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Department of Experimental Radiation Oncology (G.S., G.W., G.A.S., M.L.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Physiology (I.H), University of Turku, 20520 Turku, Finland; and Central Research & Development ASTA Medica Aktiengesellschaft (T.R.), D 60314 Frankfurt Am Main, Germany
Address all correspondence and request for reprints to: Gunapala Shetty, Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. E-mail: gshetty{at}audumla.mdacc.tmc.edu
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Introduction |
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However, treatment of LBNF1 rats with GnRH agonists, GnRH antagonists, or testosterone (T) after testicular irradiation enhances the recovery of spermatogenesis (6, 7). Similarly, GnRH agonists stimulate recovery of spermatogenesis from surviving stem cells after exposure of LBNF1 rats to the cytotoxic chemotherapeutic procarbazine (8) or of Fisher rats to an occupational toxicant metabolite, hexanedione (9). In addition, treatment with a GnRH antagonist stimulates spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice, which are depleted of all germ cells except A spermatogonia due to a genetic mutation (10).
It is not known in any of these systems whether it is the suppression of T by these treatments that is responsible for the recovery of spermatogenesis or whether suppression of T precursors and/or gonadotropin suppression are also important. Therefore the major goal of this study was to investigate the role of T in the inhibition of spermatogonial differentiation and hence recovery of spermatogenesis in these pathological systems. To do this, we studied the effects of exogenous T and/or flutamide, a specific androgen receptor antagonist, on the recovery of spermatogenesis in irradiated and irradiated GnRH analog-treated rats. Since both GnRH analogs and low doses of T stimulate spermatogenic recovery after irradiation, we investigated the effects of the combination of the two on the recovery of spermatogenesis in irradiated rats. This would determine whether T supplementation could be used to maintain the libido of patients who might be undergoing GnRH-analog treatment to enhance spermatogenic recovery after radiation or chemotherapy.
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Materials and Methods |
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Irradiation
Rats were anesthetized with 0.72 mg ketamine/kg body weight and
0.22 mg acepromazine/kg body weight (im), and the lower part of the
body was irradiated by a 60Co 
ray unit
(Eldorado 8; Atomic Energy of Canada Ltd., Ottawa, Ontario, Canada).
Rats were placed on their backs and 5 mm of tissue-equivalent bolus
material (Superflab, Mick Radio-Nuclear Instruments Inc., Bronx, NY)
was placed over the scrotum to provide a build-up layer. The
irradiation field extended anteriorly about 6 cm above the base of the
scrotum. Single doses of 5 Gy or 6 Gy were administered at a dose rate
of 0.96 Gy/min. Control animals underwent anesthesia and sham
irradiation.
Hormone treatment
Four experiments were performed. Each treatment group consisted
of a minimum of four rats. A schematic representation of the
experimental design and the sampling schedule are shown in Fig. 1
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To shorten the duration of treatment and the overall time of the
experiment, we tested the appropriate time after irradiation at which
an injection of GnRH antagonist would maximally stimulate
spermatogenesis. We injected the GnRH antagonist Cetrorelix, provided
by ASTA Medica, Frankfurt, Germany, in two forms, Cetrorelix
acetate and Cetrorelix pamoate, at the same time. To produce an
immediate effect, Cetrorelix acetate dissolved in bacteriostatic water
was injected at a dose of 1.5 mg/kg body weight. Prolonged release
(
3–4 weeks) was achieved by injecting Cetrorelix pamoate at the
same time, suspended in a medium containing 0.5% Tween 80 and 2%
sodium carboxymethylcellulose, provided by ASTA Medica, at a dose of
1.5 mg/kg body weight. Injection at 3.3 weeks after irradiation showed
higher repopulation index (RI) than at 0 or 6.6 weeks (data not shown).
This protocol was employed in Exps 2, 3, and 4 with slight modification
of the time of injection.
In the second experiment, rats were injected sc with the two forms of GnRH antagonist at 3 weeks after a radiation exposure of 5 Gy. In some of these rats SILASTIC capsules containing various doses of T were implanted during weeks 3 to 7 after irradiation. Groups of rats were killed at 5 weeks after irradiation for measurement of serum and testicular hormone levels and at 13 weeks after irradiation to measure recovery of spermatogenesis.
