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Research Article |
Institute of Reproductive Medicine,3 University Münster, 48129 Münster, Germany
Department of Cell Biology and Physiology,4 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
ABSTRACT
Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.
chorionic gonadotropin, gametogenesis, grafting, hormones, meiosis, morphometry, nonhuman primate, testis, testis development, testosterone
INTRODUCTION
Recently, the ectopic grafting of immature testicular tissues into mouse hosts has been developed as a tool for the study of testicular development and as an experimental option to mature donor tissues externally [1–6].
Ectopic transplantation of testicular tissues can be performed xenologously between individuals of different species, heterologously (between two different individuals of the same species) and autologously (within an individual functioning simultaneously as donor and host).
Application of this grafting technique to primates paves the way for novel therapeutic options for infertile patients [7, 8]. Successful germ line maturation up to mature spermatids was achieved by growth and differentiation of testicular tissues grafted into mouse hosts for several species, including the Old World monkey Macaca mulatta [1–4], but failed for tissues obtained from the neotropical marmoset monkey Callithrix jacchus [3, 6]. In a previous study we showed that even cografting with hamster testicular tissue, producing high local concentrations of testosterone, was not sufficient to overcome the early (spermatogonia stage) germ cell arrest in marmoset tubules grafted into mice [6]. The reproductive endocrinology of the male marmoset differs from that of the mouse because of peculiarities in the function of the LH/chorionic gonadotropin (CG) system [9–12], which is also found in other neotropical monkeys [10–13]. In contrast to all other primates, in the marmoset monkey CG, which was thought to play a restricted role in establishment of pregnancy, is a pituitary hormone with luteinizing activity, because LH is inactive on the LH receptor, which lacks exon 10, as is typical for the New World monkeys [10–13]. Thus, it seemed likely that the hormonal milieu provided by a mouse host based on LH could have caused developmental failure of the testicular grafts. In our previous study we tried to overcome the developmental arrest by human CG (hCG) administration to transplanted mice. However, this was not sufficient to stimulate germ cell differentiation [6], suggesting that much more complex conditions are needed to stimulate marmoset spermatogenesis in a xenologous environment.
In the present study, autologous ectopic transplantation of testicular fragments in newborn marmoset monkeys was performed with two aims: first, to determine whether the spermatogenic arrest observed in the mouse host can be overcome by grafting testis tissue autologously, i.e., in the presence of the animal's own endocrine environment, and second, to investigate whether complete spermatogenesis can be achieved in a nonhuman primate used as a preclinical model for human reproductive research. So far, germ cell differentiation in immature testicular tissues transplanted ectopically as an autologous approach has not been demonstrated in primates. Here, we show for the first time that primate testicular tissue matures and spermatogenesis starts more than one year after transplantation.
MATERIALS AND METHODS
Animals, Tissue Collection, and Transplantation Procedure
Testes were dissected from 4-wk-old marmosets (Callithrix jacchus; n = 4) obtained from the institutional breeding facilities. During surgery the monkeys were sedated with saffan (Provet AG; 0.1 ml/100 g body weight). The animals were castrated through scrotal incisions and the scrotal skin was subsequently sewed up after removal of the testes. Testicular fragments (sizes ranged from 0.5 to 1 mm3) were kept in ice-cold Dulbecco modified Eagle medium for up to 20 min until grafting (see [4]). Control animals were either castrated and sham-grafted (n = 4) or left intact (age-matched controls; n = 2).
Two skin incisions of 4–5 mm were made on either side of the dorsal midline and 4 grafts per recipient were autologously placed in pockets underneath the shaved back skin. The pockets were closed with a single suture. Throughout the experimental period, the animals were kept in their family groups, with normal diet and water available ad libitum. They were regularly weighed and blood samples (600 µl per sample) were taken during the experimental period. After 18 mo, the animals were anesthetized with saffan and killed by exsanguination. Blood was collected and stored at –20°C. Body weight was recorded and the back skin was removed. Testicular grafts were dissected from the back skin. The experimental work was performed in accordance with the German Federal Law on the Care and Use of Laboratory Animals (license No. G67/2001).
