The Telomerase Activity of Adult Mouse Testis Resides in the Spermatogonial 6-Integrin-Positive Side Population Enriched in Germinal Stem Ce
http://www.100md.com
《内分泌学杂志》
Laboratoire Gamétogenèse, Apoptose et Génotoxicité/Institut National de la Santé et de la Recherche Médicale Unité 566 (L.R., H.B., B.L., M.C., J.T., I.A., P.F.), and Laboratoire de Radiopathologie (F.D.B.), Département de Radiobiologie et Radiopathologie, Direction des Sciences du Vivant, CEA, 92265 Fontenay aux Roses, France
Abstract
Testis is one of the organs with the most telomerase activity in the adult. This activity protects chromosomes from telomere attrition and ensures the transmission of full-length chromosomes to progeny. Little is known about telomerase activity during adult germ cell differentiation, however. We demonstrate here that the telomerase activity of adult mouse testis resides in the 6-integrin-positive Side Population containing spermatogonia and enriched in spermatogonial stem cells. The telomerase activity of these cells fell upon entry into meiosis and during the subsequent spermiogenesis. In addition, the telomerase activity of cells in various stages of differentiation was unaffected by aging and, notably, remained high in the 6-integrin-positive Side Population.
Introduction
TELOMERES ARE THE nucleoprotein structures at the ends of eukaryotic chromosomes (1). Mammalian telomeres consist of several kilobases of tandem TTAGGG repeats associated with specific DNA-binding proteins. Telomeres play numerous roles in the maintenance of chromosomal integrity and cell viability; they prevent chromosome end-to-end fusions and protect chromosomes from degradation (1, 2). They are synthesized de novo by a multisubunit ribonucleoprotein known as telomerase. The telomerase core protein (TERT) (3, 4) is a reverse transcriptase that uses an internal RNA molecule (telomerase RNA component) as template to add telomeric repeat sequences onto chromosome ends (5, 6).
Telomerase is active during embryonic development and decreases after birth in somatic tissues. In adults, cells from germline tissues and most tumors express high levels of telomerase activity (7). Telomerase activity is also detected in normal somatic tissues containing cells with self-renewal capacity, such as in the hematopoietic system (8). Maintenance of telomeres by the action of telomerase has been proposed to play a crucial role in preserving genomic stability and the long-term viability of highly proliferative organs (9). Telomerase activity has been detected in extracts of human and rodent testes (7), and the enzyme is expected to remain active in germline cells during spermatogenesis to ensure the transmission of full-length chromosomes to progeny.
Spermatogenesis is a complex, stepwise process leading to the production of terminally differentiated spermatozoa constantly throughout adulthood. This differentiation process originates from a small pool of stem cells designated Asingle (As) that can self-renew or differentiate (10). When these stem cells differentiate, a phase of proliferation and differentiation occurs to generate Apr, Aal, A1–A4, In, and B spermatogonia. The transition from B spermatogonia to preleptotene spermatocyte and primary spermatocyte characterizes the entry of the cells into meiosis. At the diakinesis phase of meiosis, spermatocytes I are reduced to diploid spermatocytes II, which will divide to give haploid, round spermatids. These spermatids then undergo spermiogenesis to differentiate into elongating spermatids and, finally, into spermatozoa.
Telomerase activity was detected in spermatogonia in the testis of 9-d-old rats, indicating its possible importance during the development of testis (11). In adult rats, telomerase activity was detected in meiotic spermatocytes, but this activity decreased during spermiogenesis (11), and several studies have shown the lack of telomerase activity in epididymal spermatozoa (12, 13). Consistent with telomerase function in spermatogenesis, mice genetically deficient for the telomerase RNA (mTERC) or for telomerase reverse transcriptase (mTERT) exhibit defective spermatogenesis in the generations of animals that result from successive rounds of mating (14, 15).
To date, very little information is available about telomerase expression and regulation in adult testis, and its role in spermatogenesis remains unclear partly because it is very difficult to purify germinal cell populations from adults. Recently, our group developed a method to isolate viable germinal cell populations by using flow cytometry combined with Hoechst 33342 DNA staining (16, 17). Spermatocyte I and round and elongated spermatids can be isolated by this method (17). We showed that normal mouse testicular cells contain a Side Population (SP) that actively excludes Hoechst 33342 because of their expression of breast cancer resistance protein 1, a multidrug resistance transporter protein. We demonstrated that this 6-integrin-positive SP (6+SP) from adult testis contained spermatogonia and was enriched in germinal stem cells (16). Germinal stem cells have also been found in the SP from immature testis (18), but this germinal stem cell phenotype appears to be absent from cryptorchid testis (19). Furthermore, Leydig stem cells were recently found in the SP from cryptorchid testis (20).
Here, we analyze the telomerase activity in each purified germ cell subpopulation from adult mice. Our data indicate that the telomerase activity of adult mouse testis resides in the 6+SP fraction containing spermatogonia and germinal stem cells, suggesting a role for telomerase during the first steps of spermatogenesis and in the expansion phase of spermatogonia. In addition, the high telomerase activity of 6+SP cells is maintained throughout the lifespan of the mouse.
Materials and Methods
Mice and cell lines
C57BL/6 males (Charles River Laboratories, L’arbresle, France) were raised in our animal facilities. Testes collected from adult (2 months old, 1 yr old, and 2 yr old) mice were used immediately for cell separation. All animal procedures reported in this paper were carried out in accordance with French government regulations (Services Vétérinaires de la Santé et de la Production Animale, Ministère de l’Agriculture).
CEM1301 cells, a T cell lymphoblastic leukemia cell line, used as telomerase-positive control (21) (DakoCytomation, Glostrup, Denmark), were cultured at 0.5 x 106/ml in RPMI 1640 (Sigma) supplemented with 10% decomplemented fetal calf serum (FCS) (Invitrogen), 2 mM glutamine (Sigma-Aldrich, Saint-Quentin Fallavier, France), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Sigma).
Testicular single-cell suspensions
Cells were isolated from testis of adult mice as we previously described (16). Briefly, the seminiferous tubules were dissociated using enzymatic digestion with collagenase, followed by filtration through a 40-μm nylon mesh to remove the interstitial cells and incubation in cell dissociation buffer (Invitrogen) for 25 min at 32 C. After centrifugation, cells were resuspended in incubation medium [IM: Hanks’ balanced salt solution (HBSS) supplemented with 20 mM HEPES (pH 7.2), 1.2 mM MgSO4, 1.3 mM CaCl2, 6.6 mM sodium pyruvate, 0.05% lactate, glutamine, and 1% FCS]. Cell numbers were estimated using Trypan Blue staining (>95% viable cells). Spermatozoa were isolated by mechanical expression from the epididymis of adult mice.