In the third experiment, rats received only various doses of T implants during weeks 3–7 after irradiation. These rats were also killed 5 and 13 weeks after irradiation.
In the fourth experiment, some of the irradiated rats and some of the irradiated rats treated with Cetrorelix or Cetrorelix + 6 cm T were given an androgen receptor antagonist, flutamide, in the form of sc pellets (Innovative Research of America, Sarasota, FL) at a dose of 20 mg/kg body weight/day. Cetrorelix was injected at 3 weeks after irradiation; T capsules and flutamide pellets (4-week release time) were also implanted at week 3 after irradiation. T capsules were removed at 7 weeks after irradiation, while the biodegradable carrier for flutamide was allowed to decompose after the release of flutamide for 4 weeks. Groups of treated rats were killed at 5, 7, and 13 weeks after irradiation.
Hormone measurements
In Exp 2, weekly blood samples were collected from GnRH
antagonist (Cetrorelix)-only treated rats during weeks 7 to 13 after
irradiation. The rats were warmed under a heat lamp for 15 min to
produce vasodilation, the tail was nicked, and the blood was collected
using a 100-µl capillary tube. Histological studies indicated there
were no deleterious effects on germ cells in rats solely exposed to
similar heat treatment for 30 min and killed at 4 h and 7 days
after heat exposure.
In all experiments (weeks 10 and 20 in Exp 1, weeks 5 and 13 in Exps 2 and 3, and weeks 5, 7, and 13 in Exp 4), blood was collected from the rats by cardiac puncture under ketamine-acepromazine anesthesia, and then the rats were killed. The serum was separated and stored at -80 C. In all rats the right testis was freed of the tunica, weighed, collected on ice, and homogenized in a known amount of cold water. The tissue was not blotted so as to include most of the testicular fluid in the homogenate. In some cases, an aliquot was removed and the sperm heads counted; the remainder of all samples was stored at -80 C for ITT analysis.
Serum levels of FSH and LH were measured using in-house
immunofluorometric assays (IFMA) (Delfia, Wallac, Inc.
OY, Turku, Finland) as previously described (13, 14). The levels
of LH measured with this IFMA have been shown to correlate with its
biological activity (13). A new pair of antibodies was used in the FSH
assay, a monoclonal against recombinant human FSHß (FSH 56A) and a
polyclonal antibody against recombinant human FSH
(R93–2705), both
donated by Organon (Oss, The Netherlands). When the same
samples were run with the IFMA and the previously used RIA (6), the
IFMA gave values that were generally lower than RIA but had a greater
percentage difference between samples from different treatment groups.
The following regression equation can be used to relate the FSH values
obtained by RIA to those obtained by IFMA: FSH (IFMA) =
0.603·FSH (RIA) - 10.286.
Serum and testicular T were assayed using T antiserum-coated tubes from Diagnostics Systems Laboratories, Inc. (Webster, TX). For the serum T assay, the T standards were prepared in GnRH-suppressed rat serum that was stripped with dextran-coated charcoal (DCC) (Sigma, St. Louis, MO). The validity of the assay was demonstrated by estimating radioimmunoassayable T added in known quantities to the GnRH-suppressed rat serum before and after steroid stripping with DCC. When T was added at 0.1, 0.5, and 2.5 ng/ml to GnRH-suppressed rat serum, the assayed values, after subtraction of the values from samples lacking T addition, were 0.17, 0.59, and 3.07 ng/ml, respectively. The respective values were 0.05, 0.43, and 2.14 ng/ml when T was added to GnRH-suppressed rat serum after DCC extraction.
