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a Division of Reproductive Biology, Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205
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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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The Fas system recently has been implicated as a possible key regulator of germ cell apoptosis in the rat testis [9]. Fas (APO-1, CD95) is a type I transmembrane receptor protein that belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family [10, 11]; Fas ligand (FasL) is a type II transmembrane protein of the TNF family [10]. Fas/FasL interaction in vitro has been shown to trigger the death of cells expressing Fas [10]; binding of FasL to Fas activates the cytoplasmic death domain of Fas, which initiates a cascade of interleukin-1ß-converting enzyme family protease (caspase) activity [12]. The activated caspases cleave various cellular substrates, such as actin, fodrin, lamin, poly (ADP-ribose) polymerase, and DNA-dependent protein kinase, resulting ultimately in fragmentation of chromosomal DNA and subsequent formation of apoptotic bodies [12].
Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis [9]. Fas and FasL genes and their protein products have been shown to be up-regulated in rats exposed to Sertoli cell toxicants that induce apoptotic germ cell death [9]. This, together with the knowledge that testosterone withdrawal results in the apoptotic death of germ cells and that androgen receptor is localized to Sertoli cells [13], suggests the possibility that reduced levels of intratesticular testosterone may affect Sertoli cells in some manner that ultimately results in the activation of Fas, and thus in germ cell apoptosis. This hypothesis predicts that testicular Fas protein content would increase in response to reduced levels of intratesticular testosterone as part of a suicide pathway in germ cells.
To examine this possibility, ethane 1,2-dimethanesulfonate (EDS), an alkylating agent that selectively kills Leydig cells in adult rat testes [14], was administered to Sprague Dawley rats in order to reduce intratesticular testosterone levels [15]. The resulting apoptotic death of Leydig and germ cells was examined in situ and biochemically, and changes in Fas protein content in the testis were monitored by Western blot analysis. We show here that EDS causes Leydig cell apoptosis, that subsequently there are increases in germ cell apoptosis resulting from reduced testosterone levels, and that germ cell apoptosis is temporally related to increases in testicular Fas content.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague Dawley rats (270–300 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Rats were housed in a climate-controlled (22°C) animal room with a constant 14L:10D cycle. Rat chow (Link Klein, Baltimore, MD) and water were provided ad libitum. Animal protocols were approved by the Animal Care and Use Committee of the Johns Hopkins School of Hygiene and Public Health.
Preparation of Testosterone Capsules
Testosterone-filled polydimethylsiloxane (Silastic) capsules totaling 24 cm in length (4 x 6-cm capsules) were prepared from Dow-Corning medical Silastic tubing (i.d., 1.98 mm; o.d., 3.18 mm; Dow-Corning, Midland, MI) and sealed with Silastic medical adhesive A (Dow-Corning) according to a previously described method [16]. The estimated release rate of the testosterone capsules used was 30 µg/day per centimeter [17].
Experiment 1: Does Fas Content in the Testis Correlate with Apoptotic Germ Cell Death After Testosterone Withdrawal by Administration of EDS?
Controls (n = 3) received a single i.p. injection of vehicle (dimethyl sulfoxide [DMSO]-water, 1:3 v:v) and were killed by decapitation 10 days after injection. Experimental rats (n = 3 per group) received a single i.p. injection of EDS (85 mg/kg BW) and were killed by decapitation at 1, 3, 6, or 12 h, or 1, 2, 3, 7, or 10 days post-EDS injection. From each rat, trunk blood was collected to assay serum testosterone, and both testes were weighed. Half of each testis was immersion-fixed in 4% paraformaldehyde for 2 days and then embedded in paraffin; 6-µm sections were cut for terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labeling (TUNEL) staining. The other half of each testis was snap-frozen in liquid nitrogen and stored frozen at-80°C for subsequent analyses of genomic DNA fragmentation and for Western blot analysis of Fas protein.
