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Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center (Y.-H.L. A.P.S.H., R.S.S., A.L., C.W.), Torrance, California 90509; and California Academy of Mathematics and Science (P.I., K.S.T., T.B.), Carson, California 90747
Address all correspondence and requests for reprints to: C. Wang, M.D., Clinical Study Center, Box 16, Harbor-University of California-Los Angeles Medical Center, 1000 West Carson Street, Torrance, California 90509. E-mail: wang{at}gcrc.humc.edu
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Introduction |
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We have previously demonstrated that stage-specific loss of germ cells after acute withdrawal of gonadotropins and intratesticular T by GnRH antagonist (GnRH-A) treatment occurs exclusively by apoptosis (13, 14). Stages VII–VIII, followed later by stages IX–XI, were the first to show enhanced germ cell apoptosis between 5–7 days after deprivation of gonadotropins and intratesticular T. The objectives of this study were 1) to examine whether the heat-induced loss of germ cells in the adult rat occurs by increased apoptosis, 2) to characterize the specific germ cell types that are most sensitive to mild testicular hyperthermia, 3) to analyze the temporal and stage-specific activation of germ cell apoptosis, and 4) to establish the possible role of intratesticular T in the selective susceptibility of germ cells to programmed cell death after heat exposure.
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Materials and Methods |
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Exp 1
To examine the involvement of apoptosis in the induction of germ
cell death after mild testicular hyperthermia and to analyze the
heat-induced temporal and stage-specific changes in the kinetics of
germ cell apoptosis, scrota of groups of five adult male SD rats were
exposed to 22 C (control) or 43 C for 15 min. Animals were then killed
on days 1, 2, 9, and 56 after heat exposure.
Exp 2
The objective of the second experiment (designed after the
results of the first experiments were known) was to determine whether
intratesticular T plays a pivotal role in protecting germ cells at the
androgen-dependent (VII–VIII) stages against heat-induced cell death.
Sixteen young adult male SD rats were randomly assigned to four groups
to receive one of the following treatments. Group 1 (control) was given
a daily sc injection of a vehicle containing 3.5% mannitol and 1%
benzyl alcohol in sterile distilled water for 4 days. Group 2 (heat
only) was exposed to local testicular heating (43 C for 15 min) on day
2. Group 3 (GnRH-A only) was given a daily sc injection of Nal-Glu
GnRH-A (1.25 mg/kg BW) for 4 days to suppress gonadotropin and
intratesticular T levels without evidence of stage-specific activation
of germ cell apoptosis (13, 14). Group 4 (heat plus GnRH-A) received
both daily sc injection of GnRH-A for 4 days and a single exposure to
heat applied on day 2. All of the animals were killed at the end of day
4.
Blood collection and tissue preparation
Both control and experimental animals were injected with heparin
(130 IU/100 µg BW, ip) 15 min before being killed by a lethal
injection of sodium pentobarbital (100 mg/kg BW ip) to facilitate
testicular perfusion using a whole body perfusion technique (15, 16).
Body weight was recorded at autopsy. Blood samples were collected from
the inferior vena cava of each animal immediately after death, and
plasma was separated and stored at -20 C for subsequent hormone
assays. Also before perfusion, one testis from each rat was removed,
weighed, and, after decapsulation, divided into three portions. One
such portion of testicular parenchyma was immediately snap-frozen in
liquid N2 and stored at -70 to -80 C for subsequent
analysis of DNA fragmentation. The second portion was used for
determining the number of advanced (steps 17–19) spermatids by the
homogenization technique (17). The remaining portion of testicular
parenchyma from each rat was kept frozen at -70 to -80 C until used
for testicular testosterone (T) assay. The contralateral testes were
then fixed by vascular perfusion with 5% glutaraldehyde in 0.05
M cacodylate buffer (pH 7.4) for 30 min, preceded by a
brief saline wash. The major advantage of glutaraldehyde fixation is
that it permits recognition of apoptotic germ cells with high
sensitivity and specificity while at the same time maintaining the
excellent morphological preservation needed to resolve stage-related
susceptibility of specific germ cells to programmed cell death. The
testes were removed, cut into small (
0.2 cm) transverse slices, and
placed into the same fixative overnight. One slice from the middle
region of the testis was processed for routine paraffin embedding for
in situ detection of apoptosis.
