long telomere lengths that are maintained by the enzyme telomerase throughout the process of germ cell differentiation into spermatozoa. Alternately, telomere length may be progressively extended by the action of telomerase during the process of proliferation and differentiation of type A spermatogonia into spermatozoa. Although the expression of telomerase activity in type A spermatogonia, pachytene spermatocytes and round spermatids has been demonstrated by us previously [12], no information is available on the changes in telomere length and their relationship to telomerase activity in proliferating and differentiating male germ cells. The present study was undertaken to investigate the relationship of telomere length and telomerase activity during the process of male germ cell differentiation.
MATERIALS AND METHODS
Tissue Collection
Male Sprague-Dawley rats were purchased from the Charles River Breeding Laboratories (Wilmington, MA). Testes collected from immature (9-day) and adult (70-day) rats were either used immediately for cell separation or frozen on dry ice and stored until use at -75°C.
Isolation of Rat Germ Cell Types
Type A spermatogonia were isolated from 9-day-old male pups. Pachytene spermatocytes and round spermatids were isolated from 70-day-old adult rats. The testes were excised and decapsulated. Seminiferous epithelial cells were enzyme dispersed and separated by the method of Bellvé and colleagues [13] with minor modifications [14]. Briefly, the decapsulated testes were suspended in Dulbecco minimal essential medium (DMEM)/F12 containing collagenase (1.5 mg/ml) and DNAse (1 µg/ml), and incubated at 34°C for 15 min in a shaking water bath operated at 100 cycles/min. After two washes in DMEM/F12 medium, seminiferous cord fragments, mostly devoid of interstitial cells, were incubated in DMEM/F12 medium containing collagenase (1.5 mg/ml), hyaluronidase (1.5 mg/ml), trypsin (0.5 mg/ml), and DNAse (1 µg/ml) for 20–30 min using the conditions described above. The dispersed cells were washed twice with medium and filtered through 80-µm and 40-µm nylon mesh (Tetco Inc., Briarcliff Manor, NY), successively. The cells of the dissociated epithelium were then separated by velocity sedimentation at unit gravity at 4°C, with use of a 2–4% BSA gradient in DMEM/F12 medium. The cells were bottom-loaded into a glass chamber in a volume of 30 ml, and a BSA gradient was generated using 275 ml of 2% and 4% BSA. The cells were allowed to sediment for a standard period of 2.5 h, and then 35 fractions of 15-ml volume each were collected at 90-sec intervals. The cells in each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation, and then resuspended in DMEM/F12 medium. The purity of the cell populations and the contaminating cells were as follows: spermatogonia, 95 to 98% purity (myoid cell contamination); spermatocytes, 95% purity (spermatogonia and round spermatid contamination); spermatids, 85 to 90% purity (spermatogonia and Sertoli cell contamination).
Spermatozoa were isolated by mechanical expression from the epididymis of adult rats.
Genomic DNA Extraction
Genomic DNA was extracted from 9-day-old and 70-day-old adult rat testes and from enriched cell populations of spermatogonia, pachytene spermatocytes, round spermatids, and spermatozoa according to a standard protocol [15]. Briefly, the samples were rapidly homogenized in DNA extraction buffer (100 mM NaCl, 10 mM Tris, 25 mM EDTA, pH 8, 0.5% SDS, 0.1 mg/ml proteinase K) and incubated at 50°C for 12–18 h until most of the cellular protein was degraded. The digest was deproteinized by successive phenol/chloroform/isoamyl alcohol extractions and recovered by precipitation with 100% ethanol in 7.5 M ammonium acetate. The DNA was dissolved in Tris-EDTA buffer at 65°C for 1–3 h.
