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,
From the * Department of Experimental Radiation
Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas;
the Department of Physiology, University of
Turku, Turku, Finland; and the Institute of
Reproductive and Developmental Biology, Imperial College London, London,
United Kingdom.
| Correspondence to: Dr Karen L. Porter, US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, MD 21702 (e-mail: karen.porter{at}amedd.army.mil). |
| Received for publication August 20, 2008; accepted for publication January 2, 2009. |
Irradiation of LBNF1 rat testes induces spermatogonial
differentiation arrest, which can be reversed by gonadotropin-releasing
hormone (GnRH) antagonist–induced suppression of intratesticular
testosterone (ITT) and follicle-stimulating hormone (FSH). Although exogenous
estrogen treatment also enhanced spermatogenic recovery, as measured by the
tubule differentiation index (TDI), it was not clear whether estrogen
stimulated spermatogonial differentiation only by further suppressing ITT or
by an additional independent mechanism as well. To resolve this question, we
performed the following experiments. At 15 weeks after irradiation, rats were
treated with GnRH antagonist; some also received 17β-estradiol (E2) and
were killed 4 weeks later. GnRH antagonist treatment increased the TDI from 0%
to 8%, and addition of E2 further increased the TDI to 39%. However, E2
addition further reduced ITT from 7 ng/g testis, observed with GnRH antagonist
to 3 ng/g testis, so decreased ITT levels might have contributed to recovery.
Next GnRH antagonist–treated rats were given exogenous testosterone and
flutamide to stabilize ITT levels and block its action. This increased TDI
slightly from 8% to 13%, but the further addition of E2 significantly raised
the TDI to 27%, indicating it acted by a mechanism independent of ITT levels.
Plots of TDI for all treatment groups compared with ITT, FSH, or a linear
combination of ITT and FSH showed that treatments including E2 produced higher
TDI values than did treatments without E2. These results indicate that there
was an effect of E2 on spermatogonial differentiation because of an additional
direct action on the testis that is unrelated to its suppression of
testosterone or gonadotropins.
Key words: Irradiation, spermatogonial differentiation, testosterone
is expressed in Leydig cells, round
spermatids, and the efferent ducts, whereas estrogen receptor β is
expressed in Leydig cells, Sertoli cells, type A spermatogonia, and later
stage germ cells (Fisher et al,
1997; van Pelt et al,
1999). Evidence of estrogen's positive effects include direct
effects, as evidenced by reduced spermatogenesis in aromatase knockout mice
(Robertson et al, 1999;
Carreau et al, 2003), and
indirect effects, as evidenced by accumulation of fluid that could not be
reabsorbed in the efferent ducts and epididymis in estrogen receptor-
knockout mice (Hess et al,
1997; Zhou et al,
2001) and in rats treated with estrogen receptor antagonists
(Oliveira et al, 2001).
Estrogen, given as 17β-estradiol (E2), restores spermatogenesis in
hpg mice, which have undetectable serum gonadotropin levels, at least
in part by elevating follicle-stimulating hormone (FSH) levels through direct
stimulation of the pituitary by E2 (Ebling
et al, 2000), and was able to accelerate the onset of
spermatogenesis in immature bank voles
(Bilinska et al, 2003).
