1 Horizontal Medical Research Organization, 3 Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University and 2 The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki, Japan
4 To whom correspondence should be addressed at: Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University 606-8501, Japan. e-mail: takashi{at}mfour.med.kyoto-u.ac.jp
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Abstract |
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Key words: cryopreservation/infertility/stem cell/testis/transplantation
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Introduction |
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The development of a spermatogonial transplantation technique has provided a new treatment strategy for male infertility (Brinster and Zimmermann, 1994). Following the transplantation of dissociated testis cells into a seminiferous tubule microenvironment, the spermatogonial stem cells colonize and initiate spermatogenesis (Nagano et al., 1999
). Since spermatogonial stem cells self-renew and differentiate into proliferating spermatogonia, they provide a limitless supply of mature spermatozoa. Thus, spermatogonial stem cell transplantation may be useful for the treatment of different types of male infertility; for example, it was used to treat a mouse model of Sertoli cell-only syndrome (Ogawa et al., 2000
; Shinohara et al., 2001
). The mice that underwent transplantation recovered normal fertility and produced progeny for the rest of their lives. In addition to congenital infertility, this technique should also benefit oncology patients who are undergoing stem cell-destroying irradiation or chemotherapy, by prior isolation of stem cells and autotransplantation (Aslam et al., 2000
). Transplantation of stem cells should rescue fertility in a manner similar to that of bone marrow stem cell transplantation. Therefore, the establishment of methods to preserve stem cells and restore fertility has important clinical implications.
Brinster and colleagues first demonstrated that frozen stem cells retain the ability to carry out spermatogenesis and to produce spermatozoa (Avarbock et al., 1996). Surprisingly, in contrast to the difficulties associated with the freezing of sperm, the protocol used for freezing germline cells was very simple, and similar to those generally employed for somatic cells. Further studies indicated that the same procedure could be applied to the freezing of spermatogonial stem cells from several other species, such as rats, hamsters, cattle, primates and humans (Dobrinski et al., 1999
; 2000; Ogawa et al., 1999
; Brook et al., 2001
; Nagano et al., 2001
; 2002; Izadyar et al., 2002
; Schlatt et al., 2002a
). Despite the potential shown by this technology in initial studies, no attempts to mate recipients of cryopreserved germ cells have been reported, and it remains unknown to date whether germ cells developed from frozen stem cells are fertile. It is possible that the freezethaw procedure significantly compromises stem cell survival or proliferation, leading to infertility after transplantation. In addition, the effects of cryopreservation on stem cell viability or proliferation have not been well characterized.
In the present study, the effect of freezing on stem cell colonization efficiency was evaluated, and the feasibility of restoring fertility via the transplantation of frozen stem cells examined. It was demonstrated that cryopreserved stem cells retain significant regenerative potential. Moreover, the successful birth of healthy offspring from frozen stem cells by natural mating supports the future application of frozen stem cells in the treatment of male infertility.
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Materials and methods |
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Donor cells used for fertility experiments (second experi ments) were isolated from the testes of a transgenic mouse line C57BL/6 Tg14(act-EGFP)OsbY01 (designated Green) provided by Dr M.Okabe (Osaka University) (Okabe et al., 1997). The spermatogonia and spermatocytes of these mice express the gene for the enhanced green fluorescent protein (EGFP), the amount of which gradually decreases after meiosis. Only the male pups (6-day-old) or adults (68 weeks of age) that were positive for the transgenes were used for transplantation experiments. Cells were collected from the testes of 6-day-old Green mouse pups or from the cryptorchid testes of adult Green mice, 23 months after the operation. It has been shown that these testes are enriched for stem cells, due to the absence of differentiated germ cells (Shinohara et al., 2000
; 2001); therefore, they should improve colonization efficiency and facilitate offspring production. Cryptorchid testes were produced as previously described (Shinohara et al., 2000
).
