TOXICOLOGICAL SCIENCES 117(1), 225–237 (2010)
Advance Access publication June 27, 2010
Hormonal Suppression Restores Fertility in Irradiated Mice from both
Endogenous and Donor-Derived Stem Spermatogonia
Gensheng Wang,*,1Shan H. Shao,* Connie C. Y. Weng,* Caimiao Wei,† and Marvin L. Meistrich*
*Department of Experimental Radiation Oncology; and †Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
1To whom correspondence should be addressed at Toxicology Division, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive Southeast, Albuquerque,
NM 87108. Fax: (505) 348-4890. E-mail: email@example.com.
Received April 2, 2010; accepted June 22, 2010
Irradiation interrupts spermatogenesis and causes prolonged
sterility in male mammals. Hormonal suppression treatment with
gonadotropin-releasing hormone (GnRH) analogues has restored
spermatogenesis in irradiated rats, but similar attempts were
unsuccessful in irradiated mice, monkeys, and humans. In this
study, we tested a stronger hormonal suppression regimen (the
GnRH antagonist, acyline, and plus flutamide) for efficacy both in
restoring endogenous spermatogenesis and in enhancing coloni-
zation of transplanted stem spermatogonia in mouse testes
irradiated with a total doses between 10.5 and 13.5 Gy. A 4-week
hormonal suppression treatment, given immediately after irradi-
ation, increased endogenous spermatogenic recovery 1.5-fold, and
11-week hormonal suppression produced twofold increases
compared with sham-treated irradiated controls. Furthermore,
10-week hormonal suppression restored fertility from endogenous
surviving spermatogonial stem cells in 90% of 10.5-Gy irradiated
mice, whereas only 10% were fertile without hormonal suppres-
sion. Four- and 11-week hormonal suppression also enhanced
spermatogenic development from transplanted stem spermatogo-
nia in irradiated recipient mice, by 3.1- and 4.8-fold, respectively,
compared with those not given hormonal treatment. Moreover, the
10-week hormonal suppression regimen, but not a sham treat-
ment, restored fertility of some 13.5-Gy irradiated recipient mice
from donor-derived spermatogonial stem cells. This is the first
report of hormonal suppression inducing recovery of endogenous
spermatogenesis and fertility in a mouse model treated with
anticancer agents. The combination of spermatogonial trans-
plantation with hormonal suppression should be investigated as
a treatment to restore fertility in young men after cytotoxic cancer
Key Words: irradiation; spermatogenesis; spermatogonial
transplantation; fertility; hormonal suppression; mice.
Radiation and chemotherapy, as testicular toxicants, can lead
to temporary or permanent sterility in mammals. Indeed, cancer
therapy has induced prolonged or permanent azoospermia in
many thousands of men (Meistrich et al., 2005). The continued
increase in long-term survival and cure following cancer
treatment makes the preservation and restoration of reproduc-
tive function of increasing importance (Meistrich et al., 2005).
The prolonged depletion of mature germ cells by radiation or
chemotherapyisgenerallybelievedtobebecause ofthe killing of
stem spermatogonia. Although a small number of surviving stem
spermatogonia could regenerate spermatogenesis, it usually takes
long times for spontaneous recovery to the level required for
fertility (Meistrich et al., 1978; Pryzant et al., 1993).
Although testosterone is necessary for normal sperm pro-
spermatogenesis from surviving stem cells in some pathological
situations (Meistrich and Shetty, 2003, Review). Consequently,
protect the testis and/or stimulate recovery of spermatogenesis
following radiation or chemotherapy-induced germinal damage
(Meistrich et al., 2005). It has been demonstrated repeatedly in
rats that the suppression of intratesticular testosterone levels
induced by treatment with steroids or gonadotropin-releasing
hormone (GnRH) analogues protects against prolonged damage
to spermatogenesis if given before radiation or chemotherapy or
stimulates recovery if given after the cytotoxic damage; as
a consequence, subsequent fertility is increased (Meistrich and
Kangasniemi, 1997; Meistrich et al., 2001; Udagawa et al.,
2001). Suppression of testosterone has also been shown to
enhance the recovery of rat spermatogenesis after damage
induced by numerous environmental male reproductive tox-
icants (Meistrich and Shetty, 2003, Review).
However, the results differ between species (Shetty et al.,
forthcoming). Although the treatments improve fertility in rats,
previous attempts using hormonal suppression to protect or
simulate recovery of spermatogenesis in men (Meistrich and
Shetty, 2008, Review) and primate model systems (Boekelheide
et al., 2005; Kamischke et al., 2003) treated with irradiation
and/or cytotoxic drugs have been unsuccessful, with the
exception of one report in humans (Masala et al., 1997). In
mice, pretreatment reductions of gonadotropins with GnRH
analogues or genetic mutations also failed to protect against the
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radiation- or chemotherapy-induced disruption of spermato-
genesis (Crawford et al., 1998; da Cunha et al., 1987;
Kangasniemi et al., 1996a; Nonomura et al., 1991), and no
study has been performed to examine the stimulation of
recovery by posttreatment hormonal suppression.
The studies in rats have also shown that after cytotoxic
exposure, a significant population of surviving stem spermatogo-
Meistrich and Shetty, 2003). But in human (Kreuser et al., 1989)
or monkey (Boekelheide et al., 2005; van Alphen et al., 1988)
testis, such a radiation or chemotherapy-induced block in
spermatogonial differentiation is only transient or rare. In mice,
the spermatogonia that survive irradiation actively proliferate to
produce colonies containing differentiating cells, and very few of
the atrophic tubules contain undifferentiated spermatogonia
(Kangasniemi et al., 1996a). Because the pathophysiological
profile in the irradiated mouse testis is more similar to primates
than rat for future applications to human.
