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a Division of Physiology, Pharmacology and Toxicology, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom
b Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht,The Netherlands
c Department of Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After androgen withdrawal as a result of hypophysectomy, the number of degenerating pachytene spermatocytes and step 7 and 19 spermatids in stage VII of the seminiferous tubules of the testis increases [2]. This cell degeneration takes the form of apoptosis [7] and is attenuated by LH and FSH singly or in combination [2, 7]. The observation that testosterone can completely prevent hypophysectomy-induced cell death [7], along with the fact that GnRH antagonists and anti-LH antibodies induce the same profile of apoptotic germ cell death [4, 7–10], suggests a critical role for LH-stimulated testosterone secretion for the maintenance of germ cell viability.
Ethane dimethanesulfonate (EDS) is a unique testicular toxin with cytotoxic action confined almost exclusively to the Leydig cells [11]. EDS is a valuable tool for investigating apoptosis in the testis in response to androgen withdrawal because it selectively eliminates both basal and LH-stimulated testosterone production, thus providing a model in which there is complete androgen ablation within the testes. EDS has been used previously to investigate apoptosis in the testis [12, 13]. As with GnRH antagonists and hypophysectomy, the predominant germ cell types undergoing apoptosis as a result of androgen withdrawal induced by EDS include pachytene spermatocytes and spermatids [12].
The regulation of apoptosis is dependent upon specific gene products. The involvement of Bcl-2 in apoptosis has been well documented [14]. It is now known that Bcl-2 is only one gene in a growing multigene family whose members are thought to regulate apoptosis by the formation of hetero- and homodimers. Bcl-2, as well as its structural homologue, the long form of Bcl-x (Bcl-xl), promotes cell survival by inhibiting apoptosis [14, 15]. Other members of the Bcl-2 family can block the ability of Bcl-2 to inhibit apoptosis. These include Bax, Bak, and Bad, which in most cases promote cell death [16–18]. An alternative and often more rapid form of programmed cell death is mediated by the Fas pathway. Fas ligand is a transmembrane protein [19] that can initiate apoptosis by binding to Fas-R-expressing cells [20, 21].
It is known that Bax, Bad, Bcl-xl, and Bcl-2 are expressed in rodent testes [22–26]. Although Bak expression has not yet been reported in rodents, its presence in the human testis [27] suggests that it may also play a role in the rat testis. Studies using knockout and transgenic mice suggest that members of the Bcl-2 family may play an important role in spermatogenesis. Bax-knockout mice are infertile as a result of the accumulation of premeiotic germ cells and absence of mature haploid sperm [24], and transgenic mice expressing high levels of Bcl-xl or Bcl-2 show highly abnormal adult spermatogenesis accompanied by sterility [26]. The testis has been shown to express Fas-L at high levels [28]. It is generally accepted that Fas-L is expressed in the seminiferous epithelium of the testis [29–32], although some have shown Fas-L staining to be weak or absent [33]. However, there is a discrepancy in the precise cellular localization of Fas-L in the seminiferous tubules, with some groups [29–31] showing Fas-L localization to the Sertoli cells while others have reported that Fas-L is also expressed in the germ cells [32]. Fas-R is expressed in the germ cells [29, 32, 33], including spermatocytes [29] and spermatids [33], with some reports also showing localization in the Sertoli cells [32].
Although it is known that the Fas pathway is an important regulator of testicular apoptosis induced by environmental toxicants such as mono-(2-ethylhexyl) phthalate and 2,5-hexanedione [29], the potential role of this pathway and the Bcl-2 family in regulating apoptosis in the testis in response to androgen withdrawal has not been reported. In this study, we examine the expression of some of the Bcl-2 family members in the rat testis as well as the precise cellular localization of Fas-L and Fas-R. We have also investigated the roles of the protein products of these genes in apoptosis within the seminiferous tubules of the rat testis after EDS-induced androgen ablation.
