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Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
Address all correspondence and requests for reprints to: Kim Boekelheide, M.D., Ph.D., Department of Pathology and Laboratory Medicine, Brown University, Box G-B518, Providence, Rhode Island 02912. E-mail: Kim-Boekelheide{at}Brown.edu
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Introduction |
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In the mammalian testis, germ cells clonally expand through many rounds of mitosis before undergoing the differentiation and maturation steps that result in spermatozoa. This clonal expansion is excessive, requiring that a mechanism exist to match the number of germ cells with the supportive capacity of Sertoli cells. Overproliferation of early germ cells is tempered by selective apoptosis of their progeny (1, 2, 3). Large numbers of spermatocytes undergo apoptosis in the testis of a 4-week-old rat, and spermatogonia become the main cell type undergoing apoptosis in the adult rat (2, 3). Testicular germ cell apoptosis occurs normally and continuously throughout life (1, 3). In addition, massive testicular germ cell loss is known to result from toxicant exposure (4, 5), depletion of growth factors (6), alterations of hormonal support (testosterone or pituitary hormones including FSH and LH) (7, 8), heat exposure (9), radiation (10), or treatment with chemotherapeutic compounds (10). In many of these situations, germ cells are known to undergo apoptosis (1, 3), indicating that a specific pathway is activated when the testicular environment cannot support spermatogenesis. However, the elements that control this process have not previously been identified.
Fas (APO-1, CD95) is a transmembrane receptor protein that belongs to the tumor necrosis factor/nerve growth factor receptor family (11, 12). It contains a "death domain" and is capable of initiating apoptosis when stimulated by receptor cross-linking or binding to its ligand, FasL (CD95L) (11). FasL is a tumor necrosis factor-related type II transmembrane protein (13). The FasL-Fas interaction triggers the death of cells expressing Fas, a process best studied in lymphoid cells (11). A variety of Fas mediators that are associated with its cytoplasmic domain have been identified by the two-hybrid system (14, 15, 16). Fas-associating protein with death domain (FADD)/MORT-1 and receptor interacting protein are positive effectors of Fas-mediated apoptosis (14, 15), and Fas-associated phophatase is suggested to be an inhibitor of Fas-mediated apoptosis (16). A downstream effector protein that binds to FADD is a protease of the interleukin-1ß-converting enzyme (ICE) family, named FLICE (FADD-like ICE)/MACH (17). Activation of ICE is required for Fas-mediated apoptotic cell death (18). These data identify a Fas-mediated cell death pathway with FADD linking Fas directly to an ICE-like enzyme.
The biological importance of the FasL-Fas interaction is underscored by abnormalities seen in several strains of mutant mice, in which the Fas system is not functional (11). lpr (lymphoproliferation) mice express little or no Fas protein due to insertion of a transposon in the intron 2 region of the Fas gene (19, 20, 21). gld (generalized lymphoproliferative disease) and lprcg (lpr complementing gld) mice have a point mutation in the FasL and Fas genes, respectively, resulting in nonfunctional proteins (11, 22). All of these mice suffer from dramatic lymphoproliferation and systemic autoimmunity due to altered lymphocyte apoptosis (11, 22).
The Fas system is involved in many areas of immune regulation, including the maintenance of peripheral T and B cell tolerance, cell-mediated cytotoxicity (CTL), and control of immune-privileged sites (11, 22, 23, 24, 25, 26). The tissue distribution of Fas messenger RNA (mRNA) is universal and particularly high in thymus and spleen as well as nonlymphoid tissues, such as liver, ovary, and lung (27). FasL expression, however, is generally more restricted to lymphoid organs (13, 27). Interestingly, FasL mRNA expression is abundant in rat and mouse testis, with a suggested localization to Sertoli cells (13, 27).
In testis, the Fas system has been implicated in maintaining immune privilege (24). By transplanting Sertoli cells lacking functional FasL across immunological barriers, Bellgrau et al. showed that FasL was critical to prevent immune rejection (24). This concept has been supported by experiments in which cotransplantation of myoblasts transfected with FasL prevented islet allograft rejection (28). According to this hypothesis, FasL-expressing Sertoli cells eliminate Fas-positive activated T cells, providing general protection against rejection in the testicular environment. However, Fas expression by germ cells suggests another role for the Fas system within the seminiferous epithelium. Here, we demonstrate that Fas signal transduction is responsible for germ cell apoptosis and that this pathway is a key regulatory system for spermatogenesis.
