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From the Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas.
| Correspondence to: Dr Marvin Meistrich, Department of Experimental Radiation Oncology, Unit 066, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (e-mail: meistrich{at}mdanderson.org). |
| Received for publication September 14, 2005; accepted for publication November 28, 2005. |
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key words: Irradiation, busulfan, testis, spermatogonial transplantation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A critical factor in male germ cell transplantation is the preparation of recipients (Ogawa et al, 1999b; Brinster et al, 2003). Maximal depletion of endogenous germ cells and emptying of stem cell niches for donor cells, with minimal damage to the local spermatogenic and systemic environment, is required. Donor stem cell engraftment and spermatogenesis were more successful in recipient testes treated to ablate endogenous stem cells than they were in untreated testes (Shinohara et al, 2002).
Ablation of endogenous stem cells can be done by introducing genetic mutations or with chemical or physical treatments. Mutant mice, such as the W/Wv line (Ohta et al, 2003), provide a supportive environment for donor spermatogenesis (Ogawa et al, 2000) and have been widely used as recipients, but they must be immunocompatible with the donors (Kanatsu-Shinohara et al, 2005), and the infertility of these mice makes their production difficult (Brinster and Avarbock, 1994). No other species with comparable genetic mutations are available.
The sole effective chemical treatment used so far to prepare male germ cell transplant recipients is busulfan. Busulfan doses less than 40 mg/kg given to adult mice did not result in prolonged depletion of endogenous spermatogenesis in most tubules (Kanatsu-Shinohara et al, 2003b); higher doses often caused severe hematopoietic suppression requiring bone marrow transplantation or resulted in death (Ogawa et al, 1999a). Prenatal exposure to busulfan by treating the mother has also been done, but the dose had to be reduced to avoid pregnancy failure and thus resulted in recovery of endogenous spermatogenesis in the offspring (Brinster et al, 2003). In rats, busulfan is more toxic (Sternberg et al, 1958), and the therapeutic index is lower. When busulfan was given in a fractionated regimen to avoid severe systemic toxicity (Ogawa et al, 1999b; Zhang et al, 2003), endogenous spermatogenesis recovered in most of the tubules (Jiang, 1998), but when a high single dose was given to cause prolonged depletion of spermatogenesis in most tubules, bone marrow transplantation was required (Udagawa et al, 2001). Data on the effects of busulfan on spermatogonia in other species are limited (Stellflug et al, 1985; Anserini et al, 2002). and the doses used were close to lethal. Busulfan is given to humans only in conjunction with bone marrow transplantation. Thus, it is unlikely that busulfan will be widely used in a variety of species for recipient preparation.
Physical methods for preparing testes as recipients could avoid the systemic toxicity that usually is a problem with busulfan. Although testicular cooling depleted all germ cells from many tubules, no donor spermatogenesis was observed after transplantation into the cooled testes (Zhang et al, 2004). Localized radiation is effective at killing endogenous stem cells in mouse testes (Lu et al, 1980). However, reports showing differentiation of transplanted spermatogonia in the depleted tubules are limited. In one study (Creemers et al, 2002), only 2 irradiated mice were recipients (1 testis of each mouse) for transplantation with wild-type mouse spermatogenic cells, and normal spermatogenesis was reported in 20%-25% of tubules. In another study, transplanted mouse testicular cells colonized and showed differentiation in recipients irradiated with 3 Gy (Giuili et al, 2002), but this dose was too low to deplete a significant number of endogenous stem cells. The effectiveness of testicular irradiation for preparing recipients to efficiently support differentiation of transplanted spermatogonia has not yet been evaluated. We undertook this study to determine the ability of irradiated mouse testes to serve as recipients of mouse and rat spermatogonial transplants and support the differentiation of the transplanted cells and to compare these results with results for busulfan-treated recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of Recipients
The nude mice were irradiated without anesthesia. They were restrained in
plastic chambers (Lucite boxes) placed on a metal shield with a 3-cm diameter
hole, so that only the lower abdominal and scrotal areas were irradiated.
Radiation was delivered at a dose rate of 5.6 Gy/min using a dual-source
137Cs 
-ray unit. To deplete endogenous stem cells by
radiation, fractionated regimens are preferred over single-doses because the
testes of many strains of mice and other species in contrast to other tissues
are more sensitive to fractionated radiation
(Withers et al, 1974;
Meistrich et al, 1984).
