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Reproductive Technology |
a Department of Medical Chemistry,
b Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan 606-8501
c The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki, Japan 305-0074
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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immunology, in vitro fertilization, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immune privilege in the testis is thought to result from the unique anatomical structure of the seminiferous tubules and local immunosuppression by Sertoli cells, a major somatic cell in the seminiferous tubule [2]. In mice, Sertoli cells form the blood-testis barrier approximately 2 wk after birth [4] and separate the immunogenic haploid cells from the immune system [2]. In addition, Sertoli cells have local immunosuppressive activity, because cotransplantation of Sertoli cells with adrenal chromaffin cells [5], islet cells [6], or dopamine-secreting neurons [7] leads to acceptance of the transplants in allogeneic or xenogeneic hosts. Although the site of expression remains controversial [8], Fas ligand expressed on Sertoli cells is believed to play a key role in the induction of transplantation tolerance in the testis as well as in other immune-privileged organs [9, 10].
The body contains several self-renewing systems, including hematopoietic tissue, intestinal epithelium, and epidermis [11]. Spermatogenesis originates from spermatogonial stem cells, and the continuous production of progenitor cells supports male reproduction throughout the life of adult animals [4]. Because stem cells are considered to proliferate indefinitely, allogeneic stem cell transplantation has been a subject of intense investigation, mostly for clinical purposes. Particularly regarding the hematopoietic system, several decades of active investigation have made transplantation of allogeneic hematopoietic stem cells an accepted therapy for numerous hematological malignancies, metabolic disorders, and solid tumors [12]. However, little is known about the immunological response to transplantation of other types of allogeneic stem cells, particularly how they are affected by histoincompatibility [12, 13].
Recently, a technique to transfer spermatogonial stem cells was developed in which the stem cells are transplanted into the seminiferous tubules of infertile recipient animals [14]. The transplanted stem cells proliferate on the basement membrane and establish colonies of spermatogenesis [15]. Spermatogonial stem cells are unique among the many types of stem cells in that they reside in an immune-privileged microenvironment. In the present study, we investigated if male germ cells that develop in a histoincompatible environment are fertile, whether used for intracytoplasmic germ cell injection or for natural mating in immunosuppressed recipients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first set of experiments using adult recipients, donor cells were recovered from the testes of a transgenic mouse line B6-TgR(ROSA26)26Sor (ROSA26; H-2b haplotype) purchased from The Jackson Laboratory (Bar Harbor, ME) [16]. This mouse expresses the Escherichia coli LacZ transgene in all cells of the seminiferous tubules [15]. Donor cells used for intracytoplasmic germ cell injection experiments were isolated from the testes of a transgenic mouse line C57BL/6 Tg14(act-EGFP)OsbY01 (Green; H-2b haplotype) provided by Dr. M. Okabe (Osaka University, Osaka, Japan). The spermatogonia and spermatocytes of these mice express the enhanced green fluorescent protein (EGFP) gene, which gradually decreases after meiosis [17].
Donor cells were transplanted into the testes of C57BL/6J (B6; H-2b haplotype) and C3H/HeJ (C3H; H-2k haplotype) adult males that had been treated with busulfan (44 mg/kg) at 6 wk of age [14]. To protect from the deleterious systemic effects of busulfan (Sigma, St. Louis, MO), the recipient mice were transplanted with homologous bone marrow cells. A volume of 0.25 ml containing 0.75–1.5 x 106 cells was injected into the jugular vein of recipient mice 5–7 days after busulfan treatment. For the testicular injections, approximately 10 µl of the donor cell suspension were introduced into the seminiferous tubules of a B6 testis and 5 µl into the tubules of a C3H testis, because the latter mouse line has smaller testes. The injection filled 75–85% of the tubules in each recipient testis [18]. Transplanted donor cells, from ROSA26 or Green mice, were detected by staining for LacZ with the substrate 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal; Wako Pure Chemical Industries, Osaka, Japan) [15] or by ultraviolet (UV) light excitation [17], respectively.
