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Department of Biochemistry, Division of Reproductive Biology, School of Public Health (K.A.Y., B.A.Z., R.J.N.), and the Departments of Psychology and Neuroscience (R.J.N.), The Johns Hopkins University, Baltimore, Maryland 21218
Address all correspondence and requests for reprints to: Kelly A. Young, Department of Biochemistry, Division of Reproductive Biology, Johns Hopkins University, Baltimore, Maryland 21205-2179. E-mail: kyoung{at}ren.psy.jhu.edu
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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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During gonadal regression, a dynamic relationship between cellular growth and development and apoptotic and necrotic cell death is hypothesized to shift toward cell death. However, the time course and regulation of cell death during natural gonadal atrophy in adult rodents remain unspecified. Degeneration of germinal epithelium in rats (Rattus norvegicus) in response to hypophysectomy, gonadotropin and GnRH antibody administration, severe temperature stress, and toxin exposure is associated with increased apoptotic cell death (9, 10, 11, 12, 13, 14). Apoptotic cell death has also been implicated as the mechanism of testicular regression in peripubertal Djungarian hamsters (Phodopus sungorus) in response to short day exposure (15).
Apoptosis is a well characterized, morphologically recognizable form of cellular death that is initiated by environmental or developmental cues and involves the activation of specific genes that execute a linear cascade of death-inducing events, including characteristic intranucleosomal DNA fragmentation (16, 17, 18, 19). Distinct from the unregulated, anomalous death pathways typical of necrotic cell death, genetically mediated apoptotic cell death removes expendable or potentially harmful cells without eruptive cell lysis. This "clean" form of cellular death enlists neighboring cells to engulf apoptotically dying cells, and, in contrast to necrotic cell death, generally does not result in activation of the immune system (16). The extent to which the seasonal gonadal regression in P. leucopus is the result of apoptotic processes is unknown.
During normal spermatogenesis cycles, germ cell populations are limited
via apoptotic mechanisms (20). Under some experimental conditions,
expression of the Fas system has been implicated in mediating germ cell
apoptosis (21). The Fas receptor (APO-1, CD95) is a type I membrane
protein (45 kDa) that can mediate apoptotic cell death in targeted
cells (22, 23, 24). When bound by the Fas ligand (FasL), the conserved Fas
cytoplasmic death domain motif can rapidly (
4 h in vitro)
initiate the apoptotic cascade (22, 25). Expression of Fas messenger
RNA is widespread; high levels of expression have been detected in the
thymus, liver, ovaries, lung, and testes (21, 26). In contrast, FasL
messenger RNA expression is limited. FasL has been detected in lymphoid
and testicular tissues, primarily in activated T cells and Sertoli
cells, respectively (21, 27, 28). In the seminiferous epithelium, the
Fas system has been implicated in both maintenance of testicular immune
privilege and transduction of the apoptotic signal after toxin-induced
injury (21). Activation of the Fas system has not been previously
examined in a natural, physiological model of germ cell loss.
In the present study, the extent of apoptosis in short day-induced testicular regression after short day exposure of 2, 4, 6, 8, or 10 weeks was investigated using both histochemical localization of terminal deoxynucleotide transferase-mediated deoxy-UTP nick end-labeled (TUNEL) cells in tissue sections and quantitative DNA 3'-end-labeling autoradiography (DNA laddering). The possible involvement of the Fas system in the mediation of photoperiod-induced seasonal testicular regression was assessed using Western blot analysis and immunohistochemistry to quantify and localize Fas protein expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental protocol
Testosterone assay. At the end of photoperiod exposure,
terminal blood samples were collected into iced heparinized tubes from
the retroorbital sinus of the mice under light methoxyflurane
anesthesia (Metofane, Schering-Plough Corp., Union, NJ),
and centrifuged for 30 min at 2500 rpm at 4 C. After separation, plasma
was stored at -80 C until plasma hormone values were determined in
duplicate by a single RIA using 125I kits (ICN Biomedicals, Inc., Costa Mesa, CA). The plasma dilution was
prepared according to the instructions provided by ICN, and the RIA has
been validated for use in Peromyscus in our laboratory. The
ICN testosterone assay is specific; cross-reactions with other steroids
were less than 0.1–7.8%.