Terminal Restriction Fragment Length Measurement
The terminal restriction fragment (TRF) length measurements were performed using the TeloQuant kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. Briefly, an aliquot (10 µg) of genomic DNA was digested with a mixture of RsaI and HinfI restriction endonucleases (4 U each/µg DNA) at 37°C overnight. Aliquots (5 µg) of digested DNA were separated on a 0.6% agarose gel in TAE buffer at 1 V/cm overnight. The gels were successively washed with 0.25 M HCl and 0.4 M NaOH. Using 0.4 N NaOH as transfer buffer, DNA was Southern-transferred to a positively charged nylon membrane (Amersham Hybond-N+) at room temperature for 3–5 h. After prehybridization at 65°C for 1 h, the membrane was incubated overnight at 65°C with a specific biotinylated telomere probe (final concentration 1–10 ng/ml) that recognizes the telomeric repeat hexamer. Following the blocking of the filter at room temperature for 1 h, the membrane was further incubated in a solution of streptavidin-HP (final concentration <25 ng/ml) at room temperature for 1 h. Finally, detection of the position of the hybridized probe on the membrane was accomplished by incubation in a chemiluminescent detection system consisting of a solution of equal volumes of stable peroxide and Luminol/Enhancer. The membranes were exposed to x-ray films for 30–120 sec. An estimate of the TRF length was obtained by visually comparing the size of the signal band to the molecular size markers. For a more quantitative measure, the telomere length for each sample was estimated by calculating the mean TRF length. The TRF length analysis was performed on a densitometric scan of the autoradiogram. The mean TRF length for each sample was calculated by integrating the signal intensity above background over the entire TRF distribution as a function of length, using the formula L = 
(ODi·Li)/(ODi), where OD and L were the signal intensity and TRF length, respectively, at position i on the gel image.
Protein Extraction
Frozen testis samples (50–100 mg) were minced with a sterile blade and homogenized in ice-cold CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate) lysis buffer (100–200 µl). Isolated germ cell types were spun down by low-speed centrifugation and the medium was aspirated completely. The cells were resuspended in CHAPS lysis buffer (100–200 µl). The samples were incubated for 30 min on ice and centrifuged at 12 000 x g for 30 min at 4°C. The supernatant was collected and the protein concentration was measured by use of the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA). Aliquots were stored at -70°C.
Telomerase Repeat Amplification Protocol Assay
The telomerase repeat amplification protocol (TRAP) assay was performed by use of a modified polymerase chain reaction (PCR)-based method [16]. Five micrograms of the protein extracts were assayed in 50 µl of reaction mixture containing 50 µM dNTPs (PharMingen, San Diego, CA), 2 U Taq DNA polymerase (GIBCO-BRL, Gaithersburg, MD), 1 µg T4 gene 32 protein (Boehringer Mannheim, Indianapolis, IN), 0.1 µg TS primer (5' ATT CCG TCG AGC AGA GTT 3'), and 2 µCi 32P-labeled deoxycytidine triphosphate (Amersham, Arlington Heights, IL). After incubation for 30 min at 23°C, the reaction was heated at 90°C for 3 min. Following the telomerase-mediated extension of the TS primer, 0.1 µg CX primer (5' CCC TTA CCC TTA CCC TTA CCC TTA 3') and 1 ag internal control oligonucleotide (ITAS, 5' AAT CCG TCG AGC AGA GTT AAA AGG CCG AGA AGC GAT 3') were added to each tube. The PCR amplification was performed at 94°C for 30 sec followed by 50°C for 30 sec and 72°C for 1.5 min for 27 cycles using a DNA thermal cycler (Techne Inc., Princeton, NJ). Appropriate positive and negative controls were included in each assay. The DNA products were separated on a 12.5% nondenaturing polyacrylamide gel at 1500 V for 1 h. The gel was dried and assay products were visualized by exposing to Hyperfilm MP (Amersham, Little Chalfont, UK) with an intensifying screen for 16–18 h at -70°C.
Statistical Analyses
Telomere length analyses and determinations of telomerase activity were carried out at least five independent times for tissues and differentiated germ cells and three times for type A spermatogonial samples. A representative Southern blot and autoradiogram are shown for each of the experiments. The mean TRF was calculated as described above and data were expressed as average mean TRF ± SEM of the mean. Statistical differences between two groups (immature versus adult testis) were determined by the Student's t-test (P < 0.05). Wherever there were more than two groups (e.g., different germ cells), data were analyzed by one-way ANOVA followed by the Scheffé's F-test (P < 0.05). Because spermatogonia had the shortest telomere length compared with any other germ cells we examined, relative mean TRF was determined as the percentage of mean TRF for a given cell type sample compared with that of spermatogonia normalized to 100.
