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University of Manchester School of Biological Sciences, Manchester, United Kingdom M13 9PT; and the Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University (M.d.B.-B., K.J.T.), Utrecht 3508, The Netherlands
Address all correspondence and requests for reprints to: Dr. Ian Morris, Division of Pharmacology, Physiology, and Toxicology, G.38 Stopford Building, University of Manchester School of Biological Sciences, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: ian.morris{at}man.ac.uk
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Abstract |
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Introduction |
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Previous studies in our laboratory have shown conclusively that Leydig cells will undergo apoptosis both in vivo and in vitro in response to EDS (6, 7). Leydig cell numbers do not begin to decrease in vivo until 12–18 h after EDS administration (7). Up until and including this time, there is no change in the cellular composition of the interstitium. It is likely that the major changes in the expression of apoptosis-related gene products are occurring over this period, just before maximal Leydig cell apoptosis at 24 h (7). By 48 h nearly all Leydig cells are depleted from the interstitium (7).
Of the genes that have been shown to be involved in apoptosis, the Bcl-2 family members have been most widely studied. We have previously shown that the family members Bcl-2, Bax, and Bcl-xl appear to play no active role in the induction of apoptosis in the Leydig cell (7) in response to EDS administration. This study has led us to evaluate the potential role of other apoptotic gene products in the initiation of Leydig cell death in response to EDS.
Bak is a proapoptotic member of the Bcl-2 family of proteins that has been localized in high levels in the human testes (8). The tissue expression pattern of the bak gene (9, 10, 11) was shown to be widespread and included terminally differentiated cell types, such as the Leydig cell. In a yeast system, the Bak protein was demonstrated to bind to Bcl-xl (9), which might suggest that its mechanism of action is through homo- and heterodimerization with other Bcl-2 family members.
Recently, a new member of the Bcl-2 family, Bcl-w, was isolated through a low stringency amplification of murine macrophage and brain complementary DNA using degenerate primers encoding the S2 and S3 regions of Bcl-2, Bcl-x, and Bax (12). The way in which Bcl-w modulates apoptosis is similar to that of Bcl-2, promoting the survival of the cell rather than causing cell death. Transgenic mice lacking the Bcl-w gene exhibit testicular abnormalities during aging (13, 14).
An alternative pathway that has been shown to induce apoptosis is the Fas system. The Fas molecule (FasR) is a cell surface protein belonging to the tumor necrosis factor receptor family (15), whereas the Fas ligand (FasL) is a member of the tumor necrosis factor ligand family (16). FasL binds to FasR, which results in cysteine protease activation and target cell apoptosis; the Bcl-2 protein has been shown to inhibit this process in some cell types (17). The testes are a remarkable immune-privileged site known for the ability to support both xenogeneic and allogeneic tissue transplants (18, 19). This immune privilege of the testes has been linked to the constitutive expression of FasL (20). The cell type(s) within the testes expressing FasL is still unclear, with some reports suggesting a Sertoli cell origin (20, 21) and others suggesting a mainly Leydig cell origin (22); there are also studies which suggest that FasL is expressed on Leydig, Sertoli, and germ cells within the testes (23). The localization of FasR within the testes is thought to be associated with germ cell (21), probably spermatids (22).
We have previously shown that EDS induces Leydig cell apoptosis in vivo (6, 7). In those studies we examined the time course of EDS-induced Leydig cell apoptosis by in situ end labeling of apoptotic DNA and 3ß-hydroxysteroid dehydrogenase immunohistochemistry. We have also determined the potential roles of p53, Bcl-2, Bcl-xl, and Bax in Leydig cell apoptosis. As none of the Bcl-2 family members or p53 appears to be involved in the induction of Leydig cell apoptosis, we have investigated the potential role of other Bcl-2 family members (Bak and Bcl-w) and the Fas system in the in vivo initiation of Leydig cell apoptosis after administration of the cytotoxin EDS.
