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Research Article |
Departments of Obstetrics and Gynecology3 and of
Pharmacology and Therapeutics,4 McGill University, Montreal, Quebec, Canada H3A 1A1
ABSTRACT
In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.
aging, spermatogenesis, testis
INTRODUCTION
Stem cells are the foundation to maintain homeostasis of regenerative tissues, such as bone marrow for hematopoiesis and intestinal epithelium. These cells are defined by their biological activity to self-renew and differentiate to regenerate adult tissues [1]. Accordingly, stem cells are the source of tissue regeneration when injury is inflicted on a tissue. For example, due to the function of stem cells, hematopoiesis can be regenerated after radiation or chemotherapy for cancer [2]. Spermatogenesis, taking place in the seminiferous epithelium in the testis, is also a highly regenerative process by which a large number of male gametes are constantly produced throughout postpubertal life [3]. At the foundation of spermatogenesis lie spermatogonial stem cells (SSCs), which continuously self-renew and produce progenitor spermatogonia that are committed to differentiate and, ultimately, transform to spermatozoa [3]. Thus, it is reasonable to believe that the quantity and quality of SSCs are maintained for a lifetime to sustain spermatogenesis.
It has been reported that the production of spermatozoa and their quality gradually decline with age in humans [4, 5]. Although this observation is still controversial in human population [6], studies using rodents have provided convincing evidence that spermatogenesis can deteriorate and cease during aging, resulting in testicular atrophy [7–10]. The decline in spermatogenic activity with age suggests that SSCs may not be exempt from aging and may have a finite life span. In fact, by assaying the number of spermatogonia surviving known doses of radiation, Suzuki and Withers [7] have indicated that the number of undifferentiated spermatogonia, which include stem cells, dramatically decreases with age. If spontaneous mutations are inevitable consequences of continuous cell division, then cells should have a limited lifetime, and SSCs should be no exception, particularly because these are the cells that eventually transmit genetic information to the next generation. Elimination of SSCs that undergo many self-renewal divisions and experience mutations should be favored in evolution.
This argument, however, can be contradicted by various lines of evidence. First, stem cells in general do not frequently enter the cell cycle [1–3], which may reduce the chance of mutation events. Second, recent studies suggest that stem cells in the small intestinal epithelium continuously retain one specific DNA strand as a template at cell division, thereby protecting themselves from mutation [11, 12]. Third, cell senescence/death can be caused not only by mutations but also by the telomere shortening that occurs at cell division [13]. Telomerase activity is present in spermatogonia [14, 15] as well as in stem cells in general [13], suggesting that these cells can maintain an appropriate telomere length required for long-term cell survival. If SSCs actually do not age, then age-dependent testicular atrophy should result from aging of the environment in which SSCs reside.
To address whether age alters the characteristics of SSCs, we evaluated the number and the activity of SSCs during aging in mice. We also examined the effect of aging on the ability of the testicular environment to support spermatogenesis. To this end, we employed reciprocal SSC transplantation experiments between aged and young mice. Because a strict definition of stem cells relies on their biological activity and definitive SSC markers are not currently available, the transplantation assay is the only method at present to detect functional SSCs unequivocally.
MATERIALS AND METHODS
Donor Cell Preparation
All mice used in the present study were maintained in our animal facility for the specified experimental times. Donor cells were obtained from the testes of ROSA26 transgenic mice on the C57BL/6 (B6) x 129 genetic background (129S-Gtrosa26Sor; Jackson Laboratory), which express lacZ in virtually all types of cells in the body [16, 17]. These mice served as donors at 2 mo (2–3 mo), 6 mo (6–7 mo), 1 yr (12–14 mo), and 2 yr (23–25 mo) of age for transplantation into the testes of B6 x 129 F1 recipient mice (see below). Only animals with two normal or two atrophied testes were used as donors. In some experiments, wild-type young mice on the B6 x 129 genetic background served as donors for transplantation into the testes of aged ROSA26 recipients. Wild-type donor mice were operated on to induce experimental cryptorchidism, in which testes were sutured on the abdominal wall, at 2 mo of age and used 2 mo after operation [18]. Cryptorchid testis was used as a source of donor cells, because SSCs are concentrated in these testes [18] and, thus, more SSCs can be injected into a limited space of recipient atrophied testes. For both donor types, a single cell suspension of donor testis cells was prepared using a two-step enzymatic digestion procedure as described previously [19, 20]. Multiple testes were combined for one donor cell preparation. The number of cells recovered was measured using a hemocytometer. The viability of recovered cells was determined by trypan blue exclusion, which showed that more than 95% of the cells were viable irrespective of donor age. Donor cells were resuspended at 100 x 106 cells/ml in Dulbecco modified Eagle medium with 10% fetal bovine serum and 10% trypan blue [19, 20].