Preliminary data from our laboratory compared the affects of a single dose, 2
equal daily fractions, and 4 equal daily fractions on spermatogonial depletion
in C57BL/6 and Swiss/Ncr nude mice (G. Wilson and M. L. Meistrich, unpublished
data). Two doses of 8 Gy given 24 hours apart were most effective in reducing
tubular repopulation without other damage. Because even a small initial
radiation dose induces the remaining spermatogonial stem cells to become
radiosensitive, other investigators have used an initial dose of 1.5 Gy,
followed by a larger dose of 12-16 Gy 24 hours later
(Creemers et al, 2002). In
this study, we used 2 fractions of irradiation (8 + 8 Gy, 1.5 + 12 Gy, or 1.5
+ 14 Gy) administered 24 hours apart to mouse testes to determine an optimal
dose to deplete endogenous germ cells without severely damaging the various
somatic components of the testis.
Busulfan (Sigma, St Louis, Mo) was first dissolved in dimethyl sulfoxide (Sigma), then an equal volume of sterile water was added to obtain a final busulfan concentration of 8 mg/mL. The aqueous dilution was maintained at a temperature slightly greater than 37°C until injection to prevent the busulfan from crystallizing and to keep it in solution. Different doses were intraperitoneally injected to determine a dose of busulfan that would produce depletion of endogenous spermatogenesis similar to that of irradiation.
Mice were used for germ cell transplantation 3-5 weeks after either irradiation or injection of busulfan.
Preparation of Donor Cells
Immature mice (10-20 days old) or rats (10-11 days old) were used as
donors. To preferentially harvest single cells from the tubules, the tunica
was removed, and the tissue was sequentially digested with enzymes at 35°C
in a shaking water bath (Zhang et al,
2003). Two digestions were performed in DMEM/F12 medium (Gibco,
Carlsbad, Calif) containing DNase I (Cat #DN25; Sigma) at 100 mg/mL and 1%
fetal bovine serum (HyClone, Logan, Utah) with 0.05%-0.1% collagenase IV (Cat
#4188; Worthington Biochemical Corp, Lakewood, NJ) for 20-30 minutes and then
with 0.05%-0.1% of collagenase and 0.05% of hyaluronidase (Cat #2592;
Worthington) for 20 minutes. Tubules were finally digested with 0.1% trypsin
(Cat #3704; Worthington) and 100 mg of DNase I/mL in Dulbecco's phosphate
buffered saline (PBS, Gibco) containing 1 mM EGTA (Cat #E-4378; Sigma) for
10-15 minutes. The final pellets of cells were resuspended in DMEM/F12
containing 10% fetal bovine serum.
Trypan blue (Gibco) was added to a final concentration of 0.02%. After the cell concentration and viability was determined, the cell suspension was kept on ice until transplantation. The cell viability ranged from 91% to 99% (mean viability, 96%), and the cell concentration ranged from 3.6 x 107 to 5.8 x 107 cells/mL (mean cell concentration, 4.4 x 107 cells/mL).
Transplantation
Mice were anesthetized with a mixture of ketamine (6.7 mg/mL) and xylazine
(1.3 mg/mL) given at 0.15 mL/10 g body weight. After incision of the lower
abdomen and exposure of testis, a glass needle (tip inner diameter, 20-25
µm with a 25° angle) was inserted into the efferent duct, and donor
cells were injected into rete testes using a FemotoJet semiautomatic
microinjector (Brinkmann Instruments Inc, Westbury, NY). A mean of 8 µL
(range, 1-15 µL) of cell suspension containing approximately 40 x
104 cells was injected into each recipient testis. Trypan blue was
used as marker to monitor the success of the injection.
Macroscopic and Microscopic Assessment of Spermatogenesis
Mice were killed at different times after irradiation or busulfan
treatment, and body and testis weights were recorded. Testes were fixed in
Bouin's solution or 4% paraformaldehyde solution at 4°C and embedded in
paraffin or plastic. Sections with a thickness of 4-5 µm were cut and
stained with periodic acid-Schiff-hematoxylin for counting germ cells in the
seminiferous tubules. All tubules in a cross-section were counted (mean
number, 145 tubules per cross-section). The tubule differentiation index (TDI)
is the percentage of tubules showing differentiation, which is defined as the
presence 3 or more germ cells in a tubule reaching the B spermatogonia stage
or beyond.