Single-cell suspensions were prepared from the donor testis by two-step enzymatic digestion, as described previously (Ogawa et al., 1997). In brief, testis cells were digested with 1 mg/ml collagenase (type IV; Sigma, St Louis, Missouri, USA) for 15 min, followed by 0.25% trypsin/1 mmol/l EDTA (both from Invitrogen, Carlsbad, CA, USA) for 10 min.
Cryopreservation of testis cells
For cryopreservation, dissociated testis cells were suspended in a cell cryopreservation solution (Cellbanker; DIA-IATRON, Tokyo, Japan, www.mk-iatron.jp), which contains dimethyl sulphoxide and 10% fetal calf serum (FCS). Aliquots of 1 ml cell suspension, containing 107 donor testis cells, were transferred to 1.5-ml polypropylene cryotubes (Sumitomo Bakelite, Tokyo, Japan), placed in a freezing container, and frozen at 80°C for 1 day, after which the cryotubes were plunged into liquid nitrogen. The cryotubes were thawed in a water-bath at 37°C, and 10 ml of Dulbeccos modified Eagles medium (DMEM) containing 10% FCS (DMEM/FCS) was added drop-wise. After washing by centrifugation, the cells were suspended in DMEM/FCS and kept on ice until transplantation into the testes. Cell viability was assessed by trypan blue staining. Only live germ cells were counted for transplantation.
Recipient mice
Donor cells were transplanted into the testes of C57BL/6 (B6) mice (612 weeks old) or WBB6F1-W/Wv (W) mice (either 510 days old or 612 weeks old; see Table I for experimental design) (Silvers, 1979), which were purchased from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The B6 males were treated with busulfan (44 mg/kg bodyweight) at 6 weeks of age to destroy the endogenous germ cells, and used at least 1 month after busulfan injection. Busulfan treatment allows donor cell colonization by destroying the endogenous spermatogonial stem cells (Bucci and Meistrich, 1987
; Brinster and Zimmerman, 1994
; Shinohara et al., 2002b
). Thus, it mimics the disrupted spermatogenesis that occurs in oncology patients. Two different stages of the W recipients, namely immature (510 days old) and mature (612 weeks old) mice were also used. W mice lack endogenous spermatogenesis due to mutations in the c-kit tyrosine kinase gene (Geissler et al., 1988
), which is normally expressed on germ cells. Both chemically castrated and congenitally infertile recipients have been shown to be capable of generating spermatogenesis from transplanted fresh stem cells (Brinster and Avarbock, 1994
; Mahato et al., 2000
; Ogawa et al., 2000
; Shinohara et al., 2001
).
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In the first experiments, frozen cells were suspended in DMEM/FCS at a concentration of 7.5x106 to 3.0x107 cells/ml, whereas the concentration of fresh cells was 108 cells/ml, because the recovery of frozenthawed cells varied between experiments. In the second experiments, the frozen donor cells were suspended in DMEM/FCS at a concentration of either 108 cells/ml (B6 or mature W recipients) or 3x107 cells/ml (immature W recipients).
Microscopy
Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin wax and then cut into sections at 12-µm intervals. All sections were stained with haematoxylin and eosin. Four histological sections were taken from the testes of each mouse.
Analysis of recipient testes
In the first experiments, the recipient mouse testes were recovered 2 months after the injection of donor cells, and analysed by X-gal staining as described previously (Nagano et al., 1999). Individual blue-stained stretches of the seminiferous tubules (colonies) in the recipient testes represented spermatogenesis from transplanted stem cells, as the other testis cells could not regenerate spermatogenesis and the endogenous recipient spermatogenesis did not stain positive. Each blue colony is thought to arise from a single transplanted stem cell (Nagano et al., 1999
). Therefore, the number of blue colonies represents the number of stem cells in the injected cell population.