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MATERIALS AND METHODS |
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Adult male Sprague-Dawley rats, weighing approximately 250 g, were supplied by Charles Rivers UK Ltd. (Margate, Kent, England); Tween 20 and proteinase K were obtained from Sigma Chemical Co. (St. Louis, MO). EDS was synthesized in our laboratory from ethylene glycol and methanesulfonyl chloride according to the method described previously [34]. The anti-3ß-hydroxysteroid dehydrogenase (3ß-HSD) rabbit polyclonal antibody (dilution 1:5000) was a gift from Dr. I. Mason (University of Edinburgh, Edinburgh, Scotland). The anti-clusterin (1:100 000) rabbit polyclonal antibody was a gift from Dr. Y.C. Cheng (Rockefeller University, New York, NY). The anti-Bax (1:1000) and anti-Bcl-2 (1:10 000) rabbit polyclonal antibodies were kind gifts from Dr. A. Metcalfe [35] and Dr. G. Evans (University of London, London, England), respectively. The rabbit anti-Bcl-xl (1:500) polyclonal and the mouse anti-Bad (1:200) monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). The mouse anti-Bak (1:5000) monoclonal antibody was obtained from Calbiochem Novabiochem Ltd. (Beeston, Nottingham, England). The anti-Fas-R (1:100) and anti-Fas-L (1:100) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG) and the polyvinylidene fluoride membranes were obtained from Amersham Life Sciences (Little Chalfont, Buckinghamshire, England). For immunohistochemistry, a biotinylated goat anti-rabbit IgG was used followed by an ABC step (Vectastain kit elite; Vector Labs., Burlingame, CA). The ApopTag kit for in situ end-labeling (ISEL) was supplied by Oncor Appligene (Chester-Le-Street, Co., Durham, England). Protein standards and protein assay reagents were obtained from Bio-Rad Laboratories Ltd. (Hemel Hempstead, Hertfordshire, England).
Treatment
All animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986. Rats were housed four per cage in a light-controlled room (lights-on from 0700 to 1900 h). The animals were handled daily for at least 1 wk prior to the beginning of experimentation, and food and water were supplied ad libitum. Animals were randomly allocated into groups of four rats for each time point; they were injected (i.p.) with a single dose of 100 mg/kg EDS in dimethyl sulfoxide:water (1:3) sufficient to kill all Leydig cells within 2 days [36] and were killed by cervical dislocation 2, 5, and 8 days postinjection. Four rats injected with vehicle alone and killed after 8 days were used as controls. The testes and seminal vesicles were removed immediately and weighed.
ISEL
The left testis of each animal was fixed in Bouin's solution, processed by conventional methods, and embedded in paraffin. Apoptotic cells were identified in tissue sections (5 µm) by ISEL using an ApopTag kit as described previously [35]. For assessment of apoptosis, the percentage of seminiferous tubules with apoptotic cells was determined by scoring 75 randomly selected tubules per section (5 µm) on four sections from four different animals at each time point. The number of apoptotic cells per tubule was assessed on four sections. The number of tubules scored is indicated in the appropriate figure. The "apoptotic index" was calculated by multiplying the percentage of tubules containing apoptotic germ cells by the number of apoptotic germ cells per tubule at each time point after EDS administration.
Immunohistochemistry
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 sections were subsequently washed in 0.01 M Tris-buffered saline (TBS; pH 7.4) and then incubated with 0.01 M glycine in TBS for 30 min and rinsed with TBS. Sections were blocked with 10% normal goat serum for 30 min and then incubated at 4°C overnight with the antibodies against Fas-R or Fas-L diluted in TBS containing 0.05% acetylated BSA (Aurion, Wageningen, The Netherlands) in a humid atmosphere. After this incubation, the slides were washed with TBS and incubated for 60 min with a biotinylated goat anti-rabbit polyclonal antibody (ABC-peroxidase staining kit Elite) diluted 1:200 in TBS containing 0.05% 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 1:2000 and prepared at least 15 min prior to use. Slides were washed again in TBS; bound antibody was visualized after the addition of a 0.06 mg/ml solution of 3,3'-diaminobenzidine tetrachloride (Sigma) in TBS, to which 0.03% H2O2 was added. The slides were subsequently counterstained with Mayer's hematoxylin. Control sections, in which the primary antibody was either omitted or replaced by normal goat serum, were similarly processed.
Western Analysis
The right testis was snap frozen in liquid nitrogen and stored at -70°C until required for protein extraction [37]. SDS-PAGE and Western blotting were carried out as described previously [35]. The blots shown are representative of two animals at the time points indicated. Quantitative analysis of gene products was determined on Western blots utilizing testicular protein from four animals at the time points indicated (control, 2 and 8 days after EDS treatment). Quantification of band intensities was achieved using the Molecular Analyst package from Bio-Rad. Data are presented as percentage of controls where control values have been assigned a value of 100%.
Statistical Analysis
For the organ weight, ISEL, and gene quantification data, the results shown are the mean ± SEM (n = 4). Statistical significance was determined using the Kruskal-Wallis one-way ANOVA and the Mann Whitney U-test; p values < 0.05 (*) were considered to be significant. For the organ weight and ISEL data, bars without a common superscript are significantly different. For the gene quantification data, comparisons were made between the control group and the various times after EDS treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regression of the testis was evident as a time-dependent decrease in weight after EDS administration such that at 8 days following EDS administration the weight was only 66% of the control value (Fig. 1). Androgen ablation also reduced seminal vesicle weight, which was 16% of the control value 8 days after EDS.