T was assayed directly in the testicular homogenates, which were not centrifuged, using T standards prepared in 0.1% gelatin in PBS (GPBS). The accuracy of the assay and the lack of interference by the homogenate in the assay were confirmed by assaying the amount of T added in known quantities to the whole homogenate, the spun supernatant, and the DCC-stripped supernatant of GnRH-suppressed rat testicular homogenates. When T was added at 0.1, 0.5, and 2.5 ng/ml to the whole homogenate, the assayed values after subtraction of the values for no T addition were 0.10, 0.44, and 2.61 ng/ml, respectively. When the same amount of T was added to the supernatant of the spun homogenate, the respective values were 0.14, 0.50, and 2.31 ng/ml, and when T was added to DCC-stripped testicular homogenates they were 0.09, 0.41, and 2.28 ng/ml, respectively. The ITT was expressed as the amount per gram of testis rather than the amount per testis. This was done so as to reflect the concentration of T to which the testicular cells are exposed, which in turn determines their response.
Serum Cetrorelix concentrations were measured by a double-antibody RIA
(15). Standards were prepared from a working stock of 4 µg/ml of
Cetrorelix acetate in RIA buffer (Na-phosphate buffer, pH 7.2, 2% BSA,
1% EDTA, and 0.02% Triton X-100) by serial 1:2 dilutions. The RIA was
set up by diluting the samples or standards, iodinated Cetrorelix, and
Cetrorelix antiserum in RIA buffer and incubating at 4 C for 2 days.
Antibody-bound and nonbound radiolabeled Cetrorelix were separated by
precipitation using rabbit IgG, goat antirabbit IgG, and polyethylene
glycol, and the pellet (antibody-bound fraction) was counted in
a -counter. The lower limit of detection of Cetrorelix was
0.19–0.34 ng/ml.
Evaluation of spermatogenesis
Spermatogenesis was evaluated in rats at two times in Exp 1,
once at the end of GnRH agonist + T treatment (10 weeks after
irradiation) and the other at 20 weeks after irradiation. In Exps 2, 3,
and 4, rats were analyzed for recovery of spermatogenesis at week 13
after irradiation. An aliquot from the right testicular homogenate was
sonicated at 4 C for 4 min as described earlier (16). The
sonication-resistant sperm heads representing nuclei of 12–19
spermatids were counted on a hemocytometer.
For histological analysis, the left testis was fixed in Bouin’s fluid and embedded in paraffin or plastic (JB4, Polysciences, Inc., Warrington, PA), and 4-µm sections were cut and stained with hematoxylin. To evaluate spermatogenesis after hormone treatment, 200 seminiferous tubules in one section from each animal were scored for the most advanced germ cell stage present in each tubule. A tubule was scored as repopulating if it contained three or more spermatogonia that had reached type B or later (16). The repopulation index (RI), which is the percentage of tubules showing repopulation, was then computed.
Statistical analysis
The data are represented as arithmetic mean ±
SEM, except for sperm counts, serum T, ITT, and LH, for
which the averages and SEM were calculated on
log-transformed data. The differences between the treatment groups were
analyzed first by one-way ANOVA. If the difference was significant
(P < 0.05), a t test was performed to
determine the significance of difference between the unirradiated and
irradiated-only rats, and Dunnett’s post hoc test for multiple
comparisons was performed to determine the significance of the
difference between the treated groups and a selected control group
(irradiated only, irradiated and treated with GnRH analog alone, or
irradiated and treated with 2 cm T alone). To compare the difference
among groups in T-alone-treated irradiated rats or in GnRH
analog-treated irradiated rats with or without T, a Tukey post hoc test
for multiple comparisons was performed. In Exp 4, to specifically test
the effects of T and flutamide, an independent samples t
test was performed. A computer-assisted statistics program (SPSS, Inc., Chicago, IL) was used.
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Results |
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). Round spermatids were the most mature
type of germ cells observed at week 10 when ITT was suppressed, in
which case the terminal phase of spermatogenesis was not supported.
However, restoration of T, which occurred after the withdrawal of
agonist treatment, facilitated later stages of spermatogenic process.
The initial phase of spermatogenesis (differentiation of spermatogonia
to spermatocytes and round spermatids), which was maintained during
suppression of T, continued irreversibly for at least 10 weeks after
cessation of GnRH-agonist treatment. Consequently, sperm head count
increased from a value of 0 in the irradiated-only rats to 2.5 x
107 ± 0.6 x 107 in
agonist-treated rats at 20 weeks after irradiation, although the levels
were still below those of unirradiated control rats (1.5 x
108 ± 0.1 x 108).