Fragments of immature testicular tissue obtained for control purposes at castration and the explanted grafts were individually fixed in Bouin's solution for 24 h. Intact age-matched controls were castrated at the end of the experimental period as described above and testicular tissue fragments were fixed. All tissues were routinely embedded in paraffin and sections cut (7 µm). Periodic acid-Schiff/hematoxylin staining was used for routine analysis of histology. As previously established [6], tubular cross sections lacking germ cells were determined as Sertoli-cell-only (SCO) tubules and determination of the most advanced germ cell type in each graft was used to describe the maximum progression of spermatogenesis. All seminiferous tubules in the cross section were scored for the presence of spermatogonia, spermatocytes, and round or elongating spermatids. Histology of all tubules was analyzed morphometrically in the most central cross section of each graft and in material from 4-wk-old testes (pregraft controls) as well as from intact adult organs for control purposes. The tissue pieces from the intact age-matched controls were taken from the central portion of the testes, and the size of the fragments and the number of tubular cross sections examined were comparable to those of the transplants recovered from the experimental animals.
The size of the seminiferous lumen and the tubular diameter were analyzed with an Axiovert 200 microscope (Zeiss) and Axiovision 3.1 (Zeiss) software. Representative images were taken at magnifications of 10x, 25x, 40x, and 63x (Axiocam; Zeiss).
Immunohistochemistry
Only primary antibodies validated for Callithrix tissues were used. Boule-like (BOLL) protein in the sections was analyzed by a rabbit polyclonal antibody (diluted 1:300; [14]) and CDC25A by a rabbit polyclonal antibody (1:50, sc-97; Santa Cruz Biotechnology). We used 
-smooth muscle actin as a specific marker for peritubular cells [15], and it was detected with a specific monoclonal mouse antiserum (1:500, A2547; Sigma). To detect macrophages, a primary mouse monoclonal antibody against CD68 (1:25, M814; Dako Diagnostika) was used. The expression of 5-reductase in intratubular cells and in the interstitium was examined using a rabbit polyclonal antibody (1:100, sc-20659; Santa Cruz Biotechnology). All antibodies were applied for 60 min at room temperature in a blocking buffer. For all stainings, Dako-LSAB 2 System (K0672; Dako Diagnostika) was added for 30 min after washing, followed by an additional washing step and incubation with diaminobenzidine (DAB; Dako Diagnostika) for 20 min. Antibodies were visualized by a secondary horseradish peroxidase-labeled mixed anti-mouse and anti-rabbit IgG (1:100 dilution, K0672; Dako). DAB, used as a substrate, produced a dark brown signal. Briefly the staining process was as follows: after washing, the slides were incubated in 3% (v/v) hydrogen peroxide to suppress endogenous peroxidase activity. After washing in TBS buffer (10 mM Tris, 150 mM NaCl, pH 7.6), nonspecific background was blocked by incubation in 5% (v/v) normal goat serum diluted in incubation buffer (0.1% [w/v] BSA in washing buffer). The primary antibody was incubated in incubation buffer for more than 90 min at room temperature (RT). After extensive washing, the secondary antibodies were added and incubated as a cocktail for more than 90 min at RT. DAB was finally added for 8–10 min, followed by several washing steps. Controls were performed by omitting the primary antibody on adjacent sections.
In a final step, the slides were counterstained with hematoxylin for 10 sec and mounted under cover slips with Dako Faramount (Dako Diagnostika) before observation using a microscope (Axioskop; Zeiss) at different magnifications (objectives 10x, 25x, and 40x). Digital images of equal exposure were acquired with a CCD camera (Axiocam; Zeiss) controlled by image software (Axiovision; Zeiss).
Hormone Measurements
Serum testosterone levels were measured using a previously published RIA [16]. Each sample was processed in duplicate after extraction with diethyl ether. The intra- and interassay coefficients of variation (CVs) were 6.4% and 14.7% respectively. The detection limit of the assay was 0.68 nmol/L.