To overcome the loss of stem spermatogonia resulting from
cytotoxic therapies, spermatogonial transplantation may also be
used to supplement this cell population. When donor stem
spermatogonia are introduced into germ cell–depleted seminif-
erous tubules of host testes, they are able to colonize and
undergo complete spermatogenesis. Furthermore, hormonal
suppression significantly enhanced spermatogenesis from
transplanted spermatogonia in recipient rat testes treated with
irradiation (Zhang et al., 2007) or busulfan (Ogawa et al.,
1999) and in recipient mouse testes (Dobrinski et al., 2001;
Kanatsu-Shinohara et al., 2004; Ogawa et al., 1998; Ohmura
et al., 2003). Although hormonal suppression’s ability to
improve the success of spermatogonial transplantation was
dramatic in rat testes, the effects in mice were only moderate
and variable from different studies and seemed to be strongly
associated with the timing of treatment.
We hypothesized that a more effective hormonal suppression
regimen, such as prolonged suppression using both a GnRH
antagonist (GnRH-ant), which is more effective than GnRH
agonists, and an antiandrogen can efficiently stimulate
spermatogenesis from transplanted spermatogonia in mice.
Moreover, we examined whether this treatment regimen could
also promote the recovery of endogenous spermatogenesis and
fertility in irradiated mice.
MATERIALS AND METHODS
Animals. Adult C57BL/6Law male mice at 8–12 weeks of age, bred at
The University of Texas, M. D. Anderson Cancer Center, were used in
irradiation experiments and as transplantation recipients. Donor mice were
obtained by breeding C57BL/6-Tg(CAG-EGFP)1Osb/J mice ubiquitously
expressing green fluorescent protein (GFP) (Jackson Laboratory, Bar Harbor,
ME) with C57BL/6Law mice. The animals were maintained on a 12-h light
12-h dark cycle and were allowed food and water ad libitum. All animal
procedures were approved by The University of Texas M. D. Anderson Cancer
Center Animal Care and Use Committee.
Experimental design. Four experiments were conducted as outlined in
Figure 1. The radiation doses and timing of assays used were based on earlier
studies in which recovery of spermatogenesis in mice was measured (Meistrich
et al., 1978). Total doses of 9–12 Gy resulted in gradual recoveries of sperm
counts over the course of 45 weeks, with the mice regaining fertility at about
28 weeks after 9 Gy and failing to recover after 12 Gy. The durations of
hormone-suppressive treatments were based on studies in rats, which showed
that 4 weeks of GnRH-ant treatment, given after irradiation, with or without
flutamide, was able to stimulate spermatogenic recovery (Shetty et al., 2000),
10 weeks of GnRH-ant treatment was able to stimulate both recovery of
spermatogenesis and fertility (Meistrich et al., 2001), and that 13 weeks of
suppression stimulated differentiation of transplanted spermatogonia (Zhang
et al., 2007). In experiment (Exp.) 1, we examined effects of hormonal
suppression regimens with GnRH-ant given for different time periods on
spermatogenic recovery in mice treated with three different irradiation doses. In
Exp. 2, we determined the effect of hormonal suppression on differentiation of
endogenous stem cells and colonization of transplanted stem cells in the same
irradiated mice with two different irradiation doses. In Exp. 3, we further
examined whether hormonal suppression was able to restore fertility by
improving recovery of endogenous spermatogenesis after a total dose of
10.5 Gy, the irradiation dose that demonstrated favorable response to hormonal
suppression treatment in Exp. 1. In Exp. 4, we used a higher dose of irradiation
(13.5 Gy) to destroy nearly all the endogenous spermatogenesis and primarily
examined whether hormonal suppression could enhance donor cell colonization
and donor-derived spermatogenesis and thereby restore fertility.
Irradiation. Mice were restrained in plastic chambers and then placed into
and scrotal area of the animal was irradiated by a137Cs gamma-ray unit. The
been shown to be more effective than a single dose in depleting germ cells and
are presented as the totaldose of the twofractions throughoutthe text.Doses were
doses of irradiation (Meistrich et al., 1978).
Hormonal suppression treatment. Hormonal suppression treatments were
initiated immediately after irradiation and maintained for 4, 10, or 11 weeks in
different experiments, as indicated in Figure 1. The GnRH-ant, acyline (obtained
from the Contraceptive Development Branch of National Institute of Child Health
and Human Development, North Bethesda, MD), was prepared in sterile water
and sc injected at an initial dose of 20 mg/kg body weight and followed by
maintenance doses of 10 mg/kg body weight given every other week. For Exps
3 and 4, in which fertility tests were performed, a lower dose of 6 mg acyline/kg
body weight was given in the last injection at week 8 to allow quicker recovery of
hormonal levels. Flutamide, an androgen receptor antagonist, was delivered by
implanting two 2-cm Silastic brand silicone capsules filled with the drug. We used
two 2-cm length flutamide capsules based on our previous experiments that a total
length of 4-cm flutamide is effective in suppressing the testosterone action on the
normal testis (Shetty et al., 2006b). The effect was similar to that observed
previously with pellets releasing 1.2 mg of flutamide/day (Kangasniemi et al.,
1996a). The flutamide capsules were implanted right after completion of
irradiation (within 30 min) and were removed after 4, 10, or 11 weeks for 4-week,
10-week, or 11-week treatment groups, respectively. The controls were sham
treated by injection of sterile water and implantation of empty capsules. In Exp. 1,
the flutamide implants were found lost because of the sealing staples not being
fastened well after implantation and were removed from the housing cages within
the first few days. We thus considered the hormonal-suppressive treatment to be
GnRH-ant only in that study.