Supplementation with T suppressed the GnRH-agonist-induced
spermatogenic repopulation in a dose-dependent manner at 10 weeks and
20 weeks after irradiation. The addition of T to GnRH agonist resulted
in reduced sperm head counts at 20 weeks after irradiation (agonist + 2
cm T: 1.8 x 107 ± 0.8 x
107; agonist + 6 cm T: 1.0 x
107 ± 0.4 x
107).
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). When the GnRH antagonist
was given in combination with 2 cm or 6 cm T, the RI was reduced to
14% or 4%, respectively, with corresponding reductions in the number
of sperm heads.
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), but it fell to 4% for 6 cm T, and
1% for 24 cm T. These results were also confirmed by the stimulation
of sperm production by 2 cm T and the inverse correlation of the sperm
head counts with the T doses administered. The difference between the
present results and a previous study in which no dependence of RI on
dose of T was observed (6) may be attributable to the high variability
in the previous data.
In the present study a comparison of the RI obtained from rats treated
with GnRH antagonist + T with those in rats given T alone revealed no
significant differences in the recovery for the same doses of T (Fig. 3). Thus, the addition of T to the GnRH antagonist completely nullified
the stimulatory effect of the latter. Although the sperm head counts of
these groups of rats showed significant differences between groups
receiving same doses of T with and without GnRH antagonist, the
differences were in opposite directions with 2 cm and 6 cm T. This may
have been due to the variability in the sperm head counts when the
recovery was low.
To determine whether the inhibition of spermatogonial differentiation
after irradiation was actually due to T and if so whether it was
mediated through the androgen receptor, the androgen receptor
antagonist flutamide was used (Exp 4). Significant reductions in the
weights of seminal vesicle and prostate during the treatment period
(data not shown) confirmed the antiandrogenic action of flutamide in
the dosage used. In irradiated rats flutamide alone did not stimulate
restoration of spermatogenesis (Fig. 4).
Flutamide slightly enhanced the GnRH antagonist-stimulated repopulation
and sperm head production, but the increase was not statistically
significant. However, the inhibition of GnRH-stimulated germ cell
repopulation produced by T (RI = 5%; sperm head count =
7.9 x 103) was significantly reversed when
the latter’s action was blocked by flutamide (RI = 94%; sperm
head count = 4.7 x 106).
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), 7, and 13 (data not
shown) after irradiation, consistent with previous observations (4).
However, serum LH levels were unaltered by irradiation at week 5 (Fig. 5B), although significant increases were observed at weeks 7 and 13
after irradiation (data not shown). The ITT concentration reached
1.6-fold that of the unirradiated control value at 5 weeks (Fig. 5C)
after irradiation and 2.7-fold at 13 weeks (data not shown). The serum
T levels were not different, however, from unirradiated controls
(1–2 ng/ml) at 5, 7, and 13 weeks after irradiation.
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, A and B). In addition, both ITT and
serum T concentrations were suppressed, to 4% and 2.4% of the
irradiated control, respectively (Fig. 6, C and D).
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). Simultaneous injection of both the
soluble and depot forms of Cetrorelix rapidly produced high serum
Cetrorelix concentrations. The Cetrorelix concentrations gradually
decreased with time, reaching 3, 1.6, and 0.5–0.9 ng/ml at weeks 2, 3,
and 4, respectively, after its delivery. Analysis of time course of
Cetrorelix values and hormone levels indicated that a serum
concentration of about 1.5 ng/ml of Cetrorelix was necessary to
suppress the levels of LH in LBNF1 rats.
