Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus
E. Morales A , L. M. Pastor A E , R. Horn A , A. Zuasti A , C. Ferrer A , A. Calvo C , L. Santamaría D and M. Canteras BA Department of Cell Biology, School of Medicine, University of Murcia, 30100 Murcia, Spain.
B Department of Statistics, School of Medicine, University of Murcia, 30100 Murcia, Spain.
C Department of Histology, School of Medicine, University of Navarra, 31080 Pamplona, Spain.
D Department of Morphology, School of Medicine, Autonomous University of Madrid, 28029 Madrid, Spain.
E To whom correspondence should be addressed. email: bioetica@um.es
Reproduction, Fertility and Development 15(2) 1-10 https://doi.org/10.1071/RD02071
Submitted: 30 August 2002 Accepted: 1 February 2003 Published: 1 February 2003
Abstract
The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 ± 1.6%) compared with young animals (30.8 ± 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 ± 0.23% v. 0.77 ± 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, characterized by maturation arrest and an increase of apoptotic cells in tubular cross-sections in comparison with normal tubular cross-sections. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.
Extra keywords: ageing
Acknowledgments
We thank M. Carmen Gonzalez Ulloa (University of Murcia) for photographic and technical assistance and Manrique Cos Terrer (University of Murcia) for technical help. We are also grateful to all the members of the Servicio de Microscopía Electrónica (University of Murcia) for their technical help.
This work was supported by Fundación Séneca, Comunidad Autónoma de la Región de Murcia, PI-56/00866/FS/01. A portion of this report has been published in abstract form for the 41st American Society for Cell Biology Annual Meeting in Molecular Biology of the Cell, 12 (Suppl.) 234a–235a (2001). E. Morales is supported by a fellowship from Fundación Mapfre Medicina (Spain).
Allan, D. J. , Harmon, B. V. , and Roberts, S. A. (1992). Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during spermatogenesis in the rat Cell Prolif. 25, 241–50.
| PubMed |
Barnes, C. J. , Covington, B. W. , and Lee, M. (1999). Effect of aging and dietary restriction on rat testicular germ cell apoptosis J. Gerontol. 54, B199–204.
Beumer, T. L. , Roepers-Gajadien, H. L. , Gademan, I. S. , Lock, T. , Kal, H. B. , and De Rooij, D. G. (2000). Apoptosis regulation in the testis: involvement of Bcl-2 family members Mol. Reprod. Dev. 56, 353–9.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Blanchard, K. T. , Allard, E. K. , and Boekelheide, K. (1996). Fate of germ cells in 2,5-hexanedione-induced testicular injury. I. Apoptosis is the mechanism of germ cell death Toxicol. Appl. Pharmacol. 137, 141–8.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Blanco-Rodríguez, J. , and Martínez-García, C. (1996). Induction of apoptotic cell death in the seminiferous tubule of the adult rat testis: assessment of the germ cell types that exhibit the ability to enter apoptosis after hormone suppression by oestradiol treatment. Int. J. Androl. 19, 237–47.
| PubMed |
Brinkworth, M. H. , Weinbauer, G. F. , Bergmann, M. , and Nieschlag, E. (1997). Apoptosis as a mechanism of germ cell loss in elderly men Int. J. Androl. 20, 222–8.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Calvo, A. , Martínez, E. , Pastor, L. M. , Vázquez, J. M. , and Roca, J. (1997). Classification and quantification of abnormal sperm along the epididymal tract. Comparison between adult and aged hamsters Reprod. Nutr. Dev. 37, 661–73.
| PubMed |
Calvo, A. , Pastor, L. M. , Martínez, E. , Vázquez, J. M. , and Roca, J. (1999). Age-related changes in the hamster epididymis Anat. Rec. 256, 335–46.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Cutler, R. (1991). Human longevity and aging: Possible role of reactive oxygen species Ann. N. Y. Acad. Sci. 621, 1–28.
| PubMed |
De Kretser, D. M. and Kerr, J. B. (1994). The cytology of the testis. In
Floderus, S. (1944). Untersuchungen über ban Bau der menschlichen Hypophyse mit besonderer Berunckrichtigung der quantitative mikromorphologishen Verhaltnisse Acta Pathol. Microbiol. Scand. 53, 1–276.(in German)
Hacker, G. (2000). The morphology of apoptosis Cell Tissue Res. 301, 5–17.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Hakovirta, H. , Yan, W. , Kaleva, M. , Zhang, F. P. , Vänttinen, K. , Morris, P. L. , Söder, O. , Parvinen, M. , and Toppari, J. (1999). Function of stem cell factor as a survival factor of spermatogonia and localization of messenger ribonucleic acid in the rat seminiferous epithelium Endocrinology 140, 1492–8.
| PubMed |
Higami, Y. , and Shimokawa, I. (2000). Apoptosis in the aging process Cell Tissue Res. 301, 125–32.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Horn, R. , Pastor, L. M. , Moreno, E. , Calvo, A. , Canteras, M. , and Pallarés, J. (1996). Morphological and morphometric study of early changes in the ageing golden hamster testis J. Anat. 188, 109–17.
| PubMed |
Hsued, A. J. , Eisenhauer, K. , Chun, S. Y. , Hsu, S. Y. , and Billig, H. (1996). Gonadal cell apoptosis Rec. Prog. Horm. Res. 51, 433–55.