TUNEL and Fas immunostaining. After collection of the terminal blood sample, the left testis was separated from the epididymis, weighed, snap-frozen in a bath of dry ice-ethanol, and stored at -80 C. Animals were then perfused through the heart with 50 ml 0.9% saline, followed by 150 ml of either Bouin’s solution (n = 4–5/group) or 10% neutral-buffered formalin (n = 4–5/group) as a fixative. After fixation, the right testis was removed, weighed, and postfixed in either Bouin’s solution or 10% neutral buffered formalin for 24 or 96 h, respectively. Tissue processed with Bouin’s solution was then dehydrated in a series of ethanol solutions (50%, 70%, and 100%); tissue processed with formalin was washed in PBS and dehydrated in 70% ethanol before paraffin embedding. Formalin-processed tissue provided better resolution of the TACS Blue label. No statistically significant differences in apoptotic staining were apparent between formalin- and Bouin’s solution-fixed tissues within any photoperiod group; the groups were pooled for further statistical analyses.
For TUNEL staining, 6-µm sections were collected every 50 µm of tissue and stained for apoptotic activity with a commercially available kit (Trevigen TACS 2TdT, Gaithersburg, MD). Of the sections labeled, six randomly chosen testis cross-sections were counted per animal. Cells that incorporated the labeled biotinylated nucleotides were considered TUNEL positive (apoptotic) and were counted under brightfield illumination (x40) on a Zeiss AxioPlan 2 microscope (Carl Zeiss, Thornwood, NY) using Stereoinvestigator software (Microbrightfield, Colchester, VT). Negative control sections, processed without terminal deoxynucleotidyltransferase, showed no staining. Trevigen control slides, positive for apoptosis, were processed with experimental slides and generated apoptotic signal. Apoptotic activity was quantified by counting the number of cells positive for TUNEL staining within each testis cross-section. To control for reduction of testis size, this value was expressed as the number of apoptotic cells per total number of seminiferous tubules within each testis cross-section.
For Fas immunohistochemistry, paraffin-embedded 6-µm sections were deparaffinized in xylene and hydrated through a graded series of ethanol solutions, and endogenous peroxidases then were quenched with a 5-min incubation in 3% H2O2. Sections were placed in a blocking buffer (1.5% normal goat serum in PBS) and incubated at room temperature for 18 h with a 1:800 dilution of polyclonal antibodies to Fas (200 µg/ml; sc-716, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in normal goat serum PBS buffer. Fas antibody was detected using a biotinylated antirabbit IgG secondary antibody (Vector Laboratories, Inc., Burlingame, CA) and the Vector ABC-Elite Kit (Vector Laboratories, Inc.) and was visualized with Sigma Chemical Co. Fast diaminobenzidine substrate (Sigma Chemical Co., St. Louis, MO).
DNA isolation and analysis. The left testis was removed from
animals after 2, 4, 8, or 10 weeks of photoperiodic treatment (n =
4–5/group), immediately frozen, and stored at -80 C before
processing. DNA was isolated from frozen testis tissue using the
Genzyme Corp. (Cambridge, MA) TACS Apoptotic DNA Laddering
Kit protocol with some variations. Briefly, 0.05–0.1 g pulverized
frozen testis tissue, suspended in sample buffer, was lysed using
Genzyme Corp. lysis buffer, lightly homogenized, and
extracted. Total DNA was quantified on a spectrophotometer by
absorbance at 260 nm. Aliquots of DNA (1 µg) were processed for
3'-end labeling with [
-32P]dideoxy-CTP (10 µCi/µl;
Amersham, Arlington Heights, IL) using 5 U/µl Klenow
enzyme. Labeled samples were separated on a 1.5% Trevigel, dried for
1.5 h on a slab gel dryer, and exposed to Hyperfilm-MP
(Amersham) at -70 C for 18 h. After autoradiography,
lanes were excised from the gel, and areas that corresponded to low mol
wt DNA fractions (<15 kb) were counted in a ß-scintillation counter
to determine the extent of apoptotic activity.