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Materials and Methods |
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Animals
All animal experimentation was carried out in accordance with
the Animals (Scientific Procedures) Act 1986. Adult male Sprague Dawley
rats (250–300 g), purchased from Charles River UK Ltd. (Margate, UK),
were housed six per cage in a light-controlled room (12 h of light,
12 h of darkness; lights on at 0700 h). The animals were
handled daily for at least 1 week before the beginning of
experimentation, with food and water supplied ad libitum.
Rats were randomly distributed into eight groups of six and injected ip
with either ethane dimethanesulfonate (100 mg/kg BW) in a vehicle of
dimethylsulfoxide-water (1:3, vol/vol) or with an equivalent volume of
vehicle only (2 ml/kg BW). The animals were killed by an overdose of
anesthetic at the time points indicated, and the testes were removed.
The left testis was processed for histology after fixing in Bouin’s
solution, and an interstitial cell suspension was prepared from the
right testis and stored at -20 C until processed by SDS-PAGE.
Immunohistochemical staining
Immunohistochemistry was performed according to the method of
Teerds (26). Briefly testis sections (5 µm) fixed in Bouin’s
solution and embedded in paraffin were deparaffinized, and endogenous
peroxidase was blocked with 1% H2O2 in
methanol for 30 min. The slides were subsequently washed in 0.01
M Tris-buffered saline (TBS; pH 7.4), incubated with 0.01
M glycine in TBS for 30 min, and then rinsed with TBS.
Sections were blocked with 10% normal rabbit serum for 30 min and then
incubated at 4 C overnight with the antibodies against FasR, FasL, Bak,
or Bcl-w diluted in TBS containing 0.05% acetylated BSA (Aurion,
Wageningen, The Netherlands). After this incubation, the slides were
washed with TBS and incubated for 60 min with the corresponding
biotinylated secondary antibody (ABC-peroxidase complex staining kit,
Elite, Vector Laboratories, Inc., Burlingame, CA) diluted
1:150 in TBS containing 0.5% acetylated BSA. Sections were again
washed in TBS and subsequently incubated for at least 60 min with the
components avidin (A) and biotin (B) of the ABC staining kit. Both
components (A and B) were diluted 1:200 and prepared at least 15 min
before use. Slides were washed in TBS, and bound antibody was
visualized after the addition of a 0.06 mg/ml solution of
3,3'-diaminobenzidine tetrachloride (Sigma Chemical Co.,
St. Louis, MO) in TBS to which 0.03% H2O2 was
added. The slides were subsequently stained with Mayer’s
hematoxylin.
Interstitial cell preparation
Interstitial cells were prepared by collagenase digestion as
described previously (7). Leydig cell purity was assessed using the
3ß-hydroxysteroid dehydrogenase staining method (27, 28), with
the formation of blue-purple formazan granules in the cytoplasm
indicating the presence of Leydig cells. In all experiments
interstitial cell preparations were used that contained approximately
10% Leydig cells.
Western blot analysis
Protein determinations were carried out on interstitial cell
preparations from in vivo EDS-treated rats using the Bio-Rad
protein assay method (Bio-Rad Laboratories, Inc.,
Hercules, CA). SDS-PAGE and Western blotting were carried out as
described previously (7). Quantification of band intensities was
achieved using the Molecular Analyst package from Bio-Rad Laboratories, Inc. Data are presented as a percentage of the
control value, where control values have been assigned a value of
100%.
Statistical analysis
The results of the Western blot analysis are expressed as the
mean ± SEM (n = 6). Comparisons between the
control group and the different times of EDS treatment were made by
Kruskal-Wallis one-way ANOVA and the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b). Bcl-w
protein was also detected in control interstitial preparations;
however, the levels of this protein became undetectable 48 h after
EDS administration (Fig. 1, c and d).
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). In addition, a faint staining was
present in the cytoplasm of pachytene spermatocytes, with a strong
staining in the nuclei of round spermatids (Fig. 2, a–c,
asterisks). At 6 h (Fig. 2b) and 12–18 h (data not
shown) after EDS administration, there was no change in the staining
intensity of Bak in the interstitium, whereas at 48 h after EDS,
the Leydig cells had disappeared, although some macrophage-like cells
were then positive (Fig. 2c). The staining in the nuclei of the
spermatids did not change after EDS administration (Fig. 2, a–c). The
nuclear staining of Bak in the early round spermatids was specific, as
shown by the inclusion of serum controls (Fig. 2, g and h) and the fact
that staining disappeared as the nuclei started to elongate.