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3ß-HSD Expression
Evidence of Leydig cell death, and as a consequence androgen withdrawal, was shown by the complete absence of 3ß-HSD expression 2 days after EDS administration and at time points subsequent to this (Fig. 2).
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ISEL of Apoptotic Germ Cells
Figure 3A shows a control testis from a rat treated with vehicle for 8 days. There is a single apoptotic spermatogonium located near the base of the spermatogenic epithelium, and in other sections apoptotic spermatocytes could also be observed (not shown). There was a marked increase in germ cell apoptosis 8 days after EDS administration (Fig. 3B). The predominant cell types undergoing apoptosis following androgen withdrawal were spermatocytes and round spermatids located toward the lumen of the seminiferous tubules. Figure 4 shows the quantification for the ISEL of apoptotic germ cells in controls and after EDS administration. There appeared to be no difference between the percentage of apoptotic germ cells in control animals and the percentage 2 days after EDS administration (Fig. 4A). At 5 days after EDS administration, this percentage had risen to 16 ± 2.8 in comparison to 8.6 ± 1.5 in the controls (p < 0.05). By 8 days the percentage of tubules containing apoptotic germ cells had increased to 48.4 ± 8.9 and was significantly (p < 0.05) higher than in the control group and in the group that had received EDS for 5 days. The mean number of apoptotic germ cells per tubule was unchanged until Day 8 after EDS administration in comparison to the control value. By 8 days after EDS administration, this number had increased 4-fold, from 1.9 ± 0.5 in the control to 8.0 ± 1.1 (p < 0.05; Fig. 4B). The apoptotic index increased at Day 5 after EDS administration (29.1) in comparison to the control value (16.3). By Day 8 after EDS administration there was a 24-fold increase in the apoptotic index (386) in comparison to the control value.
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Expression of Apoptosis-Related Gene Products and Changes Following EDS Treatment
All of the apoptosis-related gene products examined in the study were present in the adult testis (Figs. 5–7). Clusterin was present in both immature and adult testes but not in any other tissue analyzed (Fig. 5). Both Bad and Bak were expressed at low levels in the thymus and spleen in comparison to the prostate and adult testis. Although Bak was present in the immature testis, Bad appeared to be absent (Fig. 5). After EDS administration there were no significant changes in the levels of clusterin (data not shown), Bcl-xl (Fig. 6 and shown graphically in Fig. 8), Bad, or Bak (Fig. 8). However, even though the expression of both Bcl-2 and Bax appeared unchanged on the Western blots (Fig. 6), densitometry showed that their levels were slightly but significantly elevated above control levels at 8 days after EDS treatment (Fig. 8). In contrast, Fas-L expression declined at 2 days after EDS treatment and was reduced further at 8 days, at which time Fas-R expression had also fallen (Figs. 7 and 8).
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Immunohistochemical Localization of Fas-L and Fas-R in the Testis
Immunohistochemistry using an antibody raised against the C-terminus of Fas-L showed the presence of Fas-L in the Sertoli cell cytoplasm, although the staining was not particularly intense. In general, spermatogonia and early spermatocytes were negative for Fas-L staining while pachytene spermatocytes showed a granule-like cytoplasmic labeling. Round spermatids also showed some staining of the cytoplasm in addition to a distinct localization of staining in the plasma membrane (Fig. 9, B and C, left-side tubules). Similar findings were observed with a Fas-L antibody raised against the N-terminus of the ligand (data not shown). Figure 9C (left-side tubule) shows a cross section of a seminiferous tubule at a stage similar to that of the control shown in Figure 9B (left-side tubule). This is from a testis of an animal treated with EDS for 8 days, and shows that the number of Fas-L-expressing germ cells had declined at this time point. In addition, the intensity of Fas-L staining had declined in the remaining Fas-L-positive germ cells. The right-side tubules in Figure 9, B and C, are of a different stage than the left-side tubules. Although Fas-L is visible on the spermatid cell membranes in these tubules, staining appears to be more localized in the cytoplasm. This suggests that in addition to changes in Fas-L expression occurring after androgen withdrawal, the localization of Fas-L is also stage dependent. In control sections in which the primary antibody was replaced by normal goat serum, no background staining could be detected, independent of the physiological state of the testis (Fig. 9A).
