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Department of Urology, Sapporo Medical University School of Medicine, Sapporo, Japan.
| Correspondence to: Naoki Itoh, Department of Urology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo, 060-8543, Japan (e-mail: nitoh{at}sapmed.ac.jp). |
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Abstract |
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Key words: Apoptosis, proliferation, Bcl-xl, aging, spermatogenesis
Several investigations of germ cell apoptosis have been reported in human testis. In infertile men with hypospermatogenesis or maturational arrest, accelerated apoptosis of germ cells has been observed (Lin et al, 1997a,b; Takagi et al, 2001). Conversely, a testis with varicocele shows a lower rate of germ cell apoptosis (Fujisawa et al, 1999). From these observations it appears that germ cell apoptosis may not be uniformly regulated in the testes of infertile men. Apoptotic potential to maintain an appropriate number of germ cells may be differently regulated, depending on the cause of the spermatogenic disorder. Therefore, the clinical relevance of germ cell apoptosis in male infertility may be variable, according to the cause of spermatogenic failure.
In this study, in order to elucidate the natural history of apoptosis of germ cells with aging, we used testicular specimens from patients who underwent bilateral orchiectomy for prostate cancer without receiving previous hormonal treatment. By using these samples, in an effort to clarify the significance of apoptosis in aging men, apoptosis of germ cells was quantitatively analyzed and compared with that in young men.
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Materials and Methods |
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Histological Examination
Testicular specimens were fixed in Bouin solution for 2 hours and in 10%
neutral-buffered formalin for 2–4 hours, embedded in paraffin, then
stained with hematoxylin and eosin. For quantitative estimation of
spermatogenesis, 20 round tubules were randomly selected, and the numbers of
Sertoli cells, spermatogonia (dark type A, pale type A, and type B), primary
spermatocytes (preleptotenes, leptotenes, zygotenes, and pachytenes), round
spermatids (secondary spermatocytes, Sa and Sb1 spermatids), and elongated
spermatids (Sb2, Sc, and Sd spermatids) were counted using light microscopy.
The ratio of each type of germ cell per Sertoli cell was then calculated. The
average ratios were determined and employed for quantitative analysis of
spermatogenesis. Observation and photographs were performed with a Nikon
microscope (Nikon Corporation, Tokyo, Japan), and photographed with a Nikon
camera using ASA 100 film. Scanned digital images were imported and reduced in
Adobe Photoshop software version 5.5 (Adobe Systems Incorporated, San Jose,
Calif).
Immunohistochemical Staining Procedure
In order to detect apoptosis, the terminal deoxynucleotidyl transferase
(TdT)-mediated deoxy-UTP biotin nick end labeling (TUNEL) technique was
performed on Bouin-fixed 5-µm sections of specimens using an
ApopTag-peroxidase kit (Oncor, Gaithersburg, Md). In brief, tissue sections
were deparaffined and dehydrated, incubated with proteinase K (20 µg/mL)
for 15 minutes at 37°C, washed in distilled water, and then treated with
3% hydrogen peroxide in phosphate-buffered saline for 5 minutes to quench
endogenous peroxidase activity. Sections were incubated with a mixture
containing digoxigenin-conjugated nucleotides and TdT in a humidified chamber
at 37°C for 1 hour, and subsequently treated with
antidigoxigenin-peroxidase for 40 minutes at room temperature. Immunoreactive
cells were detected by incubating the sections with a mixture of
3'-diaminobenzidine tetrachloride (DAB) and DAB solution buffer for
1–2 minutes under microscopy. Sections were stained in 1% methyl green
in 0.1 M sodium acetate buffer (pH 4.0) as a counterstain, dehydrated in 100%
butanol, cleared in xylene, and mounted. As a positive control for the TUNEL
assay, rat mammary gland obtained at the fourth day after weaning was used.
Intraassay and interassay coefficients of variation for the TUNEL assay were
6.3% and 11.9%, respectively. No color reaction was observed when TdT was
omitted from the procedure. To determine apoptotic rates, the number of each
type of TUNEL-positive germ cell was divided by the total number of the
corresponding type of germ cell. At least 1000 cells per cell type were
evaluated.
In order to detect Ki-67 or Bcl-xl, sections were immunostained with an anti-human Ki-67 rabbit polyclonal antibody (DAKO, Carpinteria, Calif) or monoclonal antibody against Bcl-xl protein (DAKO) by using the avidin-biotin method, followed by counterstaining with hematoxylin. Bouin-fixed sections were incubated with a mixture containing either antibody overnight at room temperature. No color reaction was observed when the specific antibodies were omitted. The Ki-67-positive rate of spermatogonia was determined by dividing the number of Ki-67-positive spermatogonia by their total number in 20 seminiferous tubules. To assess the Bcl-xl-positive rate of each cell type, the percentages of stained cells were determined by examining 20 seminiferous tubules. As a preliminary study, the expression of two other proteins besides Bcl-xl that are known to regulate apoptosis, Bcl-2 and Bax, was evaluated immunohistochemically. However, neither Bcl-2 nor Bax could be stained, therefore, only the expression of Bcl-xl was further evaluated in this study.