In the second experiments, donor cell colonization was evaluated by histological sectioning, because recipients showed extensive donor cell colonization long after transplantation. Each slide was viewed at a magnification of x400 for the analysis. To assess the level of spermatogenesis in the recipient testis, the numbers of tubule cross-sections with evidence of spermatogenesis (defined as the presence of multiple layers of germ cells in the entire circumference of the seminiferous tubule) or lacking evidence of spermatogenesis were recorded for three sections from each testis, and at least 500 seminiferous tubules were counted. Statistical analyses were performed using Students t-test.
Microinsemination
Donor-derived EGFP-positive colonies were identified under a fluorescent microscope (MZ FLIII; Leica, Tokyo, Japan), and the germ cells collected mechanically by repeated pipetting of the tubule fragments. The donor testis cell suspension was refrozen before microinsemination, as described previously (Ogura et al., 1996). Microinsemination was performed by ICSI (Kimura and Yanagimachi, 1995
) of donor testis cells into C57BL/6xDBA/2 F1 (B6D2F1) oocytes, which were collected from superovulated females. Embryos that reached the 2-cell stage after 24 h in culture were transferred to the oviducts of Day 1 pseudopregnant ICR females.
All of the animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of Kyoto University.
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Results |
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To compare the repopulation efficiency, both viable frozen and viable fresh cells were transplanted into the seminiferous tubules of congenitally infertile W recipients or busulfan-treated mouse recipients. Two experiments were performed, and the number of colonies was counted for each recipient testis, 2 months after transplantation.
The analyses of W recipient testes revealed that the extent of colony generation from frozen stem cells was 11.7-times that from fresh stem cells (62.1 versus 5.3 colonies per 3 x 105 donor cells; P < 0.05) (Figure 1a and b; Table II). Likewise, the frozen cells showed higher stem cell activities in the busulfan-treated testis (45.9 versus 9.0 colonies per 1 x 106 donor cells); the number of colonies from frozen donor cells was 5.1-times higher than that from fresh donor cells (P < 0.01) (Table II). Taken together, these results demonstrate that freezethawed testis cells had higher stem cell activities than fresh donor cells.
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The results of the fertility experiments are shown in Table III. Four of the eight (50%) W pup recipients sired offspring within 72190 days of transplantation with the frozen donor cells. Three of the fertile males received adult cryptorchid donor cells, and offspring were also obtained from one recipient that received pup donor cells. The donor cell origin of the offspring was confirmed by green fluorescence under ultraviolet (UV) light. At the time of sacrifice, the mean weight of the fertile recipient testes was significantly higher (40.7 ± 7.3 mg; n = 4) than that of the infertile recipients (22.6 ± 2.2 mg; n = 4; P < 0.05) or the untransplanted controls (10.4 ± 0.8 mg; n = 7; P < 0.01) (Figure 2a). Histological analyses also revealed more extensive donor germ cell colonization of the fertile recipient testes (76.3 ± 6.6%; n = 4) than of infertile recipient testes (32.8 ± 3.0%; n = 4; P < 0.001) (Figure 2b and c). Spermatogenesis in the recipient testes arose exclusively from donor stem cells, as the stem cells in the W recipient could not undergo spermatogenesis (Brinster and Zimmermann, 1994; Ogawa et al., 2000
; Shinohara et al., 2001
). However, the restoration of spermatogenesis occurred in all eight immature W recipient testes, and spermatozoa were observed in 87.5% (7/8) of epididymis sections, which suggests potential fertility. The four recipients that produced progeny remained fertile up to the time of analysis; that is, at least 228 days after transplantation (Figure 2d; Table IV), which indicates that the transplanted stem cells underwent continuous division and normal differentiation. Taken together, these results demonstrate that the transplantation of frozen stem cells from the testes of pups or cryptorchid adults restored normal fertility to congenitally infertile W recipients.