Hormone assays
The T concentrations in plasma and testicular homogenates were
measured by RIA, as reported previously (18). Testicular tissue was
homogenized in PBS (pH 7.4). All samples were then extracted with 10
vol of a mixture of ethyl acetate-hexane (3:2, vol/vol) before RIA. The
minimal detection limit in the assay was 0.25 ng/ml. The intra- and
interassay coefficients of variations were 8% and 11%, respectively.
Plasma FSH levels were measured by RIA, using reagents provided by the
NIDDK, as previously described (14, 18). Rat (r) FSH RP-2 reference
preparation and rFSH S-11 antiserum were used. The minimal detection
limit in the assay was 0.4 ng/ml. The intra- and interassay
coefficients of variations were 11% and 15%, respectively. Plasma LH
levels were measured by an immunoflurometric assay for rLH (19) using a
combination of monoclonal antibodies to human (Medix, Kauniainen,
Finland) and bovine LH (provided by Dr. J. F. Roser, University of
California-Davis), as described previously (14, 18). The minimal
detection limit in the assay was 0.02 ng/ml. The intra- and interassay
coefficients of variation were 6% and 8%, respectively.
Assessment of apoptosis
Our initial assessment of induction of apoptosis by heat
exposure was accomplished by detection of internucleosomal cleavage
using a DNA 3'-end labeling technique (14, 20). Briefly, genomic DNA
was isolated from frozen testicular tissue using an Easy-DNA-kit
(Invitrogen, San Diego, CA). The quality and purity of the
DNA preparations were estimated by measuring the optical density of
each sample at 260 vs. 280 nm; only samples with a ratio of
1.8 or higher were used. Aliquots of DNA (500 ng) from each sample were
processed for 3'-end labeling with [
-32P]dideoxy-CTP
(3000 Ci/mmol; ICN Pharmaceuticals, Inc., Costa Mesa, CA)
using 25 U terminal transferase enzyme (Boehringer Mannheim, Indianapolis, IN). The labeled DNA samples were
electrophoretically separated on a 2% agarose gel, and after drying,
the gel was exposed to Kodak X-Omat films (Eastman Kodak Co., Rochester, NY) at -70 C for 5–15 h.
To further validate the biochemical assessment of apoptosis and to resolve stage-related modulation of apoptosis involving specific germ cells, in situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) technique using an ApopTag-peroxidase kit (Oncor, Gaithersburg, MD). The choice of fixative was based on the results of our previous studies, which showed that glutaraldehyde fixation significantly improved both TUNEL specificity and sensitivity while maintaining excellent morphological preservation (21, 22). In brief, after deparaffinization and rehydration, tissue sections were incubated with proteinase K (20 µg/ml) for 15 min at room temperature, washed in distilled water, and then treated with 2% hydrogen peroxide in PBS for 5 min at room temperature to quench endogenous peroxidase activity. Sections were then incubated with a mixture containing digoxigenin-conjugated nucleotides and TdT in a humidified chamber at 37 C for 1 h and subsequently treated with antidigoxigenin-peroxidase for 30 min at room temperature. To detect immunoreactive cells, the sections were incubated with a mixture of 0.05% diaminobenzidine and 0.01% H2O2 for 6 min. Sections were counterstained with 0.5% methyl green, dehydrated in 100% butanol, cleared in xylene, and mounted with Permount (Fisher Scientific, Fairlawn, NJ).
Negative and positive controls were carried in every assay. As negative controls, tissue sections were processed in an identical manner, except that the TdT enzyme was replaced by the same volume of distilled water. Testicular sections from rats treated with GnRH-A for 7 or 14 days were used as positive controls (14).
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 Corp. BH-2 microscope (New Hyde Park, NY) with a x100 oil immersion objective. These stages were intentionally chosen not only to examine the whole seminiferous epithelial cycle but also to focus attention on those stages known to be classically dependent on hormones (23, 24). 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. (25) for paraffin sections. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per Sertoli cell (22, 26, 27).