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Fas-R was also present in a broad range of germ cell types including Sertoli cells, spermatocytes, and spermatids (Fig. 10A). However, it was difficult to detect Fas-R immunoreactivity in mature spermatozoa. There was no obvious decline in Fas-R staining intensity between control sections and testis sections from animals treated with EDS for 8 days (Fig. 10B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time-dependent increase in germ cell apoptosis in the seminiferous epithelium as a result of EDS administration is consistent with previous studies [4, 7, 8, 10, 12, 13]. It has been well established that apoptosis occurs in the normal testis and is associated predominantly with spermatogonia [1, 6]. On the other hand, in response to androgen withdrawal as a result of hypophysectomy [2], administration of GnRH antagonists [4, 8, 9], EDS [12, 13], or anti-LH antibodies [10], the cell types that have been shown to undergo apoptosis are pachytene spermatocytes and spermatids.
However, the molecular events governing apoptosis in the testis in response to androgen ablation have not yet been described. We have investigated the potential role of genes known to be important regulators of programmed cell death in a variety of systems in the regulation of apoptosis of the spermatogenic epithelium in response to androgen withdrawal. We chose to look at the expression of these apoptosis-related gene products 2, 5, and 8 days after EDS administration. The apoptotic index is almost twice that seen in the control group at Day 5 after EDS administration and 24-fold higher at Day 8 after EDS. Consequently, 5 and 8 days after EDS administration represent time points in which we might expect to detect changes in the levels of these proteins.
Clusterin is expressed constitutively in the Sertoli cells [41]. We did not find significant changes in clusterin expression in the seminiferous tubules after androgen withdrawal, although a previous study [42] reported that clusterin mRNA levels are transiently elevated in the testis 6 h after EDS administration. At this time point, testicular testosterone levels are still normal; thus this increase may not be directly related to local changes in androgen production but rather the result of a direct effect of EDS on Sertoli cells. Clusterin expression returned to basal levels and was unchanged until 21 days after EDS administration in the previous study, and we would suggest that levels are probably not directly influenced by androgen levels. We (this study) and others have shown that clusterin is not present in the normal prostate but—surprisingly and in comparison to observations in the testis—that it is induced during regression of the prostate after androgen ablation [43, 44]. In addition, the expression of clusterin is elevated during apoptosis of the rat testis in response to the cytotoxic compound 2-methoxy acetic acid [45].
Studies with knockout and transgenic mice suggest that the Bcl-2 family members are important regulators of apoptosis in the testis. Bax-knockout mice are infertile as a result of the accumulation of premeiotic germ cells and loss of more mature germ cells by apoptosis [24], and adult transgenic mice expressing high levels of Bcl-xl or Bcl-2 show highly abnormal spermatogenesis accompanied by infertility [26]. After androgen withdrawal there were no statistically significant changes in the levels of Bcl-xl, Bak, or Bad. This would seem to suggest that these family members are not involved in apoptosis of the germ cells in response to androgen withdrawal. However, if the decline in Bcl-xl levels was important, it could play a role, as it is a survival gene product that might be expected to be suppressed during apoptosis. On the basis of the immunolocalization of Bcl-xl in spermatocytes and spermatids [22], one would expect a decrease in Bcl-xl levels after androgen withdrawal, since these cell types specifically undergo apoptosis under these conditions. Apparently the reduction in spermatocyte and spermatid numbers is not enough to give rise to a significant decrease in Bcl-xl levels.
A rise in Bcl-2 protein levels could be explained by an increase in Bcl-2 production by the remaining mature germ cells. Perhaps physiologically, Bcl-2 is up-regulated in these cells as part of a survival mechanism to ensure that the atrophied tissue can respond to restimulation by androgen. Since Bax is a pro-apoptotic gene, it is possible that Bax is induced or that its production is increased as a result of the induction of apoptosis, thus explaining its up-regulation after androgen withdrawal. Another possibility is that a selective enrichment of Bax-expressing cell types occurs as a result of androgen withdrawal.
The most recently discovered members of the Bcl-2 family that are expressed in the testis include Mcl-1, Bcl-w, and Bok [46–48]. Both Mcl-1 and Bcl-w protect cells against apoptosis [49, 50], while Bok is involved in the induction of apoptosis [48]. We cannot exclude the possibility that these new Bcl-2 family members also play an important role in germ cell apoptosis, although Mcl-1 appears only to be expressed in the Leydig cells of the testis [46] and Bok dimerizes with Mcl-1 and not with Bcl-2, Bcl-xl, or Bcl-w [48]. Bcl-w may be important because transgenic mice in which the Bcl-w gene is missing are sterile owing to progressive testicular degeneration that is the result of loss of spermatocytes and spermatids by apoptosis [47].