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Discussion |
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The results of the first experiment showed that frozen testis cells contain significantly higher stem cell concentrations than fresh testis cells. This result was unexpected, because in a previous study the efficiency of frozen cell colonization was seen to be very low and variable (Avarbock et al., 1996). The high survival rate of stem cells contrasts with that of the more-differentiated spermatogenic cells, which undergo significant damage during the freezethaw procedure (Hafez, 1993
; Ogura et al., 1996
; Glenister and Thornton, 2000
), and probably reflects a unique biological characteristic of stem cells. It is known that stem cells are highly resistant to a variety of agents that damage the testis; the stem cell is the last cell type to be destroyed after irradiation or chemical insult, and it can regenerate via self-renewing division to complete spermatogenesis (Meistrich and van Beek, 1993
; de Rooij and Russell, 2000
). Therefore, it is reasonable to speculate that a large proportion of the stem cells survived the cryopreservation procedure based on their higher viability, while the remainder of the testicular cells underwent significant damage and died. The net result was an increase in the relative concentration of stem cells in the freezethawed mixed cell population. This also explains the higher cell recovery rates of the freezethawed pup and cryptorchid testis cells, both of which lack differentiated germ cells. Thus, the superior resistance of stem cells to cytotoxic damage can explain their high survival rates, and confers an advantage on these cells in cryopreserved populations.
The ultimate goal of the frozen stem cell technology is to generate offspring from the preserved stem cells. Since spermatogonial transplantation is a potentially effective therapy for both congenital and acquired infertility in humans, an investigation was made as to whether the technique was useful for fertility restoration in the mouse infertility models of W and busulfan-treated mice. The testicular environments are probably abnormal in both models: busulfan treatment damages the Sertoli cells, and may reduce the efficiency of spermatogenesis (Nagano et al., 1999), whereas the Sertoli cells in W mice have never been exposed to germ cells and may be incapable of physiological responses to germ-cell signal (Ogawa et al., 2000
). Thus, the seminiferous tubule microenvironments are probably different in the two models, which might affect the outcome of transplantation.
Interestingly, the immature W mice were able to sire offspring most efficiently by natural mating, although this result might reflect recipient age rather than recipient type. A previous study showed that the age of recipients was critical for the success of spermatogonial transplantation: immature W testes not only had a 10-fold higher stem cell colonization efficiency than mature W testes, but also allowed faster growth of stem cell colonies (Shinohara et al., 2001). Importantly, the higher success rate in immature recipients has a direct clinical implication for fertility protection in young cancer patients who do not have sufficient sperm for freezing purposes (Blatt, 1999
). It is encouraging to find that only a few hundred stem cells (
1% of a donor testis) can restore the fertility of immature recipients as early as 3 months after transplantation. In contrast, the restoration of fertility to an adult generally takes at least 5 months and requires higher cell numbers, even when fresh donor cells are used (Ogawa et al., 2000
; Shinohara et al., 2000
). Taken together, these results indicate that frozen stem cells are fully functional after cryopreservation, and suggest the potential advantage of immature recipients for offspring production.
On the other hand, frozen stem cell transplantation was relatively inefficient in adult recipients. Although the donor testes used in this study were relatively enriched for stem cell activity, only one of the males became fertile, at 7 months after transplantation. The absence of sperm in the epididymides of many recipient mice suggests that donor-derived sperm production was insufficient. In addition, it is also possible that sperm in the spermatogenic colonies may not be normal. Indeed, missing layers of germ cells or abnormalities in the elongation phase of spermatogenesis were found in the spermatogenic colony after spermatogonial transplantation (Russell et al., 1996), and busulfan-treated adult recipients rarely become fertile after spermatogonial transplantation (Griswold et al., 2001
; Brinster et al., 2003
). Fertility in busulfan-treated recipients was achieved in cases when a lower dose of busulfan was used to maintain some level of endogenous spermatogenesis (Brinster and Avarbock, 1994
; Kent Hamra et al., 2002
; Zhang et al., 2003
). Thus, low levels of donor-derived spermatogenesis and potential abnormalities in the sperm may account for the inefficiency of fertility restoration in the adult recipients.