Magnetic-activated cell sorting (MACS)
MACS of 6-integrin-positive cells was performed as we previously described (16). To purify 6-integrin-positive cells, 4 x 107 testicular cells were suspended in 160 μl of incubation medium and labeled with 40 μl of R-phycoerythrin-conjugated anti-6-integrin antibodies (GoH3) (BD Biosciences, Le Pont-de-Claix, France) for 15 min at 4 C. Cells were washed and resuspended in HBSS supplemented with 1 mM HEPES (pH 7.2) and 0.5% FCS (HBSS/HEPES/BSA). Labeled cells were then sorted with antiphycoerythrin antibody coupled to magnetic microbeads (Miltenyi Biotec, Paris, France) according to the manufacturer’s instructions. The 6-integrin-positive cell fraction was collected and resuspended at 106 cells/ml in IM. MACS of Thy-1-positive cells was performed using magnetic microbeads conjugated to anti-Thy-1 antibody (30-H12; Miltenyi Biotec) according to the manufacturer’s instructions.
Flow cytometry and cell sorting
For flow cytometry and cell sorting, 106 cells/ml were diluted in incubation medium and stained with Hoechst 33342 (5 μg/ml) for 1 h at 32 C. Before analysis, propidium iodide (PI) (2 μg/ml) was added to exclude dead cells. CD9 was detected by Alexa Fluor 647-R-phycoerythrin streptavidin (Molecular Probes/Invitrogen, Cergy-Pontoise, France) after staining with biotin-conjugated anti-CD9 antibody (KMC8) or isotype control antibody. MACS Thy-1-selected cells were labeled with R-phycoerythrin-conjugated anti-6-integrin antibody (GoH3) or isotype control antibody for 15 min at 4 C. Analysis and cell sorting were performed on a dual-laser FACStarPlus flow cytometer (BD Biosciences) as we described previously (16, 17). All antibodies were obtained from BD Biosciences unless otherwise stated.
For epithelial cell adhesion molecule (Ep-CAM) analysis, 107 dissociated testis cells were first stained with Hoechst 33342 as previously described. Hoechst-stained cells were washed and labeled with 15 μg of anti-Ep-CAM. (G8.8) or isotype control antibodies at 4 C for 30 min in IM. Cells were washed twice with excess IM and stained with 15 μg/ml Alexa Fluor 488 goat antirat IgG (Molecular Probes). Cells were washed twice with excess IM, and 6-integrin magnetic cell sorting was performed.
Telomeric repeat amplification protocol (TRAP)
Assays were performed using the TRAPeze ELISA telomerase detection kit (MP Biomedicals/Q-biogene, Illkirch, France) according to the manufacturer’s instructions. In brief, cell extracts were incubated with biotinylated telomerase substrate oligonucleotide at 30 C for 30 min. The extended products were amplified by PCR using Taq polymerase (Amersham Biosciences, Orsay, France), biotinylated telomerase substrate oligonucleotide, reverse primers, and a deoxynucleotide mix containing dCTP labeled with dinitrophenyl. The PCR conditions were 33 cycles of 94 C for 30 sec and 55 C for 30 sec on a Gene Amp PCR System 9700 thermocycler (Applera/Applied Biosystems, Courtaboeuf, France). The amplification products were immobilized on streptavidin-coated microtiter plates and then detected by antidinitrophenyl antibody conjugated to horseradish peroxidase. After addition of the peroxidase substrate (3,3',5,5'-tetramethylbenzidine), the amount of TRAP products was determined by measuring the absorbance at 450 and 690 nm. To confirm the results of the ELISA, amplified products were resolved by 12.5% nondenaturing PAGE in 0.5x Tris-borate, EDTA buffer. Gels were stained with SYBRgreen (Invitrogen) (dilution 1/10,000), and PCR amplification products were visualized under UV light. Furthermore, because telomerase has an essential RNA component, aliquots of samples were treated with RNase to assess the specificity of the reaction (7). Telomerase activity was measured twice on protein samples obtained from each cell fraction from two independent preparations. To compare the different germinal populations in the same experiment, assays were performed on 1800 sorted cells to be in the semiquantitative conditions (OD values between 0.2 and 1.5). The data we display are means from four adult mice. Data were statistically compared by t tests. The protein concentration was determined using the QuantiPro BCA assay kit (Sigma) and BSA as a standard.
Results
The 6+SP cells in normal adult testis express Ep-CAM, CD9, and Thy-1
We reported previously that the SP fraction from adult testis contained spermatogonia and germinal stem cells (16) (Fig. 1A). The SP is obtained from adult testis by using a two-step digestion procedure to produce a cell suspension whose interstitial cells have been discarded (see Material and Methods). To obtain a cellular suspension highly enriched in SP cells, we used a magnetic bead procedure (MACS) to select cells positive for 6-integrin (Fig. 1B). Before assessing the telomerase activity of this cell population, we first characterized the purity of the 6+SP cells from adult testis by studying by flow cytometry the expression of the spermatogonia marker Ep-CAM (22). Ninety-five percent of the cells in the 6+SP were positive for Ep-CAM, confirming that 6+SP is composed of spermatogonia (Fig. 1C). 6+SP was also positive for CD9 (97% of the cells), another marker of germinal stem cells and spermatogonia in adult mouse testis (Fig. 1D) (23).
Thy-1 is a surface maker of germinal stem cells in normal and cryptorchid adult mouse testis, and it has been reported that selection of Thy-1-positive cells by MACS enriched germinal stem cells efficiently (19, 24). Therefore, we analyzed the MACS Thy-1-positive fraction from a normal testis cell population by flow cytometry after Hoechst staining (Fig. 1E). The SP population represented 64% of Thy-1-positive diploid cells, and 55% of Thy-1-positive SP cells expressed 6-integrin (Fig. 1F). Thus, 35% (55 of 64%) of Thy-1-positive diploid cells from normal mouse testis had the phenotype 6+SP. Hence, the stem cell marker, Thy-1, is expressed in 6+SP of normal mouse testis.
Telomerase activity in adult mouse testis
We assessed telomerase activity in the extracts using TRAP (7) followed by ELISA detection. In PAGE analysis, extracts from total adult testis cell suspension and 6+SP sorted by flow cytometry exhibited telomerase activity (Fig. 2A). The intensity of the bands of the TRAP assay products was stronger in the 6+SP testis extract (lane 3) when compared with the intensity of products in the total testis suspension extract (lane 1). The specificity of the TRAP assay for telomerase activity in cell lysates was tested by pretreating lysates with RNase, which destroys the RNA component of telomerase and inactivates the enzyme. Thus, products of telomerase activity were absent in RNase-treated samples of both total and 6+SP extracts (lanes 2 and 4, respectively). Telomerase activity was not observed in the TRAP assay buffer lane (negative control, lane 5). A quantitative analysis of telomerase activity was performed by analyzing increasing amounts of protein from telomerase-positive control cells (the lymphoblastic T cell line 1301) from a fraction of total testis cell suspension and from the 6+SP of 2-month-old mice sorted by flow cytometry (Fig. 2B). The linearity of the assay was observed. The 6+SP extracts exhibited higher activity than total extracts, showing that telomerase activity in adult testis resides in the 6+SP population.