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Materials and Methods |
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Experimental protocol
Three different treatments were performed to study germ cell
apoptosis in testis. Mono-(2-ethylhexyl) phthalate (MEHP) treatment was
performed as described by Richburg et al. (5). Fischer rats
(28-day-old) received a single dose of MEHP (2 g/kg BW) in corn oil by
gavage. After 12 h, testes were removed and processed for frozen
sections and isolation of RNA. 2,5-Hexanedione (2,5-HD) treatment was
performed as described by Blanchard et al. (4). Adult
Fischer rats, weighing 150–175 g, were treated with 1% 2,5-HD
(Aldrich Chemical Co., Milwaukee, Wl) in the drinking water. At various
times after initiating exposure (0, 2, 4, and 5 weeks), rats were
killed to obtain testes for terminal deoxynucleotide
transferase-mediated deoxy-UTP nick end labeling (TUNEL) staining (29)
and isolation of RNA. For MEHP and 2,5-HD treatment, at least three
animals per time point were used. For the heat exposure experiment (9),
mice (30–38 days old) were anesthetized with sodium pentobarbital (40
mg/kg BW; Abbott Laboratories, North Chicago, IL), and their scrotal
testes were immersed in a water bath (44 ± 0.5 C) for 15 min. At
various times after immersion (0–12 h), mice were killed, and their
testes were removed.
FasL/Fas immunostaining and TUNEL
For immunostaining of FasL and Fas, 8-µm frozen cross-sections
from 28-day-old rat testis were prepared, fixed in acetone for 10 min
at -20 C, rinsed in PBS, and then incubated in 1%
H2O2 for 15 min to quench endogenous
peroxidases. Sections were blocked for endogenous biotin and then
blocked with 5% normal goat serum and 1% BSA in PBS
(PBS+). Polyclonal antibodies to FasL and Fas (sc-956 and
sc-716, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) were
added to the sections at 0.2 µg/ml in PBS+ for 1 h
at room temperature. Primary antibody was detected using a
biotin-conjugated goat antirabbit IgG secondary antibody (Calbiochem,
San Diego, CA) and the ABC-Elite Kit (Vector Laboratories, Burlingame,
CA) with 0.05% diaminobenzidine substrate. Inhibitor peptides (2
µg/ml; Santa Cruz Biotechnology) were used to verify the specific
staining of FasL or Fas. For TUNEL staining, the standard protocol for
frozen sections was followed (ApopTag, Oncor, Gaithersburg, MD). To
quantitate the incidence of apoptosis at each time point, the number of
TUNEL-positive cells within a seminiferous tubule cross-section was
counted, and the data were represented as the percentage of
seminiferous tubules containing more than three apoptotic cells of the
total number of seminiferous tubules counted in a cross-section.
Because, in control rat testis, the percentage of tubules with more
than 3 TUNEL-positive cells is less than 5%, an increase in apoptosis
is easily determined using this counting approach. In the heat
treatment experiment, a different cut-off (>4 TUNEL-positive
cells/seminiferous tubule cross-section) was applied due to the
somewhat higher incidence of apoptosis in control mouse testis. For the
MEHP and 2,5-HD experiments, about 300–400 essentially round tubules
were counted/time point.
Antisense oligonucleotide treatment in vitro
Sertoli germ cell cocultures were prepared from 21-day-old rat
testes, as previously described (30). Cells (1.5 x
106 cells/35-mm plate) were plated in DMEM mixed 1:1 with
Ham’s F-12 medium (Life Technologies, Gaithersburg, MD) plus 1 ng/ml
epidermal growth factor, 10 µg/ml ITS+ premix (containing
insulin, transferrin, selenious acid, BSA, and linoleic acid;
Collaborative Research, Bedford, MA), and 50 µg/ml gentamicin
(DMEM/F12+). Cocultures were incubated at 32 C on laminin
(Life Technologies)-coated plates (3 µg/ml). This procedure yielded a
monolayer of Sertoli cells attached to the plates and germ cells, most
of which were located on top of Sertoli cells. Taking advantage of the
distinctive appearance of germ cells by phase contrast microscopy, germ
cells were easily counted. Phosphorothioate oligonucleotides
(Eppendorf, Madison, WI) were added (10 µg/ml) 30 h after
isolation. For quantitative reverse transcription-PCR (RT-PCR), total
RNA was isolated from the cells 18 h after the addition of
oligonucleotides. The FasL antisense oligonucleotide
(5'-GGTAATTCACGGGCTGCTGCAT-3') was complementary to the translation
initiation site of the rat FasL gene. Controls included a FasL sense
oligonucleotide (5'-ATGCAGCAGCCCGTGAATTACC-3') complementary to the
FasL antisense oligonucleotide and a scrambled oligonucleotide
(5'-GCTGCATCGGGCTGGTAATTCA-3'), in which the antisense sequence was
conserved but rearranged. To quantitate survival, about 2000 germ cells
in 9 different areas/treatment group were counted by phase contrast
microscopy before the addition of oligonucleotides. Two days later,
germ cells in same areas were counted again, and the survival rate was
calculated.
