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From the Departments of * Cell Biology (Aging
Institute), Biochemistry and Molecular
Biology, and Statistics, Faculty of Medicine,
University of Murcia, Murcia, Spain.
| Correspondence to: Prof Dr Luis M. Pastor, Department of Cell Biology, Faculty of Medicine, Campus de Espinardo, University of Murcia, E-30071 Murcia, Spain (e-mail: bioetica{at}um.es). |
| Received for publication September 13, 2005; accepted for publication August 24, 2006. |
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Abstract |
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Key words: Aging, Bcl-2, Fas, photoperiod, testis
Apoptosis has been involved in testicular germ cell loss during aging in numerous species, including humans (Brinkworth et al, 1997; Kimura et al, 2003) and rodents, such as the mouse (Barnes et al, 1998), rat (Wang et al, 1999; Barnes et al, 1999) and Syrian hamster (Morales et al, 2003). In aged rats germ cell apoptosis has been related to specific stages of the seminiferous epithelium cycle (Wang et al, 1999), although this aspect has not been studied in other species. Also, short photoperiod exposure induces germ cell apoptosis in white-footed mouse (Young et al, 1999, 2000) and Syrian hamster (Morales et al, 2002). However, the specific germ cell population affected has not sufficiently been documented in both models of seminiferous epithelium atrophy.
Apoptosis can be triggered by 3 mechanisms: 1) the binding of Fas-L to Fas receptor expressing cells (Itoh et al, 1991; Oehm et al, 1992; Suda et al, 1993); 2) the activation of Bcl-2 family members (Yang and Korsmeyer, 1996; Green and Reed, 1998); and 3) the endoplasmic reticulum pathway (Nakagawa et al, 2000; Bitko and Barik, 2001; Yoneda et al, 2001). Fas/Fas-L system seems to play an important role in testicular germ cell apoptosis regulation (Pertikäinen et al, 1999). Fas is expressed in spermatogonia, spermatocytes, and spermatids, all apoptotic cells (Lee et al, 1997). Fas expression has also been related to germ cell degeneration in arrest of spermatogenesis (Eguchi et al, 2002; Francavilla et al, 2002). There is some discrepancy as to the precise localization of Fas-L in the seminiferous tubules, with some groups showing Fas-L in the Sertoli cells (Bellgrau et al, 1995; French et al, 1996; Lee et al, 1997; Koji, 2001), while others have reported that Fas-L is also expressed in the germ cells (Woolveridge et al, 1999; Francavilla et al, 2000). Bcl-2 and the long form Bcl-x (Bcl-xL) promote cell survival by inhibiting apoptosis. However, other members of the Bcl-2 family (Bax, Bak, Bcl-xS, and Bad) promote cell death (Oltvai et al, 1993). Members of the Bcl-2 family also regulate spermatogenesis, inducing apoptosis in spermatogonia, primary spermatocytes, and spermatids (Oldereid et al, 2001) and participating in the differentiation process (Oldereid et al, 2001). Also, p53 has been involved in the elimination of damaged germ cells as well as those produced in excess (Stephan et al, 1996), as well as in the control of proliferation and apoptosis in spermatogonia (Beumer et al, 1998).
Although germ cell apoptosis was implicated in aging and was seen to occur after short photoperiod exposure in a previous study in our laboratory (Morales et al, 2002, 2003), the specific cell types that display apoptosis, the relationship of apoptosis with specific stages of the seminiferous epithelium cycle, and the molecular pathways involved have not been sufficiently studied. The objectives of the present study were: 1) to study how the different populations of germ cells are affected by apoptosis caused by aging and short photoperiod exposure; 2) to analyze the induction of germ cell apoptosis during the different stages of the seminiferous epithelium cycle in aging Syrian hamster; and 3) to examine the expression of some of the Bcl-2 family members, the Fas/Fas-L system, and p53 in the seminiferous epithelium of Syrian hamsters and to investigate the roles of these proteins in the induction of testicular germ cell apoptosis in aging and short photoperiod exposure.