Telomerase activity during spermatogenesis
To assess the contribution of the various differentiated cell types to testicular telomerase activity, various germ cell populations from adult testes were isolated by flow cytometry as before: spermatogonia and germinal stem cells (SP) in the 6-integrin-positive fraction, spermatocytes I, round and elongated spermatids in the 6-integrin-negative fraction, and the total testicular cell suspension (16, 17). Spermatozoa were isolated by mechanical expression from the epididymis.
Telomerase activity was measured in the protein extracts of each differentiation stage (Fig. 3). The highest activity was found in the 6+SP fraction (1.63 ± 0.07 OD). Telomerase activity was more than 5-fold lower in extracts of spermatocytes I (0.32 ± 0.07 OD), when the cells undergo prophase I of meiosis. Haploid cells had even weaker telomerase activity that decreased further as cells progressed in spermiogenesis (0.13 ± 0.02 and 0.09 ± 0.01 OD, respectively, for round and elongated spermatids). Telomerase activity was totally absent from the extracts of epididymal spermatozoa. Telomerase activity was also absent in RNase-treated samples of all extracts and in the TRAP assay buffer (data not shown).
Telomerase activity and aging
We investigated whether telomerase activity was altered with aging in the various differentiation stages of spermatogenesis from 2-, 12-, and 24-month-old mice (Fig. 4). Telomerase activity from total testicular cell suspension did not vary with aging. High activity was detected in 6+SP cells at any age. No significant difference in telomerase activity was observed as a function of aging in 6+SP. Neither did the levels of telomerase activity change with age in spermatocytes I, in round and elongated spermatids, and in spermatozoa.
Discussion
Male fertility is highly dependent upon the ability of the adult testis constantly to expand spermatogonia. The consecutive rounds of cell division involved in this process suggest a potential role for telomerase to compensate for telomere length erosion and so to preserve normal replicative potential. The maintenance of telomeres by a functional telomerase appears to be essential for spermatogenesis, as shown by the phenotype of telomerase-deficient mice (14, 15). Our current investigation examined telomerase activity during adult male mouse germ cell differentiation. We used flow cytometry to purify viable 6+SP spermatogonial cells, spermatocytes I, round and elongated spermatids in testis (16, 17), and established that telomerase activity in adult mice resides mainly in the 6+SP.
The SP phenotype is one of the characteristics shared by a number of somatic progenitor and stem cells in various species (25). In immature testis, SP was found to be a marker of germinal stem cells (18). In adult testis, we recently demonstrated that 6+SP contains spermatogonia and is enriched in germinal stem cells (16), and this germinal stem cell population appears to be lost in cryptorchid mice (19). The Brinster group reported recently that Thy-1, a glycosyl phosphatidylinositol-anchored surface antigen, is a marker of germinal stem cells in normal and cryptorchid adult testis (24). Thus, we investigated in normal testis the SP phenotype in Thy-1-positive cells selected by MACS. We found that 35% of Thy-1+ cells coexpress the 6-integrin and the SP phenotype in normal testis, demonstrating that a fraction of 6+SP cells is also positive for Thy-1 stem cell marker, unlike a previous report showing that SP was Thy-1 negative in the cryptorchid model (19). Our data indicate that the cell populations that constitute the SP are certainly quite different in normal and cryptorchid animals. Another team recently isolated Leydig stem cells in SP from cryptorchid testis (20), raising the question of whether the SP fraction contains various types of stem cell. Taken together, the data suggest that in cryptorchid testis the SP contains Leydig stem cells but not germinal stem cells. By contrast, our data indicate that in normal testis the SP contains germinal stem cells; we have no data concerning Leydig stem cells in this fraction. Discrepancies between these studies may arise from the methods of purification of SP cells (digestion procedure or MACS enrichment), which can select preferentially some cellular subsets, and the models used (normal vs. cryptorchid) in which stem cells could regulate differently the expression of the ABC transporter Bcrp1, responsible for Hoechst dye efflux. In fact, this phenotype seems to be very sensitive to cellular microenvironment. The SP phenotype of hematopoietic stem cells may vary as a function of cell cycle status or during development, for example (26). Our SP cells from normal mouse testis (so-called 6+SP) result first from a two-step procedure to discard interstitial cells and then a selection of 6-integrin-positive cells by MACS. We show here that this 6+SP fraction is highly pure (95–97%) according to two different markers of germinal stem cells and spermatogonia in adult mouse testis (22, 23). These data and others (16, 17) confirm that the 6+SP fraction from normal, adult mouse testis contains primarily spermatogonia, including male germinal stem cells. We cannot exclude that Leydig stem cells might represent a minimal proportion of 6+SP cells, but this remains to be investigated.
The activity of telomerase in adult mouse testis resides principally in the 6+SP cellular subset corresponding mainly to germinal stem cells and spermatogonia, which are the mitotically active precursor cells in adult testis. We found that telomerase activity decreased dramatically after the entry of germ cells into meiosis; spermatocytes I had weak telomerase activity compared with spermatogonia and germinal stem cells, round and elongated spermatids displayed a progressive decrease of activity as the cells progressed through spermiogenesis, whereas mature spermatozoa obtained from the epididymis of adult mouse showed no evidence of telomerase activity. These data show that telomerase activity is markedly reduced in meiotic and postmeiotic cells during spermatogenesis, in agreement with the reported absence of telomerase activity in spermatozoa from epididymis (12) and in ejaculated spermatozoa from human (13) and rat (11). A similar down-regulation of telomerase activity was previously suggested by comparing the telomerase activity of spermatogonia from developing testis of rat pups (9 d old) to the activity of meiotic and postmeiotic cells in adult spermatogenesis (11).
