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Division of Pharmacology and Toxicology, University of Texas College of Pharmacy (J.H.R., A.N.), Austin, Texas 78712-1074; and Department of Pathology and Laboratory Medicine, Brown University (L.W., M.E., K.B.), Providence, Rhode Island 02912
Address all correspondence and requests for reprints to: John H. Richburg, Ph.D., Division of Pharmacology and Toxicology, University of Texas College of Pharmacy, Austin, Texas 78712-1074. E-mail: john_richburg{at}mail.utexas.edu
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Abstract |
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Introduction |
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The mechanisms controlling spontaneous physiological or toxicant-induced testicular germ cell apoptosis are currently the focus of many investigations (1, 4, 5, 7, 8, 13). Recent evidence indicates that the Fas/Apo-1/CD-95-mediated signaling system participates in the regulation germ cell apoptosis in rodent models (4, 14, 15) as well as in human testis (16). The Fas system is a receptor-ligand signaling system in which Fas ligand (FasL) binds to and activates the Fas receptor (Fas) to initiate a cascade of intracellular events that leads to the elimination of the Fas-bearing cells via apoptosis (for a recent review, see Ref. 17). In the rodent testis, FasL is found constituitively expressed on Sertoli cells, and Fas is localized on select germ cells (4). Experimentally induced inhibition of the expression of Sertoli cell FasL in rat Sertoli cell germ cell cocultures results in a significant reduction in the incidence of germ cell apoptosis that normally occurs with time in culture (4). Furthermore, addition of the Fas activating JO-2 antibody to mouse germ cells in vitro results in a significant increase in germ cell apoptosis (4). In addition to these rodent studies, it has recently been reported that both Fas and FasL are present in human testis, and disruption of their function leads to protection against germ cell apoptosis (16).
In the present study we use the gld (for generalized lymphoproliferative disease) mutant mice to evaluate the functional importance of the Fas system in the regulation of germ cell apoptosis in the testis. The gld mice have a point mutation in the intracellular C-terminus of FasL. This mutation abolishes the ability of FasL to bind Fas and initiate the apoptotic pathway within the cell (21, 22). These mice display diseases resulting from systemic autoimmunity and lymphadenopathy, indicating the importance of the Fas system in the function of the immune system (18, 20). In addition, these mice are fertile and display apparently normal spermatogenesis. One aim of the present study was to evaluate the spontaneous rate of germ cell apoptosis in gld mice to gain insights into the role of the Fas system in the regulation of spermatogenesis.
To further characterize the involvement of the Fas system in germ cell apoptosis, we examined the response of gld mice to two cell-specific toxicants. Exposure to mono-(2-ethylhexyl)phthalate (MEHP) specifically inhibits Sertoli cell function, leading to germ cell apoptosis (for reviews, see Refs. 23, 24, 25), and irradiation of testis specifically targets the differentiating germ cells (9). These two model systems have been shown to cause increases in both germ cell apoptosis as well as in the expression of either Fas or FasL (4, 7, 14, 15). The results of the present study implicate the Fas signaling system in the regulation of spontaneous germ cell apoptosis as well as increases in germ cell apoptosis after toxicant-induced Sertoli cell injury. However, the failure to see differences between gld and wild-type mice in the incidence of apoptosis after irradiation suggests that the Fas system participates in differentially triggering germ cell apoptosis after Sertoli cell vs. germ cell injury.
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Materials and Methods |
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MEHP exposure protocol
MEHP was purchased from TCI America (Portland, OR) and certified
to be more than 94% pure by gas chromatography. In these experiments,
prepubertal 28-day-old mice were used. Young rodents are more sensitive
to the effects of the MEHP (26, 27) and show a robust increase in germ
cell apoptosis after MEHP exposure (7). Mice received a single dose (1
g/kg) of MEHP in corn oil by gavage at a volume equal to 4 ml/kg.