Transplantation. Immature heterozygous GFP mice at 14–17 days of age
were used as donors, except for 12-Gy group in Exp. 2, in which 19- to 27-day-old
WANG ET AL.
mice were used. The stem cell spermatogonia donor cells were prepared as
previously reported (Zhang et al., 2006). Briefly, after the tunica was removed,
testicular tissue was sequentially digested, first with 0.05% type IV collagenase
for 20 min and then with combined 0.05% type IV collagenase and 0.05%
hyaluronidase for 20 min in modified Dulbecco’s Modified Eagle Medium
(DMEM)/F12 solution containing 100 lg/ml DNase at 35?C in a shaking water
bath. The tubules were washed in Dulbecco’s PBS (GIBCO, Carlsbad, CA)
and then incubated in 0.1% trypsin in D-PBS containing magnesium,
100 lg DNase/ml, and 1mM ethylene glycol tetraacetic acid. After neutralization
of trypsin with serum and filtration through a 35-lm nylon screen, the cell
suspensions were centrifuged and resuspended in DMEM/F12 solution containing
100 lg DNase/ml. Trypan blue (Invitrogen, Grand Island, NY) was added to a
final concentration of 0.04%, and the cell suspension was kept on ice until
transplantation. The average of viability of cells was 93%.
Mice irradiated with 12 or 13.5 Gy, as indicated in Figure 1, were used as
recipients for spermatogonial transplantation 3 weeks after irradiation. The
lower abdomen was opened, and the testis was withdrawn from the body
cavity. The efferent duct was identified, and surrounding fat was dissected
away under a microscope. The donor cells were transplanted into seminiferous
tubules through efferent duct injection using a glass micropipette controlled by
a FemtoJet microinjector (Brinkmann Instruments Inc., Westbury, NY). The
average injection volume was 8.2 ll, and an average of 3.1 3 105cells per
testis was injected. The success of the injection was monitored by observing the
distribution of the Trypan blue dye. In Exp. 4, the transplantation control
groups were injected with media instead of cells.
Evaluation of spermatogenesis. The mice were euthanized at different
times after irradiation as indicated in Figure 1. The weights of the body, testis,
and seminal vesicle (SV) were recorded in all experiments, and in Exps 3 and 4,
the epididymis weights were also recorded.
For histological evaluation of endogenous spermatogenic recovery, testes
were fixed in Bouin’s solution (both testes in Exp. 1, left testis in Exp. 3, and in
sham-transplanted groups of Exp. 4), embedded in paraffin, and sectioned at
5-lm thickness. The testicular cross sections were stained with hematoxylin
and periodic acid-Schiff reagent and then examined under light microscopy.
Spermatogenic recovery was evaluated as previously described (Shetty et al.,
2001) by the tubule differentiation index (TDI), which is defined as the
percentage of tubules that contain three or more differentiating germ cells at the
B spermatogonial stage or beyond.
For evaluation of spermatogenic recovery in the recipient testes (Exp. 2 and
transplanted groups of Exp. 4), both testes were removed and fixed in 4%
paraformaldehyde at 4?C for up to 24 h and embedded in paraffin. After
deparaffinization and rehydration, the testicular sections were subjected to
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started immediately after irradiation and continued for 4, 10, or 11 weeks. In Exps 2 and 4, transplantation was performed at 3 weeks after irradiation.
Schematics of the four experimental protocols used. Mice were irradiated at week 0 with total doses as indicated. Hormonal suppression treatment was
FERTILITY RESTORATION IN IRRADIATED MICE
antigen retrieval and nonspecific antibody-binding blocking. The sections were
then incubated with rabbit monoclonal anti-GFP (Cell Signaling, Danvers, MA)
at 1:300 dilution at 4?C overnight, followed by a biotinylated anti-rabbit
immunoglobulin G and an avidin-biotin-peroxidase complex reagent (Vectastain
Elite kit, Vector Laboratories, Burlingame, CA). The immunoreactivity was
visualized by incubation with the peroxidase substrate diaminobenzidine (Vector
Laboratories). The slides were counterstained with hematoxylin.
The progress of spermatogenesis in the recipient testes was evaluated by the
TDI as described above. GFP staining was used to differentiate whether
the germ cells originated from the donor cells (GFP positive) or from the
endogenous spermatogonia (GFP negative). The TDI for donor cells was
corrected for injection of different cell numbers by normalization to the average
cell numbers injected in each experiment.
The lengths of the donor cell colonies were measured in the 12-Gy study of
Exp. 2. After removal of the tunica from one testis of three or four mice from
each treatment group, the seminiferous tubules were separated using a fine
forceps under a dissecting microscope. GFP-positive colonies were identified
and imaged under a fluorescence microscope. The length of the colonies was
measured by using the image processing software Axiovision version 4.6 (Carl
Zeiss Microimaging, Inc., Go ¨ttingen, Germany).
To assess the recovery of sperm production, sperm heads were counted in
the testes and sperm were counted in the cauda epididymis. The tunica
albuginea was removed from one testis of a mouse, and the testis was weighed,
homogenized, and sonicated. The sperm heads were counted in a hemacytom-
eter (Meistrich and van Beek, 1993). For epididymal sperm counts, both cauda
epididymis were minced in 1 ml PBS and incubated at 37?C for 30 min, and the
suspension was passed though a 80-lm pore size metal filter. Sperm were
counted using a hemacytometer.
Serum testosterone measurement. Blood was collected from the axillary
vein of mice under anesthesia at euthanasia. The serum was separated by
centrifugation and stored at ?20?C until measurement of testosterone. Serum
testosterones were determined as described earlier (Shetty et al., 2000) by using
a coated tube radioimmunoassay kit (DSL-4000, Diagnostic Systems
Laboratories, Webster, TX).
Fertility test. The fertility of mice was tested starting at 11 weeks after
irradiation. Each male was housed with two ND4 Swiss Webster virgin females
(Harlan Laboratories, Indianapolis, IN) until the time assigned for euthanasia of
the males. The recovery of fertility for each mouse was defined as the date
of conception of first litter, 20 days prior to the birth date. The number and size
of litters were recorded. A group of unirradiated adult male mice were used as
positive controls for the fertility test. The pups from the transplanted mice were
examined for the expression of the GFP transgene to determine if the return of
fertility was from endogenous stem spermatogonia or from donor cells.
Because GFP heterozygous donors were used, we expected that half of pups
would be GFP positive if the sperm were derived from donor stem cells.