Telomerase is expressed in embryonic stem cells and adult stem cells from mouse bone marrow (8). Its activity is crucial for self-renewal potential as shown by serial bone marrow transplantation experiments in mice (27). In adult mice, we have shown that telomerase activity resides in the 6+SP, which is highly enriched in germinal stem cells. The ectopic expression of the telomerase catalytic component, mTERT, in undifferentiated type A spermatogonia from 6-d-old mice led to the generation of an immortalized spermatogonial cell line maintained in an undifferentiated state (28), suggesting a role for telomerase in sustaining replicative capacity and self-renewal of germinal stem cells in neonatal testis. Altogether, these data suggest that telomerase could also be involved in the maintenance of the replicative capacity of the germinal stem cell pool throughout the life of adult males, as in hematopoietic stem cells. In addition, maintenance of functional telomeres was revealed to be essential for the processes of synapsis and recombination during meiosis in intermediate late-generation (G4) telomerase-deficient mice showing decreased fertility (29). Thus, the high activity of telomerase in the 6+SP fraction in the adult, which contains mostly mitotically proliferating progenitor cells, might prevent telomere attrition that might later result in meiotic impairment caused by telomeric defects.
An age-related trend of telomere shortening and reduced telomerase activity has been reported in human leukocytes (30, 31), supporting the hypothesis that a defect in telomerase activity might contribute to telomere shortening during aging. In rodents, contradictory results have been reported concerning changes in telomerase activity of various organs with aging (32, 33). We found no significant change in the telomerase activity in testes from 2-, 12-, and 24-month-old mice. Hence, high telomerase activity is maintained during the mitotic expansion phase in spermatogenesis throughout the lifespan of the mouse. This should contribute to the maintenance of telomere length in the germinal cells (unlike the somatic cells) during the lifetime of the mouse (34) and prevent potential impairment of meiosis (29) in old mice. Likewise, this high telomerase activity in premeiotic cells should contribute to the maintenance of the telomere length in sperm (35) as a function of aging and contribute to the immortality of germ lineage.
In conclusion, our results show that telomerase activity in adult mouse testis resides in the 6+SP fraction enriched in germinal stem cells and decreases after the entry in meiosis. The telomerase would prevent telomere attrition during the phase of proliferation and differentiation of spermatogonia, which could result later in meiotic impairment. This telomerase activity remains high during aging in 6+SP. It will be interesting to clarify the role of telomerase in germinal stem cells and especially the potential involvement of telomerase in self-renewal.
Acknowledgments
We thank Dr. Céline Silva Lages for assistance with the telomerase assays and Christine Granotier for her helpful contribution. We thank P. Flament and V. Neuville for their technical assistance in animal facilities.
Footnotes
L.R. was supported by an Organon postdoctoral fellowship.
Abbreviations: Ep-CAM, Epithelial cell adhesion molecule; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; IM, incubation medium; MACS, magnetic-activated cell sorting; PI, propidium iodide; SP, Side Population; 6+SP, 6-integrin-positive SP; TERT, telomerase core protein; TRAP, telomeric repeat amplification protocol.
References
Blackburn EH 1991 Structure and function of telomeres. Nature 350:569–573
Blackburn EH 2000 Telomere states and cell fates. Nature 408:53–56
Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA 1997 hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785–795
Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB, Cech TR 1997 Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955–959
Blasco MA, Funk W, Villeponteau B, Greider CW 1995 Functional characterization and developmental regulation of mouse telomerase RNA. Science 269:1267–1270
Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J, Le S, West MD, Harley CB, Andrews WH, Greider CW, Villepouteau B 1995 The RNA component of human telomerase. Science 269:1236–1241
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW 1994 Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015
Morrison SJ, Prowse KR, Ho P, Weissman IL 1996 Telomerase activity in hematopoietic cells is associated with self-renewal potential. Immunity 5:207–216
Lee HW, Blasco MA, Gottlieb GJ, Horner 2nd JW, Greider CW, DePinho RA 1998 Essential role of mouse telomerase in highly proliferative organs. Nature 392:569–574
de Rooij DG 2001 Proliferation and differentiation of spermatogonial stem cells. Reproduction 121:347–354
Ravindranath N, Dalal R, Solomon B, Djakiew D, Dym M 1997 Loss of telomerase activity during male germ cell differentiation. Endocrinology 138:4026–4029
Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW 1996 Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18:173–179
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. Biol Reprod 56:1120–1125
Herrera E, Samper E, Martin-Caballero J, Flores JM, Lee HW, Blasco MA 1999 Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. EMBO J 18:2950–2960
Erdmann N, Liu Y, Harrington L 2004 Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc Natl Acad Sci USA 101:6080–6085
Lassalle B, Bastos H, Louis JP, Riou L, Testart J, Dutrillaux B, Fouchet P, Allemand I 2004 ‘Side Population’ cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 131:479–487
Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, Allemand I, Fouchet P 2005 Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis. Cytometry A 65A:40–49
Falciatori I, Borsellino G, Haliassos N, Boitani C, Corallini S, Battistini L, Bernardi G, Stefanini M, Vicini E 2004 Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. FASEB J 18:376–378
Kubota H, Avarbock MR, Brinster RL 2003 Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci USA 100:6487–6492
Lo KC, Lei Z, Rao CV, Beck J, Lamb DJ 2004 De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of Leydig stem cells. Endocrinology 145:4011–4015
Pennarun G, Granotier C, Gauthier LR, Gomez D, Hoffschir F, Mandine E, Riou JF, Mergny JL, Mailliet P, Boussin FD 2005 Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene 24:2917–2928
Anderson R, Schaible K, Heasman J, Wylie C 1999 Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil 116:379–384
Kanatsu-Shinohara M, Toyokuni S, Shinohara T 2004 CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 70:70–75
Kubota H, Avarbock MR, Brinster RL 2004 Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 71:722–731
Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, Johnson RP 1997 Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:1337–1345
Uchida N, Dykstra B, Lyons K, Leung F, Kristiansen M, Eaves C 2004 ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood 103:4487–4495
Samper E, Fernandez P, Eguia R, Martin-Rivera L, Bernad A, Blasco MA, Aracil M 2002 Long-term repopulating ability of telomerase-deficient murine hematopoietic stem cells. Blood 99:2767–2775
Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, Dym M 2002 Generation and in vitro differentiation of a spermatogonial cell line. Science 297:392–395
Liu L, Franco S, Spyropoulos B, Moens PB, Blasco MA, Keefe DL 2004 Irregular telomeres impair meiotic synapsis and recombination in mice. Proc Natl Acad Sci USA 101:6496–6501
Iwama H, Ohyashiki K, Ohyashiki JH, Hayashi S, Yahata N, Ando K, Toyama K, Hoshika A, Takasaki M, Mori M, Shay JW 1998 Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals. Hum Genet 102:397–402
Mariani E, Meneghetti A, Formentini I, Neri S, Cattini L, Ravaglia G, Forti P, Facchini A 2003 Telomere length and telomerase activity: effect of ageing on human NK cells. Mech Ageing Dev 124:403–408
Yamaguchi Y, Nozawa K, Savoysky E, Hayakawa N, Nimura Y, Yoshida S 1998 Change in telomerase activity of rat organs during growth and aging. Exp Cell Res 242:120–127
Suzui N, Yoshimi N, Kawabata K, Mori H 1999 The telomerase activities in several organs and strains of rats with ageing. Lab Anim 33:149–154
Prowse KR, Greider CW 1995 Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 92:4818–4822
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB 1992 Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 89:10114–10118(Lydia Riou, Henri Bastos,)
Abstract
Testis is one of the organs with the most telomerase activity in the adult. This activity protects chromosomes from telomere attrition and ensures the transmission of full-length chromosomes to progeny. Little is known about telomerase activity during adult germ cell differentiation, however. We demonstrate here that the telomerase activity of adult mouse testis resides in the 6-integrin-positive Side Population containing spermatogonia and enriched in spermatogonial stem cells. The telomerase activity of these cells fell upon entry into meiosis and during the subsequent spermiogenesis. In addition, the telomerase activity of cells in various stages of differentiation was unaffected by aging and, notably, remained high in the 6-integrin-positive Side Population.