However, in normal adult and prepubertal rats, E2 and other estrogens can
exert a negative effect on spermatogenesis by reducing testosterone levels or
action by several mechanisms (Kula et al,
2001). Estrogens can directly act on Leydig cells to down-regulate
the steroidogenic enzymes involved in testosterone biosynthesis in rat testes
(Sakaue et al, 2002); act on
the hypothalamus, pituitary, or both to reduce the levels of FSH and
luteinizing hormone (LH), thereby reducing testosterone levels
(Hossaini et al, 2003) and
down-regulating expression of the androgen receptor
(Sharpe, 2006). In fact, the
action of estrogen in inducing disorders of the male reproductive tract has
been proposed to result from a disturbance of the androgen-estrogen balance
rather than from estrogen action alone
(Rivas et al, 2003). In this study, we examined the effects of E2 on the testes of LBNF1 rats treated with radiation doses of 6 Gy. These irradiated rats display spermatogenic suppression for prolonged periods as a result of spermatogonial differentiation arrest (Kangasniemi et al, 1996). They also exhibit elevated intratesticular testosterone (ITT) and FSH levels. In contrast to the stimulatory effects of testosterone (T) and FSH on the meiotic and postmeiotic phases of spermatogenesis in normal animals, in rats that are irradiated or treated with certain other toxicants, testosterone and FSH inhibit spermatogonial differentiation (Meistrich and Shetty, 2003). Indeed gonadotropin-releasing hormone (GnRH) antagonist treatment, which lowers ITT and FSH, restores spermatogonial differentiation in irradiated rats (Shuttlesworth et al, 2000). The strong inhibitory action of testosterone on spermatogonial differentiation was demonstrated by inhibition of both the GnRH antagonist–induced recovery and the hypophysectomy-stimulated recovery by exogenous testosterone (Shetty et al, 2000, 2006). A weaker inhibition by FSH was shown by the partial inhibition of the GnRH antagonist–stimulated recovery by exogenous FSH and by the lower level of spermatogenic recovery in GnRH antagonist– and testosterone-treated rats, which have moderate FSH levels, than in hypophysectomy- and testosterone-treated rats (Shetty et al, 2006).
In contrast to the inhibitory effects of testosterone and FSH, exogenous E2 treatment alone after irradiation stimulated spermatogenic recovery (Shetty et al, 2004). Furthermore, E2 treatment further stimulated the GnRH antagonist–induced spermatogenic recovery in irradiated rats (Shetty et al, 2002; Porter et al, 2006) and partially reversed the inhibitory effects of testosterone on spermatogonial differentiation (Shetty et al, 2004).
These results seemed to indicate that E2 might directly stimulate spermatogonial differentiation, but when E2 alone suppressed ITT to a greater extent than did GnRH antagonist treatment (Shetty et al, 2004), and E2 added to GnRH antagonist treatment further suppressed ITT (Shetty et al, 2002, 2004), we recognized the possibility that E2 might only act indirectly by other mechanisms, including suppressing testosterone production. To determine whether E2 had an effect on spermatogenic recovery independent of its effects on testosterone and gonadotropin suppression, in this study we analyzed spermatogenic recovery in irradiated rats treated with combinations of E2, testosterone, and androgen receptor antagonist (flutamide) implants with or without GnRH antagonist treatment.
Materials and Methods
Experimental Design
We treated 6-Gy–irradiated rats with hormones or hormone antagonists
between 15 and 19 weeks after irradiation. Age-matched, unirradiated,
untreated rats and untreated rats killed 19 weeks after irradiation were used
as controls. Initially, 4 to 6 rats were used per group, but additional rats
were added during repeats of the experiment to increase the precision of data
points with high variability or with points that were important to address
specific tentative conclusions. All experimental groups consisted of 4 to 16
rats.
Animals and Irradiation Treatment— We obtained 8-week-old LBNF1 hybrid rats (F1 generation of Lewis and Brown-Norway strain parents) from Harlan Sprague–Dawley Inc (Indianapolis, Indiana), and they were allowed to acclimatize in our facility for 1 week before use. Rats were housed in standard lighting (12 h light, 12 h dark) and were given food and water ad libitum. All procedures were approved by the University of Texas M. D. Anderson Cancer Center Institutional Animal Care and Use Committee, and the housing facilities were approved by the American Association of Laboratory Animal Care.
Rats were anesthetized with a mixture of ketamine (0.72 mg/kg) and acepromazine (0.022 mg/kg) IM, affixed to an acrylic glass board with surgical tape, and then irradiated by a 60Co gamma ray unit (Eldorado 8; Atomic Energy Canada Ltd, Ottawa, Canada). The field extended distally from a line about 6 cm above the base of the scrotum. A single dose of 6.0 Gy was given at a dose rate of approximately 1 Gy/min (Shetty et al, 2000).