Recipient Mice
The B6 x 129 F1 mice were used as recipients for transplantation of donor cells derived from ROSA26 mice. These recipients were pretreated at 4 wk of age with busulfan (50 mg/kg body weight), which ablates endogenous spermatogenesis, and used for transplantation 4 wk or later after busulfan treatment [19, 20]. In some experiments, aged ROSA26 mice were used as recipients for transplantation of young donor cells. These recipients were 1 and 2 yr of age (five and four mice, respectively) and had bilateral atrophied testes; hence, they did not receive the busulfan treatment. Donor cells were injected into one of the two atrophied testes, chosen randomly, from an individual recipient. The other testis was left untreated as a control. For both types of recipients, approximately 7 µl of donor cell suspension were injected into recipient seminiferous tubules through the efferent ducts [19, 20]. All animal handling and care were performed in accordance with the guidelines established by the Canadian Council on Animal Care.
LacZ Staining
Two months after transplantation, recipient testes that received donor ROSA26 cells were stained with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) to visualize the colonies of donor-derived spermatogenesis, and the number of colonies was counted [19, 20]. Because one colony arises from one SSC [21], the colony number represents the number of functional SSCs derived from donors at various ages. In addition, the length of each individual colony was measured using an eyepiece micrometer under a stereomicroscope [17]. The colony length indicates the ability of SSCs to regenerate spermatogenesis.
Histology
Donor or recipient testes were fixed in 10% neutral-buffered formalin and paraffin-sectioned (7 µm thickness). Sections of donor ROSA26 mouse testes and those of aged recipient ROSA26 testes were stained with hematoxylin-eosin. For the analysis of X-gal-stained recipient testes, nuclear fast red was used for counterstaining.
Testis Weights and Serum Testosterone Levels
Testes were isolated, blotted to remove surface moisture, and weighed without removing the tunica. Serum testosterone levels were measured (in duplicate for each mouse) using Testosterone ELISA Kit (Research Diagnostic, Inc.) following the manufacturer's instruction.
Statistical Analyses
Statistical analyses for multiple comparisons were done using ANOVA followed by the Tukey post hoc test. Student t-test was used for two-group comparisons. Significance was determined when P < 0.05. All data are expressed as the mean ± SEM.
RESULTS
Testicular Atrophy Occurs in an Age-Dependent Manner
Donor testes were obtained from ROSA26 transgenic mice, which express the lacZ marker gene in all types of male germ cells [17], at 2 mo, 6 mo, 1 yr, and 2 yr of age. These mice showed a similar body weight during aging except for 2-mo-old mice, which had weights that were significantly lower (Table 1). The testis weight was similar at 2 and 6 mo of age. At 1 and 2 yr of age, however, some mice had bilateral atrophied testes (Fig. 1). Whereas no atrophied testes were found in younger age groups (2 and 6 mo), 8 of 15 mice had bilateral atrophied testes in the 1-yr group (Table 1). The incidence of testicular atrophy increased in the 2-yr group, in which six of eight mice showed bilateral testicular atrophy. These atrophied testes showed significantly lower weights than testes with normal appearance from young and aged mice, whereas the weights of normal testes were similar across age groups (Table 1). These observations indicate that spermatogenesis can cease in an age-dependent manner in ROSA26 mice. No statistically significant correlation between testicular atrophy and testosterone levels was found in the present study, although serum testosterone levels tended to decrease with age (data not shown).
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Taken together, these observations indicate that aging of spermatogenesis can occur in ROSA26 mice and is accompanied by testicular atrophy. To evaluate if these aging phenotypes in spermatogenesis coincide with altered characteristics of SSCs, we transplanted testis cells derived from ROSA26 mice at different ages into testes of wild-type young mice.
Aging Affects Both Quantity and Quality of SSCs
Prior to transplantation experiments, we examined expression of the lacZ gene in testes of aged ROSA26 mice. When reacted with X-gal, donor testes were stained strongly blue irrespective of age, indicating that expression of the marker gene did not change significantly with age (Fig. 1).