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). The hormone levels gradually rose thereafter, and by 4
weeks after GnRH injection, levels of serum LH (Fig. 7) and T were not
significantly different from the irradiated-only levels, and the ITT
still showed some significant suppression (22% of irradiated-only
rats) (data not shown). FSH was also still significantly suppressed
(42% of irradiated-only rats) at 4 weeks after GnRH antagonist
administration but was restored to the irradiated-only level by 10
weeks (data not shown). Previous studies have shown that Cetrorelix at
this dose suppressed the ITT to 4.6% of the level observed in
irradiated rats at 1 week after treatment (7). Thus, we conclude that
the GnRH-antagonist treatment rapidly suppressed serum FSH, LH, T, and
ITT to low levels, and the levels were maintained for 3 to 4 weeks.
The addition of T partially reversed the suppression of FSH induced by
GnRH agonist (Fig. 6A) but reduced serum LH levels (Fig. 6B). The
combination of GnRH agonist with 2 cm T further suppressed ITT
concentration to about 3 ng/g testis, which was 1.5% of
irradiated-only levels despite an increase in serum T level from 0.03
ng/ml to 2 ng/ml (Fig. 6, C and D). With the addition of a higher dose
of 6 cm T, the serum T levels, which were about 5 ng/ml, directly
contributed to an increase in ITT level to 14 ng/g testis. This level
was 6.5% of the value for rats given irradiation only and higher than
in the rats treated with GnRH agonist alone.
The addition of T to GnRH antagonist partially reversed the
FSH-suppressive effect of the antagonist (Fig. 5A). The extent of FSH
suppression induced by T alone also appeared to decrease with
increasing doses of T. The addition of T to GnRH antagonist in 2- and
6-cm lengths suppressed serum LH to an undetectable level as did the
treatment with different doses of T only (Fig. 5B). The levels of serum
T were increased to about 2 ng/ml and 10 ng/ml when 2 cm and 6 cm T,
respectively, were given to irradiated rats, either alone or in
combination with GnRH antagonist (Fig. 5D). Thus in the T-treated rats,
serum T levels were primarily dependent on the amount of exogenous T
supplied and independent of GnRH analog treatment. The ITT
concentrations in GnRH-antagonist treated, irradiated rats were not
further suppressed by the addition of 2 cm T to antagonist, unlike the
case for the agonist. Furthermore, the addition of 6 cm T significantly
increased the ITT concentration, to 23 ng/g testis from a GnRH
antagonist- suppressed value of 11 ng/g testis. These ITT levels of
irradiated rats treated with different doses of T combined with GnRH
antagonist were not different from the levels when respective doses of
T were given alone and both paralleled the increases in serum T.
To demonstrate that T, when given in combination
with Cetrorelix, acted directly to modulate gonadotropin levels and/or
spermatogenic recovery and did not affect Cetrorelix clearance, we
compared serum Cetrorelix levels in rats given Cetrorelix plus
different doses of T with those given Cetrorelix alone. Combined
delivery of T and Cetrorelix did not alter the serum Cetrorelix values
(Fig. 7B).
Administration of flutamide increased the serum FSH and LH levels 1.5-
and 3.7-fold, respectively, in irradiated rats (Fig. 8, A and B) as a result of inhibition of
negative feedback usually imposed by T on gonadotropin secretion.
Consistent with the rise in LH, flutamide increased the ITT and serum T
concentrations by 5- and 9-fold, respectively (Fig. 8, C and D).
Neither ITT, LH, nor serum T levels were significantly changed when
flutamide was given to GnRH antagonist- or GnRH antagonist + T-treated
irradiated rats. However, serum FSH levels were further suppressed by
the flutamide (Fig. 8A); this result is reasonable, considering the
increase of FSH levels when T was given to GnRH antagonist-treated rats
(see also Fig. 5A). However, blockade of T action by flutamide could
only partially reduce the increase in serum FSH content caused by T in
GnRH antagonist-treated irradiated rats.
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Discussion |
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The present study confirms that in the irradiated rats the resumption of the initial phase of spermatogenesis, i.e. the differentiation of spermatogonia leading to the formation of spermatocytes and some round spermatids, is triggered by the suppression of gonadotropins and T for at least several weeks. The subsequent restoration of T levels after the end of hormone treatment enabled the completion of spermiogenesis but no longer inhibited spermatogonial differentiation. The process as a whole proceeded further to restore spermatogenesis qualitatively, at least for the duration of the study.