Hormone Treatment— Hormone or hormone antagonist treatment was initiated at 15 weeks after 6-Gy irradiation. The GnRH antagonist acyline (obtained from National Institutes of Health–National Institute of Child Health and Human Development [NIH-NICHD], Bethesda, Maryland), freshly prepared in sterile water before each use, was injected into rats at 1.5 mg/kg weekly for 4 wk (Porter et al, 2006). Some rats were not given acyline. Groups of irradiated rats were also given subcutaneous Silastic implants (2 mm inner diameter, 3.2 mm outer diameter) (Dow Corning Corp, Midland, Michigan) containing E2 (0.5 cm, unless otherwise noted), T (6, 12, or 24 cm total length), or flutamide (20 cm total length, unless otherwise noted; Sigma-Aldrich, St Louis, Missouri). The release rates of T and E2 from the capsules are 30 µg/cm per day and 2.4 µg/cm per day, respectively (Robaire et al, 1979). The rationales for the various hormone combinations are given at the beginning of each section in the "Results." All the rats were killed after 4 weeks of treatment (19 weeks after irradiation) for hormone assays and analysis of spermatogenesis.
Preliminary experiments were performed to determine the length of flutamide-containing Silastic implants required for blockade of the intratesticular action of androgen. Groups of unirradiated rats were treated with weekly injections of acyline at 1.5 mg/kg and with flutamide-containing subcutaneous Silastic implants of 2.5 to 40 cm total length, with 8 cm as the maximum length of a single implant, for 2 weeks, without or with 6-cm testosterone implants. Acyline–treated rats given blank implants were used for comparison. The rats were killed at 2 weeks, the testes were weighed, and sperm heads were counted in sonicated testis homogenates with the use of a hemacytometer (Meistrich and van Beek, 1993).
Hormone Assays— Levels of testosterone in serum and testis homogenates were measured with an anti-testosterone antibody-coated tube radioimmunoassay kit (DSL 4000; Diagnostic Systems Laboratories, Webster, Texas) according to the manufacturer's instructions with modifications (Shetty et al, 2000). The testosterone standards for testis homogenate and serum analysis were prepared in phosphate-buffered saline (PBS) with 0.1% gelatin (gelatin type B G-9382; Sigma) and in dextran-coated charcoal-stripped serum from hormone-suppressed rats, respectively.
E2 levels in serum and testis homogenates were measured using an anti-E2 antibody, double antibody radioimmunoassay kit (catalog no. DSL-4800; Diagnostic Systems Laboratories), with modifications from the manufacturer's instructions designed to increase sensitivity. The sample and antibody incubation time was increased from 1 hour to overnight at 4°C, and the 125I-E2 tracer incubation time was increased from 2 to 3 hours at room temperature. The E2 standards (1.02–250 pg/mL) were prepared in PBS with 0.1% gelatin (Laboratory Grade 275 Bloom G8-500; Fisher, Fairlawn, New Jersey). The logit-log standard curves were fitted by linear regression, and values were determined from these curves. The intra-assay coefficient of variation (CV) was 10% and the interassay CV was 15%. The assay was validated for testis homogenates by performing linearity (R2 = 0.998; average absolute deviation from expected values for dilutions = 21%) and added-mass studies (average percent recovery = 114% ± 18%) with rat testis homogenate collected from the above experiments. The assay was validated for serum by performing linearity (R2 = 0.96, average absolute deviation from expected values for dilutions = 21%) and added-mass studies (average percent recovery = 144% ± 7%) using serum collected from the above experiments.
Because some samples had what appeared to be anomalous E2 values, even after verification by repeat measurements, we used the box plot method to identify outliers (National Institute of Standards and Technology, 2006). Only 8% of all testicular homogenates and 7% of all serum samples were excluded as outliers. In addition, some testicular homogenates had E2 concentrations below the limit of detection (1.02 pg/mL); therefore, that value was used in calculating the intratesticular E2 (ITE) concentration, resulting in ITE values of between 19 and 37 pg/g testis, depending on testis weight.
FSH and LH serum levels were determined by immunofluorometric assays (Delfia; Wallac, Inc OY, Turku, Finland) as previously described (Haavisto et al, 1993; van Casteren et al, 2000), having limits of detection for LH and FSH of 0.04 and 0.1 ng/mL, respectively.
Histological Analysis
The left testis was fixed in Bouin's fluid and embedded in paraffin, and
sections were stained with hematoxylin for histological examination. Tubule
differentiation index (TDI) was determined by counting the number of tubules
with differentiating germ cells (at least 3 cells that had reached the B
spermatogonial stage or later) and calculating the percentage of all tubules
that were differentiating (Meistrich and
van Beek, 1993).
