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Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center and Research and Education Institute, Torrance, California 90509
Address all correspondence and requests for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology and Metabolism, Harbor-University of California Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: swerdloff{at}gcrc.humc.edu
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Introduction |
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In additional studies (3), we further confirmed and extended earlier studies (3A ) by demonstrating that a single exposure (43 C for 15 min) of the rat testis to heat resulted in selective, but reversible, damage to the seminiferous epithelium through increased germ cell apoptosis. Heat-induced germ cell apoptosis predominantly occurred at early (I–IV) and late (XII–XIV) stages. Spermatocytes, including P at stages I–IV and XII, diplotene and dividing spermatocytes at stages XIII–XIV, and early (steps 1–4) spermatids at stages I–IV, were most susceptible to heat. Stages V–VI and VII–VIII were relatively protected from heat-induced apoptosis. We have also provided evidence indicating that mild testicular hyperthermia is able to increase germ cell apoptosis at stages VII–VIII only when intratesticular T levels were decreased by the prior treatment with GnRH antagonist. We conclude from these studies that intratesticular T plays a pivotal role in protecting germ cells at stages VII–VIII against heat-induced cell death (3).
In clinical studies to develop male hormonal methods of contraception, T administration resulted in reversible suppression of spermatogenesis. Although quite effective, the suppression was not uniform, and azoospermia was achieved in 60–90% of men (4, 5, 6) only when serum T was elevated to the upper normal range. Moreover, the time required to achieve azoospermia or severe oligozoospermia usually takes more than 12 weeks. Although there are no data to support such a contention; concern has been expressed that high doses of T may have untoward effects on prostate. To develop new regimens that could rapidly induce development of azoospermia in all men with a lower dose of T, clinical studies were designed to combine T with progestins or GnRH analogs (7, 8, 9). Some of these combined regimens achieved azoospermia in over 90% of men in 8–12 weeks. Thus, even the combined T and progestogen regimens leave room for improvement to create a faster and more complete male contraceptive approach.
In our search for optimization of an experimental suppressor of spermatogenesis, we propose that a two-hit approach, with addition of heat treatment to exogenous hormone treatment such as T, will enhance the decrease in sperm output by inducing apoptosis at all stages of seminiferous epithelial cycle. When confirmed, the identification of the mechanisms by which hormonal and physical factors induce apoptosis at different stages involving different cell types will possibly allow the replacement of the specific heat- induced effects that target pharmacological agents. Such a combination would suppress sperm counts quickly and more completely.
The objectives of the present study were 1) to document the temporal and stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous T alone, and 2) to determine whether the combination of a single testicular heat exposure with administration of a low dose of T could enhance the effect of T alone in rapidly and effectively suppressing spermatogenesis to near-complete azoospermia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T and heat treatment
T SILASTIC implants, 3 cm in length, were prepared from
polydimethylsilozane tubing (od, 3.18 mm; id, 1.98 mm; Dow Corning Corp., Midland, MI), packed with T (Sigma,
St. Louis, MO), and sealed with SILASTIC medical adhesive A (Dow Corning Corp.) based on previously described methods (10, 11).
The release rate of T from the same type of implant was estimated to be
about 30 µg/cm·day (12). A 3-cm T-filled capsule was implanted
subdermally along the dorsal surface of each rat under pentobarbital
anesthesia and kept for different periods of time. Heating of the
scrota of the adult rats was performed using a procedure described
previously (3). Briefly, rats were anesthetized with an ip injection of
sodium pentobarbital (40 mg/kg BW) and then placed in a specially
constructed holder. The scrotum containing testes and the tail were
then immersed for 15 min in a thermostatically controlled water bath at
43 C. Animal handling and experimentation were in accordance with the
recommendation of the American Veterinary Medical Association and were
approved by the Harbor-University of California-Los Angeles Research
and Education Institute animal care and use review committee.
Study protocols
Exp 1 (T treatment alone). To document the temporal and
stage-specific changes in the kinetics of germ cell apoptosis induced
by exogenous administration of T, experimental animals (four or five in
each group) were implanted subdermally with a 3-cm T capsule for 1, 2,
3, and 6 weeks, and control animals were implanted with a 3-cm empty
capsule.
