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Regular Article |
a Department of Cell Biology, UMCU, 3584 CX Utrecht, The Netherlands
b Department of Biomedicine, Biomedicum, FIN-00014, University of Helsinki, Finland
c Department of Cellular and Molecular Biology and Pathology, University of Naples, 80131 Naples, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, growth factors, male reproductive tract, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1–4 (GFR1–4), in combination with the tyrosine kinase receptor Ret [4]. Initially, GDNF was identified as a factor promoting the survival of various neuronal cell types [5–10] and mediating kidney development and generation of the enteric nervous system [11, 12].
Recently, it has become clear that many members of the GDNF signaling pathway are also expressed in the testis [9, 13–15], and GDNF has been shown to be involved in in vitro proliferation of germ cells and Sertoli cells [14, 16]. Moreover, both loss of function and targeted overexpression of GDNF in testicular germ cells have been demonstrated to have profound effects on spermatogenesis in vivo [17]. In mice with one GDNF null allele, spermatogonial stem cells are depleted, whereas in mice overexpressing human GDNF, an accumulation of cells resembling spermatogonial stem cells is noted. In the latter animals, spermatogenesis is normal for the first few weeks after birth, but at 2–3 wk of age, many tubules are found to contain large clusters of spermatogonia-like cells expressing the human GDNF (hGDNF) insert and the receptors Ret and GFR1. From these data, it has been concluded that the high levels of GDNF produced by the germ cells predisposed to stem cell renewal [17]. However, it has not yet been established whether the accumulation of spermatogonia found in the GDNF-overexpressing testes could be reproduced on transplantation of the germ cells to a different genetic environment.
A major hindrance to research focussing on stem cell-related topics is the lack of biochemical or immunologic markers that distinguish stem cells from differentiating progeny. At present, the only way of determining whether germ cells have stem cell properties is to evaluate their spermatogenic potential. To this end, the transplantation of germ cell suspensions into testes of host animals has become a routine method for assessing the presence of stem cells [18, 19]. A prerequisite in this procedure is the depletion of endogenous spermatogenesis in host testes, to enable the transplanted germ cells to move to the basement membrane and reinitiate spermatogenesis [20]. At present, the customary way of achieving this goal is via systemic administration of busulfan, a cytotoxic agent [21, 22]. However, as this agent also affects other stem cell populations, severe adverse effects occur, and bone marrow transplantation is often required [21–23]. Another source of host animals is provided by using W/W mice, mutants in which a spermatogenic defect intrinsic to the germ cells causes infertility [24, 25].
An alternative to these two approaches is provided by using local ionizing irradiation. The susceptibility of spermatogonial stem cells to fractionated ionizing irradiation [26, 27] prompted us to design a protocol to deplete the seminiferous epithelium of normal and immunodeficient mice using local fractionated gamma irradiation. We then transplanted germ cells isolated from GDNF-overexpressing mice into the depleted testes of immunodeficient host mice to investigate whether the transgenic donor cells were able to reproduce the phenotype of the donor mice, including their capability to form clusters expressing hGDNF and Ret.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal goat serum (NGS) was from Aurion (Wageningen, The Netherlands); FCS, penicillin, streptomycin, single-strength nonessential amino acids, gentamycin, minimum essential medium-Eagle (MEM), and Hepes were from Gibco (Life Technologies, Paisley, Scotland). Sodium bicarbonate, L-glutamine, hyaluronidase, and BSA (fraction V) were purchased from Sigma (St. Louis, MO). Trypsin and collagenase were from Worthington (Freehold, NJ); Percoll was from Pharmacia Biotech AB (Uppsala, Sweden). Biotinylated goat anti-rabbit antibody and horseradish peroxidase-avidin-biotin complex were from Vector (Vector Laboratories, Burlingame, CA). Rabbit anti-Ret was an affinity-purified polyclonal antibody raised against the tyrosine kinase domain of Ret, expressed in bacteria as a glutathione S-transferase fusion protein [28]. Both by immunoblotting and immunohistochemistry, the antibody does not stain Ret-negative cells such as fibroblasts and epithelial cells of various organs, whereas it reacts strongly with cells expressing Ret, such as neurons or C cells of the thyroid [29, 30]. 3,3'-Diaminobenzidine was purchased from DAKO (Carpinteria, CA). All other reagents were of analytic grade.