Statistical analysis. Simple comparisons of tissue weights, sperm counts,
and TDI among groups were performed using two sample t-tests (for two group
comparison) or ANOVA with Student-Newman-Keuls post hoc pairwise
comparisons (for three or more groups). Statistical analysis of testicular sperm
count data was performed on log-transformed data because the transformed
distributions are closer to normal. In a few cases that the TDI data were not
normally distributed, nonparametric analyses were performed, as indicated in
the figure legends, using the Wilcoxon-Mann-Whitney test for two group
comparisons and the Kruskal Wallis test for three or more group comparisons.
Time to fertility recovery was analyzed by the Kaplan-Meier estimator.
Difference between treatment groups was considered significant when p < 0.05.
These statistical analyses were performed using the SPSS version 16 statistical
software package (SPSS Inc., Chicago, IL).
We used linear mixed models (Verbeke and Molenberghs, 2000) to
examine the effects of hormone suppression treatment, source of stem cells,
radiation level, stem cell transplantation, and sacrifice time on the TDI. For
this analysis, the TDI of the hormonal-suppressed groups was normalized
against the no-hormone–treated controls by dividing all their TDI values by
the mean TDI of the mice that received no hormonal suppression with the
same combination of radiation dose, source of stem cells, and sacrifice time
point, and is referred to as the TDI ratio. To assess whether the effect of 4-week
hormonal suppression differs from that of 11-week hormonal suppression and
whether the differences between the two treatment regimens are dependent on
the source of stem cells, we fitted the linear mixed model on all data points
from Exps 1 and 2. The model included fixed effects of hormone suppression
(11 weeks vs. 4 weeks), source of stem cells (endogenous vs. donor), the
interaction between hormone suppression and source of stem cell, trans-
plantation, and radiation nested within transplantation, and a random effect of
mice (Model 1). To assess whether the effect of 10 weeks of hormone
suppression on endogenous TDI irradiated with 10.5 Gy varies over time, we
fitted a linear model with a fixed effect of sacrifice time (four levels: 16, 21, 31,
and 46 weeks) on all the data points from Exp. 3 (Model 2). Finally, to assess
whether the effect of 10-week hormone suppression on TDI is greater toward
donor cells than endogenous cells and whether the effect is time dependent, we
fitted a linear mixed model on the data points from the mice receiving stem
cell transplantation from Exp. 4, with fixed effects of source of stem cells
(endogenous vs. donor), sacrifice time (five levels: 11, 16, 21, 31, and 46 weeks),
the interaction between source of stem cell and sacrifice time, and a random effect
of animal (Model 3). All linear models were performed in SAS v 9.1 (SAS
Institute, Cary, NC).
Hormonal Suppression (Exp. 1 and Exp. 2)
In Exp. 1, mice were irradiated with total radiation doses of
10.5, 11.5, or 12.5 Gy followed by 4- or 11-week treatments with
GnRH-ant (acyline) and euthanized at 11 weeks after irradiation.
Body weights were slightly, but reversibly, reduced in the mice
under hormonal suppression treatment (Table 1). There was
a marked decrease in testis weights, SV weights, and serum
testosterone concentration with the 11-week treatment. The
reduction in SV weight with 11-week treatment clearly confirms
that testosterone activity was suppressed during the treatment.
In a subsequent experiment (Exp. 2), mice were irradiated
with total radiation doses of 12 or 13.5 Gy followed by 4- or
11-week treatments with GnRH-ant (acyline) plus antiandro-
gen and spermatogonial transplantation. Alterations of body
and tissue weights were similar to those in Exp. 1. In addition,
in a group of mice euthanized at the end of the 4-week
treatment time, both testis and SV weights were reduced
(Table 1). Although the concentration of serum testosterone
was not suppressed after 4-week treatment, the action of
testosterone was clearly reduced in those mice at 4 weeks
because the SV weights were significantly decreased. The
reduced SV weights in the absence of serum testosterone level
reductions at 4-week treatment is likely because of the effect
of antiandrogen flutamide that blocks the action of testoster-
one. Note that the tissue weights in these mice returned to
nearly the levels of control mice at 11 weeks, suggesting that
the suppression of testosterone was reversible after the
treatment ceased. With 11-week treatment, both serum
testosterone levels and SV weights were significantly re-
duced, showing that both testosterone level and action were
effectively suppressed in those animals.
WANG ET AL.
Effect of Hormonal Suppression on Recovery of Endogenous
Spermatogenesis (Exp. 1 and Exp. 2)
The hormonal suppression treatment stimulated the recovery
of endogenous spermatogenesis. In mice irradiated with 10.5 Gy,
there was a significant increase in the percentages of tubules with
differentiated germ cells after 11-week hormonal suppression, as
compared with sham-treated mice (Fig. 2A). Four-week
treatment also showed a consistent trend of an increase compared
with controls, but the effects were generally less than those with
the 11-week treatment. Similar results were observed with the
recovery of endogenous spermatogenesis in mice irradiated with
12 and 13.5 Gy and then also subjected to spermatogonial
transplantation, with significant increases in recovery after the
11-week hormonal suppression and a significant increase of
spermatogenic recovery with the 4-week treatment in mice
irradiated with 12 Gy (Fig. 2B).
effects of transplantation (p ¼ 0.61) or radiation dose (nested
hormone suppression was marginally lower in Exp. 1, in which
the flutamide capsules were lost, than in Exp. 2. The 4-week
hormone suppression treatment appeared to increase the TDI of
endogenous cells over that observed with no hormone suppres-
sion by 1.5-fold (p ¼ 0.10), whereas the 11-week treatment
significantlyincreased the TDI by twofold (p ¼ 0.025). Although
the overall effect of the 11-week hormone suppression appeared
greater than that of the 4-week treatment, the difference was not
statistically significant (p ¼ 0.25).