Recipients were killed 10 weeks after mouse-to-mouse transplantation or 13 weeks after rat-to-mouse transplantation. For recipient testes in which colonies of donor cells were to be counted, the testis was placed in cold PBS containing 0.05% collagenase, the tunica albuginea was removed, and the testis was gently teased apart at room temperature within 5-10 minutes and removed and placed in cold PBS. After the number of GFP-positive colonies in the recipient testes were counted under fluorescence, the testes were fixed in 4% paraformaldehyde solution overnight at 4°C and subjected to routine histologic processing. The other recipient testes were fixed directly in 4% paraformaldehyde solution after the tunica was removed in PBS.
Immunohistochemical Assessment of Spermatogenesis
After routine dewaxing of paraffin sections and rehydration, slides were
boiled for 3 minutes in Antigen Retrieval Citra Plus solution (Cat #HK080-9K;
BioGenex, San Ramon, Calif) on a hot plate and allowed to cool for 1 hour. The
sections were treated for 5 minutes with 0.6% hydrogen peroxide (Sigma) to
block endogenous peroxidase activity and for 1 hour with 5% bovine serum
albumin (Sigma) or serum of the same species as the second antibody to block
nonspecific background staining. Serial sections were stained with 2 different
primary antibodies: either the rat anti-mouse monoclonal anti-GCNA1 antibody
(1:100 dilution, a gift from Dr George Enders) or the rabbit polyclonal
anti-GFP (1:5000 dilution) (Cat #NB600-303; Novus Biologicals, Littleton,
Colo) was added to tissues and incubated overnight at 4°C. ABC Elite kits,
second antibodies, and 3,3'-diaminobenzidine were all ordered from
Vector Laboratories (Burlingame, Calif) and used according to procedures
recommended by the manufacturer. Sections were counterstained with
hematoxylin. For immunofluorescence staining, goat anti-rabbit IgG (Alexa
Fluor 488; Molecular Probes Inc, Eugene, Ore) was used at a 1:500 dilution.
Sections were counterstained with DAPI.
Germ cells were identified after anti-GCNA1 staining, and the total TDI was calculated. The number of GFP-positive tubules was assessed in histologic sections after anti-GFP staining. The most advanced stage of GFP-positive differentiated cells was determined in each tubule by histologic criteria. All values were obtaining by counting all tubules taken from 3 sections at least 25 µm from each other per testis.
Statistical Analysis
Testing for statistically significant differences (P < .05) between the
irradiated group and the busulfan-treated group was performed using the SPSS
statistical package (version 11.5; SPSS Inc, Chicago, Ill) and a Student's
t test, unless otherwise specified.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were given 4 different doses of busulfan and compared with another group of mice simultaneously irradiated with 1.5 + 12 Gy. We were surprised that all nude mice with this outbred genetic background survived the highest dose of 60 mg of busulfan per kg of body weight without needing bone marrow transplants, appeared healthy throughout the experiment, and showed no long-term reduction in body weight (Table 2). The response to busulfan was much more variable than the response to irradiation. Each group had clear outlier data, with TDI values of 64%-100%. Even when these data were excluded, the coefficients of variation of the TDI values were higher in the busulfan groups than in the irradiation group. Clear dose responses were seen in the reductions of TDI and testis weights with increasing busulfan dose. The lowest busulfan dose of 44 mg/kg reduced the TDI to only approximately 50% at 5 weeks after treatment, which indicated that stem cell killing was insufficient to largely eliminate endogenous spermatogenesis. TDI levels (approximately 5%) in the testes of most of the mice receiving busulfan doses of 50-55 mg/kg were similar to those in the testes irradiated with 1.5 + 12 Gy. None of the busulfan-treated testes had any calcified tubules, unlike the testes of 8 of the 10 mice irradiated with 1.5 + 12 Gy, which showed calcified tubules 5 weeks after irradiation (P <.001, by the Mann-Whitney U test) (Tables 1 and 2).
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Counts of colonies by fluorescence microscopy showed similar numbers in the irradiated and busulfan-treated testes (Table 3). Histologic analysis also showed that there were similar numbers of cross-sections in which the germ cells were immunostained for GFP in irradiated and busulfan-treated recipient testes and that the percentages of these colonies in which differentiated cells were found were the same in both treatment groups. We conclude that busulfan-treated and irradiated recipient testes demonstrated no significant differences in donor cell colonization and donor spermatogenic development.
