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Regular Article |
a Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center, and Harbor-UCLA Research and Education Institute, Torrance, California 90502
ABSTRACT
Programmed cell death occurs spontaneously during spermatogenesis and can be induced in a cell- and stage-specific manner by mild testicular hyperthermia. Studies using transgenic mice suggest the involvement of Bcl-2 proteins in regulating germ cell apoptosis. To delineate further the pathways involved, we examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in rat testes after transient exposure to heat (43°C for 15 min). Germ cell apoptosis, involving exclusively early (I–IV) and late (XII–XIV) stages, was activated within 6 h. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization within 0.5 h of heating as assessed by immunocytochemical methods. In contrast, Bcl-2 is distributed both in the cytoplasm and nucleus in those cell types susceptible to heat-induced apoptosis. Despite the striking redistribution, Bax levels remained unchanged as determined by Western analysis; Bcl-2 levels increased significantly by 6 h after heat exposure. Reverse transcription-polymerase chain reaction analysis indicated no change in either Bax or Bcl-2 mRNA levels in response to heat, suggesting the involvement of post-transcriptional rather than transcriptional mechanisms mediating their activity. The marked subcellular redistribution of Bax prior to activation of apoptosis and the increase in Bcl-2 suggest an involvement of Bcl-2 family members in heat-induced apoptotic death of germ cells.
apoptosis, spermatogenesis, stress
INTRODUCTION
Spermatogenesis entails a complex series of events in which germ cells proceed through successive rounds of mitosis, meiosis, and cellular differentiation and become mature spermatozoa. Because Sertoli cells have limited germ cell supportive capacity, overproduction of early germ cells is balanced by selective apoptosis [1–3]. In the adult rat, A2–4 spermatogonia and spermatocytes during their meiotic divisions are the main cell types to undergo apoptosis [2, 3]. The occurrence of spontaneous germ cell apoptosis can be enhanced after various testicular injuries, including withdrawal of hormonal support [4–6], radiation [7], and toxicant [8, 9] and heat exposure [10].
We have previously shown that local testicular heating (43°C for 15 min) results, within 24 h, in a marked activation of germ cell apoptosis specifically at early (I–IV) and late (XII–XIV) stages with minimum effects on the hormone-dependent stages VII–VIII [10]. Pachytene (P) spermatocytes and early spermatids at stages I–IV and P, diplotene, and dividing spermatocytes at stages XII–XIV are most susceptible to heat. The damaging effect of heat is completely reversible [10]. Increased rate of germ cell apoptosis has also been noted in adult mice after experimental cryptorchidism [11, 12]. Despite the large numbers of studies examining the effects of heat and cryptorchidism on germ cell death in the testis, the underlying mechanisms of heat-induced cell type- and stage-specific activation of germ cell apoptosis are not clearly understood.
Key regulators of apoptosis in development, autoimmunity, and disease include proteins of the Bcl-2 family [13, 14] that contain both antiapoptotic (e.g., Bcl-2 and Bcl-XL) and proapoptotic (e.g., Bax) members. The ratio of these molecules has been implicated to be a critical determinant of cell fate such that elevated Bcl-2 favors extended survival of cells and increasing levels of Bax expression accelerate cell death [15]. Although the details of how this family of proteins functions remain unclear, their ability to neutralize by homo- and heterodimerization is thought to be one of the mechanisms involved [15–17]. Most Bcl-2 family proteins have a conserved C-terminal hydrophobic domain that is necessary for insertion into the mitochondrial, endoplasmic reticulum, and nuclear membranes. Based on the subcellular localization and homology to bacterial pore-forming toxins, some Bcl-2 proteins have been implicated to function as membrane pores or channels [18]. Post-translational processes such as phosphorylation [18], cleavage [19–21], and translocation [22] of these proteins can also modulate their functions.
Studies using knockout and transgenic mice also suggest that members of the Bcl-2 family play an important role during spermatogenesis. Mice lacking the proapoptotic protein Bax displayed accumulation of premeiotic germ cells and accelerated apoptosis of mature germ cells leading to complete cessation of sperm production [23]. In addition, transgenic mice, misexpressing Bcl-2 in spermatogonia, displayed an accumulation of these cell types before puberty but during adulthood and exhibited loss of germ cells in the majority of the tubules [24]. In both cases, abnormal spermatogenesis was accompanied by infertility. Whether these proteins play a role in heat-induced germ cell apoptosis, however, remains to be determined. In this study, we examine the role of Bcl-2 and Bax in heat-induced programmed germ cell death.