RESULTS
Telomere Length Analyses in Immature and Adult Rat Testes
Figure 1 represents a Southern blot of genomic DNA isolated from immature (9-day-old) and adult (70-day-old) rat testis. The blot was hybridized with a labeled probe consisting of telomere-specific sequence. The hybridization signal appeared as a broad band both in immature and adult rat testis. A very strong signal was observed in the region corresponding to fragments >20 kilobases (kb) in adult testis DNA. In comparison, the strongest hybridization signal in immature testis was noted around 9 kb. It was apparent that the terminal restriction fragments for immature and adult testis were of different molecular sizes.
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For analytical purposes, the mean lengths of terminal restriction fragments were calculated from the relative mobility of the hybridized bands as determined by the densitometer scan (Fig. 2). The average mean telomere length in the genomic DNA of immature testis was 9.1 ± 0.9 kb (SEM) with a range of mean TRFs of 6.3–10 kb. A significant increase in the length of telomeres was observed in the genomic DNA isolated from adult rat testis (P < 0.05). The average mean telomere length for adult testis was 12.8 ± 1.4 kb (SEM) with a range of 8.9–17.3 kb.
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Telomerase Activity in Immature and Adult Rat Testes
An autoradiograph of the TRAP assay performed with the extracts of testis from immature (9-day-old) and adult (70-day-old) rats is shown in Figure 3. An extract of human choriocarcinoma 293 cells served as positive control, and as expected exhibited strong telomerase activity (lane 1). Telomerase activity was not observed in the TRAP assay buffer lane (negative control, lane 2). In lanes 3 and 5, extracts from immature and adult rat testis exhibited telomerase activity. The intensity of the bands of the TRAP assay products was very strong in the immature testis extract (lane 3) when compared with the intensity of products in the adult testis extract (lane 5). The products of telomerase activity were absent in heat-treated samples of both immature and adult testes (lanes 4 and 6, respectively).
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Telomere Length and Telomerase Activity in Isolated Type A Spermatogonia from Immature Rat Testes
Type A spermatogonia were purified from 9-day-old rat testes by the STAPUT (stable-put—velocity sedimentation at unit gravity) [17] technique and the purity of the cells ranged from 95–98%. Figure 4A demonstrates a representative autoradiogram of a Southern blot of genomic DNA from two different samples of isolated type A spermatogonia hybridized with a labeled telomere-specific probe. In both lanes, the strongest telomere-specific hybridization signal is expressed between molecular sizes 4 and 7 kb. The analysis of the hybridization signals by densitometry revealed an average mean TRF length of 10.3 ± 3.4 kb (SEM) (Fig. 4B) with a range of 3.95–15.87 kb.
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In parallel, telomerase activity was assayed in isolated type A spermatogonia (Fig. 5). Lanes 1 and 2 represent positive cell pellet and negative buffer controls, respectively. Lanes 3 and 5 represent the products of high telomerase activity exhibited by two different samples of type A spermatogonia. No products of telomerase activity were observed in heat-treated samples of type A spermatogonia (lanes 4 and 6).
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Telomere Length in Differentiating Male Germ Cells
In the present study, we used pachytene spermatocytes and round spermatids as representatives of the differentiating male germ cell population. Enriched populations of pachytene spermatocytes (80–85%) and round spermatids (70–75%) were isolated from adult rat testis by the STAPUT technique. The isolated genomic DNA was restriction digested and Southern blotted. The blot was hybridized with a labeled telomere-specific probe. A representative autoradiogram showing the hybridization signals for pachytene spermatocytes and round spermatids is presented in Figure 6. As could be observed, the spread of the signal was more concentrated in pachytene spermatocytes above 9 kb, whereas in round spermatids, the spread of the signal appeared as a broad band between 4–9 kb. A quantitative analysis of the signal revealed that average mean telomere lengths for pachytene spermatocytes and round spermatids were 11.2 ± 0.8 kb (SEM) and 9.7 ± 0.5 kb (SEM), respectively (Fig. 7). The ranges of terminal restriction fragments generated were 8.4–12.9 kb and 8–10.8 kb for pachytene spermatocytes and round spermatids, respectively.