Experiment 2: Are the Changes in Apoptotic Germ Cell Death or Fas Content That Follow EDS Administration the Result of Testosterone Withdrawal or a Direct Effect of EDS?
Controls (n = 5) received empty Silastic capsules (24 cm) s.c. and, at the same time, injections of vehicle (DMSO-water, 1:3 v:v). Rats (n = 5) of one experimental group received testosterone-filled capsules totaling 24 cm in length and simultaneous injection of vehicle; rats of a second group (n = 3) received a single i.p. injection of EDS (85 mg/kg); and rats of a third group (n = 5) received 24T and, at the same time, an i.p. injection of EDS as above. All rats were killed by decapitation 7 days postimplantation/injection. Both testes from each rat were snap-frozen in liquid nitrogen and stored frozen at-80°C for subsequent biochemical analyses; one testis was used for analysis of genomic DNA fragmentation and the other for Western blot analysis.
Serum Testosterone Concentration
Trunk blood was allowed to clot for 2 h at room temperature; serum was then separated by centrifugation (3000 rpm at 4°C for 15 min) and stored frozen at-20°C until assayed. Testosterone concentration was determined by RIA [18]. The sensitivity of the assay was 10 pg/tube.
Analysis of Genomic DNA Fragmentation
Fragmented DNA, a hallmark of apoptosis, was detected as previously described [19]. In brief, frozen tissue samples (200–300 mg) were homogenized in lysis buffer (10 mM Tris-HCl, 10 mM EDTA, and 0.2% Triton X-100 [pH 7.5]). The homogenates were centrifuged at 14 000 rpm (10 min, 4°C), and supernatants were collected and digested with ribonuclease A (Boehringer Mannheim, Indianapolis, IN; 0.6 mg/ml, 30 min, 37°C). After addition of lysis buffer, phenol-chloroform-isoamyl alcohol (25:24:1), and chloroform-isoamyl alcohol (24:1), extractions were performed, and sodium acetate (3 M, pH 5.2) was added (1:10) to the aqueous phase. Nucleic acids were precipitated by adding isopropanol (1:1, -20°C, 1 h). The DNA pellet was washed in ethanol (70%), air-dried, dissolved in TE buffer (10 mM Tris-HCl and 1 mM EDTA [pH 7.5]), and stored frozen at-20°C until assayed. After spectrophotometric quantification of DNA concentration, the DNA samples and a 1-kilobase DNA ladder molecular weight marker (Gibco BRL, Gaithersburg, MD) were electrophoresed in a 1.5% agarose gel with TAE buffer (0.04 M Tris-acetate and 0.002 M EDTA) at 70 V for 4 h. DNA was visualized by ethidium bromide staining. Photographs were taken with Polapan 667 Polaroid film (Polaroid, Cambridge, MA). As previously described [20], DNA fragmentation was quantified by scanning the Polaroid film on a Microtek flatbed scanner (Microtek International, Inc., Taiwan, ROC) and analyzing the area of the image below the 1018-base pair (bp) molecular weight marker on a Power Macintosh 7500/100 (Apple, Cupertino, CA) using NIH Image 1.59 (public domain).
In Situ Localization of Fragmented DNA (TUNEL)
DNA fragmentation in individual cells was visualized by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA using the ApopTag peroxidase kit (S7100-kit; Oncor, Gaithersburg, MD) as previously described [5]. In brief, after deparaffinization and rehydration, tissue sections were digested with proteinase K (20 µg/ml, 15 min) and quenched with 1% hydrogen peroxide in PBS. Sections were then incubated in a humidified chamber in equilibration buffer (10 min, room temperature), terminal deoxynucleotidyl transferase (TdT) enzyme (30 min, 37°C), and anti-digoxigenin peroxidase (30 min, room temperature). 3,3'-Diaminobenzidine substrate solution (Vector Laboratories, Burlingame, CA) was used to stain the sections; 0.5% methyl green was used as a counterstain. Ventral prostate tissue, 3 days postcastration, was used as the positive control [21]; for the negative control, TdT enzyme was replaced by an equal volume of water.