MATERIALS AND METHODS
Animals and Experimental Protocol
Adult (90-day-old) male Sprague-Dawley (SD) rats (350–375 g) purchased from Charles River Laboratories, Inc. (Wilmington, MA), were housed in a standard animal facility under controlled temperature (22°C) and photoperiod (12L:12D) with food and water ad libitum. Heating of the scrota of the adult rats was performed as described previously [10]. Briefly, after rats were anesthetized with an i.p. injection of sodium pentobarbitol (40 mg/kg body weight [BW]), their scrota (n = 8–10 per group) were immersed in a thermostatically controlled water bath at 43°C (treated) for 15 min and sacrificed at 0.5, 2, 6, and 24 h after heat exposure. Control rats (n = 8) were immersed in a water bath maintained at 22°C and sacrificed at 24 h after exposure. Animal handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-University of California, Los Angeles Research and Education Institute animal care and use review committee.
Blood Collection and Tissue Preparation
Both control and experimental animals were injected with heparin (130 IU/100 g BW, i.p.) 15 min before a lethal injection of sodium pentobarbitol (100 mg/kg BW, i.p.) to facilitate testicular perfusion using a whole-body perfusion technique [25]. Blood samples were collected from each animal by cardiac puncture immediately after death, and plasma was separated and stored at -20°C for subsequent hormone assays. After perfusion with saline, one testis was removed, decapsulated, weighed, snap frozen in liquid N2, and stored at -70°C for subsequent analysis. The contralateral testes were then fixed by vascular perfusion (four to five testes per group) with either 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) or Bouin solution (Sigma Diagnostics, St. Louis, MO). The testes were removed and processed for routine paraffin embedding for either in situ detection of apoptosis or immunohistochemistry.
Assessment of Apoptosis
In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the TUNEL technique [5, 10, 26] using an Apop Tag-peroxidase kit (Oncor, Gaithersburg, MD).
Enumeration of the viable Sertoli nuclei with distinct nucleoli and apoptotic germ cell population was carried out at stages I–IV, V–VI, VII–VIII, IX–XI, and XII–XIV using an Olympus BH-2 microscope (New Hyde Park, NY) with a 100x oil immersion objective. For each rat, at least 10 tubules per stage group were used. These stages were identified according to the criteria proposed by Russell et al. [27] for paraffin sections. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per Sertoli cell [5, 10, 28].
Immunohistochemical Analyses
Bouin-fixed, paraffin-embedded testicular sections were deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 2% H2O2 to quench endogenous peroxidases. Sections were blocked with 5% normal goat serum for 20 min to suppress nonspecific binding of IgG and subsequently incubated with a 1:400 dilution of Bax or 1:300 dilution of Bcl-2 affinity-purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horse radish peroxidase complex visualized with diaminobenzidine tetrahydrochloride (DAB) as per the manufacturer's instructions (rabbit Unitect Immunohistochemistry Detection System; Oncogene Science, Inc., Uniondale, NY). Slides were counterstained with hematoxylin. Bax- and Bcl-2-positive cells/Sertoli number were determined by morphologic assessment using the Olympus BH-2 light microscope. Negative (achieved by either deletion of primary antibody or peptide preadsorption) and positive controls were run in every assay. Bouin-fixed, paraffin-embedded sections of wild-type mouse spleen and small intestine were used as positive controls for Bax and Bcl-2, respectively. Testicular sections from Bax -/- mice (kindly provided by Dr. Michael Knudson, University of Iowa) were used as an additional control for Bax immunospecificity.
Western Blot Analyses
Frozen testes were thawed in 1.5 ml ice-cold RIPA buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 1% SDS in PBS, protease inhibitors: Complete Mini Protease Inhibitor Cocktail; Roche Molecular Biochemicals, Indianapolis, IN) per gram of tissue. After tissue was homogenized using a dounce homogenizer, 0.3 mg PMSF (Sigma, St. Louis, MO) per gram of tissue was added to the homogenate and further incubated on ice for 30 min. Lysates were centrifuged at 10 000 x g for 10 min at 4°C. Protein concentration of the extracts was determined by Bradford assay (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA).