As shown in Table 1 (column A), the number of cells recovered from a donor testis with normal appearance was similar regardless of age. Fewer cells were recovered from atrophied testes at 1 and 2 yr of age, however, reflecting the loss of spermatogenesis, leading to testicular atrophy.
Following transplantation, we observed colonies of donor-derived spermatogenesis in recipient testes irrespective of age or testis condition of donor mice, indicating that functional SSCs remain in atrophied testes of 1- and 2-yr-old mice (Table 1 and Figs. 1 and 2). When 106 donor cells obtained from normal testes were transplanted, a similar number of spermatogenic colonies was produced regardless of age (Table 1, column B). In addition, the number of colonies found after transplantation of 106 cells from atrophied testes of 1-yr-old mice was comparable to that produced by normal testis cells of young and aged mice, indicating that the concentration of SSCs did not change in atrophied testes of a 1-yr-old mouse. Significantly fewer colonies were observed, however, with cells from atrophied testes of 2-yr-old donors compared with those from normal testes of the same age. We then calculated the total SSC activity in a testis at different ages (Table 1, column C) by multiplying the total number of cells recovered from a testis (Table 1, column A) by the concentration of functional SSCs (Table 1, column B). The results indicated that the total SSC activity per testis was similar regardless of age as long as donor testes showed a normal appearance (Table 1, column C). The SSC activity per testis, however, was clearly lower in atrophied testes of 1- and 2-yr-old mice. A 1-yr atrophied testis was found to contain 1/6 (73.8/436.8) the activity of SSCs detected in a 2-mo testis and a 2-yr atrophied testis only 1/60 (7.4/436.8) the activity. These results demonstrate that the number of functional SSCs per testis significantly decreases with age in atrophied testes.
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Next, we measured the length of colonies that SSCs at different donor ages produced (Fig. 3). The colony length indicates the ability of SSCs to regenerate spermatogenesis (i.e., final outcome of functions that SSCs undertook after transplantation). We found that the colony length was similar regardless of age when SSCs were derived from normal testes. Interestingly, the length of colonies arising from SSCs obtained from 1-yr atrophied testes was comparable to that observed with SSCs from normal testes. This observation suggests that provided with a young testicular environment, SSCs remaining in 1-yr atrophied testes can fully exhibit normal spermatogenic activity despite the significant decrease in their number within a testis (Table 1). In contrast, colonies observed in the 2-yr atrophied group were significantly shorter than those in any other group and only measured approximately 36% of the colony length observed in the 2-yr normal group. Thus, these results indicate that the majority of SSCs in 1-yr atrophied testes have a colony-forming activity similar to SSCs in normal testes, whereas most SSCs remaining in 2-yr atrophied testes have a reduced colony-forming potential.
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Taken together, these results demonstrate that both the quantity and the quality of the SSC population decline during aging.
One-Year-Old Atrophied Testes Are Permissive for Regeneration of Spermatogenesis
In theory, the cessation of spermatogenesis during aging can result from defective germ cells, somatic cells, or both. The experiments described above focused only on the characteristics of SSCs during aging; they did not address the effect of age on the somatic environment. Thus, we next examined whether atrophied testes of aged mice can support the regeneration of spermatogenesis when SSCs from young animals are provided. To this end, we transplanted testis cells of young mice into bilateral atrophied testes of 1- and 2-yr-old ROSA26 mice.
Two months after transplantation, aged recipient testes were harvested and their weights measured (Fig. 4). We found that control testes of both ages, which did not receive donor cells, remained atrophic. Following transplantation, however, weights of 1-yr atrophied testes significantly increased, whereas weights of 2-yr atrophied testes remained similar, compared to those of control. Consistent with this finding, we observed robust regeneration of spermatogenesis in histological sections of testes from 1-yr-old, but not 2-yr-old, recipients (Fig. 5).
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These results demonstrate that atrophied testes of 1-yr-old mice can actively support the regeneration of spermatogenesis when SSCs from young animals are transplanted, whereas those of 2-yr-old mice cannot. Therefore, the function of the somatic environment in the testis also declines with age.