Animals
For irradiation protocols, 5- to 6-wk-old Nc/CpbU mice (Central Laboratory Animal Institute Utrecht, Utrecht, The Netherlands) and 5- to 6-wk-old immunodeficient NMRInu/nu (Hsd/Cpb) mice (Harlan, Horst, The Netherlands) were used. Irradiated NMRInu/nu mice were used as host animals for transplantation of transgenic and control donor cells; in both cases, donor cells were isolated at 3–4 wk of age from male offspring born through mating of wild-type FVB mice (Central Laboratory Animal Institute Utrecht, Utrecht, The Netherlands) with transgenic female mice. Transgenic animals overexpressed human GDNF under the testis-specific human translation elongation factor 1, the expression of which is confined to germ cells [17]. All animal experiments were approved of and carried out according to regulations provided by the Animal Ethical Committee of the University Medical Centre Utrecht.
Depletion of Host Testis by Ionizing Irradiation
For the initial establishment of the optimal irradiation dose, we used Nc/CpbU mice, a mouse strain commonly bred at the local animal facilities. Mice were anesthetized, and testes were irradiated using a Philips Orthovolt (0.5-mm Cu filter) (Eindhoven, The Netherlands). A tube 2 cm in diameter was used to confine radiation to the testes and their immediate surroundings. Fractionated doses of 1 + 4 (n = 4), 1.5 + 8 (n = 6), 1.5 + 12 (n = 3), and 1.5 + 16 (n = 4) Gy were given with an interval of 24 h. Using the same protocol, NMRInu/nu mice (n = 7 for analysis after 12 wk; n = 6 for analysis after 21 wk), the intended hosts for transplanted cells, were subsequently irradiated with 1.5 + 12 Gy.
For evaluation of the extent of depletion, testes were harvested 12 wk (Nc/CpbU and NMRInu/nu mice) and 21 wk (NMRInu/nu mice) after irradiation. These nontransplanted irradiated testes were used to determine the extent of endogenous, as opposed to exogenous, repopulation in testes harvested at 8 and 17 wk, respectively, after transplantation, which corresponds to 12 and 21 wk, respectively, after irradiation. Testes that had been withdrawn during irradiation into the abdominal cavity, out of the irradiation field, were not included in the analysis. Testes were fixed in Bouin solution and embedded in paraffin. Sections 5 µm in thickness were cut. The percentage of tubules containing endogenous spermatogenesis was determined in one section from the middle of the testis and in one section that was 15 sections further to the distal part of the testes, in a minimum of altogether 200 tubule cross-sections per testis. Tubules were scored as containing endogenous spermatogenesis when spermatogonia or, in addition, later cell types were present. As no significant difference was found between the two groups of sections (data not shown), the countings were combined.
Preparation of Donor Cells
Germ cells were isolated from 3- to 4-wk-old GDNF-overexpressing or wild-type mice, of which the correct genotype was confirmed by polymerase chain reaction of tail cuts. The primer sequences used for the detection of human GDNF were 5'-TGTCGTGGCTGTCTGCCTGGTGC-3' for the forward primer and 5'-AAGGCGATGGGTCTGCAACATGCC-3' for the reverse primer. Germ cells were isolated essentially as described previously [31]. Briefly, testes were decapsulated, and tubule fragments were teased apart and then subjected to 2 successive enzymatic treatments of 1 mg/ml trypsin, hyaluronidase, and collagenase and 1 mg/ml hyaluronidase and collagenase, respectively, in MEM containing 0.12% sodium bicarbonate, 4 mM L-glutamine, single-strength nonessential amino acids, 100 IU to 100 µg/ml penicillin-streptomycin, 40 µg/ml gentamycin, and 15 mM Hepes. Cells were separated from the remaining tubule fragments by centrifugation at 30 x g and, after filtration through nylon filters with 77- and 55-µm pore sizes, were pelleted and loaded onto a discontinuous Percoll density gradient. The purity of the cell suspensions, with a viability that has previously been shown to be between 95% and 99% [31], was assessed by Nomarski optics, by which cells with a high nucleus:cytoplasm ratio and one or more distinct nucleoli are characterized as spermatogonia [31]. Fractions with a purity of at least 40% spermatogonia were washed, counted, and resuspended to a concentration of cells equivalent to 106 spermatogonia per milliliter. Aliquots of 20 µl of the germ cell suspensions were kept on ice until transplantation.