In the second set of experiments, WBB6F1-W/Wv (W; Japan SLC, Hamamatsu, Japan) mice were used as recipients [19]. Donor testis cells from pups (age, 6 days) and cryptorchid adults (age, 14–20 wk) were isolated from the testes of DBA/2 (H-2d haplotype) or BALB/c (H-2d haplotype) mice (Japan SLC). Cryptorchid testes were produced as previously described [20] and used 2–3 mo after the cryptorchid operation. Single-cell suspensions from donor testes were prepared by two-step enzymatic digestion [18]. Donor testis cells were transplanted into recipient W pups (age, 5–10 days; H-2b/j haplotype) [21]. The W mice are congenitally infertile, because they lack all stages of differentiating germ cells due to mutations in the c-kit receptor tyrosine kinase [22]. Approximately 2 µl of the donor cell suspension were injected into each recipient testis through the efferent duct [18].
Donor cells were suspended at a concentration of 108 cells/ml (first experiment) or 3 x 107 cells/ml (second experiment) in Dulbecco modified Eagle medium, supplemented as described elsewhere [18]. Adult recipient mice were anesthetized by Avertin (Sigma) injection (640 mg/kg). Pup recipients were placed on ice to cause hypothermia-induced anesthesia [23].
All animal experimentation protocols were approved by the Institutional Animal Care and Use Committee at the Kyoto University.
Intracytoplasmic Germ Cell Injection and Transfer of Embryos to Foster Mothers
For intracytoplasmic germ cell injection, the testes were exposed to UV excitation light to detect EGFP-positive cells under a stereomicroscope equipped with a UV light (Nikon, Tokyo, Japan). The seminiferous tubules of the recipient testes were carefully dissected, and the germ cells were collected mechanically [24]. Intracytoplasmic germ cell injection into C57BL/6 x DBA/2 F1 (B6D2F1) oocytes was performed [24]. Embryos that reached the 4-cell stage after 48 h in culture were transferred to the oviducts of Day 1 pseudopregnant Imperial Cancer Research (ICR) females. Live fetuses retrieved on Day 19.5 were raised by lactating foster ICR mothers.
Immunosuppression of the Recipients
Several methods known to induce immunological tolerance were employed in some of the recipient W mice after transplantation. Rapamycin (Sigma) treatment consisted of 0.2 mg/kg per day i.p. for the first 3 days after transplantation, followed by 0.2 mg/kg every other day for 14 days [25]. Monoclonal antibody treatment consisted of 50 µg of anti-CD4 antibody (GK1.5) i.p. on Days 0, 2, and 4 after transplantation [26]. When indicated, recipients received additional i.p. injections of 50 µg of anti-CD8 antibody (53.67) on Days 0, 2, and 4 after transplantation [27]. Both antibodies were purchased from Pharmingen (San Jose, CA).
Analysis of Recipient Testes
To quantitate donor-derived colonies, recipient mouse testes were recovered 2 and 4 mo after donor cell transplantation and analyzed by X-gal staining [15]. This method allows the specific identification of donor germ cells, because endogenous germ cells in the seminiferous tubules of busulfan-treated recipients do not stain with X-gal (Fig. 1). A cluster of germ cells was defined as a colony when it occupied more than 50% of the basal surface of the tubule and was at least 0.1 mm in length [15]. The efficiency of colonization was evaluated by counting the total colony number and measuring the individual colony length under a stereomicroscope. Because the injected testis cell suspension contained 108 cells/ml, the number of cells injected into recipient B6 and C3H testes was 1 x 106 and 0.5 x 106 cells, respectively. Statistical analysis was performed with ANOVA followed by Turkey honestly significantly different (HSD) multiple comparisons.
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In experiments that employed W mice as recipients, four histological sections were taken at 12-µm intervals from the testes of each mouse. Each slide was viewed at 400x (to determine the level of spermatogenesis in the W testes). The number of tubule cross-sections with spermatogenesis (defined as the presence of multiple layers of germ cells in the seminiferous tubule) or no spermatogenesis was recorded for one section from each testis. All sections were fixed in 10% (w/v) neutral-buffered formalin (Wako Pure Chemical Industries) and stained with hematoxylin and eosin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the degree of donor cell colonization in an allogeneic testicular microenvironment, we transferred donor cells from the testes of fertile ROSA26 mice (B6) into the seminiferous tubules of syngeneic (B6) or allogeneic (C3H) male recipients sterilized by busulfan treatment, which allows donor cell colonization by destroying endogenous germ cells [14].