Exp 2 (T with or without concomitant heat application). In this experiment we examined whether the addition of a single testicular heat exposure to animals bearing T implant could result in more rapid and effective suppression of spermatogenesis than T alone. To establish the optimum time point of heat exposure, preliminary experiments were performed in which a single heat exposure was applied to the testis at 0, 1, 2, 3, and 4 weeks after insertion of the T implant. Animals were killed at end of 6 weeks. Based on preliminary data (see Results), subsequent experiments were performed where heat was applied 2 weeks after T implantation. Sixteen young adult male Sprague Dawley rats were randomly assigned to four groups to receive one of the following treatments. Group 1 (control) received a subdermal empty implant for 6 weeks. Group 2 (heat only) was exposed once to testicular heat of 43 C for 15 min, applied 2 weeks after empty capsule implantation. Group 3 (T only) was given a 3-cm T implant for 6 weeks. Group 4 (T+heat) received a subdermal T implant in combination with a single heat exposure applied 2 weeks later. All animals were killed at the end of 6 weeks.
Blood collection and tissue preparation
Both control and experimental animals were injected with heparin
(130 IU/100 g 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 (13, 14). 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 and weighed, and
after decapsulation, testicular parenchyma were used for determining
the number of advanced (step 17–19) spermatids by the homogenization
technique (15). In brief, testicular parenchyma were weighed and then
homogenized in the same volume (equivalent to testicular parenchyma
weight) of 0.01 M PBS (pH 7.4). An aliquot, after
appropriate dilution, was counted in a hemocytometer. Each square of
the hemocytometer with coverslip in place represents a total volume of
10-4 cm3. Results were
expressed as number of spermatids per ml or per g testis. The figure
obtained was then multiplied by the testis volume (equivalent of
testicular weight) to yield the number of spermatids per testis. The
remaining homogenized aliquots of testicular parenchyma from each rat
were kept frozen at -70 to -80 C until used for testicular 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 ventral prostates and seminal
vesicles were carefully dissected out and weighed. 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. The adjacent testicular slice from
each rat was further diced into small pieces (1 x 2 x 2
mm), postfixed in 1% osmium tetroxide-1.25% potassium ferro-cyanide
mixture, dehydrated in a graded series of ethanols, and embedded in
Arialdite. Embedded testicular specimens were sectioned with an
LKB ultramicrotome (Rockville, MD) at 2.05 µm and
stained with 1% toluidine blue for light microscopic examination and
morphometric studies (13).
Hormone assays
The T concentrations in plasma and testicular homogenates were
measured by RIA, as reported previously (16). 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 (16). 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 immunofluorometric assay for rLH (17) using
a combination of monoclonal antibodies to human LH (Medix, Kauniainen,
Finland) and bovine LH (provided by Dr. J. F. Roser, University of
California-Davis), as described previously (16). 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
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 Apop
Tag-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
(2, 3, 18, 19). In brief, after deparaffinization and rehydration,
tissue sections were incubated with proteinase K 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 out 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 antagonist for 7 or 14 days were used as positive controls (2).
Enumeration of the nonapoptotic 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 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 the heat- and hormone-sensitive stages (2, 3, 20, 21, 22). For each rat, at least 10 tubules/stage group were used. These stages were identified according to the criteria proposed by Russell et al. (23) for paraffin sections. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per 100 Sertoli cells (2, 3).
Morphometric procedures
The volume densities (Vv) of seminiferous tubules, tubular
lumens, interstitium, and Leydig cells were determined by a
point-counting method (13, 14). Five randomly selected sections per
animal in each group were examined by an American Optical Microscope
(Scientific Instruments, Buffalo, NY) with a x40 objective and
a x10 eye piece fitted with a square lattice containing 121
intersections. The results were expressed as a percentage of the testis
volume. The absolute volume of each of the testis components was then
obtained by multiplying its Vv by fresh testis volume (Vv%). The
diameters of 20 randomly selected transverse sections of seminiferous
tubules were measured across the minor axis of their profiles with an
ocular micrometer calibrated by means of a stage micrometer.