Statistical analyses
Statistical analyses were performed using the SPSS Program for
windows 6.0 (Chicago, IL). Results were tested for statistical
significance using Duncan’s multiple range test after one-way ANOVA.
Differences were considered significant if P <
0.05.
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Results |
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. The mean body
weight did not show significant changes, except for an increase in
weight in the animals studied 56 days after heat exposure. A modest
decrease in testis weight was noted 2 days after heat exposure. By 9
days, the mean testis weight was reduced to 61.3% of the values
measured in controls. At 56 days, testis weight returned to values
similar to those in the control animals. Mean testicular sperm numbers
increased transiently 1 day after heat exposure, but were not different
from the control values thereafter.
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. No significant
changes were noted in serum T levels after heat exposure (Fig. 1a).
Intratesticular T levels were significantly decreased within 2 days
after heating and were within normal values by day 9. It should be
noted that the intratesticular T levels even at their nadirs were above
the threshold previously determined to be required to maintain
spermatogenesis (28, 29). Both plasma FSH and LH levels were elevated
on day 9 after mild testicular hyperthermia, and the differences were
significant compared with values in control and heat-treated groups on
other days (Fig. 1, c and d).
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).
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). Spontaneous apoptosis of germ cells
involving primarily A2-A4 spermatogonia and a
few spermatocytes late in meiosis was seen in control rats. In
contrast, mild testicular hyperthermia within 1 and 2 days resulted in
a marked increase in the incidence of germ cell apoptosis predominantly
at early (I–IV) and late (XII–XIV) stages, with minimum effects on
other stages, including the androgen-dependent (VII–VIII) stages (Fig. 3). By day 9, the majority of the tubules were severely damaged and
displayed only a few remaining apoptotic germ cells; presumably most of
the dead cells were eliminated through phagocytosis by the Sertoli
cells. Despite the marked loss of germ cells, elongated spermatids were
still present in the majority of seminiferous tubules at all time
points. By day 56, spermatogenesis was completely recovered, and the
incidence of germ cell apoptosis was compatible to the control level.
The effect of heat on spermatogenesis was not only stage specific, but
also cell specific (Fig. 4). P
spermatocytes and early spermatids at stages I–IV and P, diplotene,
and dividing spermatocytes at stages XII-XIV were most susceptible to
heat (shaded areas in Fig. 4); differentiating
spermatogonia, advanced spermatids (steps 17–19), and Sertoli cells
remained unaffected.
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. A low incidence (0.04–0.1) of
germ cell apoptosis [apoptotic index (AI), expressed as the number of
apoptotic germ cells per Sertoli cell] was detectable at stages I–IV,
IX–XI, and XII–XIV in control rats. Germ cell apoptosis was markedly
increased (P < 0.05) on days 1 and 2 after exposure to
heat, but not on day 9, compared with that in controls. On day 1, the
mean AI was highest (AI = 5.6–5.9) at early (I–IV) and late
(XII–XIV) stages and was lowest (AI = 0.3) at stages V–VI,
whereas stages VII–VIII were intermediate (AI = 2.1). On day 2,
AI remained highest at early (I–IV) and late (XII–XIV) stages, but
the lowest AI (AI = 1.1) was noted at androgen-dependent stages
VII–VIII. The increase in AI at stages V–VI on day 2 may reflect
progression of affected germ cells programmed for death from previous
stages (I–IV).
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) as well as the total content of
testicular T (Fig. 6B) and plasma FSH (Fig. 6C) levels were markedly
reduced in GnRH-A-treated and GnRH-A- plus heat-treated groups compared
with those in either the control or heat-treated group alone. Plasma LH
levels (Fig. 6D) were also decreased in GnRH-A-treated and GnRH-A- plus
heat-treated groups compared with those in either the control or
heat-treated group alone, but the differences were not statistically
significant due to the small sample size. No differences in these
hormone levels were apparent between GnRH-A-treated and GnRH-A- plus
heat-treated groups.