Comparison of spermatogenic recovery with the ITT, serum T, and serum
FSH levels in the irradiated rats during hormone treatment (Fig. 9) revealed that ITT most closely
correlated with repopulation, and that the correlation was a negative
one. The one exception to this correlation was observed in GnRH
agonist-treated irradiated rats vs. GnRH agonist + 2 cm
T-treated ones (Fig. 9B): Greater recovery of spermatogenesis (RI
= 91%) was observed in the rats treated with the GnRH agonist alone,
in which the ITT levels were 8 ng/g testis, than in rats treated with
GnRH agonist + 2 cm T (RI = 79%), in which the ITT levels were 3
ng/g testis. It was noted that the rats with GnRH agonist alone had
serum T and FSH values of 0.3 ng/ml and 0.4 ng/ml, respectively,
whereas those treated with GnRH agonist + 2 cm T had higher serum T and
FSH values of 2 ng/ml and 3.6 ng/ml, respectively. Hence, the results
could be explained by a suppressive effect of either serum T or FSH on
repopulation when ITT levels were low.
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, C and F). This could, in part, be a result of the parallel
effects of exogenous T on ITT and on FSH levels. For example, exogenous
T reversed the GnRH analog-induced suppression of FSH. Although the
experiments using flutamide were designed to specifically test the
direct role of T, by inhibiting the action of T on the pituitary they
also produced changes in FSH levels opposite to those of T. This also
caused the negative correlation of FSH levels with spermatogenic
recovery, but there was a marked exception to this correlation in
antagonist + 6 cm T + flutamide-treated rats (Fig. 9F). In this case
the FSH level was 7.3 ng/ml and the RI was 93%. For comparison, rats
treated with antagonist + 2 cm T had similar FSH values (7.8 ng/ml) but
an RI of only 14%, and those receiving antagonist alone had lower FSH
values (5.8 ng/ml) and a lower RI of 67–78%. Further studies are
needed to ascertain whether FSH has direct role in spermatogenic
inhibition. Although our earlier studies failed to show a correlation
between recovery of spermatogenesis and FSH suppression, that
conclusion was principally based on the observation that 2 cm T
stimulated spermatogenic recovery in the irradiated rats without
altering FSH levels (6). The RIA used for FSH in that study, however,
gave variable results for this point and was not as sensitive to small
differences as the IFMA used here.
Serum T also showed a negative correlation with repopulation in the
irradiated rats (Fig. 9, A and D) but only in those with suppressed ITT
levels. Thus, in the irradiated-only rats the RI was 0% despite serum
T levels of 1–2 ng/ml, whereas rats receiving 2 cm T plus GnRH agonist
or antagonist showed spermatogenic recovery (RI = 79% and 14%,
respectively) despite higher serum T levels of 2–3 ng/ml. Furthermore,
irradiated rats treated with flutamide alone showed no repopulation
with serum T levels of 14 ng/ml, whereas those treated with antagonist
+ 6 cm T + flutamide showed 93% repopulation with only slightly lower
serum T values of 9.4 ng/ml.
Several lines of evidence showed that the levels of LH did not correlate with spermatogenic recovery after irradiation. First, suppression of the LH levels produced by adding T to GnRH agonist treatment inhibited recovery, whereas suppression of LH with the GnRH antagonist stimulated recovery. Second, GnRH agonist alone did not significantly reduce LH levels but did stimulate spermatogenic recovery. The question does arise as to whether this IFMA for LH is truly measuring its biological activity. Previous results showed that this IFMA correlates with biologically active LH (13). Correlation between immunological activity of LH using this assay and biological activity supports the validity of using IFMA. However, it is possible that the agonist affects LH pulsatility, which may also be important for its biological activity. Since the reduction in the ITT concentration by the GnRH agonist could occur by direct action on Leydig cells (17), there is not necessarily a contradiction between the LH measurements and the biological activity.