For Western blot analysis, 200 µg of protein was resolved on a 12% SDS polyacrylamide gel at 160 V in a Mini-Protean II Cell (Bio-Rad). Equal loading was examined by running a separate gel in parallel and staining with Coomassie blue. Proteins were transferred to 0.45 µm nitrocellulose membranes in transfer buffer (25 mM Tris-base, 190 mM glycine, 20% methanol) at 100 V for 1 h in the cold. Efficiency of transfer was determined using Ponceau S (Sigma). Membranes were blocked in 5% nonfat dried milk in TTBS (0.9% NaCl, 0.1% Tween 20, 100 mM Tris-HCl, pH 7.5) and then incubated in primary antibody (1:200 Bax, 1:200 Bcl-2 [Santa Cruz Biotechnology]; 1:1000 ß-actin monoclonal antibody [Sigma,]) for 1 h at room temperature. Following 3x 10-min washes in TTBS, membranes were incubated with horse radish peroxidase-conjugated donkey antirabbit (Amersham Life Science Inc., Arlington Heights, IL) or goat antimouse (Bio-Rad) secondary antibodies at a 1:2000 dilution. For immunodetection, membranes were incubated with ECL Western blotting detecting reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and exposed to Fuji x-ray film (Fuji Medical Systems, Inc., Stamford, CT). Band intensities were determined using Quantity One software from Bio-Rad. To confirm the specificity of the Bax antibody, protein extracts of spleen from wild-type and Bax -/- mice were used as positive and negative controls, respectively. For Bcl-2, Jurkat cell lysates with or without primary antibody were used as controls.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from frozen tissues using the RNeasy midi kit (Qiagen, Inc., Valencia, CA). Total RNA (1 µg) was resolved on a 1% agarose/1x TAE electrophoresis gel to confirm quality and quantity determination. First-strand cDNA was synthesized from 2.5 µg total RNA using Superscript II reverse transcriptase (Gibco Life Technologies) and random primer. One microliter of reverse transcription (RT) reaction was used as a template for polymerase chain reaction (PCR) amplification. The PCR products corresponding to nucleotides 547–843 of rat Bcl-2 and nucleotides 58–354 of rat Bax were generated using the following primers: Bcl-2, 5'-AAAGTCGACTACCGTCGCGA>>CTTTGCAGAGATG-3' and 5'-AAAGAATTCCATGCTGGGGCCATATAGTTCCACA-3'; Bax, 5'-AAAGTCGACATGAAGACAGGGGCCTTTTTGTTAC-3', and 5'-AAAGAATTCGCTAGCAAAGTAGAAGAGGGCAACC-3'.
For quantitative analysis, 18S rRNA, as an internal control, was coamplified with Bax or Bcl-2. The 18S rRNA primers and competimers (Ambion, Inc., Austin, TX) were used in a 3:7 ratio to amplify the control (18S) at a level similar to the amplicon of interest (Bax or Bcl-2). Conditions for coamplification were 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec in 1.5 mM MgCl2. The PCR products were collected at 32 cycles that was determined to be within linear range of detection.
Statistical Analyses
Statistical analyses were performed using the SigmaStat 2.0 program for Windows. TUNEL results were tested for statistical significance using Dunnett's pairwise multiple comparison test after Kruskal-Wallis one-way ANOVA. The Student t-test was used to determine statistical significance for Western blot and RT-PCR quantitative analyses. Differences were considered significant if P < 0.05.
RESULTS
Heat-Induced Stage-Specific Activation of Germ Cell Apoptosis
The occurrence of apoptosis in control and heat-treated rat testes was detected using a modified TUNEL technique. Spontaneous apoptosis of germ cells in control rats, in conjunction with previous observations, were seen primarily in type A spermatogonia and a few spermatocytes late in meiosis. In contrast, extensive numbers of TUNEL-positive cells can be observed exclusively in the early and late stages 6 h after mild testicular hyperthermia (Fig. 1). The effect of heat on spermatogenesis was not only stage-specific but also cell-type specific. Cell types most susceptible to heat included P spermatocytes and early spermatids at stages I–IV and P, diplotene, and dividing spermatocytes at stages XII–XIV.
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Temporal changes in germ cell apoptosis at various time intervals after transient heat exposure are summarized in Figure 2. The incidence of germ cell apoptosis (apoptotic index [AI], expressed as the number of apoptotic germ cells per Sertoli cell) was very low (0–0.23) in control rats. No increase in apoptosis was observed at 0.5 h of heat treatment; within 2 h after mild testicular hyperthermia, however, an increase in the incidence of germ cell apoptosis was observed exclusively at early (I–IV) and late (XII–XIV) stages. At 6 and 24 h after heat treatment, germ cell apoptosis was markedly increased (P < 0.05) at early and late stages compared to controls. Apoptotic index at stages VII–VIII was also increased by 24 h as compared to values in controls, but the difference was not statistically significant.