Six separate experiments were performed, and the results are summarized in Table 1. In each experiment, all recipients received the same donor cell population at the same time. Recipient testes were analyzed by X-gal staining 2 and 4 mo after donor cell transplantation. Because the length of time from initiation of stem cell division to formation of mature spermatozoa is 35 days in mice [4], this represents approximately 2- to 4-fold the duration of mouse spermatogenesis and should provide sufficient time to complete spermatogenesis after transplantation [15].
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In the syngeneic transplants, all 22 recipient B6 testes contained donor cell colonies (Table 1, lines 1 and 3) that gradually increased in length between 2 and 4 mo after transplantation. The colonies, which are formed by the progeny of one stem cell [15], were typically in the form of dark blue stretches of completely filled segment flanked by weakly stained regions (Fig. 2A). Spermatogenesis appeared morphologically normal at histological examination (Fig. 2, B and C).
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In the allogeneic recipients, 4 of 8 (50%) C3H allogeneic testes contained colonies 2 mo after transplantation (Fig. 2, D and E, and Table 1, line 2), but none of the 16 testes contained colonies at 4 mo after transplantation (Table 1, line 4). Colonies in the allogeneic seminiferous tubules showed varying degrees of donor cell colonization, ranging from scattered spermatogonia cells on the basement membrane (Fig. 2D) to advanced spermatogenesis (Fig. 2E). Spermatogenesis in the allogeneic recipient testes was histologically disorganized, but the donor cells were at least able to differentiate into round spermatids by 2 mo after transplantation (Fig. 2F). Focal lymphocytic infiltrates were found mostly in the intertubular region (Fig. 2G), indicating that the allogeneic donor cells were being rejected. Interestingly, not all of the colonies were accompanied by lymphocytic infiltration. Neither donor germ cells nor lymphocytes were found 4 mo after transplantation (data not shown). Thus, although some colonies were present at 2 mo, the donor germ cells were rejected by 4 mo and were incapable of completing spermatogenesis in the allogeneic environment.
Birth of Allogeneic Offspring from Adult Recipients by Intracytoplasmic Germ Cell Injection
To test whether allogeneic germ cells are fertile, we used intracytoplasmic germ cell injection (a technique commonly used to derive offspring from infertile animals or humans) [28–31]. Donor testis cells were collected from Green mice, which express the EGFP gene in their spermatogenic cells. The donor cells were transplanted into five busulfan-treated C3H recipients. At 2 mo after transplantation, two of the recipients had green fluorescent protein (GFP)-positive donor cells in both testes as identified at fluorescent microscopy. Live spermatogenic cells were recovered by repeatedly pipetting the colonized tubule fragments. Donor round spermatids, identified by a small round nucleus with a uniquely shaped chromatin mass, were subsequently injected into B6D2F1 oocytes (Fig. 3, A and B).
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Of the 69 embryos thus constructed with round spermatids, 65 (94%) progressed to the 4-cell stage by 48 h in culture. After embryo transfer into the oviducts, 52 embryos (75%) implanted in the uteri, and the two recipients gave birth to a total of 15 pups (22%; does not include one pup that was cannibalized within a few days after birth), of which 7 were male and 8 were female. The origin of the pups was confirmed by the fluorescence of GFP under UV light (Fig. 3C). All of the offspring grew into normal adults. Thus, these results indicate that donor germ cells that develop in a histoincompatible environment are fertile and can be used to produce normal offspring by intracytoplasmic germ cell injection without any immunosuppression.
Allogeneic Spermatogonial Transplantation into Infertile Pup Testes and Birth of Allogeneic Offspring by Natural Mating
In the second set of experiments, we examined whether allogeneic stem cell transplantation can restore fertility to an infertile mouse strain by natural mating. In W mice, endogenous spermatogenesis does not occur (Fig. 4A), but these mice are capable of producing offspring from transplanted donor testis cells [23, 32–34]. To improve the efficiency of allogeneic spermatogenesis, we employed three modifications in the transplantation protocol. First, we used immature pup recipients for transplantation, because they allow more colonization events for transplanted stem cells and have a higher fertility restoration rate than adults [23, 32]. Second, we used enriched stem cell populations from 6-day-old pup testes or adult cryptorchid testes (2–3 mo after the operation). These testes contain a higher concentration of stem cells than untreated adult testes do, because they lack differentiating germ cells [20, 32]. The transplantation of enriched stem cells will also improve the colonization level. Finally, several protocols were used to induce tolerance [25–27], to inhibit the rejection of allogeneic cells, as occurred in the first experiment. It was known that tolerance can be induced by using antibodies to transiently block CD4 and CD8 molecules on the T cells [26, 27]. We also used rapamycin, another immunosuppressive agent, that can induce allograft tolerance by blocking proliferative signals for T cells [25].
