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
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FERTILITY RESTORATION IN IRRADIATED MICE