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Temporal Changes in the In Vivo Expression of Bax and Bcl-2 During Heat-Induced Germ Cell Apoptosis
Changes in the in vivo pattern of Bax and Bcl-2 expression during heat-induced germ cell apoptosis were examined by immunohistochemistry (Figs. 3 and 4). In the normal testis, both these proteins were localized in the cytoplasm of germ cells in a granular or punctate pattern suggestive of association with intracellular organelles. Low to moderate Bax staining was observed in all germ cells, including spermatogonia, primary and secondary spermatocytes, round and elongated spermatids. A redistribution of Bax from a cytoplasmic (Fig. 3C) to perinuclear localization (Fig. 3D) can be observed as early as 0.5 h after heating in those selective germ cells. Also, a small percentage of spermatocytes appeared to have Bax translocation from a cytoplasmic to nuclear localization. This redistribution continued to be observed up to 24 h after heating when maximum apoptotic germ cell death was observed (data not shown).
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In contrast to Bax, Bcl-2 immunoreactivity was observed mainly in mid (VII–VIII) and late (IX–XII) pachytenes, diplotene (XIII), and secondary spermatocytes (XIV) (Fig. 4). Bcl-2 and Bax also localized to Sertoli and Leydig cells. Changes in Bcl-2 expression patterns occurred concomitant with the increased appearance of apoptotic germ cells (Fig. 4, C–H). While no apparent changes in Bcl-2 expression were noted in the nonsusceptible stages (V–VI, VII–VIII, and IX–XI), its levels were markedly increased, within 2 h of transient heat exposure, in P spermatocytes and spermatids at stages I–IV and the P, diplotene, and dividing spermatocytes at stages XII–XIV (Fig. 4, C–H). In situ analysis, within this time frame, further revealed Bcl-2 immunoreactivity in both the cytoplasm and nucleus. The observed increased expression was transient and decreased at 24 h after heat treatment (data not shown).
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Upregulation of Bcl-2 but Not Bax Following Heat Treatment
Using testicular lysates, the levels of Bax and Bcl-2 protein before and after heat treatment were examined by Western blot analysis (Fig. 5). Despite the striking redistribution that was observed by immunohistochemical analysis, Bax protein levels in total testis lysates were unchanged after heat treatment. In contrast, Bcl-2 levels began to increase by 2 h, attained statistical significance (P < 0.05) at 6 h, and decreased to pretreatment levels by 24 h after heating (Fig. 5B). These data coincide with the transient increase in Bcl-2 observed by immunohistochemistry.
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Semiquantitative RT-PCR Analysis of Bax and Bcl-2 Following Heat Stress
To determine if Bax and Bcl-2 transcription were affected by testicular heat stress, semiquantitative RT-PCR analysis with 18S and gene-specific primers was used. Results are summarized in Figure 6. Increase in the Bcl-2 protein levels, observed by 6 h based on immunohistochemical and Western blot analysis, was not accompanied by an increase in Bcl-2 amplified product compared to controls. Bax mRNA levels also were not modulated by heat stress. This was confirmed by Northern blot analysis (data not shown).
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DISCUSSION
The Bcl-2 family of proteins that contains both proapoptotic (such as Bax) and antiapoptotic (such as Bcl-2) members constitute a critical, intracellular check point that determines a cell's susceptibility to apoptosis [13, 14]. Bcl-2 and Bax have also been implicated as potential modulators of germ cell apoptosis [23, 24]. Previously, we have demonstrated that a single, transient testicular hyperthermia (43°C for 15 min) induces, as early as 24 h, stage- and cell-specific activation of germ cell apoptosis [10]. Spermatocytes, including P at stages I–IV and XII, diplotene, and dividing spermatocytes at stages XIII–XIV, and early (steps 1–4) spermatids were most susceptible to heat. Quantitative information obtained in the present study further confirms and extends those previous findings by demonstrating the same stage-related susceptibility of specific germ cells as early as 6 h of heating. The rapid induction of selective germ cell apoptosis after a single heat exposure thus provides an excellent in vivo model system for studying the underlying mechanism of programmed germ cell death in the testis. In the present study, using this model system, we examined the possible involvement of Bax and Bcl-2 in heat-induced germ cell apoptosis.