Immature Rat Testes-Derived Donor Cell Spermatogenesis in Recipient Mouse Testes
To test whether the irradiated mouse testes could support donor rat
spermatogenesis, testicular cells collected from immature rat testes
expressing GFP were successfully transplanted into the tubules of irradiated
and busulfan-treated mouse testes. Thirteen weeks later, fluorescence
microscopy of testicular tissue revealed that regions of tubules in both the
busulfan-treated (Figure 3a)
and irradiated mice (Figure 3b)
showed GFP fluorescence, which demonstrated that rat donor cells had colonized
the recipient tubules. These were tubules repopulated with donor rat cells, as
determined by anti-GFP antibody (green fluorescence was present), but both
busulfan-treated and irradiated testes also contained tubules repopulated by
host mouse cells (only DAPI staining was present)
(Figure 3c and d). This was
confirmed further when the germ cells were identified by staining serial
sections with anti-GFP and anti-GCNA1
(Figure 3e and f). Because the
anti-GCNA1 antibody was originally developed against mouse germ cells
(Enders and May, 1994), it has
species-specific sensitivity and is more reactive with mouse germ cells than
with rat germ cells. The GFP-positive tubules showed definite but weak
staining for GCNA1, demonstrating that these were indeed germ cells. Pale
GCNA1-staining of these germ cells further demonstrated that they developed
from donor rat spermatogonial stem cells. However, anti-GCNA1 staining in
tubules that were GFP negative was much stronger, proving that they were
derived from host mouse stem cells, although some tubules showed an admixture
of donor-derived and host-derived spermatogenesis
(Figure 3g, arrow). Some
tubules with donor-derived spermatogenesis differentiated incompletely, even
13 weeks after transplantation (Figure
3g, arrowhead), but elongated spermatids with the definite nuclear
shape of elongated rat spermatids could be found in some GFP-positive tubules
(Figure 3h, arrows and
inset).
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Discussion |
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An issue with busulfan treatment, which has been the most common method of preparing the recipient testis since germ cell transplantation was developed (Brinster and Zimmerman, 1994), is the strain dependence of its systemic and spermatogenic toxicity. Doses of 40-45 mg/kg were sufficient in C3H mice (Bucci and Meistrich, 1987) or C57BL/6 mice (Kanatsu-Shinohara et al, 2003b) to reduce the fraction of tubules showing recovery of spermatogenesis 5 weeks later to 1%. However, in ICR mice, there was nearly complete recovery of spermatogenesis within 12 weeks after similar doses (Choi et al, 2004). In the Swiss/Ncr nude mice used in our study, a 44 mg/kg dose resulted in endogenous recovery in approximately 50% of seminiferous tubules 5 weeks after treatment; a 60 mg/kg dose was required to maintain depletion in 99% of the tubules in most mice. In some strains, busulfan doses of 40-45 mg/kg caused the death of some mice (Bucci and Meistrich, 1987), and bone marrow transplantation was usually required for survival of other mice (Kanatsu-Shinohara et al, 2003a). In contrast, Swiss/Ncr nude mice could endure a single busulfan dose of 60 mg/kg without obvious toxicity. Nevertheless, the therapeutic index was not much improved, because higher doses were also required to deplete the seminiferous tubules of endogenous stem cells.
Our data from the irradiated mice confirmed that a small initial dose followed by a larger dose was as effective at depleting spermatogenesis as a higher total dose given in equal fractions, with a lower tendency to produce calcified tubules (Table 1). However, the fractions of tubules depleted of spermatogenesis by 1.5 + 12 Gy in our nude mice was less than the 99% and 97% depletion reported for Nc/CpbU mice and for a different strain of nude mice (NMRI, Hsd/Cpb), respectively, 12 weeks after irradiation and less than the 89% depletion for the NMRI nude mice 21 weeks after irradiation (Creemers et al, 2002).The difference was most likely due to a background strain-specific response to fractioned radiation (Meistrich et al, 1984). Our preliminary results also showed that there was greater depletion of spermatogenesis in C57BL/6 than in the nude mice at equivalent radiation doses (G. Wilson and M. L. Meistrich, unpublished data).