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, quantitative analysis of germ cell
apoptosis at various stages of the seminiferous epithelial cycle showed
no detectable changes in the incidence of germ cell apoptosis between
controls and rats treated with GnRH-A for 4 days. The selection of the
GnRH-A treatment duration of 4 days was based on the results of earlier
studies showing that the initiation of apoptosis after gonadotropin and
T deprivation does not begin until 5 days (13, 14, 30). Consistent with
the results of our first experiment, mild testicular hyperthermia
within 2 days resulted in a stage-specific activation of germ cell
apoptosis. Compared with controls or GnRH-A-treated rats (Fig. 8A), a marked increase in germ cell
apoptosis was noted in early (I–IV) and late (XII–XIV) stages, with
minimum effects on other stages, including the androgen-dependent
(VII–VIII) stages, after heat treatment (Figs. 7 and 8B). Although the
incidence of germ cell apoptosis at stages other than VII–VIII
remained unchanged between heat-treated and heat- plus GnRH-A-treated
groups, GnRH-A addition to heat resulted in a further increase in the
incidence of apoptosis at stages VII–VIII over the value measured in
the heat-treated group alone and was comparable to values at all other
stages (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that selective deprivation of gonadotropins and testicular T by GnRH-A treatment is followed by a stage-specific increase in germ cell apoptosis (13, 14). PL and P spermatocytes and round (steps 7 and 8) and elongated (step 19) spermatids were the first germ cells to undergo apoptosis detectable only 5 days after GnRH-A treatment by the same TUNEL technique. Thus, of the 14 stages in the seminiferous epithelial cycle, stages VII–VIII are the most sensitive to acute withdrawal of gonadotropins and intratesticular T. We also noted in those studies that within 2 days after GnRH-A treatment there is a dramatic decrease in T production, and this precedes the initiation of apoptosis in the germ cells. There is abundant evidence suggesting that stages VII–VIII of the rat spermatogenic cycle exhibit the highest levels of immunocytochemically detectable androgen receptor expression (34) and are considered to be androgen dependent (23, 24). These observations led us to believe that the earliest activation of germ cell apoptosis at stages VII–VIII is most likely attributable in large part to the loss of T production. Growing data from various experimental models further suggest that stages VII–VIII, to a lesser extent, are also FSH dependent (35, 36). Taken together, the general conclusion from the studies outlined above is that stages VII–VIII of the rat seminiferous epithelial cycle are mostly androgen-dependent stages.
It should be noted here that, unlike the GnRH-A-treated rat model, the
initiation of germ cell apoptosis after heat stress occurred mainly at
early (I–IV) and late (XII–XIV) stages (heat-sensitive stages) and
was not accompanied by a decrease in the circulating concentrations of
gonadotropins and T. Although intratesticular T levels were lower 2
days after heat treatment, they remained higher than the threshold
(13 ng/ml) shown to be required for the maintenance of
spermatogenesis (28, 29). These results led to the hypothesis that heat
induces germ cell apoptosis through different pathways than those
involved after gonadotropin deprivation, and intratesticular T plays an
important role in protecting germ cells at stages VII–VIII against
heat-induced apoptosis. If this hypothesis was correct, then there
would be a further increase in germ cell apoptosis also at the
androgen-dependent (VII–VIII) stages in addition to early (I–IV) and
late (XII–XIV) stages after testicular hyperthermia in rats whose
endogenous T levels were markedly suppressed by GnRH-A treatment.
Results obtained from the second experiment supported this hypothesis.
We showed that the incidence of apoptosis was low in both control rats
and animals treated with GnRH-A alone for 4 days. Consistent with the
results of our first experiment, mild testicular hyperthermia within 2
days resulted in a stage-specific activation of germ cell apoptosis in
early (I–IV) and late (XII–XIV) stages, with minimum effects at other
stages, including the androgen-dependent (VII–VIII) stages. Although
the incidence of germ cell apoptosis at stages other than VII–VIII was
not different between heat-treated and GnRH-A- plus heat-treated
groups, GnRH-A addition to heat resulted in a further increase in
apoptosis at stages VII–VIII over the values measured in group exposed
to heat alone, and this was comparable to apoptosis at all other
stages. Taken together, these results demonstrate that intratesticular
T and/or FSH (14, 35, 36) clearly play a pivotal role in protecting
germ cells at stages VII–VIII against heat-induced programmed germ
cell death. However, we have not excluded that various other factors,
including growth factors, thermoprotectants, cytokines, and various
proapoptotic and antiapoptotic proteins (37, 38), may also be involved.