Serum bioactive CG levels were measured by an in vitro bioassay based on murine Leydig cells according to a previously established method for bioactive LH measurement [17]. In brief, a Leydig cell suspension was prepared from National Medical Research Institute (NMRI) mice testes (4 mice /8 testes per assay). Mice were killed and testes were removed, decapsulated, and cut into 8–10 pieces that were placed in ice-cold Eagle medium (EM; Invitrogen Gibco) containing 0.1% sodium bicarbonate, Hepes buffer (25 mM; Serva), calf serum (1%; Gibco) and theophylline (90 µg/ml EM; Fujisawa). Testis pieces were stirred for 15 min at 4°C and filtered through nylon gauze, and the suspension was centrifuged at 80 x g for 10 min. After a preincubation, the pellet was resuspended at a cell concentration of 10000 living Leydig cells per 100 µl medium.
As hormone standard in the assay, a combined human pituitary FSH/LH reference preparation for bioassays was used (WHO standard 2nd IRP 78/549). Marmoset sera were used at three serial dilutions. Cells and standard or serum dilutions were incubated for 3 h at 37°C with shaking. Reaction was stopped by boiling the samples for 15 min. Testosterone was measured in 200 µl of the different samples by RIA. Only solutions that gave analytical responses parallel to the standard curve were used for the calculation of bioactive CG solutions. The detection limit of the assay was 7.92 U/L. The intra- and interassay CVs were 6.3% and 14.9% respectively.
Statistical Analysis
Data were analyzed by applying one-way ANOVA. Values of tubular and lumen diameter were compared by ANOVA on ranks (all pairwise, multiple comparison by Dunn's method). Computations were performed using the statistical software package SIGMASTAT 2.03 or SPSS 12.0 (SPSS Inc.). All data were expressed as mean ± SD. Values were considered significantly different if P < 0.05. Hormone analysis was performed by univariate variance analysis for repeated measurements (UNIANOVA) over the entire experimental period as well as over the last five time points (adulthood of all animals considered).
RESULTS
Histological Analysis of the Grafts
Explantation at the age of 18 mo (17 mo after transplantation) revealed surviving grafts in two of the four transplanted animals (Table 1). Under the skin, transplants were identified as small reddish elevations (diameter 2–3 mm) surrounded by subepidermal fat. In one animal, three grafts were found out of four transplanted tissue fragments (survival rate 75%); in the other, two out of four (survival rate 50%) were found (Table 1). The five testicular transplants from two successfully grafted animals were situated near the implantation sites in the surrounding subepidermal fatty tissue as distinct pieces (Fig. 1A). On average, 30 tubules per graft (range, 19–50) were recovered (Table 1). All surviving grafts showed a similar degree of development. Leydig cells and peritubular cells were arranged around the seminiferous tubules, and small vessels vascularized the grafts (Fig. 1, B and G). Neither macroscopic nor microscopic signs of inflammation were observed. Transplants exhibited about 40% of the seminiferous tubules with only Sertoli cells (no germ cells; Fig. 1, B and C). In the remaining tubules, differentiated and/or dividing spermatogonia situated at the basal membrane, or primary spermatocytes that entered the meiotic processes (Fig. 1, B and E), were found to be the most advanced germ cell type.