We noted that the recovery of endogenous spermatogenesis after irradiation was not significantly affected by transplantation. In testes transplanted with immature mouse testicular cells, 73% of tubules showed recovery, but approximately 9% (ie, the percentage of GFP-positive tubule cross-sections [16%] multiplied by the percentage of GFP-positive tubule cross-sections with differentiated germ cells [55%]; Table 3) could be attributed to donor spermatogenesis, leaving approximately 64% of the tubules showing endogenous spermatogenic recovery. Similarly, in testes transplanted with immature rat testicular cells, 69% of the tubules showed recovery, but 26% (ie, the percentage of GFP-positive tubule cross-sections [35%] multiplied by the percentage of GFP-positive tubule cross-sections with differentiated germ cells [76%]; Table 4) could be attributed to the donor, leaving an endogenous recovery of aproximately 42%. These values are similar to the 53% recovery observed without transplantation (Table 1).
The presence of calcification in a small percentage of tubules was the only negative effect of irradiation. This was specific to the Swiss/Ncr nude mice; very little calcification was observed after irradiation of C57BL/6 mice with equivalent doses (G. Wilson and M. L. Meistrich, unpublished data). Although we were concerned that calcification might affect the ability of the transplanted cells to flow through the seminiferous tubules, the numbers of tubules or tubule cross-sections containing donor germ cells were still quite high. Calcified tubules have also been observed in rats treated with busulfan (Udagawa et al, 2001) and with dibromo-chloropropane (Meistrich et al, 2003) but not in busulfan-treated mice or irradiated rats (Kangasniemi et al, 1996).
By showing that irradiation is as good as other commonly used methods for preparing mouse testes for spermatogonial transplantation, our data support the consideration of irradiation for testing in species for which genetic mutants are not available or that have a narrow therapeutic window for spermatogenic vs systemic toxicity with chemical treatments. Indeed, the effects of radiation on the testes have been described in many species, and dose-response data are available for such an approach. In the rat, although relatively low doses of irradiation caused spermatogenic arrest in certain strains (Kangasniemi et al, 1996), the surviving stem cells differentiated and repopulated the seminiferous tubules in other strains (Dym and Clermont, 1970; Delic et al, 1986), as was the case in the mouse. Even in a strain in which radiation caused spermatogenic arrest, hormonal treatment to suppress intratesticular testosterone was able to stimulate the differentiation of endogenous surviving stem cells (Meistrich and Kangasniemi, 1997), and the same should apply to transplanted cells (Ogawa et al, 1999b; Zhang et al, 2003). Doses for killing stem cells and the subsequent repopulation of tubules have been described in the dog (Lushbaugh and Casarett, 1976), rabbit (Lyon and Cox, 1975), ram (van Vliet et al, 1988), boar (Erickson and Martin, 1984), bull (Erickson et al, 1972), rhesus macaque (van Alphen et al, 1988; van Alphen et al, 1989), cynomolgus macaque (Foppiani et al, 1999; Kamischke et al, 2003), stump-tailed macaque (Boekelheide et al, 2005), and humans (Clifton and Bremner, 1983). For application to different species, the physical dosimetry is straightforward, and differences in pharmacokinetics, which are an issue with chemical agents such as busulfan, do not have to be considered. Furthermore, local irradiation of testes avoids the systematic toxicity that often occurs in busulfan-treated animals.
Two studies have reported the use of irradiation to deplete cells from testes of other species for use as hosts for transplantation. In one case, monkey testes irradiated with 2 Gy were used as a host for autologous transplantation of germ cells (Schlatt et al, 2002). At this dose, there was significant recovery of endogenous spermatogenesis, but only 2 of the 5 monkeys showed significantly increased numbers of differentiated spermatogenic cells in the transplanted testis, compared with the saline-injected control monkey. In a study of autologous transplantation into calf testes, the animals were hemicastrated and irradiated with 10-14 Gy, and cells from the contralateral testis were injected into the irradiated testis; 5 of 6 animals showed enhanced spermatogenesis (Izadyar et al, 2003). It should be noted that neither of these studies included a marker to unequivocally distinguish donor germ cells from the endogenous germ cells.
In summary, irradiation consistently produced well-functioning germ cell-depleted recipient mouse testes. The irradiated testes could support complete donor spermatogenesis derived from either donor mouse or rat spermatogonial stem cells (as confirmed with the use of a GFP marker), with an efficiency similar to that of busulfan-treated recipient mouse testes. Our results suggest that irradiation is worthwhile to investigate as a possible effective method for preparing recipient testes in other species.
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Acknowledgments |
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Footnotes |
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Present address: Monash Institute of Medical Research, Centre for
Reproduction and Development, Clayton, Victoria, Australia. 
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