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Materials and Methods |
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In Situ Germ Cell Apoptosis Detection and Quantification
Left testes were fixed in methacarn (methanol:chloroform: acetic acid
6:3:1), and representative samples, which were chosen randomly, were
dehydrated, immersed in toluene, and embedded in Paraplast Plus (Panreac
Química SA, Barcelona, Spain). Germ cell apoptosis was examined
following the protocol of TACS TdT in situ Apoptosis Detection kit (TUNEL
reaction; R&D Systems Inc, Minnesota, Minn). For this, 5-µm-thick
sections were deparaffinized, hydrated, washed, and incubated with proteinase
K (1 mg/mL). The peroxidase activity was quenched with 10%
H2O2. The samples were immersed in 1x TdT labeling
buffer (1 mol TACS Safe-TdT Buffer, 0.5 mg/mL BSA, 0.6 mmol
2-mercaptoethanesulfonic acid) at 18°–24°C for 5 minutes and
incubated for 1 hour at 37°C with TdTdNTP Mix (0.25 mmol biotinylated
dNTP), 50x Mn+2 (1 µL), TdT Enzyme (1 µL), and 1x
TdT labeling buffer. The reaction was stopped with TdT stop buffer (0.1 mol
EDTA, pH 8.0). Subsequently, the samples were washed in PBS and incubated with
streptavidin-horseradish peroxidase for 10 minutes at 18°–24°C.
After washing in PBS, the samples were stained with TACS Blue Label and
incubated with Contrast C solution. Negative control sections, processed
without TdT, did not show positive labeling.
The apoptotic germ cells were identified according to their position within the seminiferous epithelium, the cell size and nuclear morphology, and stage of the seminiferous epithelium. The quantity of apoptosis and the percentage of apoptotic germ cells were recorded in the populations of spermatogonia (SG) and spermatocytes (SC). For this purpose, 5-µm-thick sections were collected every 50 µm of tissue and processed for in situ germ cell apoptosis detection. Of the sections processed, 4 randomly chosen testis cross-sections were counted per animal. Within each section, 25 random fields were chosen by systematically moving the microscope lens across the tissue section without overlap and selecting every second field and were analyzed (1 field = 0.018 mm2). In each field of study the number of TUNEL-positive and negative germ cells was scored in the populations of spermatogonia and spermatocytes. The following apoptotic indices of each population of germ cells, expressed as the percentage of TUNEL-positive cells, were calculated: 1) the total apoptotic index (% of TUNEL-positive SG+SC); 2) the total apoptotic index in spermatogonia (% of TUNEL-positive SG); and 3) the total apoptotic index in primary spermatocytes (% of TUNEL-positive primary SC).
Also, in 6- and 24-month-old hamsters the apoptotic activity was examined during the different stages of the seminiferous epithelium cycle. The identification of stages was based on the description of Leblond and Clermont (1952) and Tiba et al (1992). Similarly to previous studies (Wang et al, 1999; Sinha Hikim et al, 2003), the stages were grouped into 4 groups: I–IV, V–VI, VII–VIII, and IX–XIII. For quantification of apoptosis during the seminiferous epithelium cycle, at least 100 randomly selected perpendicular seminiferous tubule cross sections on 4 sections from each animal of each group were analyzed. In each tubular section, the number of TUNEL-positive and negative germ cells was scored in the populations of spermatogonia, preleptotenes, pachytenes, and leptotenezygotene spermatocytes. The apoptotic index of each population of germ cells during the different stages of the seminiferous epithelium cycle was expressed as the percentage of TUNEL-positive cells.
Immunohistochemistry
Five-µm-thick sections fixed in methacarn and embedded in paraffin were
deparaffinized in xylene, hydrated, and transferred to PBS for 10 minutes.