Jo-2 antibody treatment in vitro
Sertoli germ cell cocultures were prepared from testes of
21-day-old mice, as previously described with minor modifications (30).
Cells (1.5 x 106 cells/35-mm plate) were initially
plated in DMEM/F12+. The cocultures were incubated at 32 C
on laminin-coated plates (3 µg/ml). Two days after incubation,
culture medium was collected from the plates, and the cells were washed
with DMEM/F12+. As the Sertoli cells remained attached to
the plates after washing, germ cell-enriched cultures resulted from
centrifugation and replating (1 x 105 cells/35-mm
plate) of the cells in the culture media and the washing solutions.
Jo-2 antibody (PharMingen, San Diego, CA) or hamster IgG antibody
(PharMingen) as a control were added to the culture medium (5 µg/ml),
and the viable cells were counted by trypan blue exclusion after 0, 3,
6, and 12 h For each time point, plates were prepared in
triplicate.
Quantitative RT-PCR
Total RNA was isolated from tissues using TRIreagent (Molecular
Research Center, Cincinnati, OH). First strand complementary DNA was
made using 1 µg total RNA in the presence of Superscript II reverse
transcriptase (Life Technologies) and random primer. One microliter of
reverse transcription reaction buffer was used as a template for the
subsequent PCR reaction. PCR products of rat FasL (238 bp), rat Fas
(969 bp), mouse Fas (314 bp), and ß-actin (389 bp) were amplified
using the following primers: rat FasL,
5'-GGAATGGGAAGACACATATGG-AACTGC-3' and
5'-CATATCTGGCCAGTAGTGCAGTAATTC-3'; rat Fas,
5'-CTGTGGATCATGGCTGTCCTGCCT-3' and 5'-CTCCAGAC-TTTGTCCTTCATTTTC-3';
mouse Fas, 5'-GAGAATTGCTGAAGAC-ATGACAATCC-3' and
5'-GTAGTTTTCACTCCAGACATTGTCC-3'; and ß-actin,
5'-AGGCATCCTGACCCTGAAGTAC-3' and 5'-TCT-TCATGAGGTAGTCTGTCAG-3'.
All PCR products were verified by restriction enzyme analysis. For quantitative analysis, ß-actin mRNA, as an internal control, was coamplified with FasL or Fas mRNA by using ß-actin primers (0.1–0.2 µM) and FasL/Fas primers (1 µM). PCR products were collected between 25–40 cycles, and the exponential increase in PCR products was confirmed. Conditions for coamplification of rat FasL and ß-actin were 94 C for 1 min, 65 C for 1 min, and 72 C for 1 min for 35 cycles in 1.0 mM MgCl2; conditions for coamplification of rat Fas and ß-actin were 94 C for 1 min, 65 C for 1 min, and 72 C for 1 min for 35 cycles in 1.5 mM MgCl2; conditions for coamplification of mouse Fas and ß-actin were 94 C for 1 min, 53 C for 1 min, and 72 C for 1 min for 35 cycles in 1.5 mM MgCl2.
Western blot analysis
Tissues were weighed and decapsulated into homogenation buffer
(10 mM Tris-HCl, 1 mM EDTA, 2.5% SDS, 1
mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, and 1
µg/ml pepstatin). After being homogenized and boiled at 95 C for 5
min, samples were centrifuged at 10,000 x g for 2
h. Supernatants were collected, and the protein concentration was
determined by Bradford assay. Proteins (0.5–1 µg/µl) were loaded
into SDS-PAGE gel (Phast Gel, Pharmacia, Uppsala, Sweden) and
electrophoresed, then transferred to nitrocellulose. Two anti-Fas
antibodies (sc-716 and sc-1024; Santa Cruz Biotechnology; 0.2 µg/ml)
were used. For detection of primary antibody, an ECL detection Kit
(Amersham, Aylesbury, UK) was used. Crude lysates from CTLL-2, a mouse
cytotoxic T cell line, were used as a positive control.