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, d and e), and staining in the seminiferous tubules was
negligible. At 6 h (Fig. 2e) and 12–18 h (data not shown) after
EDS, Leydig cells remained positive for Bcl-w, and at 48 h after
EDS administration when the Leydig cells had disappeared, no staining
in the interstitial space was observed (Fig. 2f).
FasR and FasL expression in the interstitial cell preparations and
Leydig cells after EDS treatment
FasR and FasL proteins exhibited similar expression patterns after
EDS administration. By 6 h postinjection, both proteins were
elevated in interstitial preparations. FasL protein levels continued to
rise until 12 h after EDS treatment, after which time they started
to decrease until 48 h postadministration when they were almost
undetectable (Fig. 3, a and b). FasR
protein levels rose until 18 h after EDS administration, declining
thereafter to undetectable levels at 72 h (Fig. 3, c and d).
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). There was no positive staining
of other interstitial cells within the interstitium. After 6 h of
EDS administration, all Leydig cells stained positively for FasL, with
an increase in the staining intensity (Fig. 4b). At 12 and 18 h
postinjection, the majority of Leydig cells were still positively
stained for FasL (data not shown). By 48 h after EDS
administration there were no FasL positively staining cells within the
interstitium (Fig. 4c). Throughout the time course only Leydig cells
showed positive staining for FasL. Macrophages (identified by their
nuclear morphology) and fibroblasts were negative with both FasL
antibodies used in this study, as shown in the Leydig cell-depleted
interstitium (Fig. 4c).
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). Six hours after EDS
administration, all Leydig cells exhibited positive staining for FasR,
with the intensity of staining being stronger than that in control
testes (Fig. 5b). As Leydig cells became apoptotic at 12–24 h after
EDS, the FasR staining intensity became weaker, with severely apoptotic
Leydig cells becoming negative for FasR (data not shown). Forty-eight
hours post-EDS injection, no FasR-positive Leydig cells were observed
in the interstitium, and remaining macrophages were also negative (Fig. 5c).
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and 5d). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In other systems it has been shown that the Bcl-2 family of proteins regulates the apoptotic phenomenon by either improving cell survival (Bcl-2, Bcl-xl, and Bcl-w) or enhancing cell death (Bax, Bad, and Bak). In a previous study we investigated the potential roles of Bcl-2, Bcl-xl, and Bax in Leydig cell apoptosis in response to EDS (7). There was an absence of both Bcl-2 and Bax protein in Leydig cells, and Bcl-xl protein levels initially increased up to 12 h postinjection and then returned to basal levels. These data indicated that none of these proteins was involved in initiating the apoptotic signal; however, the antiapoptotic Bcl-2 family member Bcl-xl was probably elevated in an attempt by the Leydig cells to attenuate the severity of the cell death signal. In the present study we have examined two other members of the Bcl-2 family (Bak and Bcl-w) to determine whether these proteins were involved in initiating Leydig cell apoptosis in response to EDS administration.
The bak gene is a proapoptotic member of the family that has been localized in the testes by immunohistochemistry (8). Western blot analysis of Bak protein in interstitial cell preparations showed that although Bak protein was easily detected, there was no change in protein levels between control interstitial cell preparations and those up to 72 h after EDS administration. However, as the interstitial cell preparations used for Western blotting are contaminated with other cell types, such as pachytene spermatocytes and round spermatids, the lack of change in Bak expression could be due to the overriding signal generated by these other testicular cells. Immunohistochemistry showed that Bak is expressed in the round spermatids, and staining intensity in these cells does not change after EDS treatment. This may explain why Bak levels in the interstitial cell preparations do not to change. However, staining for Bak in the Leydig cells is unchanged at 6–18 h after EDS treatment, whereas at 48 h, the Leydig cells are dead, and staining is absent, apart from that in a few macrophages. Thus, it is unlikely that Bak plays a role in either the initiation or propagation of the apoptotic signal in Leydig cells.