Statistical Analysis
Results were presented as either 
± SEM calculated from untransformed data or as the
± SEM calculated from
log-transformed data (for LH, intratesticular testosterone, intratesticular
estradiol, and sperm head counts) obtained from individual rats. The
statistical significance of the differences were determined with SPSS v.11.5
software (Lead Technologies, Chicago, Illinois) by Student's t test,
with P < .05 being considered significant. If more than 2 groups
were being compared, ANOVA was used first, with P < .05 being
considered significant.
Results
Flutamide-Containing Silastic Implants Block Action of Intratesticular Testosterone
To determine the length of flutamide-containing Silastic implants required
for complete intratesticular androgen blockade, we treated unirradiated rats
with the GnRH antagonist acyline, with or without additional flutamide.
Whereas GnRH antagonist treatment decreased testis weights from 1.6 g in
control rats to 0.62 g, additional treatment with 20 cm total length of
flutamide capsules further reduced the testis weights to 0.40 g (P
> .05; Figure 1A).
Similarly, the GnRH antagonist treatment alone also reduced sperm head counts
from 2.9 x 108 in control rats to 2.8 x 107
sperm/testis, and additional treatment with 20-cm flutamide capsules further
reduced the counts to 9.7 x 105
(Figure 1B). Variation of the
flutamide capsule length from 10 to 40 cm did not significantly change the
reductions in testis weights or sperm head counts (P > .05); the
independence of the inhibitory effect within this range of flutamide doses
indicated that 10-cm flutamide capsules were sufficient to completely block
endogenous ITT levels, which were about 8 ng/g testis in these GnRH
antagonist–treated, unirradiated rats
(Figure 1C). Because rats
treated with exogenous testosterone would have higher ITT levels, 20-cm
flutamide capsules were chosen to block these higher testosterone levels in
all subsequent experiments.
|
Estrogen Treatment Increases Intratesticular E2 Levels
The levels of E2 in the serum or testes of groups of irradiated rats that
were treated with E2 for 4 weeks were measured. Additional treatments with
acyline or flutamide had no effect on the E2 levels in the testis or the
serum. Additional treatments with testosterone also had no effect on E2 levels
in the serum and produced no increase in ITE levels, indicating that
contributions to the E2 levels from aromatization of testosterone were
negligible. Hence, data from groups receiving each dose of E2 were pooled
separately, irrespective of the acyline, flutamide, or testosterone doses. E2
treatment with 0.5-cm capsules raised the level of E2 in the serum of
irradiated rats to 31 ± 3 pg/mL, compared with 12 ± 0.4 pg/mL in
irradiated rats with no E2 treatment; the latter was similar to the value of
12 ± 0.4 pg/mL for control rats. A longer capsule length of 2 cm raised
the serum E2 level to 81 ± 7 ng/mL. E2 treatment with 0.5-cm capsules
elevated the ITE levels from 57 ± 3 pg/g testis in irradiated rats to
71 ± 3 pg/g testis (P < .05). Treatment with 2-cm E2
capsules further raised the ITE levels significantly, to 136 ± 9 pg/g
testis (P < .05).
Estrogen Enhances Spermatogonial Differentiation in Irradiated Rats
To assess the ability of E2 alone to stimulate spermatogonial
differentiation, irradiated rats were treated with 0.5-cm capsules of E2, and
the results were compared with GnRH antagonist treatment. Irradiation markedly
reduced the TDI in rat testes to near 0
(Figure 2A), despite the
presence of type A spermatogonia
(Shuttlesworth et al, 2000),
indicating that spermatogonial arrest had occurred. Treatment with E2
increased the TDI from near 0 to 25%. GnRH antagonist treatment and the
resulting suppression of testosterone and FSH raised the TDI of irradiated
rats to only 8%.
|
These results indicate that E2 treatment restores spermatogonial differentiation in irradiated rats, possibly through suppression of ITT. Although FSH also inhibits spermatogonial differentiation in irradiated rats (Shetty et al, 2006), its suppression does not appear to be responsible for the greater stimulation by E2 since its levels were higher in the E2-treated rats than in the GnRH antagonist–treated ones.