Control mice received a similar volume of corn oil vehicle. The primary
cellular site of MEHP toxicity is the Sertoli cell, and this exposure
model has been extensively employed in the investigation of testicular
injury that results from the disruption of Sertoli cell function (for
review, see Ref. 23). Control and MEHP-treated mice were killed by
CO2 asphyxiation after 0, 3, 6, 12, and 24
h, and the testes were quickly removed. One testis was immersed in
Tissue-Tek OCT embedding medium (Miles Laboratories, Inc., Elkhart, IN)
and quickly frozen via submersion in liquid nitrogen and then stored at
-80 C until analyzed. The other testis was submerged and stored in
neutral buffered formalin after gently perforating the tunica with a
27.5-gauge needle.
Radiation exposure
Adult 8-week-old wild-type C57 or gld mice were used
for experiments. Mice were given half-body irradiation to a single dose
of 5 Gy without anesthesia at a dose rate of 98.5 rads/min using a
Philips 250kVp x-ray machine. Mice were restrained in polystyrene
chambers, and the upper body was shielded using lead. Mice were killed
by carbon dioxide asphyxiation 12 h after irradiation, and the
testes were excised. This treatment protocol has been previously been
shown to stimulate massive apoptosis of spermatogonia and spermatocyte
germ cells (9).
In situ terminal deoxxynucleotidyltransferase-mediated deoxy-UTP
nick end labeling assay (TUNEL) staining and quantitation
Germ cell apoptosis was detected in 8-µm cryosections of
fresh-frozen testis by the TUNEL labeling method using the ApopTag kit
(Intergen, Purchase, NY). Tissue was counterstained with
methyl green. Tissues sections were viewed using a Nikon
E800 microscope (Melville, NY) using differential interference contrast
microscopy. The images were captured with a Kodak DC120
digital camera equipped with a MDS120 adapter (Eastman Kodak Co., Rochester, NY) and processed using Adobe Photoshop 5.0
software (Adobe, San Jose CA). TUNEL-positive germ cells were
quantitated in each tissue section by counting the number of
TUNEL-positive cells in each essentially round seminiferous tubule. For
each testis section, approximately 100–200 tubules were counted from
each of three different mice. The incidence of apoptosis was then
categorized into either of two groups, defined as zero to three or more
than three TUNEL-positive germ cells per seminiferous tubule
cross-section. In the control mouse testis, the percentage of
seminiferous tubules with more than three TUNEL-positive cells is less
than 5%, so that an increase in apoptosis is easily determined using
this data presentation. The data, calculated as a percentage of the
total, are expressed as the mean ± SEM.
Testicular sperm head counts
Testicular sperm head numbers were assessed by the procedure of
Blazak et al. to evaluate the numbers of mature elongate
spermatids in the testis (28). Briefly, testes were homogenized in an
8-ml solution of 0.9% NaCl and 0.05% Triton X-100, and sperm heads
were counted using a hemocytometer. Each sample was counted four times
and averaged.
Histopathology
To examine the morphological appearance of the seminiferous
tubules after MEHP treatment, testes were fixed in 10% neutral
buffered formalin and embedded in glycol methacrylate using a
Historesin embedding kit (Reichert-Jung, Heidelberg, Germany). Sections
(2 µm) were stained with periodic acid-Schiff reagent and
hematoxylin.
Statistics
Significance between groups (P < 0.05) was
determined by single factor ANOVA with Fisher’s least significant
differences test comparison using StatView software (SAS Institute, Inc., Cary, NC).
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Results |
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). A significant
difference in both the body and testis weights of adult gld
mice was observed compared with that in wild-type controls. The
gld mice had an increase in both body and testis weights of
approximately 10%. Measurement of testicular sperm head counts in the
testis, a useful indicator of differences in the size of the germ cell
population in the testis (28), also revealed an approximately 10%
increase in the number of spermatid heads per testis in the
gld mice compared with wild-type animals.