In the present study, the infertility of the busulfan-treated adult recipients was successfully overcome by microinsemination (Palermo et al., 1992; Kimura and Yanagimachi, 1995
), demonstrating that at least some spermatozoa in the busulfan-treated recipients are functionally normal. An important advantage of this technique is that it allows fertilization from a small number of spermatozoa that are generated in a tiny segment of the seminiferous tubule. Therefore, it allows offspring production from infertile recipients that have a level of spermatogenesis that is insufficient for natural mating. Clearly, the restoration of natural fertility is one of the advantages of spermatogonial transplantation. However, as the results of the present study show, not all recipients become fertile after transplantation, and the success of the technique depends on extensive colonization by donor cells and long periods of stem cell proliferation, particularly in adult recipients. In contrast, microinsemination allows offspring production at 23 months, when mature sperm are first observed in spermatogenic colonies in mice (Nagano et al., 1999
), thereby shortening the period for fertility restoration. Given that the technique is well established in humans (Palermo et al., 1992
; Silber, 1995
), microinsemination will be useful in overcoming the slow growth of stem cells in humans, and will complement spermatogonial transplantation in case of long-term infertility.
It is necessary to note here that the present success in mice may not be simply extrapolated to humans due to the different structure and biology of the human testis. In fact, the ability to restore fertility using spermatogonial transplantation has not been demonstrated for any species except mice and rats (Brinster and Avarbock, 1994; Kent Hamra et al., 2002
; Zhang et al., 2003
). Nonetheless, the present results highlight the promising aspects of using frozen stem cell technology in humans. As the results show, the technique will most likely benefit prepubertal patients in terms of fertility protection (Aslam et al., 2000
), while adult patients may require microinsemination to obtain offspring. At present, there are other approaches to preserve male germ cells, such as testis piece freezing (Nugent et al., 1997
; Hovatta, 2001
; Honaramooz et al., 2002
; Schlatt et al., 2002b
; Shinohara et al., 2002a
) or fresh tissue grafting (Honaramooz et al., 2002
; Schlatt et al., 2002b
; Shinohara et al., 2002a
), and healthy isogenic (Shinohara et al., 2002a
; Schlatt et al., 2003
) or xenogenic offspring were born with these procedures (Shinohara et al., 2002a
). However, spermatogonial transplantation is the only method that restores natural fertility to the host, and it will be important to identify circumstances when this technique may be applied in clinical situations.
The results from the present study showed clearly that it is essential to achieve extensive donor cell colonization in order to restore normal fertility, and that immature recipients have better success rates. Clearly, another critical factor to increase colonization is the number of stem cells in the donor cell population. The donor testes used in this study were relatively enriched for stem cell activity (Shinohara et al., 2000; 2001). However, the percentage of stem cells in the normal adult testis is significantly lower; stem cells comprise approximately 0.02% of the adult testis cell population (Meistrich and van Beek, 1993
). As only a limited number of stem cells can be recovered from a small biopsy sample from a patient (Brook et al., 2001
), the enrichment of stem cells alone does not necessarily ensure high stem cell recovery. From this viewpoint, one of the approaches to overcome this problem would be to develop methods to expand spermatogonial stem cells in vitro. If it is possible to obtain large number of stem cells by in-vitro culture from a small biopsy, this will greatly enhance the level of donor cell colonization and offspring production. Studies are now in progress to culture stem cells to increase their number (Nagano et al., 1998
; Hasthorpe et al., 2000
; Izadyar et al., 2003
). Once established, the combination of in-vitro culture method with transplantation and freezing technologies of spermatogonial stem cells will be a powerful technique for fertility restoration.
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Acknowledgements |
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Submitted on March 24, 2003; resubmitted on May 27, 2003; accepted on August 19, 2003.