Endogenous peroxidase was blocked with 1% H2O2 in PBS
for 30 minutes. After washing in PBS, samples were blocked with 1.5% normal
rabbit or goat serum (Jackson ImmunoResearch, West Grove, Pa), depending on
the origin of the secondary antibody. Subsequently, the samples were washed in
PBS and incubated overnight at 4°C, with the primary antibody diluted in
PBS/BSA. The primary antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif), and the concentrations used were the following: anti-Fas
(1:30) (sc-7886/FL-335), anti-Fas-L (1:50) (sc-834/N-20),
anti-Bcl-xL (1:100) (sc-8392/H-5), anti-Bcl-xS/L (1:100)
(sc-1041/L19), anti-Bcl-2 (1:100) (sc-492/N-19), anti-Bax (1:100)
(sc-526-P19), and anti-p53 (1:20) (sc-6243/FL-393). The samples were washed in
PBS and then incubated for 45 minutes with the corresponding biotinylated
secondary antibody (CHEMICON International, Temecula, Calif) diluted 1:500 in
PBS/BSA. Sections were again washed in PBS and subsequently incubated for 45
minutes with HRP-streptavidin (Dako, Glostrup, Denmark) diluted 1:300 in
PBS/BSA. Samples were washed again in PBS; bound antibody was visualized after
the addition of 0.05% solution of 3,3'-diaminobenzidine tetrachloride in
TBS, to which 0.03% H2O2 was added. The slides were
subsequently counterstained with hematoxylin. Control sections, in which the
primary antibody was replaced by PBS, were similarly processed. For all the
antibodies analyzed, no immunostaining was detected in control sections from
which the primary antibody had been omitted.
Western Blot Analysis
The right testis was snap frozen in liquid nitrogen and stored at
–70°C until required for protein extraction. Tissue was homogenized
in 1% SDS in PBS and a tablet of protease inhibitors Complete Mini, EDTA-free
(Roche, Mannheim, Germany) for each 10 mL of homogenization solution
(Pentikäinen et al,
1999). One mL of homogenization solution was used for each 100 mg
of fresh tissue. After centrifugation at 17 000 x g for 30
minutes at 4°C, the supernatants were collected and their protein
concentrations determined by BCA method (Pierce, Rockford, Ill). The
equivalent amounts of protein were mixed with sample buffer (0.5 mol Tris-HCl
pH 6.8, 20% glycerol, 10% SDS, 0.1% bromophenol blue, and 0.5%
ß-mercaptoethanol) and incubated at 95°C for 5 minutes. Proteins
(10–15 µg) were loaded in 8%–12% SDS-polyacrylamide gels, and
electrophoresis was performed in the presence of marker standards with
molecular weights between 200 and 6.5 kd (Bio-Rad Laboratories, Hercules,
Calif) at 15 mA during stacking and 25 mA during the separation. The proteins
were transferred to Immobilon-P membranes (Millipore, Bedford, Mass) by
semidry electrophoretic transfer for 1 hour at room temperature in transfer
buffer (50 mmol Tris, 40 mmol glycine, 10% SDS and 20% methanol, pH 9.2) at 22
V. Subsequently, the membrane was blocked with 5% nonfat milk in PBS for at
least 1 hour at room temperature. Before blocking, the marker standards were
separated and labeled with Amido Black solution (0.1% Amido Black, 45%
methanol, and 10% acetic acid) to check the protein molecular weight. After 3
washes of 10 minutes in PBS containing 0.1% Tween 20 (PBST), the membrane was
incubated overnight at 4°C using the same primary antibodies used for
immunohistochemistry diluted 1:1000 in a solution containing 5% albumin in
PBST. After 3 washes of 10 minutes in PBST, the membranes were incubated for 1
hour at room temperature with the same biotinylated secondary antibodies used
for immunohistochemistry diluted 1:10 000 PBST. Subsequently, the membranes
were washed with PBST and incubated with HRP-streptavidin (Dako) diluted
1:5000. Immunoreactive bands were located with the enhanced chemiluminescence
detection Kit (Amersham Biosciences, Buckinghamshire, United Kingdom) and
Hyperfilm ECL (Amersham Biosciences). The semiquantitative study was performed
for each antibody with an automatic image analyzer (MIP version 4.5;
Consulting Image Digital, Barcelona, Spain). After digitalization and
inversion of grey level image, the bands were delimited out manually, and the
area and medium grey were calculated for each band in a range of 0–255.
Labeling density was obtained by multiplying the area times medium grey and
was taken as an index of band intensity. The densitometrical analysis is
present as a bar diagram with arbitrary unit (the densitometrical minor value
was the unit in each bar diagram).
Statistical Analysis
The mean values obtained in aged and photoinhibited hamsters were compared
with data obtained in young hamsters (control group) using Student's
t test and, for measurements lacking equal variance, by a Welch test.
The tests were performed using log-transformed data. Data were
back-transformed following analyses and are presented as means ± SEM.