Transplantation
Germ cell suspensions (20 µl) were transplanted via efferent duct microinjection [32] into NMRInu/nu mice, 4–5 wk after their local fractionated irradiation with 1.5 + 12 Gy. Six mice were transplanted with transgenic germ cells, and 2 mice received wild-type germ cells. For every mouse the nontransplanted, contralateral testis served as a negative control. After 8 wk, host mice were killed by CO2 asphyxia. Testes were fixed in Bouin solution for histology and Ret immunocytochemistry and in 4% paraformaldehyde for in situ hybridization. Samples were embedded in paraffin, and sections 5 µm in thickness were cut. In 2 random sections from the middle part of the testis, a minimum of 200 tubule cross-sections were evaluated for the presence of normal spermatogenesis or clusters of spermatogonial cells.
In Situ Hybridization of hGDNF
Radioactive in situ hybridization for hGDNF was performed as described previously [33]. Antisense and sense cRNA probes were synthesized from the 636-base pair full-length coding region of hGDNF cDNA using appropriate RNA polymerases and 35S-labeled UTP. The hybridization temperature was 52°C, and autoradiography slides were exposed at 4°C for 2–4 wk. Slides were counterstained for histologic structures by hematoxylin staining and photographed with an Olympus Provis microscope equipped with a CCR camera (Photometrics Ltd., Tucson, AZ). In Adobe Photoshop 4.0 (Adobe Systems Inc., San Jose, CA), the dark-field images were inverted, artificially stained red, and recombined with the bright-field images.
Immunolocalization of Ret in Transplanted Testes
For Ret localization, testes with transplanted cells were fixed in Bouin fluid, embedded in paraffin, and sectioned. Sections were deparaffinized, and endogenous peroxidase was blocked by incubation in 3.5% H2O2 in PBS for 10 min. The slides were washed in PBS, and nonspecific binding was blocked by a 1-h incubation in 10% NGS in PBS. Incubation with 30 ng/ml anti-Ret or 1000 ng/ml rabbit control isotype immunoglobulin G (IgG), diluted in PBS containing 5% NGS and 5% BSA, was performed overnight at room temperature in a humidified chamber. After the sections were washed extensively in PBS, they were incubated for 60 min with biotinylated goat-anti-rabbit diluted 1:200 in PBS containing 5% NGS and 5% BSA. After the sections were washed in PBS, the horseradish peroxidase-avidin-biotin complex reaction was performed according to the manufacturer's protocol. To visualize antibody binding, sections were washed and exposed to 0.05 M Tris-buffered saline (pH 7.6) containing 0.3 mg/ml 3,3'-diaminobenzidine and 0.01% H2O2. Sections were counterstained with Mayer hematoxylin.