The numbers of germ cells and Leydig cells per testis (n = 3 per group) labeled by the TUNEL method were determined after scoring stained tissue sections, as follows: TUNEL-stained germ cells and Leydig cells in 6-µm sections of whole testicular cross-sections were counted under a x40 objective by moving an ocular grid across entire sections in a non-overlapping fashion and counting cells within its boundaries. Typically, approximately 150 fields were counted per section. The average number of labeled cells per ocular grid was then determined. The numbers of TUNEL-stained germ cells and Leydig cells per testis were computed as follows: avg. no. stained cells/ocular grid volume x no. ocular grid volumes/testis = no. stained cells/testis. The ocular grid volume was defined by the dimensions of the ocular grid (253 µm x 253 µm) and the thickness of the section (6 µm). The number of ocular grid volumes per testis was calculated by dividing the volume of the testis (essentially the testis weight) by the ocular grid volume. It should be noted that this quantification method may not reflect actual numbers of cells, given that larger cells span more sections than smaller ones. The method is useful, however, for direct comparisons between groups, which was our objective.
Western Blot Analysis
Protein extraction and immunoblot analysis were performed as previously described [22]. In brief, frozen tissue samples were homogenized in lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 0.28 U/ml aprotinin, 50 µg/ml leupeptin, and 0.7 µg/ml pepstatin) and then centrifuged at 14 000 x g (20 min, 4°C). Protein concentrations were determined in supernatants by the Bio-Rad protein assay (Bio-Rad Laboratories). Laemmli buffer (double-strength: 100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, and 20% glycerol) was added (1:1) to the remaining supernatants, which were then boiled in a water bath (5 min). After the addition of 1% Bromophenol Blue (1:100), samples were stored frozen at-80°C until assayed.
Protein samples and Benchmark Prestained Protein Ladder molecular weight markers (Life Technologies, Gaithersburg, MD) were separated by SDS-PAGE using a 12% acrylamide gel under reducing conditions as described by Laemmli [23]. Electrophoretic transfer to Hybond nitrocellulose membrane (Amersham, Arlington Heights, IL) was performed according to Towbin et al. [24]. The membrane was blocked (1 h, room temperature) with 5% nonfat dry milk (Bio-Rad Laboratories) in TBS-Tween (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, and 0.01% Tween-20) and then incubated (1 h, room temperature) in anti-Fas antibody (sc-716; Santa Cruz Biotechnology, Santa Cruz, CA; 0.2 µg/ml). After being washed in TBS-Tween, the membrane was incubated (1 h, room temperature) in horseradish peroxidase-labeled secondary antibody (Amersham; 1:3000 dilution). Antibody binding sites were visualized on Hyperfilm using the ECL detection system (Amersham). Jurkat cells (clone E6–1; American Type Culture Collection, Rockville, MD), a human leukemia T-cell line, were used as a positive control for Fas. The Fas band was localized at a molecular mass of 37.6 kDa, which is consistent with the molecular weight of 36 000 calculated from the amino acid sequence of Fas [25]. Fas content was quantified by scanning the film on a Microtek flatbed scanner and analyzing the image on a Power Macintosh 7500/100 using NIH Image 1.59 (public domain).
Statistical Analysis
Data are expressed as the mean ± SEM. Results were analyzed on a Power Macintosh 7500/100 using StatView 4.51 (Abacus Concepts, Berkeley, CA). Statistical significance was determined by ANOVA. Post-hoc comparisons between treatment group means were made using Fisher's protected least-significant-difference test. Differences were considered significant if p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EDS administration resulted in DNA laddering patterns characteristic of apoptosis (Fig. 2). DNA laddering was observed at 1, 7, and 10 days post-EDS treatment (Fig. 2A). Quantification of the DNA laddering revealed that maximal DNA fragmentation, an 18-fold increase over the control level, occurred 1 day post-EDS treatment, with smaller (5.3-fold) increases occurring at 7 days and 10 days post-EDS treatment (Fig. 2B). Increases in the numbers of cells per testis stained by the TUNEL method after EDS administration were consistent with increases in DNA fragmentation (Fig. 3). At 12 h to 2 days post-EDS, the number of Leydig cells stained by TUNEL was significantly higher than control numbers, with an abrupt increase at 1 day post-EDS treatment (Fig. 3A). At 7 days and 10 days post-EDS, when no Leydig cell apoptosis was evident, the number of germ cells undergoing apoptotic death was significantly higher than control numbers (Fig. 3B).