Western blot analysis. Fas activity was assessed in testes
collected after 2, 4, 8, or 10 weeks of exposure to the experimental
photoperiod (n = 4–5/group). The left testis was removed,
immediately frozen, and stored at -80 C before processing for protein
extraction. Minced tissue (0.1 g) was sonicated (twice, 5 sec each
time) in 2 vol buffer [1 M Tris-HCl, pH 7.4; 0.5
M EDTA, pH 8.0; and 10% SDS] containing proteolytic
inhibitors 4-(2-aminoethyl)-benzenesufonyl fluoride (AEBSF), 1
µg/ml leupeptin, and 1 µg/ml pepstatin. Samples were incubated at 0
C for 30 min and centrifuged for 30 min at 14,000 rpm. The supernatant
was extracted and boiled for 5 min at 95 C with an equal volume of
Laemmli buffer (containing 10% ß-mercaptoethanol), and protein
concentration determined by Bradford assay. Proteins were loaded 20
µl/lane at 25 µg/ml into SDS-PAGE gels (12% solution),
electrophoresed, and then transferred onto nitrocellulose (Hybond ECL,
Amersham, Aylesbury, UK). Benchmark prestained protein
ladder (Life Technologies, Gaithersburg, MD) was used to
determine transfer efficiency and to estimate protein size. After a 1-h
blocking period, blots were incubated for 1 h with anti-Fas
antibodies (sc-716, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) diluted to 1:1000. Primary antibodies were detected by an ECL
detection kit (Amersham, Aylesbury, UK). Crude lysates
from the Jurkat cell line were used as a positive control.
Statistical analysis
Parametric statistical evaluation of mean differences between
experimental groups was performed by ANOVA; mean differences in
nonparametric data were evaluated with a Kruskal-Wallis ANOVA on ranks
using the SigmaStat software package (Jandel Scientific, San Rafael,
CA). Dunn’s method was used to isolate significant differences between
groups. Mean differences were considered statistically significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Significant
reductions in testosterone concentrations occurred in short day animals
after 6, 8, or 10 weeks of exposure, decreasing 44.7%, 52.6%, and
57.6%, respectively, compared with those in long day animals (Table 1). Relative testis mass (milligrams per g body mass) increased with
time in animals housed in long days (Table 1) as did absolute testis
mass (data not shown). With 10 weeks of short day exposure, relative
testis mass decreased significantly, falling to 52.1% of the relative
testis mass in week 10, long day animals (Table 1). No changes in body
mass were observed among either short or long day animals with
continued photoperiod exposure (Table 1).
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, A and B), and no obvious change with 2
weeks of short days. In contrast, in males maintained in short
photoperiods for 4, 6, 8, or 10 weeks, there was an obvious increase in
apoptotic staining (Fig. 1, C and D). Compared to the seminiferous
tubules of the long day animals that showed normal, complete
spermatogenesis, testes of short day animals housed for 6, 8, or 10
weeks showed a reduction in number of spermatozoa, spermatids, and
spermatocytes. By week 10 of short photoperiod exposure, tubules were
reduced in size and generally contained only spermatogonia, Sertoli
cells, and spermatocytes, with few remaining spermatids (Fig. 1C).
Apoptotic staining was primarily limited to germ cells; little
endothelial staining was noted. Based on their position within the
seminiferous epithelium and the size of their nuclei, stained cells
were identified predominantly as spermatocytes.
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). In contrast, the number of stained
cells in cross-sections from animals exposed to 6, 8, and 10 weeks of
short days was significantly higher than that in animals exposed to
corresponding long day photoperiods (Fig. 2). Mice killed after 4 weeks
of short day exposure showed the first significant increase in
TUNEL-positive cells compared with the average long day value
(post-hoc analysis; P < 0.025; Fig. 2).
Compared with the average number of apoptotic cells in long day
animals, the number of apoptotic cells labeled in animals after 4, 6,
8, and 10 weeks of short days increased 2.00-, 3.26-, 3.21-, and
2.83-fold, respectively.