Statistical Analysis
Data are presented as the mean ± SEM. Statistical analysis was performed by nonparametric ANOVA or the Mann-Whitney test. P < 0.05 (two-tailed) was assumed to indicate statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 12 wk after irradiation, a dose-dependent decrease in the percentage of tubule cross-sections containing spermatogenic cells was found in testes of Nc/CpbU mice (P < 0.01; nonparametric ANOVA). Using 1 + 4 Gy, only 3.1% ± 1.1% (n = 4) of the tubules were depleted, which increased to 71.6% ± 9.6% (n = 6) using 1.5 + 8 Gy, whereas 1.5 + 12 Gy resulted in near complete removal of germ cells, with 99.2% ± 1.1% (n = 3) of the tubules having only Sertoli cells. Because the dose of 1.5 + 16 Gy (n = 4) resulted in skin lesions characterized by erythema and transudation, this regimen was not pursued further. For the NMRInu/nu mice, the mice intended as recipients for transplanted germ cells, the dose of 1.5 + 12 Gy resulted in a depletion of 96.9% ± 1.7% (n = 7) at 12 wk and 89.4% ± 3.7% (n = 6) at 21 wk after irradiation (not significant; Mann-Whitney test).
Occasionally, immediately after irradiation, the testes were observed to have been withdrawn into the abdominal cavity. The final radiation dose applied to the testes will be unknown in these cases; therefore, before and immediately after the irradiation procedure, mice should be checked for withdrawal of their testes. Alternatively, withdrawal can be prevented by the crosswise application of tape to the lower abdomen.
Transplantation of GDNF-Overexpressing Germ Cells
Two months after surgery, transplantation of germ cell suspensions isolated from GDNF overexpressors had resulted in the formation of spermatogonial clusters in all host testes (n = 6), and the percentage of tubules containing clusters was 10.3% ± 2.5%. Clusters were of variable size and could be recognized by the accumulation of relatively large cells with a high nucleus:cytoplasm ratio and a characteristic distribution of heterochromatin, resembling A spermatogonia [34] (Fig. 1A, arrow). There was no evidence of a chimeric histologic pattern similar to the pattern found at 2–3 wk by Meng et al. [17], who found many tubules with apparently normal spermatogenesis adjoining tubules with cluster cells. Clusters and a small number of more differentiated germ cells, usually spermatocytes (arrowheads in Fig. 1), were frequently found together in the same tubule cross-sections (Fig. 1, A and B). In addition to cluster-containing tubules, 4.5% ± 0.6% of tubules contained normal spermatogenesis, as defined by the presence of an intact seminiferous epithelium throughout the tubule cross-section without evidence of abnormalities. Analysis of the nontransplanted contralateral control testes revealed a repopulation of 3.1% ± 0.6%, which was not significantly different from the extent of normal spermatogenesis found in cluster-containing testes. In addition to cluster-containing tubules, some tubules contained only a rim of spermatogonia (Fig. 1C). Strikingly, often Sertoli cell nuclei were found at a considerable distance from the basement membrane at the periphery of clusters (designated by S in Fig. 1, A and B) or overlying rims of spermatogonia (S in Fig. 1C).
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In the testes of the two mice transplanted with wild type cells, neither clusters nor rims of spermatogonial cells were noted (data not shown). In these mice, normal spermatogenesis was found in 24.5% and 20.5% of the seminiferous tubules, respectively, and germ cells in these tubules usually had advanced to the pachytene spermatocyte stage, in line with the results of previous transplantation studies [22]. The contralateral controls of these transplanted testes showed a repopulation of 1.7% and 2.8%, respectively.
In Situ Hybridization of hGDNF
With the radiolabeled antisense probe, a clear signal was observed over the clusters of cells found in transplanted testes (Fig. 2A, in situ signal displayed in red), whereas sense controls showed hardly any label (Fig. 2B).