Statistical evaluation of apoptotic indices during the different stages of the
seminiferous epithelium was performed by analysis of variance in conjunction
with a least significant difference (LSD) test. Mean differences were
considered statistically significant when P is less than .05.
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Results |
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In photoinhibited hamsters the germ cells undergoing apoptosis were predominantly early spermatocytes and, especially, pachytene spermatocytes (Figure 1J and K) and, on some occasions, spermatogonia and early round spermatids. Apoptotic spermatocytes were observed in different meiotic phases: preleptotenes, early, middle, and late pachytenes (Figure 1J and K), and leptotene-zygotenes.
Aging and Short Photoperiod Exposure Increase Apoptotic Indexes in the Seminiferous Epithelium of Syrian Hamsters
Quantitatively, aging induced an increase in the percentage of both
spermatogonia and spermatocytes in apoptosis. During the cycle of the
seminiferous epithelium, germ cell apoptosis (% of apoptotic SG+SC) was higher
in aging in stages I–IV, V–VI, and VII–VIII (P <
.05) (Table 1). The percentage
of apoptotic spermatogonia was significantly increased in stages I–IV
and V–VI (P < .05), with no differences in stages
VII–VIII and IX–XIII (Table
1). The percentage of spermatocytes in their preleptotene phase
and pachytene in apoptosis was significantly higher in aged than in young
hamsters (P < .05), with no differences in leptotene-zygotene
spermatocytes. The percentage of pachytene spermatocytes in apoptosis was
significantly increased in stages I–IV, V–VI, and VII–VIII
(P < .05), with no differences in stages IX–XIII.
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After 8 weeks of short photoperiod exposure, the percentage of apoptotic spermatocytes was significantly increased with respect to animals maintained in a normal photoperiod (P < .05). But no differences in the percentage of apoptotic spermatogonia were found between groups (Table 2).
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Immunohistochemistry
Fas/Fas-L System—
Immunohistochemistry showed the presence of Fas-L in cytoplasmic
prolongations of Sertoli cells (Figure
2A) and in Leydig cells of young, aged
(Figure 2B), and photoinhibited
hamsters (Figure 2C). Also,
spermatozoon tails were positive for Fas-L in young
(Figure 2A) and aged hamsters.
Fas expression was observed in Leydig cells of young and aged hamsters
(Figure 2D and E), and a slight
immunoreactivity was also present in the tails of mature spermatozoa
(Figure 2D and E). However, in
photo-inhibited hamsters a strong expression of Fas was observed in
spermatocytes (Figure 2F) and
occasionally in Leydig cells and early round spermatids.
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Bcl-2 Family Proteins— Staining for Bax was observed in round spermatid acrosomes of young and aged hamsters (Figure 2J and K). In photoinhibited hamsters an intense immunoreactivity for Bax was observed in spermatocytes (Figure 2L) and some staining was observed in Leydig cells. In young and aged hamsters, the seminiferous tubules were negative for Bcl-2 staining (Figure 3A and B). However, in photoinhibited hamsters Bcl-2 was intensively expressed by Sertoli and Leydig cells (Figure 3C).
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Germ cells of young hamsters, including spermatogonia, spermatocytes, spermatids, and spermatozoa, showed an reactivity to Bcl-xL during the different stages of the seminiferous epithelium (Figure 3D). Also, some immunoreactivity was observed in Sertoli cells. However, both in aged and photoinhibited hamsters the Bcl-xL staining declined in germ cells and Sertoli cells (Figure 3E and F). Bcl-xS/L was present in germ cells and Leydig cells of young and aged hamsters (Figure 3G). Also, in aged hamsters, an intense immunoreactivity for Bcl-xS/L was observed in spermatocytes of the seminiferous tubules which were in maturation arrest of spermatogenesis (Figure 3H). In photoinhibited hamsters a strong immunoreactivity for Bcl-xS/L was observed, especially in spermatocytes (Figure 3I).