These possibilities clearly merit further investigations.
The mechanisms by which testicular hyperthermia induces germ cell apoptosis are not known. It is likely that apoptosis will be controlled in a cell type-specific fashion, but the basic elements of the death machinery may be universal. A distinct genetic pathway is apparently shared by all multicellular organisms. The Bcl-2 family of proteins, which contains both proapoptotic (such as Bax) and antiapoptotic (such as Bcl-2) family members, constitutes a central checkpoint within this pathway (39, 40, 41, 42). Bcl-2 and Bax have also been implicated as potential modulators of germ cell apoptosis (43, 44, 45). The present study does not provide data on the molecular mechanisms by which local testicular heating induces germ cell apoptosis. Additional studies are ongoing to elucidate the roles of Bax, Bcl-2, and other intratesticular factors in heat-induced germ cell apoptosis.
In summary, this study has demonstrated that single exposure of the adult rat testis to heat results in selective, but reversible, damage to the seminiferous epithelium. P spermatocytes and early spermatids at stages I–IV and P, diplotene, and dividing spematocytes at stages XII–XIV are the most vulnerable (by undergoing apoptosis) to heat stress. Intratesticular T plays a pivotal role in protecting germ cells at the androgen-dependent (VII–VIII) stages against heat-induced programmed germ cell death. Furthermore, these results suggest that physical insult, such as mild heat, can induce germ cell apoptosis, possibly through pathways different from those involved in the activation of apoptosis by gonadotropin deprivation. The concept of additive or synergistic effects of two distinct stimuli in inducing germ cell apoptosis at different stages and cells in the spermatogenic process, possibly through different mechanisms, may have potential application for male contraceptive development.
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Acknowledgments |
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Footnotes |
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Received September 18, 1998.
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L. Somwaru, S. Li, L. Doglio, E. Goldberg, and B. R. Zirkin Heat-Induced Apoptosis of Mouse Meiotic Cells Is Suppressed by Ectopic Expression of Testis-Specific Calpastatin J Androl, July 1, 2004; 25(4): 506 - 513. [Abstract] [Full Text] [PDF]
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G. Martin, O. Sabido, P. Durand, and R. Levy Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm Biol Reprod, July 1, 2004; 71(1): 28 - 37. [Abstract] [Full Text] [PDF]
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Y. Vera, M. Diaz-Romero, S. Rodriguez, Y. Lue, C. Wang, R. S. Swerdloff, and A. P. Sinha Hikim Mitochondria-Dependent Pathway Is Involved in Heat-Induced Male Germ Cell Death: Lessons from Mutant Mice Biol Reprod, May 1, 2004; 70(5): 1534 - 1540. [Abstract] [Full Text] [PDF]
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H. Izu, S. Inouye, M. Fujimoto, K. Shiraishi, K. Naito, and A. Nakai Heat Shock Transcription Factor 1 Is Involved in Quality-Control Mechanisms in Male Germ Cells Biol Reprod, January 1, 2004; 70(1): 18 - 24. [Abstract] [Full Text] [PDF]
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E. Carlsen, A.-M. Andersson, J. H. Petersen, and N. E. Skakkebaek History of febrile illness and variation in semen quality Hum. Reprod., October 1, 2003; 18(10): 2089 - 2092. [Abstract] [Full Text] [PDF]