Numerical densities (Nv) of Sertoli and germ cells (number per unit volume of the seminiferous tubule) at stages VII–VIII of the cycle was determined by accepted stereological techniques as described previously (13, 14). For each rat, 10 round cross-sections of seminiferous tubules were used. The Floderus equation Nv = NA/(T + D - 2 h) was used to calculate the Nv of germ cell nuclei and Sertoli cell nucleoli, where NA is the number of nuclei or nucleoli counted per unit area of the seminiferous tubule profile, T is the section thickness, D is the average diameter of a given germ cell nucleus or the Sertoli cell nucleolus, and h is the height of the smallest recognizable nuclear or nucleolar profile in the section. The nuclear profile of each germ cell (A1 spermatogonia, PL and P spermatocytes, and step 7 and 8 spermatids) and the number of Sertoli cell nucleoli (thereby cells, as only 1 typical nucleolus is present per nucleus) in the seminiferous tubules were counted under a x1000 magnification using an oil immersion objective. The seminiferous tubule profile area (a) was determined by point counting using the equation: a = p x u2, where p is number of points per tubular profile, and u is the distance between 2 neighboring point in terms of the magnification used to measure the area. The mean diameters of Sertoli cell nucleoli and germ cell nuclei were obtained by direct measurements of their largest cross-sectioned profiles in serial sections. Even though the profiles of A1 spermatogonial nuclei were somewhat ellipsoidal, their eccentricity did not reach levels that would produce serious error. The height of the smallest recognizable nuclear or nucleolar profile was assumed to be 1/10th of the diameter of the structure. The Nv of a given cell type (number per unit volume of fixed tissue) was corrected by multiplying a factor of 0.855 to provide the number of cells per unit volume of the fresh tissue. The absolute number of these cells was then determined by multiplying their Nv by the fresh volume of the testis. Cell counts were finally expressed as the number of germ cells per Sertoli cell (germ cell/Sertoli cell ratios).
Statistical analysis
Statistical analyses were performed using the SigmaStat 2.0
Program (Jandel Corp., San Rafael, CA). Results were tested for
statistical significance using the Student-Newman-Keuls test after
one-way repeated measures ANOVA. Differences were considered
significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. No significant differences in the mean
body weight and ventral prostate and seminal vesicle weights were
observed among all treatment groups. In contrast, a significant
(P < 0.05) decrease in testis weight was noted as
early as 3 weeks after T administration. By 6 weeks, the mean testis
weight was reduced to 57.8% of the values measured in controls. Mean
testicular sperm content was also significantly decreased to 29.4%
(106/testis) of the control values at 6 weeks
after T treatment.
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. Plasma levels of FSH and LH
decreased to 51.1% and 3.1% of control values, respectively, by 1
week and remained suppressed thereafter throughout the treatment
period. Plasma T levels remained at the normal range throughout the
entire treatment duration. Intratesticular T levels were rapidly and
significantly reduced to 2.9% (ng/testis) of control levels as early
as 1 week after T administration. No further decrease in
intratesticular T levels was noted thereafter.
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. A low incidence of germ cell apoptosis
(AI = 1.59–9.52) was detected at stages I–IV, IX–XI, and
XII–XIV in control rats. The incidence of germ cell apoptosis was
significantly increased exclusively at stages VII–VIII at the earliest
time point examined, 1 week (AI = 21.43) after T administration. A
further increase in the incidence of apoptosis (AI = 56.30) was
noted by 6 weeks, at which time the mean incidence of germ cell
apoptosis became significantly elevated also at stages IX–XI (AI
= 5.99). The earliest response (1 week) of the seminiferous epithelium
to T implant involved primarily P spermatocytes and step 7 spermatids
and occasionally step 19 spermatids at stages VII–VIII (Fig. 1, A–C).
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). Accordingly, this combined treatment
regimen was used for subsequent experiments for detailed examination of
apoptosis and histomorphometry. The data presented below represent the
results of these experiments.
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) and testicular
sperm numbers (Fig. 2B) were markedly decreased in both heat- and
T-treated groups compared with those in control animals. Notably,
T+heat further significantly reduced testis weight to 31.1% of control
levels and specifically decreased sperm counts to almost zero. The
effects of heat exposure and T administration, either alone or in
combination, on plasma levels of FSH, LH, T, and testicular tissue
levels of T in adult rats are summarized in Table 5. Plasma LH levels were decreased in the
T and T+heat groups compared with those in either the control or
heat-treated group. In contrast, plasma FSH levels were increased in
the heat-treated group, but significantly decreased in both T and
T+heat groups. Plasma LH and FSH levels were not different between T
and T+heat groups. No significant difference in plasma T levels was
noted among the groups. The total content of testicular T was markedly
reduced in both T and T+heat groups compared with that in either the
control or heat-treated group. No differences in intratesticular T
levels were apparent between T and T+heat groups.