Introduction
TELOMERES ARE THE nucleoprotein structures at the ends of eukaryotic chromosomes (1). Mammalian telomeres consist of several kilobases of tandem TTAGGG repeats associated with specific DNA-binding proteins. Telomeres play numerous roles in the maintenance of chromosomal integrity and cell viability; they prevent chromosome end-to-end fusions and protect chromosomes from degradation (1, 2). They are synthesized de novo by a multisubunit ribonucleoprotein known as telomerase. The telomerase core protein (TERT) (3, 4) is a reverse transcriptase that uses an internal RNA molecule (telomerase RNA component) as template to add telomeric repeat sequences onto chromosome ends (5, 6).
Telomerase is active during embryonic development and decreases after birth in somatic tissues. In adults, cells from germline tissues and most tumors express high levels of telomerase activity (7). Telomerase activity is also detected in normal somatic tissues containing cells with self-renewal capacity, such as in the hematopoietic system (8). Maintenance of telomeres by the action of telomerase has been proposed to play a crucial role in preserving genomic stability and the long-term viability of highly proliferative organs (9). Telomerase activity has been detected in extracts of human and rodent testes (7), and the enzyme is expected to remain active in germline cells during spermatogenesis to ensure the transmission of full-length chromosomes to progeny.
Spermatogenesis is a complex, stepwise process leading to the production of terminally differentiated spermatozoa constantly throughout adulthood. This differentiation process originates from a small pool of stem cells designated Asingle (As) that can self-renew or differentiate (10). When these stem cells differentiate, a phase of proliferation and differentiation occurs to generate Apr, Aal, A1–A4, In, and B spermatogonia. The transition from B spermatogonia to preleptotene spermatocyte and primary spermatocyte characterizes the entry of the cells into meiosis. At the diakinesis phase of meiosis, spermatocytes I are reduced to diploid spermatocytes II, which will divide to give haploid, round spermatids. These spermatids then undergo spermiogenesis to differentiate into elongating spermatids and, finally, into spermatozoa.
Telomerase activity was detected in spermatogonia in the testis of 9-d-old rats, indicating its possible importance during the development of testis (11). In adult rats, telomerase activity was detected in meiotic spermatocytes, but this activity decreased during spermiogenesis (11), and several studies have shown the lack of telomerase activity in epididymal spermatozoa (12, 13). Consistent with telomerase function in spermatogenesis, mice genetically deficient for the telomerase RNA (mTERC) or for telomerase reverse transcriptase (mTERT) exhibit defective spermatogenesis in the generations of animals that result from successive rounds of mating (14, 15).
To date, very little information is available about telomerase expression and regulation in adult testis, and its role in spermatogenesis remains unclear partly because it is very difficult to purify germinal cell populations from adults. Recently, our group developed a method to isolate viable germinal cell populations by using flow cytometry combined with Hoechst 33342 DNA staining (16, 17). Spermatocyte I and round and elongated spermatids can be isolated by this method (17). We showed that normal mouse testicular cells contain a Side Population (SP) that actively excludes Hoechst 33342 because of their expression of breast cancer resistance protein 1, a multidrug resistance transporter protein. We demonstrated that this 6-integrin-positive SP (6+SP) from adult testis contained spermatogonia and was enriched in germinal stem cells (16). Germinal stem cells have also been found in the SP from immature testis (18), but this germinal stem cell phenotype appears to be absent from cryptorchid testis (19). Furthermore, Leydig stem cells were recently found in the SP from cryptorchid testis (20).
Here, we analyze the telomerase activity in each purified germ cell subpopulation from adult mice. Our data indicate that the telomerase activity of adult mouse testis resides in the 6+SP fraction containing spermatogonia and germinal stem cells, suggesting a role for telomerase during the first steps of spermatogenesis and in the expansion phase of spermatogonia. In addition, the high telomerase activity of 6+SP cells is maintained throughout the lifespan of the mouse.
Materials and Methods
Mice and cell lines
C57BL/6 males (Charles River Laboratories, L’arbresle, France) were raised in our animal facilities. Testes collected from adult (2 months old, 1 yr old, and 2 yr old) mice were used immediately for cell separation. All animal procedures reported in this paper were carried out in accordance with French government regulations (Services Vétérinaires de la Santé et de la Production Animale, Ministère de l’Agriculture).
CEM1301 cells, a T cell lymphoblastic leukemia cell line, used as telomerase-positive control (21) (DakoCytomation, Glostrup, Denmark), were cultured at 0.5 x 106/ml in RPMI 1640 (Sigma) supplemented with 10% decomplemented fetal calf serum (FCS) (Invitrogen), 2 mM glutamine (Sigma-Aldrich, Saint-Quentin Fallavier, France), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Sigma).
Testicular single-cell suspensions
Cells were isolated from testis of adult mice as we previously described (16). Briefly, the seminiferous tubules were dissociated using enzymatic digestion with collagenase, followed by filtration through a 40-μm nylon mesh to remove the interstitial cells and incubation in cell dissociation buffer (Invitrogen) for 25 min at 32 C. After centrifugation, cells were resuspended in incubation medium [IM: Hanks’ balanced salt solution (HBSS) supplemented with 20 mM HEPES (pH 7.2), 1.2 mM MgSO4, 1.3 mM CaCl2, 6.6 mM sodium pyruvate, 0.05% lactate, glutamine, and 1% FCS]. Cell numbers were estimated using Trypan Blue staining (>95% viable cells). Spermatozoa were isolated by mechanical expression from the epididymis of adult mice.