Statistical Analysis
The Mann-Whitney U-test and one-way analysis of variance (ANOVA)
were used for statistical analyses. Statistically significant differences were
confirmed when the P value was less than .05. For ANOVA, significant
differences were evaluated by using the Tukey-Kramer significant difference
test for multiple comparisons. The StatView statistical program (Abacus
Concepts, Berkeley, Calif) was used to analyze all data.
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Results |
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The age distribution of subjects was so wide (53–88 years) that the condition of sperm production might have been different between younger and older subjects. Subjects were categorized into two age groups (53–69 and 70–88 years) so that the time span would be comparable with that of the control group (Table 2). The ratios of round spermatids and elongated spermatids per Sertoli cell in each age group were significantly decreased compared with that of controls. However, no significant difference in the ratio of each type of germ cell to Sertoli cells in seminiferous tubules was recognized between the two age groups.
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Apoptotic Rates of Germ Cells
To clarify the cause of the decreased ratios of germ cells to Sertoli cells
except for spermatogonia, the apoptotic rate of each germ cell type was
studied (Table 3). The
apoptotic rates of spermatogonia in the aged testis were significantly lower
than in controls (0.11% ± 0.06% vs 0.34% ± 0.21%, P
< .0001) (Fig. 1C and D).
This finding indicated that spermatogonial apoptosis in aged testes was
suppressed compared with that of controls. However, this seemed strange
because the ratio of spermatogonia to Sertoli cells was not different between
them (Fig. 2). In contrast to
the low frequency of spermatogonial apoptosis, the apoptotic rate of primary
spermatocytes in aged testes was significantly elevated compared with that of
controls (0.60% ± 0.54% vs 0.22% ± 0.12%, P =
.003).
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To determine whether increased apoptosis in primary spermatocytes was correlated with decreases in the numbers of those cells, simple regression analysis was performed. A negative correlation was found between the apoptotic rate of primary spermatocytes and the number of primary spermatocytes per Sertoli cell (r = -.54, P < .05; Fig. 2). The apoptotic rates of both round and elongated spermatids were higher in aged testes than in controls, however, the difference did not reach statistical significance.
Balance of Spermatogonial Proliferation and Apoptosis
Although spermatogonial apoptosis was significantly decreased in aged men,
the number of spermatogonia per Sertoli cell was similar to that of controls
(Tables 1 and
3). We hypothesized that the
reason for this is that the proliferative potential of spermatogonia was, like
apoptosis, also decreased. To examine this hypothesis, the expression of Ki-67
in spermatogonia was examined as a marker of cell proliferation
(Fig. 3A and B). The positive
staining rate of Ki-67 in aged testes (18.6% ± 6.0%) was significantly
lower than in controls (24.9% ± 3.3%), indicating that spermatogonial
proliferation was diminished in aged men
(Table 4). The number of germ
cells was believed to be dependent on the balance between cell proliferation
and apoptosis. The ratio of spermatogonial proliferation to apoptosis (Ki-67
positive rate:apoptotic rate) showed no difference between aged men and
controls (Table 4). This was
assumed to be one of the reasons why the ratio of spermatogonia to Sertoli
cells was not changed in aged men compared with that of controls.
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Expression of Bcl-xl in Testes of Aged Men
Many proteins that regulate apoptosis have been reported. Clarification of
the expression of some proteins in seminiferous tubules in aged men seemed
likely to provide new insights into the regulatory system of apoptosis in
spermatogenic failure. However, as mentioned earlier, the expression of Bcl-2
and Bax could not be found in our preliminary study. Thus, in this study, only
the expression of Bcl-xl protein was examined.