Very good correlations between ITT levels in the irradiated hormone-treated rats and RI were obtained both in the absence and presence of flutamide. However, in the latter case the curve was markedly shifted to the right. This result is consistent with the inhibition of spermatogenesis by T binding to the androgen receptor. Although the irradiated rats treated with flutamide alone failed to show any recovery, this was most likely due to the inability of the flutamide to prevent the androgenic action of the very high ITT level (666 ng/g testis).
The inhibition of spermatogenesis by T and FSH reported here appears at first to be contradictory to their well studied roles in stimulating and maintaining spermatogenesis. High ITT is required for maintaining spermatogenesis in normal rats (18, 19), and T and FSH are both needed for restoring spermatogenesis after hormonal deprivation (20). Although T and FSH have overlapping and synergistic or additive effects on spermatogenesis (21), the role of T is more pronounced in supporting the differentiation of spermatids (spermiogenesis) past step 7 (22), and the role of FSH is more pronounced in supporting spermatogonial numbers and differentiation (23). Whereas in the case of low ITT and absence of gonadotropins, spermiogenesis is blocked at step 8 (22), spermatogonial differentiation proceeds. In such cases the numbers of A and intermediate spermatogonia may be reduced, but at most by a factor of 2 (24). In irradiated rats, gonadotropins and ITT have a similar role in spermiogenesis, as shown by the failure to find cells that had developed past the round spermatid stage in rats treated with a GnRH agonist for 10 weeks after irradiation (6). But, unlike normal rats in which spermatogonial survival and differentiation are qualitatively independent of ITT and FSH, in irradiated rats the survival and differentiation of A spermatogonia are inhibited by moderate ITT levels and possibly also by FSH.
The inhibition of GnRH-stimulated recovery of spermatogenesis by T observed here in irradiated rats is reminiscent of a different situation, in which T also reversed the effects of GnRH analogs on spermatogenesis. In normal rats, monkeys, and humans GnRH agonists (25, 26, 27) or antagonists (28, 29) suppressed sperm production, but sperm production was restored by concomitant administration of low doses of T. GnRH analogs and T appear to play opposite roles in irradiated rats in supporting and inhibiting spermatogonial differentiation, respectively. Although the germ cells that are the targets of these effects, the spermatids in the case of normal rats and undifferentiated spermatogonia in irradiated rats, are different, there might be features in common to both examples with regard to the mechanism of the effect of T. One explanation for T’s ability to reverse the GnRH analog-mediated suppression of sperm production in rats and monkeys was based on the ability of FSH to support spermatogenesis when ITT production is reduced. T reverses the GnRH analog-mediated suppression of FSH by directly up-regulating FSHß gene transcription, circumventing the need for hypothalamic stimulation of FSH production (30). In the present study, similar increases in FSH secretion during GnRH-induced suppression were produced by T (and also decreases in FSH upon addition of flutamide). Thus, the decrease in the spermatogenic recovery caused by exogenous T could be due in part to its role in stimulating FSH.
It is possible that T and FSH act additively to inhibit spermatogonial differentiation in the irradiated rat, as when they stimulate the reinitiation of spermatid development in the unirradiated testis under situations involving hormone deficiencies (20, 23), by affecting gene expression in the somatic cells. Radiation, either directly or indirectly through the depletion of germ cells, appears to switch the spermatogenic regulation of these hormones to the negative side for some unknown reason. A direct effect of radiation could be due to induction of specific regulatory genes (31), which can change the response pattern of downstream androgen/FSH-responsive genes in a target cell. Sertoli cells are the likely targets since they contain both FSH and androgen receptors, which are absent in spermatogonia, and the Sertoli cell can affect germ cells through growth factors or cytokines. In vitro studies have shown that irradiation of rat Sertoli cells increases their production of IL-6, which is an inhibitor of meiotic DNA synthesis (32).