Fas-L has been localized to the Sertoli cells [29–31]. Nevertheless, it has also been reported that Fas-L is expressed in germ cells [32], while it has also been shown that Fas-L expression is virtually absent in the both the Sertoli cells and germ cells [33]. Using two different antibodies we observed that although Fas-L is expressed in the Sertoli cell cytoplasm, it is predominantly localized in the pachytene spermatocytes and spermatids. It is not very likely that the staining of Fas-L on spermatid cell membranes is associated with the Sertoli cell plasma membrane, since we found membrane staining for Fas-L only at the round to slightly elongating spermatid stage around these cells, not around spermatogonia or elongating spermatids in the same tubules. Nevertheless, we cannot completely exclude the possibility that Fas-L is also present on the Sertoli cell membrane. Fas-R was found in Sertoli cells, spermatocytes, and spermatids. This is in agreement with observations by other groups [29, 32, 33].
The expression of Fas-L declined considerably at 2 days post-EDS treatment and was barely detectable by 8 days. The initial drop in Fas-L expression is attributable to the loss of Leydig cells from the interstitial compartment because it is known that these cells express Fas-L [32, 33,51]. However, the significant further fall in expression between 2 days and 8 days after EDS treatment must be due to changes in the seminiferous epithelium, because Leydig cells do not regenerate until at least 14 days after EDS treatment [52]. This was confirmed by the immunohistochemical data showing that the number of germ cells expressing Fas-L and the intensity of Fas-L staining in the remaining germ cells were reduced by Day 8 after EDS in comparison to the control value. The Western data showed that Fas-R expression also declines at 8 days after EDS administration. As with Bcl-xl, it perhaps not surprising that the expression of Fas-L and Fas-R declines after androgen ablation, as the cell types that are lost during apoptosis are those expressing these genes. Nonetheless, as the colocalization of Fas-L and Fas-R correlates with the germ cell types that die after androgen withdrawal, the potential does exist for apoptosis in the rat spermatogenic epithelium to be regulated by the Fas pathway. If this was the case, there are several mechanisms whereby Fas-L can mediate apoptosis through binding to its receptor. Fas-L also exists in a soluble form that can move freely among cells and can induce apoptosis in an autocrine manner by binding to Fas-R present on its own cell surface or on adjacent cells by a paracrine mechanism [53]. At this point in time it is not known whether soluble Fas-L is present in seminiferous tubules. If Fas-L is not released from the surface of cells, it could bind to abutting cells that express the Fas-R and induce fratricide.
However, if the Fas pathway were important in apoptosis in the seminiferous epithelium of the testis after androgen withdrawal, one might expect to see an up-regulation in these gene products at a time before the majority of apoptosis occurs as has been observed in other situations in reproductive tissues in response to a variety of physiological and pharmacological conditions [29, 44, 51,54–56]. It is possible that Fas-L and Fas-R are up-regulated in the germ cells of the testis after androgen withdrawal just prior to maximal apoptosis. The fact that there was no discernible up-regulation in expression may reflect the fleeting nature of changes in the regulation of these genes that trigger the cascade of downstream events leading to programmed cell death. These transient and early changes in gene regulation may be detectable only with carefully timed sampling.
Fas-mutant mice are a useful tool for investigating apoptosis in the testis and have proved important in the investigation of apoptosis in cryptorchidism [29, 55, 56]. Although apoptosis is still observed in the cryptorchid testes of Fas-mutant mice [29, 55, 56], Fas may be involved, because the decline in testis weight following cryptorchidism of Fas-mutant mice is less than in controls [56]. Use of Fas-R- or Fas-L-mutant mice may provide further insight into the role of the Fas pathway in apoptosis in the testis in response to androgen withdrawal.
In conclusion, the induction of Bax after androgen withdrawal suggests that it may play a role in germ cell apoptosis. The concomitant elevation in Bcl-2 expression may represent a physiological survival mechanism to ensure that germ cell-depleted testis can respond to restimulation by androgen. In addition, because the colocalization of Fas-L and Fas-R correlates with the germ cell types that undergo apoptosis, it is possible that programmed cell death in the rat spermatogenic epithelium induced by androgen withdrawal could be regulated by the Fas pathway. This initial study would also suggest that the expression of Fas-L is stage dependent. Consequently, it is likely that stage-dependent changes in the expression of these apoptosis-related gene products occur after androgen withdrawal. Future studies should be aimed at addressing this issue.
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FOOTNOTES |
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2 Correspondence: Ian Woolveridge, G38 Stopford Building, Manchester University, Oxford Road, Manchester, M13 9PT, UK. FAX: 0161 275 5600; ian.woolveridge{at}man.ac.uk
Accepted: September 22, 1998.
Received: July 20, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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