DISCUSSION
In the present study, we investigated the effect of aging on the number and the activity of SSCs as well as the effect on the somatic environment using reciprocal germ cell transplantation between young and aged mice. The results demonstrate that the cessation of spermatogenesis can occur during aging in ROSA26 mice and that both SSCs and somatic environment are involved in the aging process. To our knowledge, the present study is the first to analyze aging of SSCs based strictly on their biological function rather than their morphology.
Aging of SSCs is evidenced by the following observations. First, atrophied testes appear more frequently with age, and in these testes, the total SSC number declines in an age-dependent manner (Table 1). Second, the reduction of SSC numbers becomes evident at 1 yr of age in an atrophied testis, although SSCs remaining in these testes retain normal colony-forming activity (Table 1 and Fig. 3). By 2 yr of age, SSCs in atrophied testes show a significant decline not only in their numbers but also in the length of colony they can form. Thus, both quantity and quality of SSCs decline in an age-dependent manner in this mouse strain.
Aging also significantly affects the function of the testicular environment. Provided with young SSCs, atrophied testes at 1 yr can support the regeneration of spermatogenesis, but those at 2 yr cannot (Fig. 4). Hence, the function of somatic environment in a testis also declines with age.
An interesting finding of the present study is that 1-yr atrophied testes contain reduced numbers of functional SSCs (Table 1) yet retain a functional environment for spermatogenic regeneration (Fig. 4). Therefore, it can be suggested that the life span of SSCs is finite and that some SSCs die, or start losing their function, by 1 yr of age, before deterioration of the somatic environment. By 2 yr of age, the loss of functional SSCs and the cessation of spermatogenesis that follows may result in significant damage of the testicular environment and cause a further decline in SSC quantity and quality. This simplified interpretation of the results leads to a conclusion that the basis for age-dependent testicular atrophy is primarily the aging of SSCs.
We need to be cautious, however, when we correlate the results of SSC aging and environmental aging. In the present study, SSC numbers and function were examined for individual SSCs, and the parameters measured were highly quantitative. The transplantation assay allowed us to delineate precisely the number of functional SSCs and the length of colonies. Nevertheless, the function of testicular environment was examined only at a whole-testis level, and the number of SSC niches (microenvironments where SSCs reside) was not investigated. Hence, the aging of SSCs and their environment were analyzed at different levels, rendering it difficult to correlate these two phenomena directly. This difficulty arises from the fact that we currently do not have a means of assessing directly the quantity and quality of SSC niches, thus limiting our understanding of niche biology.
Therefore, it is possible to interpret the results of the present study in favor of environmental aging as a primary mechanism of age-dependent testicular atrophy. When transplanted into a young testicular environment, a small population of SSCs remaining in atrophied testes of 1- and 2-yr-old animals is capable of establishing spermatogenesis (Table 1 and Fig. 2). This observation could indicate simply that SSC aging may spread gradually among the SSC population in an aging testis but also could imply that defects in the environment may appear progressively along the seminiferous tubules and precede the decline in SSC numbers. For instance, the environment may first become defective by 1 yr and cause the loss of functional SSCs, which is detectable by the transplantation assay. The region of tubules damaged by this time, however, may be limited relative to the total length of tubules in a testis. At a whole-testis level, therefore, a sufficient length of seminiferous tubules may remain permissive, even if the tubules are suboptimal, for spermatogenic regeneration. If this is the case, then the effect of aging on individual SSC niches should be masked and undetectable; this would not allow us to distinguish the aging of individual SSCs and their niches at 1 yr of age.
The results reported in previous studies appear to favor the theory that testicular environment, rather than SSCs, is the primary mechanism of age-dependent testicular atrophy, although not without reservation. Ogawa et al. [22] sequentially transplanted SSCs derived from 3- to 4-mo-old mice into testes of successive populations of young recipients and found that SSCs sustained functional integrity for more than 2 yr. Whereas this observation may support the notion that SSCs do not age, sequential transplantation into testes of young recipients may have resulted in a continuous selection of functionally intact SSCs as well as their activation and expansion over time, which masks the age effects on a total population of SSCs. The results obtained by Ogawa et al. [22], however, clearly indicate that given a young testicular environment, some aged SSCs can fully retain their activity to regenerate spermatogenesis for at least the lifetime of a mouse (2 yr).