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In contrast to the immature control tissues fixed at the time point of transplantation (Fig. 1C), the grafted material showed differentiated germ cells, actively dividing spermatogonia (Fig. 1F), mature Sertoli cells (Fig. 1, E and G), mature peritubular cells (Fig. 1, E–G), and Leydig cells (Fig. 1, B and G) with identical morphology compared to the somatic cell types of the age-matched controls, whereas these somatic cell types exhibited immature characteristics in the pregraft controls. In the grafts, Sertoli cells exhibited an elongated shape and nuclei were orientated at the basal sites. Leydig cells with nuclei located centrally in the cytoplasm were situated between the tubules in the intertubular spaces, grouped together in loose formation. This histology reflected the situation found in the intact adult control tissues. In contrast, Sertoli cells in the immature pregraft controls were smaller, of roundish shape, and with a more centrally located nucleus, and the Leydig cells with central nuclei, surrounded by a smaller amount of cytoplasm, were grouped in close association. In the pregraft controls, only gonocytes positioned in the middle of the cords and spermatogonia at the basal membrane were observed (Fig. 1C). The mature control tissues showed complete spermatogenesis, including all germ cell types up to the level of elongated spermatids, in every tubular cross section examined (Fig. 1D). Thus, the grafted material revealed a spermatocyte maturation arrest at the pachytene stage (Fig. 1E).
In accordance with this arrested developmental state, the tubular and luminal diameters in the transplants were at an intermediate size compared to immature controls and adult testes. All differences were statistically significant (Fig. 2, B and C). Whereas in the immature control tissue 62.4% ± 4.3% of the tubules contained gonocytes or early spermatogonia as the most advanced germ cell type, complete spermatogenesis up to elongated spermatids was found in 100% of the tubules from the adult testes. Comparing immature control with graft tissue showed that the percentage of seminiferous tubules with spermatogonia as the most advanced germ cell type was significantly smaller in the grafts (35.4% ± 6.5%; Fig. 2A) and that 23.9% ± 19.2% of the grafted tissue tubules presented primary spermatocytes (Fig. 2A).
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Immunohistochemistry
The peritubular cells in the transplants exhibited a strong specific staining for -smooth muscle actin, indicating that these cells possess mature, functional contractile elements (Fig. 3A). In the transplants, expression of 5-reductase showed a pattern identical to that seen in both mature and immature control tissues, i.e., in Leydig and Sertoli cells (Fig. 3, B–D). Adark spermatogonia and spermatocytes were positive, indicating the expression of the enzyme in the tubules of both grafts and adult controls. No CD68-positive cells were found in the recovered transplants (data not shown). BOLL staining was detected in the primary spermatocytes of the transplants, indicating that the germ cells had entered meiosis. All germ cells in the immature controls were negative for BOLL, whereas the primary spermatocytes in the adult tissues were positive (Fig. 3, E–G). However, CDC25A staining was negative in the explanted material. Because BOLL activates CDC25A expression, this finding pinpointed the spermatogenic arrest in the grafts at early meiosis. Control tissues from the adult intact monkeys exhibited positive CDC25A staining, and no staining was present in the seminiferous tubules of the immature controls (Fig. 3, H–J).
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During normal spermatogenic development in the marmoset, CDC25A expression is first detectable in pachytene spermatocytes because of the close relationship to its regulator BOLL, and lasts up to elongated spermatids (data not shown). In earlier stages (preleptotene and zygotene spermatocytes), both proteins are absent.
Hormones and Puberty Onset
The body weight gain in all animals was normal over the experimental period (Table 1). All monkeys were healthy and abnormal behavior was not observed. The onset of puberty was ascertained in the age-matched, intact controls by a remarkable increase in serum testosterone levels from values below 10 nmol/L to values above 80 nmol/L, whereas serum bioactive CG was low in these monkeys (Fig. 4C). Serum testosterone values were significantly higher than those measured in the grafted and in the sham-operated animals, which exhibited castrate levels (Fig. 4A). In the castrated sham-grafted and in the grafted animals, CG levels increased during the period designated as puberty by comparison with the age-matched intact controls (Fig. 4B). Puberty onset varied individually among the animals and started between the ages of 10 and 13 mo. Phenotypically, the fur color pattern reflected this developmental change. In general, the castrated monkeys had significantly higher serum CG levels compared with intact controls, because of insufficient inhibition of gonadotropin secretion by the low androgen levels (Fig. 4B). Serum testosterone and serum CG levels of sham-grafted castrates did not differ from graft-positive or graft-negative animals at any time point (Fig. 4, A–C).