Effects of Hormone Suppression on Weights and Testosterone Levels
irradiation doseHormonal suppression
(weeks after irradiation)
4 weeks GnRH-ant
11 weeks GnRH-ant
4 weeks GnRH-ant þ flutamide
4 weeks GnRH-ant þ flutamide
11 weeks GnRH-ant þ flutamide
29 ± 1a
27 ± 1a
25 ± 1b
28 ± 0d
25 ± 0e
27 ± 0d
25 ± 0e
25 ± 1a
24 ± 1a
14 ± 1b
29 ± 1d
14 ± 0e
27 ± 1d
11 ± 0f
300 ± 12a
260 ± 7b
22 ± 2c
256 ± 12d
21 ± 3e
241 ± 7d
10 ± 1e
2.8 ± 0.8a
3.2 ± 0.9a
0.5 ± 0.1b
1.2 ± 0.3d
2 ± 0.9d
0.9 ± 0.2d
0.1 ± 0.0e
Note. The data of three irradiation doses from Exp. 1 were pooled because ANOVA analysis showed no significant difference among them. The data from the
two irradiation doses from Exp. 2 were pooled because t-test analysis showed no significant difference between them except for the SV weights of 11-week
treatments (9 ± 0 mg at 12 Gy vs. 13 ± 2 mg at 13.5 Gy), both of which were dramatically lower than those in control mice. N was between 9 and 29 for weights
and between 5 and 12 for serum testosterone concentrations. For a given parameter, values that are significantly different from each other (p < 0.05) within each
experiment are indicated by different letters.
Exp. 1 (A) and Exp. 2 (B). The TDI is the percentage of tubule cross sections with more than three differentiated germ cells. ‘‘Endogenous’’ indicates the
differentiated germ cells that were derived from surviving stem spermatogonia of the recipient. ‘‘Donor’’ indicates the differentiated germ cells that were derived
from GFP-positive transplanted stem spermatogonia. The statistical analyses of 12- and 13.5-Gy donor TDI data in panel (B) were conducted by a nonparametric
method because the data are not normally distributed. N ¼ 3–4 (panel A) and N ¼ 4–18 (panel B). ‘‘a’’ and ‘‘b’’, p < 0.05 versus the sham- or 4-week–treated
Hormonal suppression improved spermatogenic recovery from both endogenous and donor-derived stem spermatogonia in irradiated mouse testis in
FERTILITY RESTORATION IN IRRADIATED MICE
Effect of Hormonal Suppression on Colony Development
from Transplanted Stem Spermatogonia (Exp. 2)
Gross examination of recipient testes under fluorescence
microscopy showed that transplanted spermatogonia success-
fully colonized the testes in all treatment groups (Figs. 3A–C).
Immunohistochemical staining for GFP was then performed on
tissue sections (Figs. 3D–F) to determine which tubules were
repopulated with spermatogenic cells derived from donor versus
endogenous stem cells. We noted that the overall efficiency of
donor-derived stem cell colonization was lower in the 12-Gy
study than that in the 13.5-Gy study (Fig. 2B). This discrepancy
is attributable to the older age of the donor animals used in the
12-Gy study as previous observations showed that the efficiency
of colonization by cells from 28-day-old donors was only half
that of cells from 12-day-old donors (McLean et al., 2003).
When testosterone was suppressed for either 4 or 11 weeks
in the irradiated recipient mouse testes, the recovery of
spermatogenesis from transplanted stem spermatogonia was
in irradiated recipient testes that were sham treated (A) or given 4-week GnRH-ant plus flutamide (B) or 11-week GnRH-ant plus flutamide (C) treatments.
Immunohistochemical staining for GFP in fixed tissue cross sections from (D) sham-treated mice, (E) 4-week GnRH-ant plus flutamide treatment, or (F) 11-week
GnRH-ant plus flutamide treatment was performed to distinguish tubules with donor-derived spermatogenesis (brown) from those with endogenous
spermatogenesis (blue). Note that there is some nonspecific staining of cytoplasm of the late spermatids. GFP-positive tubules were separated from recipient testis
and imaged by fluorescence (G) for measurement of colony length or by bright field (H) to clearly show the tubule. Bars, 200 lm (A, B, C, G, and H) and 100 lm
(D, E, and F).
Evaluation of the recipient testes. GFP-expressing donor cell colonization was visualized at 11 weeks after transplantation by fluorescence microscopy
WANG ET AL.
consistently improved in two separate studies with different
doses of irradiation (Fig. 2B). The results from Model 1
showed that the 4-week hormonal suppression treatment
significantly (p < 0.0001) increased the TDI of donor cells
over that observed with no hormonal suppression by 3.1-fold,
whereas the 11-week treatment significantly (p < 0.0001)
increased the TDI of donor cells by 4.8-fold. Furthermore, the
increase in TDI with 11-week therapy was greater than that
with the 4-week treatment (p ¼ 0.002). The effects of hormonal
suppression appeared to be greater toward donor than toward
endogenous stem cells (p for interaction term ¼ 0.07), and the
fold increases in TDI with both 4- and 11-week hormonal
suppressions were greater toward the donor than toward the
endogenous cells (p ¼ 0.002 and p < 0.0001 for 4- and
11-week hormonal suppression, respectively).
Because the relationship between the TDI, which is the
percentage of tubule cross sections with differentiating germ
cells, and the actual number of developing donor germ cell
colonies is dependent on the length of those colonies, we
measured the colony lengths (Fig. 3G) after the different
hormonal treatments. The length of donor cell colonies (12-Gy
dose, Exp. 2) was 512 ± 12 lm (n ¼ 9 colonies from three
testes) in sham-treated mice, 498 ± 14 lm (n ¼ 36 from four
testes) in mice receiving 4-week hormonal suppression, and
464 ± 34 lm (n ¼ 26 from four testes) in mice receiving
11-week hormonal suppression. These values were not signif-
icantly different from each other. These results demonstrate that
the TDI values were proportional to numbers of colonies and
that the higher TDI in the 11-week hormonal suppression group
was actually because of increased colony numbers.