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Figure 4A shows a Western blot of testicular Fas protein in testes from 0 h to 10 days post-EDS treatment, with protein from Jurkat cells as a positive control. Figure 4B shows quantification of Fas protein. There was no increase in Fas content at 1 day post-EDS, indicating that the Leydig cell apoptosis that occurs at that time (Fig. 3A) is independent of Fas. Thereafter, Fas content increased over time post-EDS treatment and was significantly higher (2.2- to 2.8-fold) than control levels from 2 to 10 days post-EDS treatment. The significant increases in Fas seen at Days 2 and 3 occurred before any decrease in testis weight was seen; mean testis weight in the controls was 1.5 ± 0.1 g, and testis weights at Days 2 and 3 post-EDS were 1.4 ± 0.04 and 1.4 ± 0.02 g, respectively. Significant decreases in testis weight were seen at Days 7 (1.3 ± 0.04 g) and 10 (1.3 ± 0.1 g).
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To determine whether the observed increases in testicular apoptotic cell death and in Fas content were the result of testosterone withdrawal, or rather, were a direct effect of EDS itself, rats received injections of EDS and simultaneously received testosterone-filled Silastic capsules 24 cm in total length. The testes from these rats were examined 7 days later. Controls received either empty capsules, EDS alone, or 24-cm testosterone capsules alone. The 24-cm testosterone-filled capsules were used to achieve intratesticular testosterone concentrations in vivo known to be high enough to quantitatively maintain spermatogenesis [26]. There was substantial testicular DNA fragmentation 7 days post-EDS (Fig. 2), seen consistently. In contrast, no DNA fragmentation was observed in rats that received 24-cm testosterone-containing capsules at the time of EDS injection, or in the groups that received empty capsules or testosterone-containing capsules alone (Fig. 5). Consistent with the results of DNA fragmentation analyses, Fas content was elevated 7 days after EDS treatment (Fig. 4), but not if testosterone was administered at the time of EDS injection (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Only after testosterone levels were depressed for 3–7 days were significant increases in apoptotic germ cell death observed. Testicular Fas content was significantly higher than control levels as early as 2 days post-EDS treatment and continued to rise until the tenth day. Thus, increases in Fas preceded germ cell apoptotic death, suggesting that germ cell Fas may have to reach a threshold level before apoptotic death occurs.
The conclusion that Fas may play a role in germ cell apoptosis following testosterone withdrawal is consistent with the results of previous studies demonstrating Fas-mediated apoptosis in the prostate and epididymis after testosterone withdrawal [27]. Observations of increased germ cell apoptosis following cryptorchidism in lpr mice lacking functional Fas [28, 29] are not consistent with a role for Fas, although it has been reported that testes of lpr mice express Fas, and that germ cell apoptosis and Fas up-regulation occur to the same extent after heat exposure in both control and lpr mice [9]. Our study provides further evidence of a possible relationship of Fas to germ cell apoptosis and offers insight into the possible regulation of testicular Fas by physiological factors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Barry R. Zirkin, Department of Biochemistry, Division of Reproductive Biology, The Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205. FAX: 410 614 2356; brzirkin{at}jhsph.edu
Accepted: February 16, 1999.
Received: November 3, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement. Endocrinology 1998; 139:3005–3113.This article has been cited by other articles:
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