Leydig cell apoptosis might be regulated not by increased synthesis of a proapoptotic member of the Bcl-2 family, but by the down-regulation of a survival gene. A recently discovered family member that, like Bcl-2, can inhibit apoptosis is Bcl-w (12). Knockout mice that lack the Bcl-w gene exhibit progressive testicular cell degeneration, including Leydig cells (13, 14). Any non-Leydig cell-expressed apoptosis proteins in the interstitial cell preparations should show up in Western blots at 72 h and later times after EDS administration when Leydig cells are absent. Bcl-w protein could be detected in control interstitial cell preparations and at times up to 24 h after EDS administration. However, at 48 and 72 h postinjection, Bcl-w protein was virtually undetectable. The Western blots fit with the immunohistochemical data. Bcl-w expression in the Leydig cells was unchanged at 6–18 h after EDS, and staining was absent in the interstitium at 48 h when all Leydig cells are dead. This would suggest that Bcl-w protein is Leydig cell specific, and that as the Leydig cells are removed from the interstitium, the Bcl-w protein also disappears. The lack of changes in Bcl-w protein before its loss from the interstitium would suggest that it is not involved in the initiation of Leydig cell apoptosis.
It is well known that in the ovary, developing follicle numbers are reduced by atresia. Several recent reports have suggested that follicular atresia is mediated by the Fas system (29, 30, 31). Some studies have shown that the Fas system is only responsible for the degeneration of granulosa cells within atretic follicles (30), whereas other studies indicate that both granulosa and luteal cells are removed by Fas-mediated apoptosis (29). Within the reproductive system, Fas-mediated apoptosis has also been implicated in regression of the vaginal epithelium during the estrous cycle and after ovariectomy (32), regression of the glandular epithelium of the ventral prostate after androgen ablation (33, 34), and germ cell death after toxic insult to the Sertoli cells (21). FasL messenger RNA has also been localized in high amounts in the testes using in situ hybridization (15).
In testis sections from control animals, most, but not all, Leydig cells express FasR and FasL proteins. FasL expression in our study agrees with some reports (22, 23), although others failed to show FasL expression in the interstitium (21). FasR expression in Leydig cells, on the other hand, is in disagreement with the findings of several studies, although Sugihara et al. have recently shown that FasR is expressed in Leydig cells (23). Within 6 h after EDS administration, all Leydig cells expressed FasR and FasL protein. The Western blot analysis shows that both proteins were significantly elevated from 6–18 h postinjection. These increases in the Fas proteins occur before any detectable Leydig cell apoptosis, which we have previously shown to be maximal at 24 h after EDS administration (7). This suggests that activation of FasR initiates the apoptotic signal. The disappearance of FasR and FasL proteins at 48 h after EDS administration, as shown by Western blotting, is consistent with the immunohistochemical data showing that these proteins are only expressed on Leydig cells within the interstitium. Forty-eight hours after EDS administration, nearly all of the Leydig cells have been eliminated, and there is no staining for FasL or FasR, which would suggest that it is not present in the non-Leydig cell component of the interstitium. The initial increase in Bcl-xl levels seen previously (7) might account for why apoptosis is not maximal at a time when both FasL and FasR protein levels have more than doubled. This ability of Bcl-xl to protect cells from Fas-mediated apoptosis has been documented previously in both B and T cells (35, 36).
In conclusion, the Leydig cells of the testes initiate programmed cell death after the administration of EDS. Leydig cell apoptosis would appear to be mediated through the actions of the Fas receptor and its ligand, which are both up-regulated early after EDS administration. The Bcl-2 family of proteins does not appear to play an initiating role in Leydig cell apoptosis.
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Footnotes |
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Received May 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-3ß-hydroxysteroid-dehydrogenase during the foetal
and neonatal development of rat Leydig cells. Andrologia 15:392–397[Medline]
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