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Telomerase Activity in Differentiating Male Germ Cells
The telomerase activity in the extracts of pachytene spermatocytes and round spermatids is depicted in Figure 8. In lane 1, the positive cell pellet control (human choriocarcinoma 293 cells) showing the products of telomerase activity is shown. Absence of the telomerase activity can be seen in lane 2 (negative buffer control). Lanes 3 and 5 represent the telomerase activity in pachytene spermatocytes and round spermatids, respectively. The intensity of the signals of telomere bands for round spermatids was much stronger than that observed for the pachytene spermatocytes. Heat-treated samples of the extracts of both pachytene spermatocytes (lane 4) and round spermatids (lane 6) did not show any products of telomerase activity.
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Telomere Length and Telomerase Activity in Terminally Differentiated Spermatozoa
The Southern blot of the restriction enzyme-digested genomic DNA from terminally differentiated spermatozoa obtained from the rat epididymis probed with labeled telomere-specific sequence exhibited a very strong hybridization signal at a higher molecular size (>23 kb) (Fig. 9A). A quantitative analysis of the signal revealed that the average of mean TRF was 15.2 ± 1.8 kb (SEM), and the range of TRF distribution was between 13.2 and 18.2 kb (Fig. 9B). In parallel, the extracts of terminally differentiated spermatozoa were assayed for telomerase activity. Terminally differentiated spermatozoa did not exhibit any telomerase activity (data not shown).
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DISCUSSION
The present investigation is the first study examining telomere length changes during male germ cell differentiation. Following spermatogonial proliferation and differentiation into the tetraploid pachytene spermatocytes, there appears to be an elongation of the telomeres. Telomere length either remains stable or marginally decreases during the transition of pachytene spermatocytes into the haploid round spermatids. During the final process of the morphological differentiation of round spermatids into spermatozoa and then their maturation in the epididymis, further lengthening of the telomeres ensues. These data indicate that telomere length increases during male germ cell differentiation (see comparative analysis in Fig. 10).
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All eukaryotic chromosomes possess repetitive noncoding sequences known as telomeres that cap their ends and protect the chromosomes from aberrant recombination and degradation during divisions [2, 18]. They consist of evolutionary conserved repetitive DNA and its associated specific DNA binding proteins. In most vertebrates, telomeric DNA consists of tandem repeats of short G-rich sequences [1]. As conventional DNA polymerase cannot complete the replication of chromosome ends, in the absence of an active elongation mechanism, the telomeres shorten with each cell division [5]. Normal human somatic cells lose terminal repeats of TTAGGG at a rate of 50–200 base pairs (bp) per population doubling [5]. To balance for chromosome shortening, telomeric repeat sequences can be added de novo onto chromosome ends by the enzyme telomerase. Telomerase is a reverse transcriptase enzyme containing an RNA template for the telomeric sequence, and it synthesizes telomeres de novo by specifically adding them to the chromosome 3' ends [19].
During the process of spermatogenesis, type A spermatogonial stem cells undergo DNA synthesis and replication either to renew themselves or to differentiate into intermediate and type B spermatogonia. Type B spermatogonia proliferate and differentiate into preleptotene spermatocytes by mitosis. Preleptotene spermatocytes undergo meiotic divisions to ultimately produce tetraploid pachytene spermatocytes. Subsequently, in the absence of DNA synthesis, reductional meiotic divisions occur to yield haploid round spermatids that eventually transform into spermatozoa. Very little is known about the effect of these divisions on the telomere length dynamics during spermatogenesis. In an initial experiment (Fig. 1), we observed that the mean telomeric length in the genomic DNA isolated from whole immature rat testis (9 days old) was 9.1 kb and this was significantly shorter than the mean telomeric length of adult rat testis (12.8 kb). In immature rat testis, at this age, the only germ cell type present is the type A spermatogonia [14, 20]. In comparison, adult rat testis consists of all the differentiating forms of germ cell types including the differentiated late spermatids in addition to type A spermatogonia. The testicular somatic cells present in both immature and adult testes are Sertoli cells, peritubular myoid cells, and Leydig cells. In addition, macrophages, fibroblasts, and vascular endothelial cells are also present. Thus, it was difficult to assess the telomeric length of germ cell types specifically using whole testis. A highly purified population of type A spermatogonia was isolated from immature rat testis and characterized by the technique standardized in our laboratory [14, 21]. Terminal restriction fragment analysis of genomic DNA from the population of the isolated type A spermatogonia revealed that the telomere length was 10.3 kb. Despite high levels of telomerase activity observed both in immature testis and in type A spermatogonia [12] (present study), the telomeres were of a relatively short length. In comparison with the immature testis, the adult testis consisting of a variety of germ cell types (spermatogonia, spermatocytes, early and late spermatids), exhibited a significantly longer mean telomere length (12.8 kb). This suggested that the differentiating germ cell types that outnumber the somatic cells in the adult testis might possibly have longer telomeres. Longer telomere lengths in adult mouse testis compared to neonate testis have also been reported previously [22]. Interestingly, the adult testis exhibited relatively low telomerase activity in comparison with the immature testis or type A spermatogonia [12] (present study). Thus, there appears to be an inverse correlation between the expression of telomerase activity and the length of telomeres in both immature and adult testes.