Effect of Hormonal Suppression on Regeneration of
Reproductive Tissues and Spermatogenesis (Exp. 3 and
Because hormonal suppression was able to stimulate the
initiation of spermatogenesis from both endogenous and donor-
derived stem spermatogonia, we tested whether the spermato-
genic recovery was maintained after treatment and whether
spermatozoa were produced.
We first examined the time course of the recovery of the
androgen-dependent reproductive accessory organs after either
10.5-Gy irradiation (Exp. 3) or 13.5-Gy irradiation (Exp. 4).
Because androgen is required for the development of
spermatozoa and mating behavior, we monitored the re-
versibility of testosterone suppression by SV weights. In the
mice receiving GnRH-ant (acyline) and flutamide for the first
10 weeks after irradiation, the SV weights remained low at
16 weeks but returned to levels observed in the non–hormone-
suppressed mice by 21 weeks (Figs. 4A and 4F). Note that the
epididymal weights, which were reduced primarily by hormonal
suppression, although sperm numbers may also have some effect,
took between 31 and 46 weeks to recover to the levels observed
in the irradiated mice that did not receive hormonal suppression
(Figs. 4B and 4G).
Spermatogenesis gradually recovered in 10.5-Gy irradiated
testes not receiving hormonal suppression treatment, as
indicated by gradual increases in testis weights, testicular
sperm head counts, and TDIs (Figs. 4C and 4D and 5A). The
suppression of testosterone significantly improved the progress
of spermatogenic recovery, as shown by the significant
increases of testis weights (Fig. 4C), testicular sperm head
count (Fig. 4D), and TDI (Fig. 5A) at the 16-, 21-, and 31-week
postirradiation time points. Model 2 showed that the TDI ratio
was overall significantly greater than 1 (p < 0.0001); it
appeared to be highest at early time points (week 16, 1.78-fold)
and showed a downward trend (p ¼ 0.08) with time. The cauda
epididymal sperm counts were lower in the hormone-
suppressed mice than in controls at week 16 as a result of
the residual effect of the hormonal suppression. However, they
were significantly increased in the previously hormone-
suppressed group by week 21 and were significantly higher
than those in the sham-treated controls on weeks 31 and 46
(Fig. 4E). However, it should be noted that epididymal sperm
counts are more variable than testicular sperm head counts, can
be affected by sperm storage or hormone levels because of
changes in transport rate, and show an attenuated response to
changes after irradiation (Meistrich and Samuels, 1985).
We then examined the time course of restoration of sper-
matogenesis in mice irradiated with a higher dose (13.5 Gy) to
deplete endogenous spermatogenesis and subjected to the
hormonal suppression treatment and/or germ cell transplantation
(Exp. 4); four treatment groups: sham, hormonal suppression
only, transplantation only, and the combination of hormonal
suppression and transplantation were analyzed. In 13.5-Gy
irradiated testes in which only sham transplantation was
performed, hormonal suppression improved the spermatogenic
recovery from endogenous spermatogonia, as indicated by
elevated sperm head count in the testis (Fig. 4I) and increased
endogenous TDIs (Figs. 5B and C) at nearly all time points, with
the differences being statistically significant at several of these
Transplantation of germ cells alone, without hormonal
suppression, resulted in the formation of donor colonies in up
to 10% of the tubules at the 21-, 31-, and 46-week time points
(filled squares, Fig. 5C), but these donor colonies did not
produce enough cells to significantly increase testis weights
(filled inverted triangles, Fig. 4H). However, in these trans-
planted mice, 10-week suppression of testosterone markedly
enhanced recovery of spermatogenesis as measured by
significant increases in testis weights during weeks 16–31
after irradiation (open inverted triangles, Figs. 4H and 4I).
Testicular sperm head counts, which were only measured at
week 46, were also increased (triangles, Fig. 4I). This was
clearly a result of enhanced spermatogenesis from transplanted
spermatogonia, compared with mice receiving transplantation
but not hormonal suppression, as shown by significant
increases of donor TDI (open squares, Fig. 5C) at all time
FERTILITY RESTORATION IN IRRADIATED MICE
Model 3 showed that the 10-week hormone suppression
significantly increased the TDI ratio for both donor and
endogenous stem cells in this experiment and that the TDI ratio
for donor stem cells was greater than for endogenous stem cells
(p ¼ 0.0003). Although effect of time of assessment was not
statistically significant, the increases in donor TDI ratios were
between 4.7-fold (at the 11-week time point) and approxi-
mately sevenfold (later time points).
In general, the spermatozoa numbers in cauda epididymis in
all treatment groups were relatively low and variable from
animal to animal (Fig. 4J). However, during the 21- to 46-week
time period, most of the mice with the highest sperm counts
measured by SV weight (A and F), epididymal weight (B and G), testis weight (C and H), testicular sperm head count (note log scale) (D and I), and cauda
epididymal sperm count (E and J). Groups are designated as Sham, Hormone (GnRH-ant þ flutamide only, no transplantation), Transplant (transplantation only),
Hormone þ Transplant (GnRH þ flutamide and spermatogonial transplantation). N ¼ 10 for 46-week time point data and 5 for all other time points for both
irradiation doses. ‘‘a,’’ ‘‘b,’’ and ‘‘c’’, p < 0.05 versus values in the sham group, hormone group, and transplant group, respectively. Ellipses in (K) identify those
mice that were fertile.
Time course of accessory sexual organ or spermatogenic recovery in mice irradiated with 10.5 Gy (A–E) from Exp. 3 or 13.5 Gy (F–J) from Exp. 4, as
WANG ET AL.
were those treated with both hormonal suppression and germ
Effect of Hormonal Suppression on Restoration of Fertility
(Exp. 3 and Exp. 4)
The recovery of spermatogenesis by hormonal suppression
treatment observed in Exp. 3 was indeed translated into
function as fertility was restored in 90% of the treated mice
during 20–40 weeks after irradiation. This was significantly
higher than the recovery of fertility in the mice without
hormonal suppression, in which only 10% recovered (p <
0.001) and the recovery did not occur until week 37 (Fig. 6A).