| PubMed |
Kierszenbaum, A. L. (2001). Apoptosis during spermatogenesis: the thrill of being alive Mol. Reprod. Dev. 58, 1–3.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Lee, J. , Richburg, J. H. , Younkin, S. C. , and Boekelheide, K. (1997). The Fas system is a key regulator of germ cell apoptosis in the testis Endocrinology 138, 2081–8.
| PubMed |
Li, L. H. , Wine, R. N. , and Chapin, R. E. (1996). 2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testis: an in vitro comparison J. Androl. 17, 538–49.
| PubMed |
Loveland, K. L. , and Schlatt, S. (1997). Stem cell factor and c-kit in the mammalian testis: lessons originating from mother nature’s gene knockouts J. Endocrinol. 153, 337–44.
| PubMed |
Lue, Y. , Sinha-Hikim, A. P. , Swerdloff, R. S. , Im, P. , Taing, K. S. , Bui, T. , Leung, A. , and Wang, C (1999). Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity Endocrinology 140, 1709–17.
| PubMed |
Meistrich, M. L. (1993). Effects of chemotherapy and radiotherapy on spermatogenesis Eur. Urol. 23, 136–41.
| PubMed |
Morales, E. , Pastor, L. M. , Ferrer, C. , Zuasti, A. , Pallarés, J. , Horn, R. , Calvo, A. , Santamaría, L. , and Canteras, M. (2002). Proliferation and apoptosis in the seminiferous epithelium of photoinhibited Syrian hamsters (Mesocricetus auratus) Int. J. Androl. 25, 281–7.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Obregón, E. B. , and Ramírez, O. (1997). Ageing and testicular function in Octodon degus. Andrologia 29, 319–26.
| PubMed |
Olaso, R. , Pairault, C. , and Habert, R. (1998). Expression of type I and II receptors for transforming growth factor β in the adult rat testis. Histochem Cell Biol. 110, 613–18.
| Crossref | GoogleScholarGoogle Scholar |
Paniagua, R. , Nistal, M. , Amat, P. , Rodriguez, M. C. , and Martín, A. (1987). Seminiferous tubule involution in elderly men Biol. Reprod. 36, 939–47.
| PubMed |
Paniagua, R. , Martinez-Onsurbe, P. , Santamaría, L. , Sáez, F. J. , Amat, P. , and Nistal, M. (1990). Quantitative and ultrastructural alterations in the lamina propria and Sertoli cells in human cryptorchid testes Int. J. Androl. 13, 470–87.
| PubMed |
Pentikäinen, V. , Erkkilä, K. , and Dunkel, L. (1999). Fas regulates germ cell apoptosis in the human testis in vitro Am. J. Physiol. 276, 310–16.
Rosiepen, G. , Weinbauer, G. F. , Schaltt, S. , Behre, H. M. , and Nieschlag, E. (1994). The duration of the cycle of the seminiferous epithelium, estimated by the 5-bromodeoxyuridine technique, is identical in laboratory and feral rats J. Reprod. Fertil. 100, 299–306.
| PubMed |
Russell, L. D., Ettlin, R. A., Sinha-Hikim, A. P. and Clegg, E. D. (1990). Mammalian spermatogenesis. In
Salama, M. , Tsuji, M. , Tamura, M. , and Kagawa, S. (1998). Impact of aging and diabetes mellitus on the expression of the proliferating cell nuclear antigen in rat testicular tissue Arch. Androl. 40, 95–107.
| PubMed |
Sanders, E. J. (1997). Methods for detecting apoptotic cells in tissues Histol. Histopathol. 12, 1169–77.
| PubMed |
Santamaría, L. , Martín, R. , Codesal, J. , and Paniagua, R. (1995). Myoid cell proliferation in rat seminiferous tubules after ischaemic testicular atrophy induced by epinephrine. Morphometric and immunohistochemical (bromo-deoxyuridine and PCNA) studies Int. J. Androl. 18, 13–22.
| PubMed |
Schoenfeld, H. A. , Hall, S. J. , and Boekelheide, K. (2001). Continuously proliferative stem germ cells partially repopulate the aged, atrophic rat testis after GnRH agonist therapy Biol. Reprod. 64, 1273–82.
| PubMed |
Sinha-Hikim, A. P. , Wang, C. , Leung, A. , and Swerdloff, R. S. (1995). Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin-releasing hormone antagonist treatment Endocrinology 136, 2770–5.
| PubMed |
Swanson, L. J. , Desjardins, C. , and Turke, F. W. (1982). Aging of the reproductive system in the male hamster: behavioral and endocrine patterns Biol. Reprod. 26, 791–9.
| PubMed |
Wang, C. , Sinha-Hikim, A. P. , Lue, Y. , Leung, A. , Baravarian, S. , and Swerdloff, R. S. (1999). Reproductive aging in the Brown Norway rat is characterized by accelerated germ cell apoptosis and is not altered by luteinizing hormone replacement J. Androl. 20, 509–18.
| PubMed |
Yan, W. , Suominen, J. , and Toppari, J. (2000). Stem cell factor protects germ cells from apoptosis in vitro J. Cell. Sci. 113, 161–8.
| PubMed |