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shows the morphological appearance of the stage VII seminiferous
tubules from a control (A), heat-treated (B), T-treated (C), and T-
plus heat-treated (D) rats. In heat-treated animals (4 weeks after heat
treatment), the majority of the seminiferous tubules revealed a picture
of partial recovery of spermatogenesis (Fig. 3B). Histological
examination further revealed a mild spermatogenic damage in T-treated
animals, with the occurrence of degenerating germ cells in basal and
midepithelial levels at stages VII–VIII (Fig. 3C). Compared with T or
heat alone, T+heat clearly resulted in a marked impairment of
spermatogenesis, and the seminiferous tubules were smaller in diameter
and exhibited fewer P spermatocytes and round spermatids and complete
absence of step 19 spermatids (Fig. 3D).
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. Treatment with either T or heat
resulted in a significant decrease in seminiferous tubular diameter and
the volume of the seminiferous tubules and their lumens, with the
greatest decrease after T in combination with heat. The volumes of
interstitium and Leydig cells were also markedly reduced both in T and
T+heat groups compared with those in either the control or heat-treated
group. No differences in volume of Leydig cells were apparent between T
and T+heat groups.
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) and PL spermatocytes (Fig. 4B) were
apparent among various treatment groups. There was, however, a
significant decrease in the number of P spermatocytes (Fig. 4C) and
round spermatids (Fig. 4D) in heat, T, and T+heat groups compared with
controls. Notably, the number of P spermatocytes and round spermatids
in T+heat group were decreased to 46.36% and 28.74% of control
values, respectively, and also significantly decreased compared with
those in either the heat or T treatment group.
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). In the heat alone group, the
marked reduction of apoptosis was noted at early (I–IV) and late
(XII–XIV) stages. The spermatogenesis had been partially recovered 4
weeks after heat exposure. In the T+heat group, the seminiferous
tubules were smaller in diameter and exhibited a marked loss of germ
cells. A few remaining apoptotic cells were detected in this group
(Fig. 5B). Most of the apoptotic cells had been lost through
phagocytosis by Sertoli cells. Spermatogonia and PL spermatocytes were
unaffected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that a single transient local testicular heat exposure induces germ cell apoptosis in a stage-specific and cell-specific fashion. Early (I–IV) and late (XII–XIV) stages are more sensitive to heat. We also demonstrated in that study that a combination of a single heat exposure with selective deprivation of gonadotropins and intratesticular T by GnRH antagonist treatment further results in the activation of apoptosis at both hormone- and heat-sensitive stages (3). Thus, we hypothesized that the combination of these two proapoptotic signals (hormone deprivation and heat stress) may surpass the efficacy of each individual signal and result in a rapid and marked suppression of spermatogenesis to complete azoospermia. Results obtained from the second experiment supported this hypothesis. Compared with individual apoptotic stimuli given alone (either heat or T treatment), T in combination with heat clearly resulted in marked suppression of spermatogenesis to almost azoospermia. The observed azoospermia most likely is attributed to increased germ cell apoptosis and their eventual phagocytosis by Sertoli cells. The reasons why T in combination with heat results in marked suppression of spermatogenesis can be best explained as follows. Exogenous T (hit 1) suppresses LH and FSH levels, lowers intratesticular T levels, and allows apoptosis at a moderate rate to occur in the hormone-sensitive (VII–VIII) stages without affecting heat-sensitive stages (I–IV and XII–XIV). The addition of heat exposure (hit 2) to the testes with low intratesticular T levels induced by exogenous T administration results in marked acceleration of apoptosis at both hormone- and heat-sensitive stages, i.e. the window of protection seen with heat treatment alone had disappeared with prior hormone administration. Heat exposure accelerated the apoptosis caused by T treatment alone. In addition, we provided evidence indicating that the increased programmed germ cell death was independent of suppression of spermatogonia proliferation, as shown in this study and also demonstrated earlier in GnRH antagonist-treated or GnRH-immunized rats (13, 31, 32). This suggests that spermatogenesis inhibited by T in combination with heat will most likely attain full recovery after withdrawal of treatment. Unlike previous reports of combined treatment of T and estradiol in rats (28, 33), we did not observe sloughing of germ cells in the present study with T implants. This observation is consistent with our previous finding that no sloughing of germ cells occurred even after 4 weeks of GnRH antagonist treatment when the rat became completely azoospermia (2, 13). As expected (11), treatment with a low dose of T alone does not adequately withdraw the hormonal support, FSH in particular, required for optimum suppression of spermatogenesis. At present we are unable to determine the precise mechanism that causes the much greater proportional decrease in the level of LH compared with FSH after exogenous T administration. However, it is clear that the marked suppression of spermatogenesis in the heat- plus T-treated group is most likely not influenced by gonadotropins and intratesticular T, as these parameters are not different between the T and T+heat groups.