Western Blot Analysis
Similar levels of Fas-L (38 kd) were found in the testes of young, aged,
and photoinhibited hamsters (Figure
4A). A slight expression of Fas (48 kd) was observed in young and
aged animals. However, in hamsters exposed to a short photoperiod a strong
expression of Fas was observed (Figure
4B). In hamsters exposed to a short photoperiod, the levels of p53
were higher than in young hamsters maintained in a normal photoperiod
(Figure 4C). Aged hamsters
showed levels of p53 similar to young hamsters. Bax (23 kd) levels were
similar in young, aged, and photoinhibited hamsters
(Figure 4D). Western analysis
demonstrated similar levels of Bcl-2 (28 kd) in the testes of young and aged
hamsters, but a strong expression was found in photoinhibited hamsters
(Figure: 4E). In aged hamsters
and those exposed to a short photoperiod, the levels of Bcl-xL (31
kd) showed lower intensity than young hamsters maintained in normal conditions
(Figure 4F).
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Discussion |
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Aging and exposure to a short photoperiod provoked a significant increase in apoptosis in the seminiferous epithelium of Syrian hamsters. However, aging induced increase in apoptotic spermatogonia and spermatocytes, whereas short photoperiod exposure encouraged an increase in apoptotic spermatocytes without affecting the population of spermatogonia.
Aging increased germ cell apoptosis in stages I–IV, V–VI, and VII–VIII of the seminiferous epithelium cycle of Syrian hamsters. In previous studies in rats, an increase in germ cell apoptosis was observed in all the stages of the seminiferous epithelium cycle, although the increase was only statistically significant in stages XII–XIV (Wang et al, 1999). Other experimental situations, including heat exposure and testosterone deprivation, have led to differences in the incidence of germ cell apoptosis during the stages of the seminiferous epithelium cycle. After gonadotrophin deprivation and intratesticular testosterone in the rat, apoptosis occurred in stages VII–VIII, whereas hyperthermia induced germ cell apoptosis in stages I–IV and XII–XIV (Sinha Hikim et al, 2003). These studies indicate that apoptosis depends on the stages of the seminiferous epithelium cycle and that it differs according to the trigger stimuli even in the same animal species.
In the Syrian hamster, aging increased apoptosis in the spermatogonia in stages I–IV and V–VI, while apoptosis was absent in stages VII–VIII and IX–XIII, similar to the results observed in young animals. As mentioned above, these results probably reflect the existence of an optimal and constant regulation of germ cell density by an increasing apoptosis of spermatogonia in areas where these cells are too numerous (De Rooij and Janssen, 1987; De Rooij and Lok, 1987). Also, aging induced an increase in apoptotic pachytene spermatocytes over to the low rate found in young hamsters. It is known that control points are not confined to cells that divide by mitosis, but also operate during meiosis. Of particular note is the control checkpoint at the end of the pachytene phase of the meiotic prophase, when the meiotic recombination and chromosome synapse are incomplete (Roeder, 1997), preventing the production of aneuploid gametes (Roeder and Bailis, 2000). Our results in aging hamsters suggest that this increase in apoptosis in pachytene spermatocytes provides a control mechanism of meiosis that could eliminate aberrant aged germ cells. The strong coincidence between spontaneous and age-induced apoptosis in the same germ cell types and in the same stages of the seminiferous epithelium cycle suggests that aging causes an exacerbation of apoptosis at checkpoints where the germ cells are eliminated spontaneously. It is reasonable to think that the high degree of synchronization required during the spermatogenic process imposes a strict control over germ cell apoptosis, both in normal situations and in situations that imply a deteriorated spermatogenic capacity of the epithelium, such as aging.
Germ cell types that suffer apoptosis during testicular regression after exposure to a short photoperiod may differ between species. In photoinhibited Syrian hamster the main apoptotic germ cells observed are the spermatocytes and occasionally spermatogonia and round spermatid. Similar results to these presented have been reported in the mouse and birds (Sinha Hikim et al, 1997; Young et al, 1999, 2001). The present study is the first to demonstrate this phenomenon quantitatively, and to indicate that apoptosis does not increase in the population of spermatogonia. Testis exposed to short photoperiod shows a pattern of cell death similar to pharmacological deprivation of gonadotrophins (Sinha Hikim et al, 1997; Young and Nelson, 2001). This indicates that spermatocytes are more susceptible to perturbations of the seminiferous epithelium environment during short photoperiod exposure, which is characterized by severe testosterone deprivation (Furuta et al, 1994; Calvo et al, 1997).