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Allogeneic donor testis cells were collected from either BALB/c or DBA/2 testes and transplanted into W pup recipients (age, 5–10 days) using modified transplantation protocols. Approximately 3 x 104 allogeneic testis cells (
2% of the cells recovered from a donor testis) were introduced into each W pup recipient testis, and 37 testes of 24 pups were injected with donor cells under four different recipient conditions (Table 2). Two months after transplantation, all of the recipients were housed with two untreated females to determine whether spermatogonial transplantation had restored fertility. The testes of the recipients were analyzed for the presence of spermatogenesis between 184 to 219 days after transplantation.
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Histological analyses revealed that a restoration of spermatogenesis occurred in six of eight (75%) untreated W recipient testes (Fig. 4B and Table 2, line 1), indicating that spermatogonial stem cells can colonize and complete spermatogenesis when transplanted into an immature allogeneic environment. Interestingly, no inflammatory cells were found in the recipient testes with allogeneic spermatogenesis (Fig. 4C). Spermatogenesis in the recipient testes originated only from the donor cells, because the W recipients were incapable of generating spermatogenesis from the defective endogenous stem cells [19]. In the most successful case, the testes contained as many as 59 cross-sections of seminiferous tubules (45% of all tubules), which hosted various stages of spermatogenesis (Fig. 4, B and C). However, spermatozoa were not found in the epididymis of the recipient animals, and none of the untreated recipient males became fertile.
In contrast, 4 of the 18 (22%) immunosuppressed recipient males produced progeny, beginning 123 days after donor cell injection (Fig. 4D and Table 3). Consistent with these observations, the immunosuppressive treatments increased both the testicular weight and the colonization level (Fig. 4E and Table 2). In total, epididymides of 9 of the 18 (50%) immunosuppressed recipients contained spermatozoa, suggesting that they had the potential to be fertile (Table 2, lines 2–4). Furthermore, the testes of the fertile males were significantly heavier than those of the untreated, infertile males, indicating the extensive donor cell colonization (Tables 2 and 3). The four recipients that produced progeny remained fertile up to the time of analysis, at least 219 days after transplantation (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first experiment demonstrated that allogeneic donor cells can be rejected in adult seminiferous tubules. Although donor germ cells established colonies in the allogeneic seminiferous tubules, they could not complete spermatogenesis and were rejected by 4 mo after transplantation. This result was unexpected, because previous transplantation studies using rats or mice suggested that allogeneic spermatogonial stem cell transplantation within the same species is possible between nonimmunosuppressed, histoincompatible males [32, 35, 36]. However, the animals used in these studies were either random-bred [35, 36] or the donors and recipients shared major histocompatibility antigens [32]. Therefore, determining the effect of immunological factors from these experiments is difficult. Our results clearly show that histocompatibility between donors and recipients is a prerequisite for the successful spermatogonial transplantation in adult recipients.
Although rejection occurred by 4 mo after transplantation, the donor cells were able to complete meiosis by 2 mo after transplantation and to differentiate into round spermatids. Previous studies have shown that most transplanted germ cells are rapidly eliminated from the seminiferous tubule through phagocytosis by Sertoli cells and that only stem cells can colonize the recipient testis [15]; therefore, it is reasonable to believe that transplanted stem cells differentiate into round spermatids that can be used for intracytoplasmic germ cell injection. Intracytoplasmic germ cell injection is widely used to treat infertility in both animals and humans [28–31], and it allows the production of offspring from testes without a level of spermatozoa sufficient for normal fertilization. This technique contributed to the success of the present study for two reasons. First, although intracytoplasmic germ cell injection was originally used with mature spermatozoa [30, 31], recent progress has allowed the production of healthy offspring using immature germ cells, such as round spermatids or even primary spermatocytes, for fertilization [37, 38]. Therefore, use of mature spermatozoa is not essential to obtain offspring. Second, unlike natural mating, intracytoplasmic germ cell injection requires only a small number of spermatogenic cells. Therefore, the completion of meiosis in a few donor cell-derived colonies was sufficient for offspring production in these experiments. Our results demonstrate that intracytoplasmic germ cell injection is a powerful method for overcoming immunological barriers in allogeneic spermatogonial transplantation.