Estrogen Enhances GnRH Antagonist–Induced Spermatogonial Differentiation in Irradiated Rats
In an approach to determining whether the E2-induced stimulation of
spermatogonial differentiation could be separated from its effect on
suppression of ITT, some rats were treated with GnRH antagonist to suppress
ITT levels and some were treated with both GnRH antagonist and exogenous
testosterone to further stabilize ITT levels; subgroups of these rats were
also treated with E2. E2 in 0.5-cm capsules, given to GnRH
antagonist–treated rats, induced recovery of spermatogenesis in 39% of
the tubules, which was significantly greater than the 8% observed with GnRH
antagonist alone (P < .05;
Figure 3A). Increasing the E2
capsule length to 2 cm did not further enhance recovery; in fact, the TDI was
only 25% ± 2% when 2-cm E2 capsules were used. Exogenous testosterone
treatment, given in 6-cm capsules to GnRH-treated rats, significantly reduced
the TDIs in the subgroups both without and with additional E2 treatment.
Nevertheless, the 0.5-cm E2 capsules still increased the TDI in the presence
of exogenous testosterone from 1% to 11%. Increasing the E2 capsule length to
2 cm again did not enhance recovery in the presence of GnRH antagonist and
testosterone, producing a TDI of only 6% ± 1%.
|
The changes in other hormones produced by these treatments were analyzed to determine whether they could account for the stimulatory effect of E2 on spermatogonial differentiation (Figures 3B through E). Addition of E2 to GnRH antagonist treatment increased serum FSH levels in these rats, as was previously observed with E2 treatment of hpg mice (Ebling et al, 2000). Hence, serum FSH levels could not account for the increase in TDI (Figure 3B). LH levels were suppressed to baseline in most rats with GnRH antagonist alone or combined with testosterone, E2, or both (Figure 3C). Nevertheless, the combination of GnRH antagonist and E2 further suppressed ITT concentrations from the level of 7 ng/g testis in GnRH antagonist–treated rats to 2 ng/g testis (Figure 3D). Supplementation of GnRH antagonist–treated rats with 6-cm testosterone implants increased ITT levels to 14 ng/g testis. However, additional E2 treatment still significantly reduced ITT levels in GnRH antagonist–treated plus testosterone-treated rats from 14 to 10 ng/g testis. Differences in ITE levels were not significant between the irradiated, irradiated GnRH antagonist–treated, and irradiated GnRH antagonist plus testosterone–treated rats, but exogenous E2 treatment with 0.5-cm capsules significantly increased ITE levels (Figure 3E).
The data to this point indicated that addition of E2 enhanced recovery of spermatogonial differentiation, even in the presence of GnRH antagonist, without or with additional exogenous testosterone treatment. However, it was still not possible to rule out that the additional suppression of ITT was responsible for the recovery.
Estrogen Enhances GnRH Antagonist–Induced Spermatogonial Differentiation in the Presence of Flutamide
Because ITT levels were still reduced by E2 treatment despite suppression
with GnRH antagonist and exogenously administered testosterone, we treated
irradiated GnRH antagonist–treated rats with the same combinations of
testosterone and E2 as above, but this time with 20-cm flutamide-containing
Silastic implants during the same time period. Addition of flutamide to GnRH
antagonist treatment significantly increased the TDI to 14%
(Figure 4A) from 8% observed
with GnRH antagonist treatment alone (P < .05;
Figure 3A). Addition of
flutamide to the GnRH antagonist treatment
(Figure 4A) also prevented the
reduction of TDI that occurred after addition of 6 cm of testosterone to the
GnRH antagonist regimen. Thus the 20-cm flutamide implant was sufficient to
block the effects of the added androgen in irradiated rats. E2 treatment still
markedly increased spermatogonial differentiation in GnRH antagonist plus
flutamide–treated irradiated rats, producing a TDI of 43%. This result
supports the notion that E2 acts independently on spermatogonial
differentiation, because although the E2 treatment did reduce ITT
(Figure 4D) despite no change
in LH (Figure 4C), the
flutamide should have blocked the effects of changes in testosterone.
Furthermore, changes in FSH were not responsible for the stimulation of
spermatogonial differentiation induced by E2 because, in fact, E2 induced an
increase rather than a decrease in FSH
(Figure 4B).
|
An increase in ITE levels was apparent when E2 was added to the GnRH antagonist, flutamide, and testosterone treatment, which was significant compared with the other groups by a t test; however, ANOVA did not indicate that the E2 levels in the 4 treated groups were significantly different (Figure 4E).