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) gave a basal and spoke-like pattern,
characteristic of localization to Sertoli cells, whereas Fas staining
(Fig. 1C) was limited to germ cells, mostly spermatocytes. Rat FasL and
Fas proteins were also detected by Western blots using crude lysates of
testis (data not shown). TUNEL analysis was performed to detect
programmed cell death in situ (Fig. 1E). As with Fas
immunohistochemistry, the TUNEL technique (Fig. 1, C and E) stained
only spermatocytes with a similar incidence. To further explore this
association, we modulated the frequency of apoptosis by toxicant
treatment. Rats were treated with MEHP, a widely studied Sertoli cell
toxicant known to increase germ cell apoptosis within 12 h of
exposure (5, 31). After injury, FasL expression became more pronounced
in areas of Sertoli cell cytoplasm adjacent to spermatocytes (Fig. 1B),
and the number of spermatocytes expressing Fas markedly increased (Fig. 1D). This up-regulated expression of FasL and Fas correlated with
increased apoptosis detected by TUNEL analysis (Fig. 1F) and nuclear
DNA fragmentation (data not shown).
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). After exposure, the number of TUNEL-positive
cells was dramatically increased. Quantitative RT-PCR was performed on
total RNA from control and MEHP-treated testes to evaluate the
expression level of FasL and Fas mRNA. The induction of FasL and Fas in
the MEHP-exposed testis was evident compared with that in controls
(Fig. 2B). 2,5-HD was used as another model of toxicant-induced
testicular injury. 2,5-HD exposure in the rat produces a well
characterized sequence of Sertoli cell dysfunction leading to death of
the dependent germ cell population by apoptosis (32, 33, 34). The incidence
of germ cell apoptosis was quantitated by counting TUNEL-positive cells
(Fig. 2C). By 2 weeks after the start of exposure, the incidence of
germ cell apoptosis started to increase and peaked at 5 weeks. Using
quantitative RT-PCR, FasL and Fas mRNA expression were shown to be
dramatically induced by 2,5-HD exposure (Fig. 2D), and the levels of
expression corresponded with the increased incidence of apoptosis.
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). Cocultures of rat Sertoli
cells and germ cells contain approximately 15% germ cells, most of
which die within 1 week (36, 37). Many germ cells in cocultures were
stained by the TUNEL technique (data not shown); therefore, this
coculture system may be useful for the study of germ cell apoptosis.
Oligonucleotides were added to Sertoli-germ cell cocultures, and the
level of FasL mRNA was determined after 18 h. Only antisense
oligonucleotide-treated cultures showed a marked decrease in FasL mRNA
(Fig. 3B), as determined by quantitative RT-PCR. After 2 days of
antisense oligonucleotide exposure, germ cell survival in cocultures
was increased by about 40% compared with that in untreated controls or
cocultures treated with scrambled or sense oligonucleotides (Fig. 3A).
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20–30%) incubated with Jo-2 antibody (5 µg/ml)
survived compared to nontreated cells or control IgG antibody-treated
cells (Fig. 4).
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), suggesting that germ cells from
control and lpr mice behave similarly after heat exposure.
Then, the expression of Fas after heat exposure was determined by
examining both mRNA levels (Fig. 5B) and protein levels (Fig. 5C).
Surprisingly, similar expression of Fas was detected in testes from
control and lpr mice. Furthermore, Fas expression was
dramatically up-regulated in both after heat exposure.
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). Primers were designed to amplify the
3'-region to the transposon insertion locus in the Fas gene (22).
Consistent with previous data (21), high expression of Fas mRNA in
liver and thymus from control mice was observed, with little or no
expression in their lpr counterparts. Fas mRNA was also
detected in brain from control, but not from lpr, mice.
Again, testes showed similar expression of Fas mRNA in control and
lpr mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell type-specific expression of FasL in Sertoli cells and of Fas in germ cells implies the presence of a paracrine mechanism similar to that of Fas-dependent cytotoxicity, in which cytotoxic T cell lymphocytes expressing FasL kill Fas-positive infected cells (23). As FasL staining in MEHP-exposed testis became intense and diffuse in areas of Sertoli cell cytoplasm surrounding spermatocytes, and the number of Fas-positive spermatocytes markedly increased after MEHP exposure, FasL may directly interact with Fas and induce apoptosis. The function of FasL as an initiator of germ cell death was evaluated in two ways: 1) transient blocking of FasL by antisense oligonucleotides, and 2) mimicking FasL using an agonist of Fas, Jo-2 antibody. The fact that germ cell apoptosis in vitro could be modulated by interrupting or potentiating the Fas-mediated pathway argues that the Fas system is a key regulator of germ cell apoptosis, at least in vitro.