Most somatic tumors express high levels of telomerase activity [23]. Furthermore, these tumors generally have telomere lengths that are shorter or similar to those found in normal somatic cells [6, 8]. The induction of differentiation of immortalized somatic cells leads to a repression of telomerase activity without a major change in telomere length [24–27]. Human testicular germ cell tumors that are undifferentiated also exhibit short mean telomere lengths with high telomerase activity [28]. Surprisingly, germ cell tumor differentiation into teratomas leads to a repression of telomerase activity and an increase in telomere length [28]. We hypothesized that there is an increase in telomere length and a repression of telomerase activity during the formation of spermatozoa from spermatogonial stem cells. To test this hypothesis, we measured the telomere lengths and telomerase activity in pachytene spermatocytes and round spermatids. The pachytene spermatocyte, a postmitotic cell with increased ploidy, exhibited a significant increase in its telomeric length in comparison with the telomeric length of type A spermatogonia. Mean telomere length was consistently shorter (not statistically significant) in postmeiotic haploid round spermatids compared with pachytene spermatocytes.
Elongation of telomere length in pachytene spermatocytes and maintenance or marginal reduction in the telomere length in round spermatids was accompanied by reduction and increase in the level of telomerase activity, respectively. This observation is in agreement with previously published results from our laboratory [12] and others [29] on the expression of telomerase activity in germ cells. Although in the previous report, we attributed the presence of activity in pachytene spermatocytes and round spermatids to possible contamination with spermatogonial stem cells [12], it appears that telomerase activity is present and necessary for elongation of telomere length in these cell types. This is further confirmed by the fact that epididymal spermatozoa exhibit significantly longer telomere length in comparison to type A spermatogonia, pachytene spermatocytes, and round spermatids. However, we do not know whether telomere lengthening occurs during spermiogenesis in the testis or during maturation of spermatozoa in the epididymis. As has been reported by others and by our group, spermatozoa do not express telomerase activity [11, 12, 30]. The inverse correlation between telomere length and telomerase activity during the entire process of spermatogenesis is shown schematically in Figure 11.
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The increased length of telomere repeats in sperm compared to somatic cells has been reported [7, 9, 22, 31]. Long telomeres in the sperm are brought to the zygote during fertilization and may help to maintain a critical telomere length during subsequent cell divisions in early blastocyst development. Thus, an elongated telomere length in terminally differentiated spermatozoa may be necessary for maintaining the species-specific telomere length in the newborn.
In summary, this study establishes that telomere length increases during the process of germ cell differentiation from spermatogonial stem cell to spermatozoa. In marked contrast, telomerase activity decreases during the process of germ cell differentiation and is completely repressed in spermatozoa. Thus, these findings indicate that unlike somatic cells, telomere length increases in terminally differentiated germ cells following mitotic and meiotic divisions. Furthermore, telomere length and telomerase activity are inversely correlated in male germ cell types.
FOOTNOTES
First decision: 5 January 2000.
1 This work was supported in part by NIH grants HD 33728 (M.D.) and HD 00627 (M.V.A.). 
2 Correspondence: Martin Dym, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20007. FAX: 202 687 9864; dymm{at}gunet.georgetown.edu
Accepted: March 23, 2000.
Received: December 1, 1999.
REFERENCES
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  S. Schaetzlein, A. Lucas-Hahn, E. Lemme, W. A. Kues, M. Dorsch, M. P. Manns, H. Niemann, and K. L. Rudolph Telomere length is reset during early mammalian embryogenesis PNAS, May 25, 2004; 101(21): 8034 - 8038. [Abstract] [Full Text] [PDF]
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