In another study, Schoenfeld et al. [23] observed undifferentiated spermatogonia that were actively dividing in atrophied testes of aged Brown-Norway rats. They also found, at a whole-testis level, that a pharmacological reduction of intratesticular testosterone levels in aged rats partially restored spermatogenesis. In addition, rat Sertoli cells were found to exhibit, in an age-dependent manner, an increased expression of the secreted form of Steel factor compared to the membrane-bound form of Steel factor; this latter form is more potent than the secreted form in supporting spermatogenesis [24]. These observations led the authors to conclude that testicular atrophy in aged Brown-Norway rats is caused by environmental aging, not by SSC aging. Because SSCs were defined in this rat study [23] by morphology rather than by their function, the identity of actively dividing spermatogonia as SSCs was not formally confirmed.
Further studies to address SSC proliferation during the postnatal life of mice may provide interesting insights regarding SSC biology in light of previous studies with hematopoietic stem cells. It has been reported that hematopoietic stem cells in aged mice divide more frequently, show diminished lymphoid potential, and upregulate the expression of genes involved in myeloid specification compared to those in young mice; these features may be related to increased leukemia incidents in the aged human population [25, 26]. It would be interesting to investigate if a similar situation applies to SSCs.
In the present study, ROSA26 mice were used as donors and recipients for transplantation. It should be noted that this mouse strain is not inbred; thus, a possibility remains that genetic factors contribute to the age effect in individual ROSA26 mice. An additional contributing factor that could affect aging of SSCs may be integration of the ROSA26 transgene in the genome and presence of the gene product in germ cells.
In summary, we have demonstrated that both SSCs and testicular environment can age in ROSA26 mice, leading to testicular atrophy by 1 yr of age. The results of the present study also indicate that to better understand the mechanism of age-dependent testicular atrophy, the effect of aging needs to be evaluated simultaneously for both SSCs and testicular environment. To pursue SSC aging further, determination of definitive SSC markers and purification of SSCs are necessary, because the biological properties of SSCs that we can address using the transplantation assay are limited. In addition, although the present study focused on SSCs, further studies of nonstem germ cells in aged ROSA26 mice, from undifferentiated spermatogonia to epididymal sperm, may provide valuable information about germ cell aging. For aging of the testicular environment, further investigations regarding age-dependent alterations in Sertoli and Leydig cell characteristics should lead to a better understanding. Such studies on Leydig cells are actively ongoing [27], but Sertoli cell aging appears to be addressed less intensively. Sertoli cells can support a limited number of germ cells during spermatogenesis, yet they do not divide and are not replenished for a lifetime after maturation around the time of puberty [28, 29]. Thus, these cells may be prone to aging. Taken together, such studies of both germ and somatic cell lineages in the testis should improve our understanding on the mechanism that induces age-dependent testicular atrophy and senescence in the male reproductive system.
ACKNOWLEDGMENTS
We thank Trang Luu for the testosterone assay, Frances Clerk for editing this manuscript, the members of the Central Animal Facility at the Royal Victoria Hospital for the maintenance of mice, and those of the Bone Center of McGill University and the Histology Core of the Montreal Children's Hospital Research Institute for the preparation of paraffin sections.
FOOTNOTES
1 Supported by the Canadian Institutes of Health Research (MOP-49444 to M.N. and MOP-57882 to B.R. and M.N.) and the Canadian Foundation for Innovation (4177 to M.N.). K.T.E. is supported by The Stem Cell Network. M.N. is a Fondation pour la Recherche en Sante du Quebec scholar. 
2 Correspondence: Makoto Nagano, Royal Victoria Hospital, Rm F3.07, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. FAX: 514 843 1662; makoto.nagano{at}muhc.mcgill.ca
Received: 15 July 2005.
First decision: 8 August 2005.
Accepted: 13 September 2005.
REFERENCES
This article has been cited by other articles:
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  K. T. Ebata, X. Zhang, and M. C. Nagano Male Germ Line Stem Cells Have an Altered Potential to Proliferate and Differentiate During Postnatal Development in Mice Biol Reprod, May 1, 2007; 76(5): 841 - 847. [Abstract] [Full Text] [PDF]
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 J. Ehmcke, B. Joshi, S. D Hergenrother, and S. Schlatt Aging does not affect spermatogenic recovery after experimentally induced injury in mice Reproduction, January 1, 2007; 133(1): 75 - 83. [Abstract] [Full Text] [PDF]
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0.025) is detected between 2-yr atrophied testis group and all other groups. Data are presented as the mean ± SEM