lpr and gld mice show apparently normal spermatogenesis and are fertile (38), although spermatogenesis in these mice has not been thoroughly studied. However, our in vitro results suggest that disruption of FasL or Fas in vivo should result in hyperplasia of early germ cells or abnormal spermatogenesis due to inadequate germ cell apoptosis. There are three possible explanations for this discrepancy: 1) the Fas system is not necessary for initiating apoptosis during spermatogenesis; 2) an alternative pathway is activated in the mutant mice, which assumes the Fas function of apoptosis initiation; or 3) a salvage pathway is activated in the mutant mice that restores the Fas system. There are many examples of knock-out mice in which an essential gene is knocked out, but apparently normal phenotypes are seen (39, 40). It is generally assumed that an alternative pathway(s) takes over the function of the knocked out gene in these circumstances. The apparently normal spermatogenesis in gld mice and Fas knock-out mice (41) may be explained in this context. Although the third possibility initially seemed unlikely, our data support activation of a salvage pathway that "rescues" Fas expression in the lpr testis. The incidence of testicular germ cell apoptosis in control and lpr mice was similar, and after heat exposure, up-regulation of Fas, corresponding to the increased incidence of apoptosis, was similar in both control and lpr mice. These data indicate that the lpr testis is not Fas deficient, suggesting that a salvage mechanism(s) restores Fas expression.
The transposon element inserted into intron 2 of the Fas gene in the lpr mouse results in premature transcript termination or a larger chimeric transcript that incorporates the transposon (19, 20), both of which are nonfunctional. In thymus and liver from lpr mice, functional Fas mRNA and protein were expressed at less than 10% of the normal level (21). However, our data show normal expression of testicular Fas in lpr mice. Two anti-Fas antibodies used in our experiments were raised against peptides corresponding to amino acids 295–314 (for sc-1024) or 308–327 (for sc-716) mapping at the carboxyl-terminus of mouse Fas. These antibodies detected the same size of proteins in both control and lpr testes, excluding the possibility that the Fas protein seen in lpr testes is the product of an abnormal transcript. Among several organs tested, including liver, brain, and thymus, testis was the only organ that expressed Fas at the same level in control and lpr mice.
The unique testicular expression of Fas may be explained by the fact that the lpr mouse is a spontaneous mutant with a leaky mutation. As such, compensatory mechanisms unique to the testis may result in normal Fas expression. For example, overcrowding of germ cells in the seminiferous epithelium could result in insufficient Sertoli cell support. Germ cells may sense this inadequate environment and activate a self-elimination pathway, in which Fas is involved. In this situation, the transcriptional inefficiency of the Fas gene in lpr mice might be overcome, at least in testis, by repeated transcription of the Fas gene. If so, our results strongly argue for the importance of the Fas system in testicular homeostasis. Alternatively, the lpr mutation may have little or no influence on the expression of the Fas gene in lpr testis because of testis-specific mechanisms of transcriptional regulation. Many genes are known to be regulated in a germ cell-specific way (42). In this context, an unknown mechanism(s), developed by natural selection of a fertile lpr mutant, could result in normal expression of Fas in lpr testis. To fully assess these alternative explanations of normal Fas expression in the lpr testis will require an extensive series of experiments, including a determination of the relative ratios of different Fas transcripts, including premature transcript, chimeric transcript, and normal transcript seen in lpr mice. The ratio of premature transcript, chimeric transcript, and normal transcript seen in lpr mice may be tissue dependent, which may explain the unique Fas expression in lpr testis.
Cross-talk between Sertoli and germ cells via cell to cell contact or secretion of paracrine factors was supported by several observations (43, 44). Sertoli cells, the only supportive cells in the seminiferous epithelium, respond to and provide the hormonal and nutritional needs of germ cells. Therefore, alterations in Sertoli cell function may result in germ cell loss. The modes of action of MEHP and 2,5-HD are known to be different, but both selectively target the Sertoli cell (31). Thus, up-regulation of FasL in both models of toxicant-induced testicular injury suggests an endogenous activation of the Fas-mediated pathway, resulting from injured Sertoli cells within the seminiferous tubule. We hypothesize that in the normal state Sertoli cells express FasL and signal the killing of Fas-positive germ cells, limiting the size of the germ cell population to numbers they can support. After injury, Sertoli cells increase FasL expression to reach a new equilibrium state, which matches the reduced supportive capacity of the dysfunctional Sertoli cells with fewer germ cells. Up-regulation of Fas in germ cells is thus seen as a self-elimination process for cells that are destined to die because of inadequate support. The Fas-mediated pathway in the testis, in which active culling is initiated from Sertoli cells, may show a different aspect of paracrine control between Sertoli and germ cells, that is a negative selection process of germ cells as opposed to positive support.
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Acknowledgments |
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Footnotes |
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2 Burroughs Wellcome Fund Scholar in Toxicology.
Received September 30, 1996.
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