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A. P. S. Hikim, Y. Lue, C. M. Yamamoto, Y. Vera, S. Rodriguez, P. H. Yen, K. Soeng, C. Wang, and R. S. Swerdloff Key Apoptotic Pathways for Heat-Induced Programmed Germ Cell Death in the Testis Endocrinology, July 1, 2003; 144(7): 3167 - 3175. [Abstract] [Full Text] [PDF]
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R. A. Anderson and D. T. Baird Male Contraception Endocr. Rev., December 1, 2002; 23(6): 735 - 762. [Abstract] [Full Text] [PDF]
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Y.-H. Lue, B. L. Lasley, L. S. Laughlin, R. S. Swerdloff, A. P. S. Hikim, A. Leung, J. W. Overstreet, and C. Wang Mild Testicular Hyperthermia Induces Profound Transitional Spermatogenic Suppression Through Increased Germ Cell Apoptosis in Adult Cynomolgus Monkeys (Macaca fascicularis)( J Androl, November 1, 2002; 23(6): 799 - 805. [Abstract] [Full Text] [PDF]
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J. Tesarik, F. Martinez, L. Rienzi, M. Iacobelli, F. Ubaldi, C. Mendoza, and E. Greco In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium Hum. Reprod., July 1, 2002; 17(7): 1811 - 1819. [Abstract] [Full Text] [PDF]
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D. J. McLean, L. D. Russell, and M. D. Griswold Biological Activity and Enrichment of Spermatogonial Stem Cells in Vitamin A-Deficient and Hyperthermia-Exposed Testes from Mice Based on Colonization Following Germ Cell Transplantation Biol Reprod, May 1, 2002; 66(5): 1374 - 1379. [Abstract] [Full Text]
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R. W. Bailey, B. Aronow, J. A.K. Harmony, and M. D. Griswold Heat Shock-Initiated Apoptosis Is Accelerated and Removal of Damaged Cells Is Delayed in the Testis of Clusterin/ApoJ Knock-Out Mice Biol Reprod, April 1, 2002; 66(4): 1042 - 1053. [Abstract] [Full Text] [PDF]
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 J. S. Tash, D. C. Johnson, and G. C. Enders Long-term (6-wk) hindlimb suspension inhibits spermatogenesis in adult male rats J Appl Physiol, March 1, 2002; 92(3): 1191 - 1198. [Abstract] [Full Text] [PDF]
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M. Anzar, L. He, M. M. Buhr, T. G. Kroetsch, and K. P. Pauls Sperm Apoptosis in Fresh and Cryopreserved Bull Semen Detected by Flow Cytometry and Its Relationship with Fertility Biol Reprod, February 1, 2002; 66(2): 354 - 360. [Abstract] [Full Text] [PDF]
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J. C. Rockett, F. L. Mapp, J. B. Garges, J. C. Luft, C. Mori, and D. J. Dix Effects of Hyperthermia on Spermatogenesis, Apoptosis, Gene Expression, and Fertility in Adult Male Mice Biol Reprod, July 1, 2001; 65(1): 229 - 239. [Abstract] [Full Text] [PDF]
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Y. Lue, P. N. Rao, A. P. Sinha Hikim, M. Im, W. A. Salameh, P. H. Yen, C. Wang, and R. S. Swerdloff XXY Male Mice: An Experimental Model for Klinefelter Syndrome Endocrinology, April 1, 2001; 142(4): 1461 - 1470. [Abstract] [Full Text]
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C. M. Yamamoto, A. P. Sinha Hikim, P. N. Huynh, B. Shapiro, Y. Lue, W. A. Salameh, C. Wang, and R. S. Swerdloff Redistribution of Bax Is an Early Step in an Apoptotic Pathway Leading to Germ Cell Death in Rats, Triggered by Mild Testicular Hyperthermia Biol Reprod, December 1, 2000; 63(6): 1683 - 1690. [Abstract] [Full Text]
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Y. Lue, A. P. Sinha Hikim, C. Wang, M. Im, A. Leung, and R. S. Swerdloff Testicular Heat Exposure Enhances the Suppression of Spermatogenesis by Testosterone in Rats: The "Two-Hit" Approach to Male Contraceptive Development Endocrinology, April 1, 2000; 141(4): 1414 - 1424. [Abstract] [Full Text] [PDF]
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