Magnetic-activated cell sorting (MACS)
MACS of 6-integrin-positive cells was performed as we previously described (16). To purify 6-integrin-positive cells, 4 x 107 testicular cells were suspended in 160 μl of incubation medium and labeled with 40 μl of R-phycoerythrin-conjugated anti-6-integrin antibodies (GoH3) (BD Biosciences, Le Pont-de-Claix, France) for 15 min at 4 C. Cells were washed and resuspended in HBSS supplemented with 1 mM HEPES (pH 7.2) and 0.5% FCS (HBSS/HEPES/BSA). Labeled cells were then sorted with antiphycoerythrin antibody coupled to magnetic microbeads (Miltenyi Biotec, Paris, France) according to the manufacturer’s instructions. The 6-integrin-positive cell fraction was collected and resuspended at 106 cells/ml in IM. MACS of Thy-1-positive cells was performed using magnetic microbeads conjugated to anti-Thy-1 antibody (30-H12; Miltenyi Biotec) according to the manufacturer’s instructions.
Flow cytometry and cell sorting
For flow cytometry and cell sorting, 106 cells/ml were diluted in incubation medium and stained with Hoechst 33342 (5 μg/ml) for 1 h at 32 C. Before analysis, propidium iodide (PI) (2 μg/ml) was added to exclude dead cells. CD9 was detected by Alexa Fluor 647-R-phycoerythrin streptavidin (Molecular Probes/Invitrogen, Cergy-Pontoise, France) after staining with biotin-conjugated anti-CD9 antibody (KMC8) or isotype control antibody. MACS Thy-1-selected cells were labeled with R-phycoerythrin-conjugated anti-6-integrin antibody (GoH3) or isotype control antibody for 15 min at 4 C. Analysis and cell sorting were performed on a dual-laser FACStarPlus flow cytometer (BD Biosciences) as we described previously (16, 17). All antibodies were obtained from BD Biosciences unless otherwise stated.
For epithelial cell adhesion molecule (Ep-CAM) analysis, 107 dissociated testis cells were first stained with Hoechst 33342 as previously described. Hoechst-stained cells were washed and labeled with 15 μg of anti-Ep-CAM. (G8.8) or isotype control antibodies at 4 C for 30 min in IM. Cells were washed twice with excess IM and stained with 15 μg/ml Alexa Fluor 488 goat antirat IgG (Molecular Probes). Cells were washed twice with excess IM, and 6-integrin magnetic cell sorting was performed.
Telomeric repeat amplification protocol (TRAP)
Assays were performed using the TRAPeze ELISA telomerase detection kit (MP Biomedicals/Q-biogene, Illkirch, France) according to the manufacturer’s instructions. In brief, cell extracts were incubated with biotinylated telomerase substrate oligonucleotide at 30 C for 30 min. The extended products were amplified by PCR using Taq polymerase (Amersham Biosciences, Orsay, France), biotinylated telomerase substrate oligonucleotide, reverse primers, and a deoxynucleotide mix containing dCTP labeled with dinitrophenyl. The PCR conditions were 33 cycles of 94 C for 30 sec and 55 C for 30 sec on a Gene Amp PCR System 9700 thermocycler (Applera/Applied Biosystems, Courtaboeuf, France). The amplification products were immobilized on streptavidin-coated microtiter plates and then detected by antidinitrophenyl antibody conjugated to horseradish peroxidase. After addition of the peroxidase substrate (3,3',5,5'-tetramethylbenzidine), the amount of TRAP products was determined by measuring the absorbance at 450 and 690 nm. To confirm the results of the ELISA, amplified products were resolved by 12.5% nondenaturing PAGE in 0.5x Tris-borate, EDTA buffer. Gels were stained with SYBRgreen (Invitrogen) (dilution 1/10,000), and PCR amplification products were visualized under UV light. Furthermore, because telomerase has an essential RNA component, aliquots of samples were treated with RNase to assess the specificity of the reaction (7). Telomerase activity was measured twice on protein samples obtained from each cell fraction from two independent preparations. To compare the different germinal populations in the same experiment, assays were performed on 1800 sorted cells to be in the semiquantitative conditions (OD values between 0.2 and 1.5). The data we display are means from four adult mice. Data were statistically compared by t tests. The protein concentration was determined using the QuantiPro BCA assay kit (Sigma) and BSA as a standard.
Results
The 6+SP cells in normal adult testis express Ep-CAM, CD9, and Thy-1
We reported previously that the SP fraction from adult testis contained spermatogonia and germinal stem cells (16) (Fig. 1A). The SP is obtained from adult testis by using a two-step digestion procedure to produce a cell suspension whose interstitial cells have been discarded (see Material and Methods). To obtain a cellular suspension highly enriched in SP cells, we used a magnetic bead procedure (MACS) to select cells positive for 6-integrin (Fig. 1B). Before assessing the telomerase activity of this cell population, we first characterized the purity of the 6+SP cells from adult testis by studying by flow cytometry the expression of the spermatogonia marker Ep-CAM (22). Ninety-five percent of the cells in the 6+SP were positive for Ep-CAM, confirming that 6+SP is composed of spermatogonia (Fig. 1C). 6+SP was also positive for CD9 (97% of the cells), another marker of germinal stem cells and spermatogonia in adult mouse testis (Fig. 1D) (23).
Thy-1 is a surface maker of germinal stem cells in normal and cryptorchid adult mouse testis, and it has been reported that selection of Thy-1-positive cells by MACS enriched germinal stem cells efficiently (19, 24). Therefore, we analyzed the MACS Thy-1-positive fraction from a normal testis cell population by flow cytometry after Hoechst staining (Fig. 1E). The SP population represented 64% of Thy-1-positive diploid cells, and 55% of Thy-1-positive SP cells expressed 6-integrin (Fig. 1F). Thus, 35% (55 of 64%) of Thy-1-positive diploid cells from normal mouse testis had the phenotype 6+SP. Hence, the stem cell marker, Thy-1, is expressed in 6+SP of normal mouse testis.
Telomerase activity in adult mouse testis
We assessed telomerase activity in the extracts using TRAP (7) followed by ELISA detection. In PAGE analysis, extracts from total adult testis cell suspension and 6+SP sorted by flow cytometry exhibited telomerase activity (Fig. 2A). The intensity of the bands of the TRAP assay products was stronger in the 6+SP testis extract (lane 3) when compared with the intensity of products in the total testis suspension extract (lane 1). The specificity of the TRAP assay for telomerase activity in cell lysates was tested by pretreating lysates with RNase, which destroys the RNA component of telomerase and inactivates the enzyme. Thus, products of telomerase activity were absent in RNase-treated samples of both total and 6+SP extracts (lanes 2 and 4, respectively). Telomerase activity was not observed in the TRAP assay buffer lane (negative control, lane 5). A quantitative analysis of telomerase activity was performed by analyzing increasing amounts of protein from telomerase-positive control cells (the lymphoblastic T cell line 1301) from a fraction of total testis cell suspension and from the 6+SP of 2-month-old mice sorted by flow cytometry (Fig. 2B). The linearity of the assay was observed. The 6+SP extracts exhibited higher activity than total extracts, showing that telomerase activity in adult testis resides in the 6+SP population.