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). Quantification by ß-scintillation
counting of [-32P]dideoxy-ATP-labeled DNA
revealed no detectable differences in the degree of 3'-end labeling of
total testis DNA among animals exposed to 2, 4, 8, and 10 weeks of long
and 2 and 10 weeks of short days (Fig. 3B). In contrast, an increase in
low mol wt (
15-kb) DNA was seen in animals exposed to 4 and 8 weeks
of short days compared with that in animals exposed to long days (Fig. 3B), with increases of 52.2% and 94.3%, respectively, compared with
the average degree of low mol wt labeling for long day animals.
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).
Quantification of Fas protein expression revealed significant increases
in testes from mice housed in short days for 4, 8, and 10 weeks of
2.1-, 1.6-, and 1.8-fold, respectively (Fig. 4B). This up-regulation of
Fas protein in the testis was not seen among animals exposed to either
long photoperiods or with 2 weeks of short day exposure. Observation of
testis sections revealed that Fas staining was localized to cells whose
positions within the seminiferous tubules were consistent with those of
spermatocytes and spermatids (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western blot analysis revealed an up-regulation of Fas, an apoptotic death cascade initiator, at 4, 8, and 10 weeks of short day photoperiod exposure. Observations of immunohisotological staining for Fas suggested localization primarily to spermatocytes and spermatids, a pattern generally consistent with the observed spermatocyte localization of TUNEL-positive apoptotic labeling. We detected few or no examples of necrotic cell death in testis sections, and thus suggest that the apoptotic death of germ cells is an important mechanism underlying testicular regression after long term housing in short days.
Testis mass did not significantly decrease until 10 weeks of short day exposure, but the peak number of apoptotic cells per seminiferous tubule was observed in males housed for 6 or 8 weeks in short photoperiods. Indeed, the first significant increase in the number of apoptotic cells, as indicated by DNA laddering, was observed in testes of short day males as early as 4 weeks. This preceded significant reductions in plasma testosterone concentrations, which were not detected until week 6 of short day exposure. Thus, the initiation of testicular regression, mediated by apoptotic processes, appears to be independent of testosterone concentrations and probably reflects reduced FSH concentrations (15). Indeed, apoptotic DNA fragmentation is suppressed in hypophysectomized rats administered exogenous FSH, and injection of FSH antiserum results in apoptotic germ cell death within 24 h (14, 29). A reduction in the serum FSH concentration has been shown to be coincident with apoptotic DNA fragmentation in peripuburtal Djungarian hamsters exposed to inhibitory photoperiods (15). Peak apoptotic activity in short day male P. leucopus correlated with significant drops in plasma testosterone; a reduction of testosterone has been shown to enhance germ cell apoptosis (11, 12).
Photoperiod-responsive species such as white-footed mice (P. leucopus) undergo dramatic physiological changes, specifically in reproductive function, in response to short photoperiods. Apoptosis has been established as the form of cellular death in the testis after severe alterations in hormone concentrations, temperature, and exposure to toxins (9, 10, 11, 12, 13, 30). A significant increase in apoptotic low mol wt DNA has also been observed with short day exposure in peripuburtal Djungarian hamsters (15).
Taken together, the results of the present study support the hypothesis that the rate of apoptosis increases in the testes of white-footed mice housed in short days, and that evidence of apoptosis precedes detectable reductions in spermatogenesis and steroidogenesis. Our study is the first to examine multiple markers of apoptosis over the time course of photoperiod-induced adult testicular regression. Naturally occurring gonadal regression in P. leucopus provides an excellent animal model to study the cellular bases of and individual differences in seasonal changes in reproductive capability. Additional studies are necessary to determine whether vernal testicular recrudescence reflects the down-regulation of apoptotic activities in the gonads, or whether gonadal regression in response to other ambient factors, such as low temperature or reduced food availability, is mediated by testicular apoptosis or necrotic cell death.
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Acknowledgments |
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Footnotes |
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Received January 6, 1999.
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