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DISCUSSION
Induction of spermatogenesis up to the level of spermatozoa has been described in xenologously transplanted immature rhesus monkey testes [2], but never demonstrated for marmoset tissues [3, 6]. Although the xenologous [6] and the heterologous transplantation (unpublished results) of the marmoset testis fails, testicular tissues transplanted autologously are capable of surviving in ectopic locations. In this study, we have demonstrated for the first time that marmoset testis grafts can develop at least up to meiosis when autotransplanted. This means that transplants can maintain the differentiating potential of all testicular cell types for at least 1 year, i.e., during the complete so-called infantile "quiescent phase" [18]. In contrast to a study in which the onset of puberty was demonstrated to occur around wk 75 of age [18], our animals already showed puberty at the age of 55–60 wk, similar to results reported by others that demonstrated testicular maturity during this period [19]. Because we explanted the grafts after a period of one to two spermatogenic cycles after puberty, the disrupted spermatogenesis in the grafts must be attributable to developmental arrest and not to immaturity.
The morphometric data were consistent with the observation of spermatogenic arrest. Tubular diameters and luminal widths in the transplanted tissues were intermediate between immature and adult tissue values. Compared to our previous inability to induce marmoset spermatogenesis in combined hamster and monkey xenografts [6], the autologous grafting presented here was successful in stimulating germ cell differentiation. This indicates that endogenous gonadotropin secretion, which in the marmoset is based on CG rather than on LH, might be instrumental in stimulating germ line proliferation and differentiation before puberty [6, 10, 12], far beyond the neonatal period of pituitary testicular activity, which seems to have only minor effects [20]. However, serum testosterone levels rose only marginally in the transplanted animals compared to age-matched intact controls. Possibly, the grafts were too small and the number of Leydig cells secreting testosterone too few to secrete normal serum androgen levels. The low testosterone levels could be assumed to account for the developmental arrest at meiosis, because testosterone is known to be essential for postmeiotic expression of germ cell-specific genes and thus for the maturation of fertile male gametes in mammals [21]. In rats, the conversion of round to elongated spermatids is attributed to testosterone [22, 23], and suppression of testosterone and FSH results in remarkably reduced spermiation [24].
Despite the abnormal androgenization, we found that the somatic cells of the grafts differentiated to the mature state after puberty [25]. Histologically, position and shape of Sertoli, peritubular, and Leydig cells were identical to those in the testes from age-matched intact controls and differed from the immature somatic cells found in the pregraft controls. Immunohistochemistry revealed normal functional development of the somatic component of the transplanted testicular tissues. We found that the peritubular myoid cells positively stained for -smooth muscle actin, indicating that its contractile apparatus developed appropriately during the experimental period. Sertoli cells exhibited expression of the 5-reductase protein, suggesting their ability to metabolize testosterone [26]. Activity of 5-reductase was also observed in Leydig cells and in germ cells (Adark spermatogonia and spermatocytes) of the transplants. Thus, the machinery for the conversion of testosterone was available in the ectopically located tissues [26, 27] so that the 5-reduced metabolites, which are the predominant androgens in the pubertal testis, could have been provided [28, 29]. Although data reported from Sertoli cell-specific androgen receptor knockout mice indicate that Sertoli cells can proliferate and support spermatogenesis up to meiosis without an androgen receptor, and thus without direct androgen signaling, the cells must be surrounded by normal peritubular cells mediating the testosterone effects [30]. Therefore, at least the normal maturational state of the peritubular cells suggests the presence of sufficient local testosterone concentrations in the transplanted tissues, because peritubular cell development requires androgen action [30].
In our previous study [6], cografting of monkey with hamster testicular tissue was performed to provide local testosterone support, but the development of the marmoset germ line was not stimulated, possibly because of inappropriate gonadotropin supply in the mouse host. In the present study we suggest that the observed meiotic arrest was caused neither by a malfunction of intratesticular testosterone conversion nor (given the negative staining for the macrophage marker CD68) by an inflammatory process [31, 32]. Taking this together with our previous results [6], we postulate that the high gonadotropin levels induced low intratesticular androgen concentrations in the grafted tissue, which allowed adequate somatic cell maturation but was insufficient to drive spermatogenesis to completion.