Role of Fas/Fas-L System, Bcl-2 Family, and p53 in Germ Cell Apoptosis in Aging and After Short Photoperiod Exposure
In the Syrian hamster Fas-L expression was observed in Sertoli and Leydig
cells as well as in spermatozoon tails of young and aged animals. Similar
results have been documented in humans
(Francavilla et al, 2000) and
rodents, including the mouse (French et
al, 1996) and rat (Lee et al,
1997). Fas-L expression in the normal testis may be related to the
fact that the testis is an immunologically privileged organ, and Fas-L would
be involved in the elimination of active and infiltrated T cells that express
Fas (Bellgrau et al, 1995).
Other authors have described Fas-L expression in postmeiotic germ cells, such
as mature spermatozoa in the rat and mouse
(D'Alessio et al, 2001) and
even in humans (Francavilla et al,
2002), which is consistent with the present results obtained in
the Syrian hamster. These results have led us to propose an alternative
hypothesis concerning the role of Fas-L in the reproductive system, whereby
Fas-L may represent an important molecule in the complex mechanism developed
by male gametes to escape immunological reaction both in the male genital
tract (autoimmune reaction against auto antigens of sperm cells) and the
female genital tract (D'Alessio et al,
2001; Riccioli et al,
2003).
In young and aged Syrian hamsters, a slight expression of Fas was observed, mainly in Leydig cells, which agrees with previous results reported in rodents, such as mouse (Koji et al, 2001) and rat (Lee et al, 1999), as well as in humans (Francavilla et al, 2000, 2002). These results suggest that there is no evidence to support a relationship between the Fas system and germ cell apoptosis in the testis of young and aged hamsters, as was found in the mouse (Koji, 2001). Although the Fas system is essential for germ cell apoptosis in several pathological situations, it is irrelevant in normal conditions (Koji, 2001). However, Western blot analysis revealed a significant increase in Fas expression in photoinhibited testes. Also, immunohistochemically Fas was strongly expressed by the spermatocytes of hamsters exposed to a short photoperiod, which is coincident with the predominantly TUNEL-positive germ cells observed in the spermatocytes of these animals. In Fas-mediated apoptosis, the binding of Fas-L to Fas antigen is a prerequisite (Koji et al, 2001). So the temporal and spatial association between Fas and Fas-L expression in hamsters exposed to short photoperiod suggests that the Fas system is a mediator of germ cell apoptosis induction after short photoperiod exposure. Although the Fas system has been correlated with germ cell degeneration in situations of meiotic and postmeiotic arrest of spermatogenesis in man (Eguchi et al, 2002; Francavilla et al, 2002), our immunohistochemical results in aged hamsters suggest that there is no connection between TUNEL-positive germ cells and Fas expression. Also, Western blot analysis revealed no changes in the expression of Fas and Fas-L in aged animals, which suggests that the Fas/Fas-L system is not involved in apoptosis induction of germ cells in the testes of aged Syrian hamsters.
Another widely known regulatory system of apoptosis is the Bcl-2 family composed of anti- and proapoptotic proteins which regulate apoptosis by controlling the release of cytochrome c and other mitochondrial changes (Yang and Korsmeyer, 1996; Green and Reed, 1998). In the present study, Western blot analysis and the immunohistochemical study demonstrated an expression of the antiapoptotic form of Bcl-xL in germ cells of young hamsters, whereas aged and photoinhibited hamsters showed a lower degree of expression. On the other hand, proapoptotic form Bcl-xS was expressed in all the groups (young, aged, and photoinhibited). With respect to the localization of both proteins, Bcl-xL was expressed in all the populations of germ cells in young hamsters. Based on the decrease of Bcl-xL expression in germ cells of photoinhibited and aged hamsters, as shown by Western blot analysis and immunohistochemistry, we can affirm that when Bcl-xS/L antibody is used, what we see predominantly is the expression of Bcl-xS in germ cells in these 2 groups of animals. Accordingly, and based on the immunohistochemical results, in animals exposed to a short photoperiod using Bcl-xS/L antibody we found a strong expression of Bcl-xS predominately in the spermatocytes, which is concordant with TUNEL-positive germ cells as well as with other proapoptotic proteins studied in the present report. Also the seminiferous epithelium of aged hamsters showed germ cells positive for Bcl-xS. As suggested by other authors, a balance of anti- and proapoptotic members of the Bcl-2 family is critical for regulating the survival of testicular germ cells (Oltvai et al, 1993; Yang and Korsmeyer, 1996; Yamamoto et al, 2001; Sakamaki, 2003). Our results indicate that the lower expression of the anti-apoptotic form Bcl-xL in germ cells of aged and photoinhibited hamsters may be due to a predominance of proapoptotic Bcl-xS forms, similar to that observed by immunocytochemistry in aging human testes (Kimura et al, 2003). This imbalance between members of the Bcl-2 family, with a predominance of proapoptotic forms in germ cells, seems to be responsible for the increase in germ cell apoptosis found in aged hamsters activating the mitochondrial pathway of apoptosis; the Fas/Fas-L system and p53 also participate in the increase of germ cell apoptosis in hamsters exposed to a short photoperiod.