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). Significant differences between the
gld and wild-type mice in the incidence of TUNEL-positive
germ cells were evident. The gld mice showed a significant
increase in the percentage of seminiferous tubules with greater than
three TUNEL-positive cells (7.24%) relative to wild-type mice
(3.29%).
MEHP-induced histopathology in wild-type and gld mice
Histopathology was evaluated in 2-µm cross sections of
plastic-embedded testis stained with periodic acid-Schiff reagent and
hematoxylin. The seminiferous epithelium appeared similar in untreated
wild-type and gld mice (Fig. 1, A and D, respectively). At 12 h
(Fig. 1, B and E) and continuing to 24 h (Fig. 1, C and F) after
MEHP exposure, a similar increase in the incidence of characteristic
features of MEHP-induced Sertoli cell injury (germ cell sloughing into
the lumen, enlarged lumen size, and increases in the incidence of
Sertoli cell vacuoles) was evident in both wild-type and gld
mice, respectively. However, at higher magnification, germ cells from
wild-type mice, but not gld mice, display the characteristic
apoptotic morphology of cell shrinkage and chromatin condensation after
MEHP exposure (data not shown, see Fig. 2).
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, A–D). This increase in
apoptosis resulted in a marked reduction in the number of cells evident
in the seminiferous epithelium by the 24 h point (Fig. 2D). In
gld mice, exposure to MEHP did not result in an increase in
the incidence of TUNEL-positive germ cells (Fig. 2, F–H). The testis
cross-sections from the gld mice 24 h after MEHP
exposure showed no apparent decline in the numbers of cells in the
seminiferous epithelium (Fig. 2H).
To quantitatively evaluate the amount of germ cell apoptosis above
baseline induced by MEHP treatment, the difference in the percentages
of seminiferous tubules displaying greater than three apoptotic cells
per seminiferous tubule were compared (Fig. 3). After MEHP treatment of wild-type
mice, a time-dependent increase in TUNEL-positive germ cells was
evident. A sharp increase in apoptosis occurred between 3–12 h after
MEHP exposure and leveled off by 24 h after exposure. In
MEHP-treated gld mice, only a small increase in
TUNEL-positive cells was observed (Fig. 3). A small increase in germ
cell apoptosis occurred between 3–12 h, although the peak incidence of
apoptosis at 12 h was approximately 50% less than that in the
wild-type mice.
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).
Approximately 50% of the seminiferous tubules in wild-type and
gld mice had more than three TUNEL-positive cells compared
with approximately 5% in untreated mice (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and D, respectively). These findings suggest that either the
mutant gld mice have adapted to the FasL-deficient system,
perhaps by using a parallel apoptosis regulatory pathway, or that the
Fas signaling system is not required for spermatogenesis.
The observations of this study that gld mice have
significantly increased testicular weights and testicular spermatid
heads per testis indicate that the numbers of germ cells in the
gld testis are maintained at a higher baseline level than in
wild-type animals. However, the low absolute expansion of the germ cell
population (
10%) suggests that the number of the germ cells in the
testis is tightly limited. The observation of an increased incidence of
baseline apoptosis in gld mice implies that apoptosis is
serving as a mechanism to modulate an expanded population of the germ
cells in these mice. These findings suggest that germ cell apoptosis in
the gld testis is triggered by an alternate mechanism or
that the Fas system in testis is only involved in triggering apoptosis
of a small subpopulation of germ cells in the testis.