To conclude, the results indicate GnRH analogs stimulate recovery of spermatogenesis after cytotoxic insult by reducing the inhibitory effects of T, which were mediated through androgen receptor in the testis and possibly in the pituitary as well. The fact that the addition of T nullifies the stimulatory effect of GnRH analogs on spermatogenesis, whether it be by direct androgen action or by reversal of FSH suppression, has practical implications. Although it may appear advisable that during GnRH treatment of humans who were treated with chemo- or radiotherapy, T should be given so as to maintain libido, it may have negative effects on the action of the GnRH analog, in this case the stimulation of spermatogenesis. Hence, our findings could explain the failure of some of the earlier attempts to enhance recovery of testicular function during cytotoxic therapies in humans in which a combination of GnRH analog + T was given during chemotherapy (33).
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Acknowledgments |
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Footnotes |
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Received December 7, 1999.
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G. Shetty, C. C. Y. Weng, K. L. Porter, Z. Zhang, P. Pakarinen, T. R. Kumar, and M. L. Meistrich Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (jsd) Mice with Androgen Receptor or Follicle-Stimulating Hormone Mutations Endocrinology, July 1, 2006; 147(7): 3563 - 3570. [Abstract] [Full Text] [PDF]
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 G. Grafstrom, B.-A. Jonsson, A. M. El Hassan, J. Tennvall, and S.-E. Strand Rat testis as a radiobiological in vivo model for radionuclides Radiat Prot Dosimetry, April 1, 2006; 118(1): 32 - 42. [Abstract] [Full Text] [PDF]
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K. L. Porter, G. Shetty, and M. L. Meistrich Testicular Edema Is Associated with Spermatogonial Arrest in Irradiated Rats Endocrinology, March 1, 2006; 147(3): 1297 - 1305. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, S. J. Meachem, O. U. Bolden-Tiller, Z. Zhang, P. Pakarinen, I. Huhtaniemi, and M. L. Meistrich Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats Endocrinology, January 1, 2006; 147(1): 472 - 482. [Abstract] [Full Text] [PDF]
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 H. Jimenez-Severiano, M. L. Mussard, L. A. Fitzpatrick, M. J. D'Occhio, J. J. Ford, D. D. Lunstra, and J. E. Kinder Testicular development of Zebu bulls after chronic treatment with a gonadotropin-releasing hormone agonist J Anim Sci, September 1, 2005; 83(9): 2111 - 2122. [Abstract] [Full Text] [PDF]
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 K. Boekelheide, H. A. Schoenfeld, S. J. Hall, C. C. Weng, G. Shetty, J. Leith, J. Harper, M. Sigman, D. L. Hess, and M. L. Meistrich Gonadotropin-Releasing Hormone Antagonist (Cetrorelix) Therapy Fails to Protect Nonhuman Primates (Macaca arctoides) From Radiation-Induced Spermatogenic Failure J Androl, March 1, 2005; 26(2): 222 - 234. [Abstract] [Full Text] [PDF]
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 G. Shetty and M. L. Meistrich Hormonal Approaches to Preservation and Restoration of Male Fertility After Cancer Treatment J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 36 - 39. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, O. U. Bolden-Tiller, I. Huhtaniemi, D. J. Handelsman, and M. L. Meistrich Effects of Medroxyprogesterone and Estradiol on the Recovery of Spermatogenesis in Irradiated Rats Endocrinology, October 1, 2004; 145(10): 4461 - 4469. [Abstract] [Full Text] [PDF]
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) with or
without 2 cm T (
) and 6 cm T (
) (Exp 1). All rats received 6 Gy
of testicular irradiation (n = 4 in each group). Significance of
the difference from the values in rats treated with GnRH agonist alone
(Dunnett’s test): **, P < 0.01; ***,
P < 0.001. Significance of difference among
agonist-treated, irradiated rats with or without different doses of T
(Tukey test): §, P < 0.05.
, P < 0.05; 
and 
; n =
8) (data from Ref.7) or irradiated with 5 Gy and received Cetrorelix at
3 weeks (n = 4) (•, 
), or GnRH agonist + 6 cm T (filled hexagon).
The lower panels (Exps 2, 3, and 4) show serum T (D),
ITT (E), and serum FSH (F) from unirradiated control (
), 6 cm T (•), 24 cm T
(+), flutamide (
), antagonist + flutamide (open
hexagon) or antagonist + 6 cm T + flutamide (