A striking result of the second set of experiments was that immunosuppression allowed restoration of fertility in infertile recipients that had received histoincompatible donor germ cells. The long-term spermatogenesis in the recipients indicates continuous donor stem cell proliferation and normal differentiation in the allogeneic environment. It is noteworthy that only 2% of the donor testis cells, which is equivalent to the number capable of restoring fertility in syngeneic transplants [23], was sufficient to produce allogeneic offspring. Therefore, transplantation into immunosuppressed immature testis provides a system to produce allogeneic offspring by natural mating, which would allow stable offspring production and more experimental opportunities.
An important question posed by the second set of experiments is the immunological status of the prepubertal testis. The successful allogeneic spermatogenesis in the untreated testes does not appear to reflect the neonatal tolerance phenomenon [39]. In short, newborn animals are immunologically immature and accept allografts. However, the critical period is very short in mice, and the immune system is tolerant on the day of birth but loses this tolerance 1–2 days later [2]. The W pup recipients used in the present study were 5–10 days old and already capable of rejecting allogeneic bone marrow cells [40]; therefore, the successful spermatogenesis in these mice more likely resulted from the special immunological microenvironment or "immune privilege" in the testis rather than from the neonatal tolerance phenomenon. In addition, the observation that no spermatozoa were in the epididymis, despite the presence of complete spermatogenesis in the untreated testis, also suggests that the testis and epididymis have different immunological microenvironments. Perhaps the allogeneic germ cells were able to survive only in the special immunological environment of the testis but the allogeneic spermatozoa were rejected during transport from the testis to the epididymis.
On the other hand, the results obtained in immunosuppressed recipients clearly suggest the involvement of immunological factors in the development of allogeneic spermatogenesis. Although there may have been too few recipients in the present study to conclude the effect of each treatment, monoclonal antibody treatments (particularly anti-CD4 antibody) not only allowed transport of spermatozoa to the epididymis but also improved the level of allogeneic spermatogenesis in the testis. This ultimately resulted in the restoration of fertility in the recipient animals. Therefore, the results from these experiments are at odds with the hypothesis that the prepubertal testis is immunologically special. Currently, it is difficult to reconcile these apparently conflicting results, and more extensive studies using spermatogonial transplantation are required to examine the immunological environment of the seminiferous tubules. The unique immunological status of the prepubertal testis might be related to the anatomical or endocrinological differences from the adult testes, such as the absence of tight junction between Sertoli cells or endocrinological immaturity [41]. Understanding the mechanisms behind these differences will likely allow improvements in the level of spermatogenesis obtained in the allogeneic recipients.
The results reported here may be useful for the manipulation of male germ lines from many animal species. Spermatogonial stem cells are unique among adult tissue stem cells, because they can pass genetic information to the next generation. Furthermore, the recent development of spermatogonial transplantation has provided the ability to manipulate this valuable cell population [14]. For example, the technique can be used to preserve germ lines of economically valuable animals, older animals that are unable to breed naturally, and endangered animals [42]. In addition, it has now been demonstrated that spermatogonial stem cells provide an excellent vehicle for the genetic modification of male germ lines and the production of transgenic animals [32]. Although spermatogonial transplantation was originally developed in mice [14], the cell transfer technique is now beginning to be established in several other species, including humans [43, 44]. However, immunological incompatibility between the donor and recipient animals will be a clear impediment to such transplantation studies, because immunodeficient animals do not exist in most species. Our successful production of allogeneic offspring has demonstrated methods to overcome this problem and will facilitate the application of technologies established in the mouse system to a broad range of animal species. Thus, allogeneic spermatogonial transplantation can be used in the study of the unique immunological nature of the testis and can provide new possibilities in biotechnology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takashi Shinohara, Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, Japan 606-8501. FAX: 81 75 753 4388; takashi{at}mfour.med.kyoto-u.ac.jp
Received: 19 June 2002.
First decision: 10 July 2002.
Accepted: 5 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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