The mechanisms by which these hormonal and nonhormonal factors govern germ cell apoptosis are not well understood. It is likely that apoptosis will be regulated in a cell type-specific fashion, but the basic element of the death machinery may be universal. A distinct genetic pathway is apparently shared by all multicellular organisms (34). 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. Bcl-2 and Bax have also been implicated as potential modulators of germ cell apoptosis (35). It has been reported that some members of the Bcl-2 family are involved in apoptosis after withdrawal of androgen support of the testis after treatment with ethane dimethanesulfonate, a Leydig cell cytotoxin (36). The Fas system is also a widely recognized apoptosis signal transduction pathway in which a ligand-receptor interaction triggers the cell death pathway. This system has recently been implicated in the activation of germ cell apoptosis in response to a variety of proapoptotic stimuli, including testicular hyperthermia and T withdrawal (37, 38). The present study did not address whether the same molecular mechanisms by which testicular hyperthermia or androgen withdrawal induces germ cell apoptosis are involved. Ongoing additional studies will elucidate the roles of Bax, Bcl-2, Fas, Fas ligand, and caspases in germ cell apoptosis triggered by these hormonal and nonhormonal regulatory stimuli.
These studies of the induction of germ cell apoptosis in rats are very likely applicable to humans. We and others have also demonstrated that spontaneous loss of germ cells occurs by apoptosis in adult human testis (39, 40). Other data suggesting a role for intratesticular T in suppressing apoptosis in the human testes include the following: cessation of hCG treatment for cryptorchidism in prepubertal life increases apoptosis in the human testis (41); T regulates apoptosis in adult human seminiferous tubules in vitro (42); and apoptotic germ cells are present in testes from patients with prostate cancer who received short term antiandrogen treatment (43). Heat has long been recognized as a risk factor responsible for decreased sperm counts in men. Efforts have been made to harness these effects as an antifertility measure. Unfortunately, the effects have been incomplete or transient. Our group showed that an increase in human scrotal temperature of 0.8–1 C induced by polyester-lined athletic supports is insufficient to cause significant suppression of spermatogenesis or alteration of sperm function (44). We believe that this is due to failure to attain the critical (43 C) testicular temperature (45, 46) and the maintenance of normal or near-normal levels of intratesticular T after heat exposure. Based on the data provided from this study, in which administration of a low dose of T (hit 1) in combination with testicular warming (hit 2) rapidly suppresses spermatogenesis in rats, we postulate that the combination of hormone deprivation and heat could be applied as an induction strategy in the human. The advantages of this combination will be 1) shortening the duration of onset of suppression of spermatogenesis, resulting in azoospermia; and 2) lowering the dosage of T and diminishing the potential adverse effects of higher doses of T administration on prostate. In addition, the concept of a two-hit strategy will undoubtedly lead to fundamental studies of the mechanisms responsible for the induction of germ cell apoptosis by distinct pathways. This, in turn, will allow targeting of these pathways by pharmacological means to result in more rapid and complete azoospermia. Thus, this concept of the additive or synergistic effects of two distinct stimuli inducing germ cell apoptosis probably by different molecular cascades may have important applications for male contraceptive development.
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Acknowledgments |
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Footnotes |
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Received October 25, 1999.
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