Although a high level of fertility was restored in the irradiated,
hormonally suppressed mice, their fecundity was only at most
a quarter of that of unirradiated, non-hormonally treated control
mice (Fig. 6C). During the 21- to 46-week postirradiation time
periods, only 83% of the treated males were fertile, litter size
was 5.2 compared with 8.9 in normal controls, and the number
of litters they produced per 5-week period was only half that of
Although mice irradiated with 13.5 Gy (Exp. 4), even if they
were treated with either hormonal suppression or transplantation,
were sterile up to 46 weeks after irradiation, combined treatment
with hormonal suppression and transplantation successfully
restored fertility of three mice between 15 and 30 weeks after
irradiation (Fig. 6B) (p ¼ 0.09). However, it is not clear why
there was a trend of declining fecundity and also testis weights
(Fig. 4H) at week 46, but not decreases in donor cell TDI
(Fig. 5C). In the hormonally treated, transplanted mice, the
fecundity was only 5% of controls (Fig. 6D), as at most 20% of
mice were fertile during any time period; average litter sizes were
only 6.9; and the frequency of litters was 40% of that in controls.
In every litter produced by the transplanted, hormonally
suppressed mice, both GFP-positive and GFP-negative pups
were present, and of the total of 76 pups from 11 litters, 43
were GFP positive. Because the donor animals we used were
hemizygous for GFP, we expected that half of spermatozoa
from donor stem spermatogonia would carry the GFP trans-
gene. Thus, nearly all the spermatozoa in those fertile mice
must have developed from transplanted stem spermatogonia.
In the current study, for the first time, we demonstrated that
hormonal suppression given after irradiation successfully
accelerated the recovery of spermatogenesis and significantly
shortened the time to return of fertility in irradiated mice.
Moreover, the suppression regimen also enhanced the efficiency
of transplanted cell colonization in irradiated mouse recipient
testes and resulted in the production of progeny mice derived
from the donor cells.
The duration, timing, and degree of hormonal suppression all
appeared to be important for successful recovery from
spermatogenic injury in the irradiated mouse testis. In previous
attempts to stimulate spermatogenic recovery following
cytotoxic damage in mice, hormonal suppression was generally
given prior to or during irradiation or chemotherapy (da Cunha
et al., 1987; Kangasniemi et al., 1996a; Nonomura et al.,
1991). The durations of hormonal suppression treatment were
generally short, being less than 4 weeks in these studies. Two
studies (da Cunha et al., 1987; Nonomura et al., 1991) used
treatment with a GnRH agonist, which is not as effective as
GnRH-ant at hormonal suppression (Meistrich et al., 2001). In
a third study, hypogonadal mice with a null mutation in the
GnRH gene were used (Crawford et al., 1998), but there was
still some basal production of testosterone from the Leydig
cells in these mice (Singh et al., 1995). Thus, the androgen
ablation in most of the studies was incomplete. Only one of the
studies employed GnRH-ant and antiandrogen to produce total
hormonal suppression. Furthermore, in the transplanted testes, the TDI of the endogenous cells and donor cells were scored separately. N ¼ 10 for 46-week time
point group and 5 for all other time point groups for both irradiation doses. The statistical analysis of 46-week TDI data in panel (B) was conducted by
a nonparametric method because the data are not normally distributed. ‘‘a’’ and ‘‘c’’, p < 0.05 versus values in the sham group and transplant-only group,
TDIs in testes of mice irradiated with 10.5 Gy (A) from Exp. 3 or 13.5 Gy (B, endogenous and C, transplanted) from Exp. 4, with and without
FERTILITY RESTORATION IN IRRADIATED MICE
androgen ablation, but the duration of treatment was only
2 weeks (Kangasniemi et al., 1996a). The success of our
current treatment strategy is likely attributable to the
suppression of testosterone immediately in the postirradiation
period, a relatively prolonged treatment period, and perhaps the
use of an antiandrogen to completely inhibit the action of the
In comparison to the less than twofold stimulation of
endogenous colony formation observed here in the mouse,
hormonal suppression in the rat using GnRH-ant alone for as
short as 4 weeks (Shetty et al., 2000) or GnRH-ant plus
flutamide for 2 weeks, either prior to or after cytotoxic therapy
(Kangasniemi et al., 1995; Shetty et al., 2006a), produced
much greater stimulation of endogenous colony formation.
Because hormonal suppression stimulates recovery of sper-
matogenesis in irradiated rats by reversing the block to
spermatogonial differentiation (Meistrich and Shetty, 2003;
Meistrich et al., 2000), this interspecies difference in
stimulation appears to be related to differences in the
magnitude of the differentiation block that is produced by the
The duration, timing, and degree of hormonal suppression
are also of importance in enhancing colonization of trans-
planted spermatogonia in recipient mouse testes. In the present
study, the hormonal suppression was always started 3 weeks
before transplantation. When the hormonal suppression was
continued for only 1 week more after transplantation, there was
an average of a 3.1-fold stimulation of the TDI (Exp. 2), and
when it was continued for 7 or 8 weeks (Exp. 4 or Exp. 2,
respectively) after transplantation, the TDI was increased
significantly to 4.8-fold. Analysis of three previous studies
using hormonal suppression with GnRH agonists (Dobrinski
et al., 2001; Ogawa et al., 1998; Ohmura et al., 2003) given for
4 weeks indicates a consistent enhancement of donor
spermatogenesis by about 1.9-fold. GnRH agonist suppressive
treatments lasting 8–10 weeks stimulated increases in coloni-
zation by about threefold (Dobrinski et al., 2001; Kanatsu-
Shinohara et al., 2004). By comparison, our hormonal
suppression strategy with the GnRH-ant and antiandrogen
appears to be somewhat more effective.