Estrogen Enhancement of Spermatogonial Differentiation Is Independent of Other Hormone Levels
To determine whether there was an effect of E2 on the TDIs observed in the
variety of treatment groups that was independent of intratesticular
testosterone and FSH levels, we plotted the data from the various treatments
described in the previous figures, plus some additional treatment groups given
implants of 12- or 24-cm testosterone capsules, as 2-dimensional graphs.
Because we had initially observed a good correlation between the TDI and ITT,
which represents the inhibitory effect of testosterone
(Shetty et al, 2000), we first
plotted the TDI data for different treatments of irradiated rats that did not
include flutamide, and therefore the action of ITT was unopposed
(Figure 5A), against ITT.
However, we later showed that FSH is also inhibitory and obtained the best
correlation of the data by plotting TDI against a linear combination of ITT
and FSH, the value of which was obtained by taking the ITT concentration (ng/g
testis) and adding 3 times the FSH concentration (ng/mL;
Shetty et al, 2006). Thus we
next plotted the TDI values for different treatments of irradiated rats that
did not include flutamide against the ITT plus 3xFSH
(Figure 5B). A single curve
could not be fitted to all of the points. However, when the points
representing rats not treated with E2 were fitted separately from those
representing rats treated with E2, excellent fits showing an inverse
relationship were obtained, with the curves for groups with E2 treatment
displaying higher TDI values than did the curves for the groups without E2 at
equivalent ITT and FSH levels. The separation between the curves is clearly
apparent in Figure 5B,
indicating that E2 stimulated spermatogonial differentiation independently of
its suppression of testosterone or FSH.
|
The TDIs for treatment groups receiving flutamide were also plotted against ITT plus 3xFSH, the relationship that was developed to represent the inhibition of spermatogonial differentiation in irradiated rats not receiving flutamide (Figure 5C). In addition, on the basis of observations that 20-cm flutamide implants could block the inhibitory effects of testosterone on spermatogonial differentiation in GnRH antagonist–treated irradiated rats (Figure 4A), the TDI values were also plotted against FSH alone (Figure 5D). In both cases, the data points for these rats treated with flutamide showed, even more clearly than in the rats not treated with flutamide, that it was not possible to fit a single curve through all of the data. However, when a separate curve was fitted to the data from rats treated with E2, an excellent inverse correlation was obtained. The TDI values for groups treated with E2 were well above the values obtained from groups not given E2 treatment, both at equivalent levels of FSH or of a linear combination of ITT and FSH. Thus, the conclusion that E2 treatment resulted in increased TDI values, independent of changes it might have induced in ITT or FSH levels, must be valid, even if the flutamide only partially inhibited the effects of testosterone.
Discussion
In this study, we have shown that treatment of irradiated rats, after irradiation, with capsules containing E2 consistently induced recovery of spermatogenesis, even when given in conjunction with a variety of other hormonal manipulations. These results can be compared with studies of recovery of spermatogenesis in which the estrogen treatment was started before the cytotoxic exposures. Our results are consistent with a study that showed that treatment with the E2 agonist/antagonist clomiphene citrate also enhanced recovery of spermatogenesis from gonadotoxic chemotherapy (Weissenberg et al, 1995). However, they differ from 2 studies in which E2 given before radiation (Morris et al, 1988) or gonadotoxic chemotherapy (Weissenberg et al, 1995) did not enhance recovery of the seminiferous epithelium, despite observed enhanced recovery when GnRH analogs were given in the same order (Ward et al, 1990). In any case, our model will be simpler to use to elucidate mechanisms involved because hormone treatment is given only after the cytotoxic treatment, whereas these other studies involved hormonal perturbation both before and after doses of the cytotoxic agents.