Telomerase activity during spermatogenesis
To assess the contribution of the various differentiated cell types to testicular telomerase activity, various germ cell populations from adult testes were isolated by flow cytometry as before: spermatogonia and germinal stem cells (SP) in the 6-integrin-positive fraction, spermatocytes I, round and elongated spermatids in the 6-integrin-negative fraction, and the total testicular cell suspension (16, 17). Spermatozoa were isolated by mechanical expression from the epididymis.
Telomerase activity was measured in the protein extracts of each differentiation stage (Fig. 3). The highest activity was found in the 6+SP fraction (1.63 ± 0.07 OD). Telomerase activity was more than 5-fold lower in extracts of spermatocytes I (0.32 ± 0.07 OD), when the cells undergo prophase I of meiosis. Haploid cells had even weaker telomerase activity that decreased further as cells progressed in spermiogenesis (0.13 ± 0.02 and 0.09 ± 0.01 OD, respectively, for round and elongated spermatids). Telomerase activity was totally absent from the extracts of epididymal spermatozoa. Telomerase activity was also absent in RNase-treated samples of all extracts and in the TRAP assay buffer (data not shown).
Telomerase activity and aging
We investigated whether telomerase activity was altered with aging in the various differentiation stages of spermatogenesis from 2-, 12-, and 24-month-old mice (Fig. 4). Telomerase activity from total testicular cell suspension did not vary with aging. High activity was detected in 6+SP cells at any age. No significant difference in telomerase activity was observed as a function of aging in 6+SP. Neither did the levels of telomerase activity change with age in spermatocytes I, in round and elongated spermatids, and in spermatozoa.
Discussion
Male fertility is highly dependent upon the ability of the adult testis constantly to expand spermatogonia. The consecutive rounds of cell division involved in this process suggest a potential role for telomerase to compensate for telomere length erosion and so to preserve normal replicative potential. The maintenance of telomeres by a functional telomerase appears to be essential for spermatogenesis, as shown by the phenotype of telomerase-deficient mice (14, 15). Our current investigation examined telomerase activity during adult male mouse germ cell differentiation. We used flow cytometry to purify viable 6+SP spermatogonial cells, spermatocytes I, round and elongated spermatids in testis (16, 17), and established that telomerase activity in adult mice resides mainly in the 6+SP.
The SP phenotype is one of the characteristics shared by a number of somatic progenitor and stem cells in various species (25). In immature testis, SP was found to be a marker of germinal stem cells (18). In adult testis, we recently demonstrated that 6+SP contains spermatogonia and is enriched in germinal stem cells (16), and this germinal stem cell population appears to be lost in cryptorchid mice (19). The Brinster group reported recently that Thy-1, a glycosyl phosphatidylinositol-anchored surface antigen, is a marker of germinal stem cells in normal and cryptorchid adult testis (24). Thus, we investigated in normal testis the SP phenotype in Thy-1-positive cells selected by MACS. We found that 35% of Thy-1+ cells coexpress the 6-integrin and the SP phenotype in normal testis, demonstrating that a fraction of 6+SP cells is also positive for Thy-1 stem cell marker, unlike a previous report showing that SP was Thy-1 negative in the cryptorchid model (19). Our data indicate that the cell populations that constitute the SP are certainly quite different in normal and cryptorchid animals. Another team recently isolated Leydig stem cells in SP from cryptorchid testis (20), raising the question of whether the SP fraction contains various types of stem cell. Taken together, the data suggest that in cryptorchid testis the SP contains Leydig stem cells but not germinal stem cells. By contrast, our data indicate that in normal testis the SP contains germinal stem cells; we have no data concerning Leydig stem cells in this fraction. Discrepancies between these studies may arise from the methods of purification of SP cells (digestion procedure or MACS enrichment), which can select preferentially some cellular subsets, and the models used (normal vs. cryptorchid) in which stem cells could regulate differently the expression of the ABC transporter Bcrp1, responsible for Hoechst dye efflux. In fact, this phenotype seems to be very sensitive to cellular microenvironment. The SP phenotype of hematopoietic stem cells may vary as a function of cell cycle status or during development, for example (26). Our SP cells from normal mouse testis (so-called 6+SP) result first from a two-step procedure to discard interstitial cells and then a selection of 6-integrin-positive cells by MACS. We show here that this 6+SP fraction is highly pure (95–97%) according to two different markers of germinal stem cells and spermatogonia in adult mouse testis (22, 23). These data and others (16, 17) confirm that the 6+SP fraction from normal, adult mouse testis contains primarily spermatogonia, including male germinal stem cells. We cannot exclude that Leydig stem cells might represent a minimal proportion of 6+SP cells, but this remains to be investigated.
The activity of telomerase in adult mouse testis resides principally in the 6+SP cellular subset corresponding mainly to germinal stem cells and spermatogonia, which are the mitotically active precursor cells in adult testis. We found that telomerase activity decreased dramatically after the entry of germ cells into meiosis; spermatocytes I had weak telomerase activity compared with spermatogonia and germinal stem cells, round and elongated spermatids displayed a progressive decrease of activity as the cells progressed through spermiogenesis, whereas mature spermatozoa obtained from the epididymis of adult mouse showed no evidence of telomerase activity. These data show that telomerase activity is markedly reduced in meiotic and postmeiotic cells during spermatogenesis, in agreement with the reported absence of telomerase activity in spermatozoa from epididymis (12) and in ejaculated spermatozoa from human (13) and rat (11). A similar down-regulation of telomerase activity was previously suggested by comparing the telomerase activity of spermatogonia from developing testis of rat pups (9 d old) to the activity of meiotic and postmeiotic cells in adult spermatogenesis (11).
Telomerase is expressed in embryonic stem cells and adult stem cells from mouse bone marrow (8). Its activity is crucial for self-renewal potential as shown by serial bone marrow transplantation experiments in mice (27). In adult mice, we have shown that telomerase activity resides in the 6+SP, which is highly enriched in germinal stem cells. The ectopic expression of the telomerase catalytic component, mTERT, in undifferentiated type A spermatogonia from 6-d-old mice led to the generation of an immortalized spermatogonial cell line maintained in an undifferentiated state (28), suggesting a role for telomerase in sustaining replicative capacity and self-renewal of germinal stem cells in neonatal testis. Altogether, these data suggest that telomerase could also be involved in the maintenance of the replicative capacity of the germinal stem cell pool throughout the life of adult males, as in hematopoietic stem cells. In addition, maintenance of functional telomeres was revealed to be essential for the processes of synapsis and recombination during meiosis in intermediate late-generation (G4) telomerase-deficient mice showing decreased fertility (29). Thus, the high activity of telomerase in the 6+SP fraction in the adult, which contains mostly mitotically proliferating progenitor cells, might prevent telomere attrition that might later result in meiotic impairment caused by telomeric defects.