Expression of Bcl-xl was identified in all types of germ cells in the testes of aged men (Fig. 3C and D). A significant inverse correlation was found between the Bcl-xl-positive rate and apoptotic rate of primary spermatocytes (r = -.44, P < .019; Table 5). In other cell types, no correlation was found between Bcl-xl expression and apoptosis.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A greater rate of apoptosis has been postulated to be one of the causes of germ cell loss, possibly contributing to male infertility. A higher apoptotic rate was demonstrated in testes with spermatogenic arrest and hypospermatogenesis than in obstructed testes with normal spermatogenesis (Lin et al, 1997a,b). It has also been reported that an imbalance of spermatogonial proliferation and apoptosis caused by up-regulated apoptosis of spermatogonia decreases their number in hypospermatogenesis (Takagi et al, 2001). On the other hand, in the varicocele testis, it has been reported that both germ cell apoptosis and cell proliferation are suppressed compared with that in normal men. Decreased apoptosis of primary spermatocytes in varicocele may be a consequence of the failure of differentiation from spermatogonia because decreased DNA synthesis in preleptotene spermatocytes has been demonstrated (Tanaka et al, 1996; Fujisawa et al, 1999). Hence, germ cell apoptosis in such conditions may not be induced as a cell-loss mechanism, but it may compensate for diminished proliferation. Based on these findings, it appears that the role of apoptosis in the testis in infertile men is not uniform.
As in male infertility, many aged testes have spermatogenic damage to some extent (Johnson 1986). It has already been reported that apoptosis has a crucial role in the degeneration of spermatogenesis in the aged testis (Brinkworth et al. 1997). However, no up-regulation of germ cell apoptosis was found in that study, in which the apoptotic rate was defined by the total number of apoptotic germ cells divided by the total germ cell number. To elucidate the role of apoptosis of each germ cell type, we analyzed the apoptotic rate of each cell type. In addition, in the study by Brinkworth et al (1997), many patients had already received androgen-deprivation treatment, which may have influenced apoptotic potential. In the current study, no subjects received androgen-deprivation treatment. Our results thus seemed to properly reflect the condition of the aged testis.
Quantitative analysis revealed a lower number of late primary spermatocytes in the testes of elderly men compared with that of young men (Johnson 1986). In this study, accelerated apoptosis of primary spermatocytes was detected and correlated well with the cell decrease. It was speculated that apoptosis of primary spermatocytes might be the most relevant cause of impaired spermatogenesis in the aged testis.
Another interesting result of our study was the downregulated apoptosis of spermatogonia. Diminished spermatogonial proliferation was also found concomitant with low spermatogonial apoptosis. We hypothesized that the decline of spermatogonial apoptosis might reflect a compensatory role of apoptosis in these cells for the diminished proliferation that occurred during aging. Otherwise, because it has been reported that germ cell apoptosis is highly stage-dependent (Blanco-Rodriguez and Martinez-Garcia, 1996; Blanco-Rodriguez, 1998), when the number of proliferating spermatogonia decreases, the number of spermatogonia entering certain cell cycles in which apoptosis is induced might also decrease. It seems that regulation of spermatogonial apoptosis is closely related to the proliferative status of such cells.
Apoptotic rates of round spermatids and elongated spermatids showed no significant elevations, whereas quantitative analysis revealed a reduction in their number. Sertoli cells might already have digested many apoptotic spermatids at the time of the detection of DNA fragmentation, because those cells are phagocytosed in the early phase of the apoptotic process in the rat testis (Henriksen et al, 1996). Therefore, the apoptotic rate of spermatids in the present study might have been underestimated.
Bcl-xl is one of Bcl-2 family members and is believed to inhibit apoptosis and to promote cell survival by heterodimerization with Bcl-2 family members or by itself (Minn et al, 1999). Transgenic mice that express high levels of Bcl-xl show highly abnormal spermatogenesis accompanied by sterility due to the prevention of an early and massive wave of apoptosis (Rodriguez et al, 1997). It is strongly suggested that Bcl-xl is involved in the regulation mechanisms of normal maturation steps of spermatogenesis in mouse testis (Rodriguez et al, 1997). Little has been clarified as to the role of Bcl-xl in human spermatogenesis. Following short-term and long-term antiandrogen treatment, expression of Bcl-xl on human testicular sections was investigated (Woolveridge et al, 1998). Bcl-xl expression declined in testes that received long-term treatment. In the current study, a declining expression level of Bcl-xl was associated with a higher apoptotic rate of primary spermatocytes of the human aged testis. Bcl-xl might be involved in the survival mechanism of primary spermatocytes.
We conclude that accelerated apoptosis of primary spermatocytes was one of the causes of germ cell loss with aging. In order to control apoptosis of primary spermatocytes, Bcl-xl is believed to be one of the survival factors of germ cells. Suppression of apoptosis in spermatogonia might have a compensatory role for their diminished proliferative potential. The current investigation is the first report in which apoptotic potentials of germ cells in the aged testis have been clarified in detail; however, to understand the exact regulatory mechanisms of germ cell apoptosis, investigations of expression patterns of apoptotic proteins are required.
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Acknowledgments |
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References |
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Blanco-Rodriguez J. A matter of death and life: the significance of germ cell death during spermatogenesis. Int J Androl.1998; 21:236 –248.[Medline]
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