Because with in vivo studies of the effects of E2 on spermatogenesis it is often difficult to distinguish between the nontesticular effects of E2 due to pituitary feedback and the direct testicular effects and, among the testicular effects, between the consequences of lowering ITT levels and other paracrine factors or direct actions on germ cells, we employed various treatment regimens so that we could identify the active pathways. Action of E2 on LH levels did not appear to be a factor in the stimulation of spermatogonial differentiation. E2 treatment alone produced greater stimulation of spermatogonial differentiation than did GnRH antagonist treatment, despite similar or possibly less suppression of LH (Figure 2), and E2 treatment in combination with other hormones or antagonists enhanced spermatogonial differentiation without altering LH, which was already at baseline levels (Figures 3 and 4). Furthermore, a previous study showed that spermatogonial differentiation could be stimulated by suppression of testosterone and FSH, without LH suppression (Shetty et al, 2000). We also ruled out the possibility that E2 stimulated recovery simply by reducing the levels of FSH, a hormone that inhibits spermatogonial differentiation in this model system. GnRH antagonist given alone produced more suppression of FSH than did E2 given alone or in combination with GnRH antagonist, but it produced less stimulation of spermatogonial differentiation (Figures 2 and 3). In addition, GnRH antagonist and flutamide produced more suppression of FSH than did E2 in combination with these agents, but they produced less stimulation of spermatogonial differentiation (Figure 4). Plotting spermatogonial differentiation against FSH levels in irradiated rats treated with GnRH antagonist and flutamide showed that E2-treated rats had higher TDI values than did rats not treated with E2 at equivalent FSH levels (Figure 5D). We thereby ruled out the possibility that E2 stimulated spermatogonial differentiation by action on the pituitary.
Even though suppression of ITT levels indeed appeared to be one factor in the stimulation of spermatogonial differentiation by E2 treatment in irradiated rats given GnRH antagonist or GnRH antagonist and testosterone (Figure 3), stimulation of differentiation was greater by E2 when the TDI values were compared at equivalent ITT or ITT/FSH values (Figure 5A and B). The effects of ITT levels were further reduced or eliminated when flutamide was also given. Flutamide completely blocked the action of ITT levels of 15 ng/g testis or less, as indicated by the failure of 6-cm testosterone capsules to inhibit spermatogonial differentiation in irradiated rats treated with GnRH antagonist and flutamide (Figure 4A and D). Although 20-cm flutamide capsules completely inhibited testosterone action in irradiated rats implanted with 6-cm testosterone capsules, this does not conflict with the partial inhibition observed in unirradiated rats because, in the latter case, ITT values were higher at 26 ng/g testis. Thus, the the higher TDI values in irradiated rats treated with E2 in addition to GnRH antagonist and flutamide, whether they received exogenous testosterone or not, than in rats not receiving E2 (Figure 5C and D) demonstrates that E2 can directly stimulate spermatogonial differentiation at the testicular level, independently of its effects on ITT levels.
We have overcome the confounding effects of pituitary feedback and testosterone suppression on in vivo studies of E2 action on the testis by characterizing the effect of ITT and FSH on spermatogonial differentiation and accounting for these effects. We have thereby shown that E2 must have a direct action on spermatogonial recovery independent of any effect on either ITT or serum FSH levels.
Direct action of exogenous E2 on the testis is indeed a possible
explanation for our results because the treatments with E2 implants raised ITE
levels in a dose-responsive manner. Treatment with 0.5-cm E2 implants resulted
in an ITE level of 261 pM, which compared with the
Ki values of estrogen receptor (ER)- and
ERβ for E2 of 130 and 120 pM, respectively, is sufficient to trigger and
nearly saturate receptor-mediated effects
(Kuiper et al, 1997). The
observation that spermatogonial differentiation did not increase when the E2
capsule length was increased from 0.5 to 2 cm is consistent with a
receptor-mediated mechanism for E2-enhanced recovery.
We had previously shown that the block in spermatogonial differentiation in
irradiated rats is due to damage to the somatic cells of the testis, not the
spermatogonia (Zhang et al,
2007). We had also shown that this block can be reversed by
suppression of testosterone and FSH, which must target the somatic cells of
the testis that express androgen or FSH receptors
(Kliesch et al, 1992;
Bremner et al, 1994). However,
the action of E2 on the testis to enhance spermatogonial differentiation could
be either the result of direct action on the spermatogonia, which express
ERβ, or mediated by somatic cells, which express both ER and
ERβ (Fisher et al, 1997;
van Pelt et al, 1999).
Although E2 (Miura et al,
1999) or ER β-agonist
(Wahlgren et al, 2008)
treatment in vitro has been shown to stimulate spermatogonial proliferation
and inhibit later germ cell apoptosis
(Pentikainen et al, 2000),
action of E2 on the somatic cells cannot be ruled out because whole testis or
intact seminiferous tubules were used. However, in studies done with purified
gonocytes (Li et al, 1997) and
mouse spermatogonial GC-1 cells (Sirianni
et al, 2008), E2 has been shown to stimulate proliferation in
isolated germ cells directly.