Immunocytochemistry revealed that Bax is localized in the cytoplasm of spermatogonia, spermatocytes, and spermatids during the normal seminiferous epithelial cycle. Initiation of germ cell apoptosis after heat stress was, however, preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization as early as 0.5 h after heating in those selective germ cells before their eventual apoptosis at later time intervals. As the mRNA and protein levels of Bax do not change during activation, it seems plausible that a post-translational activation of Bax occurs during apoptosis. Thus, although the cytosolic Bax expression per se is not lethal to germ cells, the redistribution of Bax may represent an important step in the pathway by which this proapoptotic protein induces cell death. Bax translocation from the cytosol to either close proximity of the nucleus or into the nucleus has been observed in various cell lines upon induction of apoptosis [29]. In addition, overexpression of the PML (promyelocytic leukemia) gene, has been shown to recruit Bax into nuclear PML-oncogenic domains (PODs) and subsequently induce rapid cell death independent of caspase-3 activation in rat embryo fibroblasts [30]. Movement of Bax and Bcl-XL from the cytosol into membranes has been observed in thymocytes induced to undergo apoptosis by dexamethasone treatment [31]. Specifically, the insertion of Bax into mitochondrial membranes has been shown to play an essential role in releasing cytochrome c from the mitochondrial intermembrane space to the cytosol [20, 32]. Together with other cytosolic factors, cytochrome c can subsequently trigger the activation of caspase-3, -6, and -7 through the autoproteolytic activation of caspase-9 [33]. This notion is further supported by another line of evidence showing that removal of a COOH-terminal hydrophobic domain (a key element in membrane docking) from a green fluorescent protein (GFP)-tagged Bax prevents Bax redistribution and abrogates its proapoptotic activity [16].
Bcl-2 displayed a diffuse cytoplasmic staining in germ cells, Sertoli, and Leydig cells, and was moderately abundant in late pachytene spermatocytes. While no apparent changes in Bcl-2 expression was noted by 0.5 h post-heat treatment, its levels, as assessed by immunocytochemistry, were markedly increased in the susceptible germ cells 2 h after heat treatment. Consistent with our findings, increased myocyte apoptosis has also been observed in the decompensated human heart in spite of enhanced expression of Bcl-2 [34]. The observed increase in Bcl-2 may indicate a proapoptotic role for Bcl-2 in heat-induced germ cell apoptosis. For example, recent in vitro studies have demonstrated that Bcl-2 is a substrate for a key executioner caspase such as caspase-3 that cleaves the C-terminal domain of the Bcl-2 during apoptosis and abolishes its antiapoptotic activity [21]. Furthermore, the Bcl-2 cleavage product has been reported to possess a proapoptotic activity similar to that of Bax and can trigger cell death through activation of downstream caspases [21]. Thus, a feedback loop may exist between Bcl-2 and caspases, and future studies will show whether cleavage of Bcl-2 plays a role in the susceptibility of germ cells to heat-induced apoptosis. Western blot analysis of Bcl-2 levels within this time frame fully corroborated the immunocytochemical data. However, no significant change in Bcl-2 mRNA levels was detected. Taken together, these findings suggest that upregulation of Bcl-2 expression during heat-induced germ cell apoptosis occurs possibly at post-transcriptional and/or post-translational levels.
In summary, we have provided evidence for the involvement of Bax and Bcl-2 in heat-induced germ cell apoptosis and hypothesize that redistribution of Bax may represent an important step in the pathway by which members of this family may regulate programmed germ cell death. However, the possible involvement of other Bcl-2 family members that have been identified in testis, including Bak [35], Bok [36], Mcl-1 [37], Bcl-x [38], and Bcl-w [39, 40] has yet to be determined. Mild testicular hyperthermia, thus, provides an in vivo model system to study the regulation of a form of stress-induced germ cell apoptosis and can be used to examine the possible protein-protein interactions within the Bcl-2 family as well as outside the family (death factors, death receptors, and caspases). Further understanding of the regulation of germ cell apoptosis may allow new targeted approaches to male contraceptive development and treatment of some forms of male infertility.
FOOTNOTES
First decision: 19 April 2000.
1 This study was supported by a National Institutes of Health training grant DK07571. 
2 Correspondence: Ronald S. Swerdloff, Department of Medicine, Harbor-UCLA Medical Center, 1000 W. Carson St., Torrance, CA 90502. FAX: 310 533 0627; swerdloff{at}gcrc.humc.edu
Accepted: July 25, 2000.
Received: March 2, 2000.
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