Several processes are needed for the development of
spermatogenesis from transplanted stem spermatogonia. Hom-
ing involves moving to the basement membrane of seminifer-
ous tubules to already existing niches or formation of new
niches. Individual stem cells must then proliferate, undergoing
self-renewing divisions to increase their numbers (de Rooij,
2001). Finally, the stem cells must reach a steady state of self-
renewalanddifferentiationdivisions to producelater
suppression (A) or irradiated with 13.5 Gy with or without hormone suppression or spermatogonial transplantation (B) by Kaplan-Meier survival analysis. Relative
fecundity (fraction of males that were fertile 3 litters per fertile male per 5-week time period 3 average litter size) for male mice irradiated with 10.5 Gy, with or
without hormone suppression (C), or irradiated with 13.5 Gy, with or without hormone suppression or spermatogonial transplantation (D). Note, for comparison,
that the calculated Relative fecundity for our unirradiated, non-hormonally treated control mice was 21.4. Symbols in panels (A and B) represent times at which
individual mouse became fertile or were censored (i.e., the time of euthanasia before they became fertile). *p < 0.05 compared with values of sham-treated group.
#p < 0.05 compared with values of sham, hormonal suppression, or transplant groups.
Time course of fertility recovery expressed as probability of having returned to fertility for male mice irradiated with 10.5 Gy with or without hormone
WANG ET AL.
spermatogenic cells while maintaining their populations. The
use of two different hormonal suppression times and the
evaluation of both endogenous and donor-derived spermato-
genesis in the same animals allowed us to determine the
relative effect of hormonal suppression on homing versus
The greater stimulation of tubule repopulation by transplanted
cells, which were introduced after 3 weeks of hormonal
suppression, than by endogenous stem cells must have been
due primarily to an increase in the homing efficiency of the
donor cells. The 3.1-fold stimulation by the 4-week hormonal
suppression treatment was primarily because of enhancement of
the homing step. The additional stimulation to 4.8-fold from
continuing the treatment to week 11 can be attributed to effects
on proliferation/differentiation. Previous observations showing
that hormonal suppression prior to but not after transplantation
stimulates recovery (Dobrinski et al., 2001) are consistent with
the effect on the homing step but not the additional stimulation
we observed with continuing the hormone suppression after
The enhancement of the abilities of stem cells to both
produce differentiated cells and maintain their numbers,
independent of the homing process, by hormonal suppression
must be responsible for the stimulatory effect of the 4-week
suppression on the endogenous TDI and the greater effective-
ness of the longer than the shorter treatment on both
endogenous and donor TDI. Previously, a 1.8-fold increase
in donor cell colony number was observed even when the
hormonal-suppressive treatment was not started until 4 weeks
after transplantation (Ohmura et al., 2003).
The molecular and cellular mechanisms by which hormonal
suppression facilitates homing of transplanted stem cell
spermatogonia and enhances their proliferation and/or differen-
tiation are unclear. Because cellular alterations produced by
irradiation that inhibit the differentiation of both endogenous
and transplanted spermatogonia in the rat are in the somatic
environment (Zhang et al., 2007), which contains the cells that
have the receptors for testosterone and the gonadotropic
hormones, it is most likely that hormonal suppression enhances
germ cell development from spermatogonial stem cells by action
on the supporting somatic environment. Recently, b1-integrin,
expressed in both the stem spermatogonia and the Sertoli cells,
has been identified as an essential adhesion receptor for the
homing of mouse transplanted stem spermatogonia (Kanatsu-
Shinohara et al., 2008). However, analysis of gene expression
changes in irradiated rat testes (Zhou et al., 2010) and SCARKO
mouse testes (Wang et al., 2009) (Zhou, Wang, Small, Liu,
Weng, Yang, Griswold, and Meistrich, submitted) showed that
neither b1-integrin nor its targets, the various laminin genes, is
upregulated by hormonal suppression or abrogation of androgen
action on Sertoli cells and therefore do not appear to be involved
in the enhancement of homing. Further studies of the hormonal
regulation of stem spermatogonial homing and proliferation/
differentiation are necessary to understand this phenomenon.
The combined treatment of hormonal suppression and
spermatogonial transplantation appears to be necessary for
promoting recovery of spermatogenesis and fertility, especially
after relatively high radiation doses. Although hormonal
suppression alone restored fertility after 10.5-Gy irradiation,
it did not increase endogenous spermatogenesis to the level
necessary for fertility in 13.5-Gy irradiated mice. Spermato-
gonial transplantation supplemented the recipient testes with
stem spermatogonia, providing an additional source for
repopulation of the testes.
Our results demonstrated that functional spermatozoa de-
veloped after hormonal suppression and/or transplantation. The
fecundity was low compared with wild-type mice, as expected
because the testicular sperm counts were below or barely above
the cutoff of 2 3 106(15% of control) necessary for recovery
of fertility after irradiation (Meistrich et al., 1978). This result
also illustrates the interspecies difference, as in irradiated rats
hormonal suppression for 10 weeks restored endogenous
testicular sperm production to 86% of control and nearly
completely restored fecundity (Meistrich et al., 2001).
The application of the hormonal suppression treatment and
spermatogonial transplantation can be considered as treatment
for men exposed to testicular toxicants. Although clinical trials
of hormonal suppression alone have not been very successful,
it is likely in several of these studies that the gonadal damage
was so severe that there were too few stem cells to yield
significant recovery (Meistrich and Shetty, 2008). Combined
treatment of hormonal suppression and spermatogonial trans-
plantation should be more promising because hormonal
suppression more significantly stimulated homing of the
transplanted germ cells than it did recovery of endogenous
spermatogenesis. The development of methods for improve-
ment of transplantation efficiency are most important for
fertility preservation in prepubertal boys who are subjected to
gonadal damage from a cytotoxic cancer treatment but are too
young to be able to produce sperm for storage. The mouse may
be a better model than the rat for preclinical trials because the
stimulation of recovery occurs without reversal of a major
block of spermatogonial differentiation as occurs in the rat.
and R01 ES-008075 to M.L.M.); Cancer Center Support (Grant
number CA-016672 to M. D. Anderson Cancer Center).
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guidance in statistical analysis, and Mr Walter Pagel for his
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