The mechanism by which E2 acts on the irradiated testis to stimulate
spermatogonial differentiation is not known, but several targets are under
investigation. We have recently shown that the block in spermatogonial
differentiation in irradiated rats is associated with the development of
testicular interstitial edema (Porter et
al, 2006). Whereas the suppression of testosterone with GnRH
antagonist partially reduces the edema and restores spermatogonial
differentiation, addition of E2 to the GnRH antagonist treatment further
reduces the interstitial fluid volume and further stimulates spermatogonial
differentiation, suggesting that E2 might affect spermatogonial
differentiation by reducing interstitial fluid levels. This fluid volume
reduction is unlikely to be due to E2-induced increases in fluid absorption in
the efferent ductules (Cho et al,
2003) because we found that seminiferous tubule fluid volume was
not affected by E2 (Porter et al,
2006). Rather, the reduction could be the result of E2's direct
effects on the vasculature, which expresses high levels of ERβ and lower
levels of ER< (Nakamura et al,
2005), or indirect effects through the Leydig cells, which express
ER. Although E2 might act through nongenomic pathways as well
(Sirianni et al, 2008), we are
currently analyzing changes in gene expression that occur in the testes of
GnRH antagonist plus flutamide–treated rats on further treatment with
E2.
In this study, we have shown that E2 stimulates spermatogonial differentiation in irradiated rat testes independently of its effects on gonadotropin or testosterone levels. Elucidation of the mechanism by which this occurs might yield a better understanding of the action of estrogens in normal testicular function and spermatogenesis. We have also shown that E2 is more potent than GnRH antagonists in stimulating spermatogonial recovery in irradiated rats. Although enhancement of recovery of spermatogenesis in patients treated with radiation or chemotherapy by suppression of intratesticular testosterone levels with GnRH analogs, systemic low doses of testosterone, an antiandrogen, or a weakly androgenic progestin was reported in only 1 of 8 clinical trials, the application of hormonal treatment to enhancement of spermatogenic recovery from endogenous surviving stem spermatogonia or transplanted spermatogonia remains a possibility for these patients (Shetty and Meistrich, 2005), and the added effectiveness of E2 in the rat studies suggests a possible novel approach. However, E2 alone is not a good candidate for treatment of cytotoxic therapy–induced azoospermia in men because it could produce gynecomastia or cardiovascular side effects (Henriksson et al, 1999). Instead, selective estrogen receptor modulators with a similar action on the testis but without the other estrogenic side effects should be investigated for possible clinical application in the reversal of azoospermia induced by cancer therapies (Morris and Shalet, 1990).
Acknowledgments
We thank Dr R. P. Blye and Dr Hyun K. Kim of NICHD for providing the acyline, Dr Olga Bolden-Tiller for assistance with initial experiments that used flutamide-containing capsules, Gene Wilson and Taina Kirjonen for excellent technical assistance, Kuriakose Abraham for histological preparations, and Walter Pagel for editorial assistance.
Footnotes
Supported by NIEHS grant ES-08075, NIH Cancer Center Support grant CA-16672, NIH Radiation Oncology Training Program T32 CA-77050, and NIH Mammalian Reproduction Training Program T32 HD-007324.
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.05); L, 1 or more samples within the group had ITE levels
below the limits of detection for the assay. The dashed line in panel C
indicates the limits of detection for the LH assay (n = 5–24). The
values for groups of irradiated rats with different letters (a, b, c) are
significantly different from each other (P < .05), and groups with
the same letter are not.
) no hormone treatment; (
) A; (
) A+6T;
(
) A+24T; (•) E; (
) A+E; (
) A+6T+E; (
) A+24T+E;
(
) A+F; (
)
A+F+6T; (
) A+F+E;
(
) A+F+6T+E;
(
) A+F+12T+E; (#) A+F+24T+E. Data points for groups treated with
estradiol-17β (filled symbols) and groups not treated with estrogen (open
symbols) were fitted separately with sigmoidal 3-parameter regression curves,
(n = 4–24).