The present results indicate that the levels of Bcl-2 are low in the testis both of young and aged hamsters. These results are congruent with previous studies obtained in mouse (Hockenbery et al, 1991; Knudson et al, 1995) and humans (Beumer et al, 2000; Sakamaki, 2003), which suggested that Bcl-2 plays no function in the male gonad in normal conditions, unlike in the ovary, where this protein is critical for primordial ovarian follicle formation (Ratts et al, 1995). On the contrary, using Western blot an overexpression of Bcl-2 by Sertoli and Leydig cells was observed in hamsters exposed to short photoperiod compared with young animals. Studies in transgenic animals have indicated that the overexpression of Bcl-2 in somatic cells of the testis produces alterations in spermatogenesis, including the inhibition of gamete formation, spermatid malformations, vacuolization of the epithelium, loss of germ cells, and increased apoptosis, while seminiferous tubules are characterized by an accumulation of spermatogonia, Sertoli cells, and apoptotic germ cell in the meiotic prophase (Knudson et al, 1995; Furuchi et al, 1996; Rodríguez et al, 1997; Yamamoto et al, 2001). These spermatogenic defects found in transgenic animals are comparable with the modifications observed in hamsters exposed to a short photoperiod, which suggests that Bcl-2 plays an important role in the atrophy of the seminiferous epithelium of Syrian hamsters after exposure to the short photoperiod.
Previous studies have reported that Bax is expressed predominantly in primary spermatocytes and spermatids (Oldereid et al, 2001). Our immunohistochemical study revealed that Bax was expressed by the spermatid acrosomes of young and aged hamsters, which suggests that Bax plays a role in the maturation and differentiation process of the acrosome in the hamster, as has been suggested in other species (Oldereid et al, 2001). Also, Bax has been classified as a proapoptotic member of the Bcl-2 family and has been related to the induction of apoptosis of spermatocytes and spermatids in humans (Oldereid et al, 2001). Based on our immunohistochemical results, Bax does not seen to be involved in germ cell apoptosis in young or aged hamsters. However, in hamsters exposed to short photoperiod, the expression of Bax by the spermatocytes was concordant with the expression of Fas and p53 as well as with TUNEL-positive cells. These results suggest that Bax is involved in germ cell apoptosis induction after exposure to a short photoperiod.
Strong immunostaining with p53 was found in spermatocytes and occasionally in spermatogonia of photoinhibited hamsters. These results indicate that p53 expression is concordant with Fas and Bax expression as well as with TUNEL-positive germ cells in photoinhibited hamsters, which suggests that p53 is involved in germ cell apoptosis induction after short photoperiod exposure.
In summary, aging of the seminiferous epithelium in the Syrian hamster is characterized by an increase in germ cell apoptosis in the populations of spermatogonia and spermatocytes and is dependent on the stage of the cycle. After short photoperiod exposure, however, the increase in apoptosis is only observed in the spermatocytes. Different molecular pathways are triggered to induce germ cell apoptosis in aged animals and those exposed to a short photoperiod. The results obtained show both the intrinsic and extrinsic pathways being activated after short photoperiod exposure, but only the intrinsic pathway during aging.
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Footnotes |
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A portion of this report has been communicated in abstract form for the 44th American Society of Cell Biology Annual Meeting.
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