Boule is a highly conserved key player in the regulation of meiosis. In boule-deficient fruit flies, spermatogenesis is arrested at the meiotic step [33, 34] and the same is observed in infertile men [35]. Here we found BOLL expression in primary spermatocytes of the transplanted tissues with a pattern identical to that found in intact controls and in various other primate species [36]. Interestingly, CDC25A, a protein phosphatase regulated by BOLL during meiosis, was absent from the grafts but was present in tissue from the intact controls. CDC25A in general has a crucial function for the completion of the meiotic M-phase [37–39].
Our data indicate that the arrest of spermatogenesis in transplants occurs at a well-defined point and that meiosis I cannot proceed because of the lack of CDC25A expression. It is believed that in the absence of CDC25A, the maturation-promoting factor cannot be dephosphorylated, halting meiosis at the spermatocyte state [40].
In contrast to nude mouse hosts, the implantation sites of the autologous grafts under the back skin are covered by fur. It appears likely that temperature at the implantation site is higher than it would be in the scrotum or under the back skin of a nude mouse. Therefore, because of their ectopic position, the grafts might have suffered from hyperthermia or conditions similar to those experienced in cryptorchidism. Spermatogenic arrest after meiosis (during spermiation) is a typical feature of heated testicular tissue, or when testes do not descend to the scrotal position [41–47]. Thus, one could speculate that increased temperature contributed to the observed arrest in the transplants, although most of the changes reported in the literature did not result in a distinct meiotic arrest at the stage we found in the transplants. Further studies varying the implantation sites will clarify this point.
In addition to hormonal milieu and local temperature, the structural organization of marmoset spermatogenesis should be considered when attempting to explain the spermatogenic arrest. The marmoset is the only species that has as yet been used for xenografting to exhibit a multistage organization of the seminiferous epithelium (see [13]). It cannot be excluded that the multistage organization of the marmoset seminiferous epithelium, obviously related to differences in spermatogonial clonal expansion, is incompatible with ectopic developmental progress. This would be of clinical relevance, because the seminiferous epithelium of the human is organized in a similar way [13].
In summary, we have demonstrated for the first time that autologous transplantation is suitable for maintaining immature testicular tissue and for inducing spermatogenesis during puberty up to meiosis in the nonhuman primate model Callithrix jacchus. Gonocytes migrated to the basal membrane and formed a pool of spermatogonia, including a spermatogonial stem cell population that repopulates spermatogonia and differentiating cells (spermatocytes). Thus, the meiotic division, the necessary key step for maturation of fertile spermatozoa, was achieved before spermatogenic arrest. The arrest reflects insufficient local levels of testosterone, organization of seminiferous epithelium, and/or hyperthermia. More studies have to be performed to overcome the meiotic arrest in order to explore the model further and to develop novel therapeutic options.
ACKNOWLEDGMENTS
The authors are indebted to Reinhild Sandhowe-Klaverkamp, Ingrid Upmann, Jutta Salzig, and Margret Kloth for technical assistance. We thank Martin Heuermann and Günter Stelke for animal caretaking and assisting in the operations. Finally, we are grateful to Trevor G. Cooper, Ph.D., and Susan Nieschlag, M.A., for language editing.
FOOTNOTES
1 Supported by the German Research Foundation (DFG; grant SCHL 394/6–1). 
2 Correspondence: Joachim Wistuba, Institute of Reproductive Medicine, University Münster, Domagkstrasse 11, 48129 Münster, Germany. FAX: 49 251 835 6093; joachim.wistuba{at}ukmuenster.de
Received: 21 October 2005.
First decision: 17 November 2005.
Accepted: 15 December 2005.
REFERENCES
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|
 |
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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