The decreased sensitivity of gld mice to MEHP-induced germ cell apoptosis provides strong evidence that the Fas signaling system plays a direct role in initiating germ cell apoptosis after Sertoli cell injury. The Sertoli cells are widely recognized to maintain the viability of the germ cells by providing appropriate nutritional, hormonal, and physical support. Therefore, injury to Sertoli cells could compromise their supportive capacity. A potential explanation for the decreased sensitivity of germ cells to apoptosis after MEHP exposure could be that Sertoli cells of gld mice are insensitive to MEHP-induced injury. The similar MEHP-induced changes in testicular histopathology in wild-type and gld mice shown in this report argue that the Sertoli cells of the gld mice are not protected from MEHP-induced injury. We have previously reported increases in the expression of both Fas and FasL in young rats and mice after MEHP exposure (4, 14, 15). The protection of young gld mice against MEHP-induced apoptosis further argues for the participation of the Fas system in triggering germ cell apoptosis after Sertoli cell injury. These data also indicate that the injured Sertoli cells themselves, via the expression of FasL, are actively involved in eliminating germ cells. We hypothesize that the reduction in the number of germ cells via apoptosis occurs as a mechanism to match their numbers to the reduced supportive capacity of the injured Sertoli cells and preserve functional spermatogenesis by the remaining germ cells.
To further examine the role of the Fas system in germ cell apoptosis after toxicant-induced injury, we used a radiation exposure model that directly injures mitotically active spermatogonia and spermatocytes (9). Increases in Fas, but not FasL, messenger RNA (mRNA) in rats and mice have been observed 6 h after irradiation with 5 Gy (14), a time point just before the observation of large increases in apoptosis (14, 32). Although these data suggest the participation of Fas in radiation-induced germ cell apoptosis, no differences in the massive radiation-induced increases in germ cell apoptosis were observed between wild-type and gld mice. The experiments presented in this report exposed adult mice to radiation. However, preliminary experiments in our laboratory irradiating young gld mice show the same massive increase in germ cell apoptosis as that in wild-type mice (unpublished observations). Direct damage to germ cells elicited by radiation may activate an autocrine pathway within the germ cell that is Fas independent. The reason for the up-regulation of Fas mRNA after irradiation and the role that it may play in elimination of germ cells is uncertain. However, an increase in expression of Fas mRNA in germ cells occurs after exposure to a variety of toxicants whose primary targets are Sertoli cells or germ cells (14). The expression of Fas by the germ cells may ensure their elimination after various testicular injuries by the constitutive expression of FasL by the Sertoli cells. In the case of radiation exposure, the damage induced by 5 Gy may initiate a signaling pathway that overrides the effects of the Fas signaling system.
Although the participation of the Fas signaling system in the regulation of testicular germ cell apoptosis has been proposed by several investigators (4, 14, 30, 33), an understanding of the exact populations of cells that are regulated by this pathway is lacking. The subtypes of germ cells that undergo apoptosis after MEHP or radiation exposure are not the same. The germ cells that undergo apoptosis after MEHP exposure are primarily spermatocytes and early spermatids, whereas after irradiation the majority of cells undergoing apoptosis are differentiating spermatogonia. Therefore, it is possible that the Fas system plays a more predominant role in triggering apoptosis in certain subtypes of germ cells. A differential role of the Fas signaling system in various germ cell types may explain the protection of gld mice against germ cell apoptosis induced by MEHP, but not against that caused by radiation.
In summary, the present study demonstrates the following three novel observations: 1) testis of gld mice are larger, with more spermatid heads; 2) gld mice are insensitive to MEHP-induced germ cell apoptosis; and 3) germ cells of wild-type and gld mice are similarly sensitive to the radiation-induced increases apoptosis. The increase in testis weights and spermatid head counts in gld mice suggests the involvement of the Fas signaling system in triggering the spontaneous germ cell apoptosis associated with normal testicular homeostasis. The insensitivity of gld mice to MEHP-induced germ cell apoptosis further underscores the participation of the Fas system in the regulation of germ cell apoptosis after MEHP-induced Sertoli cell injury. Finally, our findings also reveal that the Fas system participates differentially in the cell-specific regulation of germ cell apoptosis that occurs as a consequence of Sertoli cell vs. germ cell injury.
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Acknowledgments |
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Footnotes |
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Received July 29, 1999.
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