An age-related trend of telomere shortening and reduced telomerase activity has been reported in human leukocytes (30, 31), supporting the hypothesis that a defect in telomerase activity might contribute to telomere shortening during aging. In rodents, contradictory results have been reported concerning changes in telomerase activity of various organs with aging (32, 33). We found no significant change in the telomerase activity in testes from 2-, 12-, and 24-month-old mice. Hence, high telomerase activity is maintained during the mitotic expansion phase in spermatogenesis throughout the lifespan of the mouse. This should contribute to the maintenance of telomere length in the germinal cells (unlike the somatic cells) during the lifetime of the mouse (34) and prevent potential impairment of meiosis (29) in old mice. Likewise, this high telomerase activity in premeiotic cells should contribute to the maintenance of the telomere length in sperm (35) as a function of aging and contribute to the immortality of germ lineage.
In conclusion, our results show that telomerase activity in adult mouse testis resides in the 6+SP fraction enriched in germinal stem cells and decreases after the entry in meiosis. The telomerase would prevent telomere attrition during the phase of proliferation and differentiation of spermatogonia, which could result later in meiotic impairment. This telomerase activity remains high during aging in 6+SP. It will be interesting to clarify the role of telomerase in germinal stem cells and especially the potential involvement of telomerase in self-renewal.
Acknowledgments
We thank Dr. Céline Silva Lages for assistance with the telomerase assays and Christine Granotier for her helpful contribution. We thank P. Flament and V. Neuville for their technical assistance in animal facilities.
Footnotes
L.R. was supported by an Organon postdoctoral fellowship.
Abbreviations: Ep-CAM, Epithelial cell adhesion molecule; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; IM, incubation medium; MACS, magnetic-activated cell sorting; PI, propidium iodide; SP, Side Population; 6+SP, 6-integrin-positive SP; TERT, telomerase core protein; TRAP, telomeric repeat amplification protocol.
References
Blackburn EH 1991 Structure and function of telomeres. Nature 350:569–573
Blackburn EH 2000 Telomere states and cell fates. Nature 408:53–56
Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA 1997 hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785–795
Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB, Cech TR 1997 Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955–959
Blasco MA, Funk W, Villeponteau B, Greider CW 1995 Functional characterization and developmental regulation of mouse telomerase RNA. Science 269:1267–1270
Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J, Le S, West MD, Harley CB, Andrews WH, Greider CW, Villepouteau B 1995 The RNA component of human telomerase. Science 269:1236–1241
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW 1994 Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015
Morrison SJ, Prowse KR, Ho P, Weissman IL 1996 Telomerase activity in hematopoietic cells is associated with self-renewal potential. Immunity 5:207–216
Lee HW, Blasco MA, Gottlieb GJ, Horner 2nd JW, Greider CW, DePinho RA 1998 Essential role of mouse telomerase in highly proliferative organs. Nature 392:569–574
de Rooij DG 2001 Proliferation and differentiation of spermatogonial stem cells. Reproduction 121:347–354
Ravindranath N, Dalal R, Solomon B, Djakiew D, Dym M 1997 Loss of telomerase activity during male germ cell differentiation. Endocrinology 138:4026–4029
Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW 1996 Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18:173–179
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. Biol Reprod 56:1120–1125
Herrera E, Samper E, Martin-Caballero J, Flores JM, Lee HW, Blasco MA 1999 Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. EMBO J 18:2950–2960
Erdmann N, Liu Y, Harrington L 2004 Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc Natl Acad Sci USA 101:6080–6085
Lassalle B, Bastos H, Louis JP, Riou L, Testart J, Dutrillaux B, Fouchet P, Allemand I 2004 ‘Side Population’ cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 131:479–487
Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, Allemand I, Fouchet P 2005 Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis. Cytometry A 65A:40–49
Falciatori I, Borsellino G, Haliassos N, Boitani C, Corallini S, Battistini L, Bernardi G, Stefanini M, Vicini E 2004 Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. FASEB J 18:376–378
Kubota H, Avarbock MR, Brinster RL 2003 Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci USA 100:6487–6492
Lo KC, Lei Z, Rao CV, Beck J, Lamb DJ 2004 De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of Leydig stem cells. Endocrinology 145:4011–4015
Pennarun G, Granotier C, Gauthier LR, Gomez D, Hoffschir F, Mandine E, Riou JF, Mergny JL, Mailliet P, Boussin FD 2005 Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene 24:2917–2928
Anderson R, Schaible K, Heasman J, Wylie C 1999 Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil 116:379–384
Kanatsu-Shinohara M, Toyokuni S, Shinohara T 2004 CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 70:70–75
Kubota H, Avarbock MR, Brinster RL 2004 Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 71:722–731
Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, Johnson RP 1997 Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:1337–1345
Uchida N, Dykstra B, Lyons K, Leung F, Kristiansen M, Eaves C 2004 ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood 103:4487–4495
Samper E, Fernandez P, Eguia R, Martin-Rivera L, Bernad A, Blasco MA, Aracil M 2002 Long-term repopulating ability of telomerase-deficient murine hematopoietic stem cells. Blood 99:2767–2775
Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, Dym M 2002 Generation and in vitro differentiation of a spermatogonial cell line. Science 297:392–395
Liu L, Franco S, Spyropoulos B, Moens PB, Blasco MA, Keefe DL 2004 Irregular telomeres impair meiotic synapsis and recombination in mice. Proc Natl Acad Sci USA 101:6496–6501
Iwama H, Ohyashiki K, Ohyashiki JH, Hayashi S, Yahata N, Ando K, Toyama K, Hoshika A, Takasaki M, Mori M, Shay JW 1998 Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals. Hum Genet 102:397–402
Mariani E, Meneghetti A, Formentini I, Neri S, Cattini L, Ravaglia G, Forti P, Facchini A 2003 Telomere length and telomerase activity: effect of ageing on human NK cells. Mech Ageing Dev 124:403–408
Yamaguchi Y, Nozawa K, Savoysky E, Hayakawa N, Nimura Y, Yoshida S 1998 Change in telomerase activity of rat organs during growth and aging. Exp Cell Res 242:120–127
Suzui N, Yoshimi N, Kawabata K, Mori H 1999 The telomerase activities in several organs and strains of rats with ageing. Lab Anim 33:149–154
Prowse KR, Greider CW 1995 Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 92:4818–4822
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB 1992 Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 89:10114–10118(Lydia Riou, Henri Bastos,)