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Ret Expression of Cluster Cells in Transplanted Testes
In the nontransgenic wild-type testis, the presence of the Ret receptor appeared to be confined to early spermatogenic cells (Fig. 2C), as only germ cells up to leptotene spermatocytes were positive for the protein. Spermatogonia occasionally displayed a concomitant staining of nucleus and cytoplasm (inset), and spermatocytes showed a more distinct membrane-bound labeling (arrows). In the clusters present in the testes of 5-wk-old transgenic mice (Fig. 2D) as well as in the testes transplanted with transgenic germ cells, a distinct labeling for Ret was found, frequently in both nucleus and cytoplasm (Fig. 2E). Although most cluster cells showed strong staining, the intensity was variable, further supporting the view that the clusters did not consist of interconnected cells [17]. Negative controls processed with nonimmune rabbit IgG did not yield any staining in tissue sections from wild-type, transgenic, or transplanted mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For both strains of mice irradiated, a dose of 1.5 + 12 Gy was found to eliminate virtually all endogenous stem cells, as histologic analysis revealed that only a small percentage of tubule cross-sections showed germ cell repopulation 12 wk after irradiation. At lower doses of irradiation, depletion was insufficient. After 21 wk, repopulation was somewhat more pronounced and also showed more variation, but was still limited to one tenth of the tubule cross-sections. Thus, in addition to the 8-wk interval used in our transplantation experiments, harvesting testes at later time points after transplantation is also possible.
An important advantage of using local irradiation is the lack of harmful side effects for the animals, in contrast to depletion achieved with the administration of busulfan or whole body irradiation. Sertoli cells and Leydig cells are known to be relatively insensitive to irradiation [35, 36] and did not appear to be affected in the current study, as growth and maintenance of transgenic and wild-type germ cells was supported on their transplantation into irradiated testes (discussed subsequently). Importantly, as depletion of recipient testes proved to be very efficient, almost all repopulation of the recipient testis after transplantation will originate from the transplanted donor cells, precluding the need for specific markers identifying donor cells. Although in this respect W/W mice, which are infertile because of a complete lack of male germ cells, are most suitable, the advantage of the current method is that it departs from common and readily available mouse strains without breeding defects.
In testes transplanted with germ cells isolated from GDNF-overexpressing mice, but not with germ cells from wild-type mice, large clusters of spermatogonia were found as well as seminiferous tubules containing only a rim of these cells. Both of these phenomena were observed previously in the original transgenic animals [17]. Moreover, the cluster cells in transplanted testes expressed the insert hGDNF and showed the same size and nuclear heterochromatin pattern as that found for cluster cells in the transgenic host testes. Further support for their identical behavior was provided by the localization of the Ret receptor. In both transgenic testes and testes of mice transplanted with overexpressing cells, an intense and heterogeneous staining of cells inside the clusters was noted, whereas in wild-type mice, staining was confined to spermatogonia and spermatocytes lying at the basal lamina. It should be noted that the localization of the Ret protein currently found deviates from that of its mRNA observed previously, which is limited to a small subset of spermatogonia [17]. Although nonspecific immunostaining can never be fully excluded, one possible explanation may lie in a long half-life of Ret protein, enabling its prolonged presence during subsequent steps along the spermatogenic lineage. A similar discrepancy between mRNA and protein expression has been reported during embryonic kidney and ureteric development [37]. In that study, a nuclear association for Ret protein was also found [37], which we likewise observed for some wild-type germ cells and in cluster cells. Although this localization seemingly contradicts its feature of a membrane-bound protein, evidence is accumulating for the hypothesis that under certain circumstances, various membrane-bound tyrosine kinases display a nuclear association, as has been demonstrated for the fibroblast growth factor receptor 1 [38], the vascular endothelial growth factor receptor Flk-1/KDR [39], and the growth hormone receptor [40]. It has been postulated that this type of localization might be linked to mitogenesis [41], which would coincide with our observation that in meiotic spermatocytes, Ret is solely expressed at the plasma membrane.
In contrast to the phenotype found in the transgenic donors, tubules containing normal spermatogenesis in the transplanted animals were scarce. Transgenic animals of approximately 3 wk of age have a chimeric pattern of tubules containing clusters and tubules with normal spermatogenesis [17]. It is possible that in young transgenic animals the concentration of hGDNF produced by the transgenic cells had not yet reached levels sufficient to cause an overall block of spermatogenesis. However, differentiating cells, usually spermatocytes, were observed in some tubules of transplanted testes containing spermatogonial clusters producing GDNF. Moreover, despite the low percentage (previously discussed) of cluster-containing and thus hGDNF-producing tubule segments, no normal spermatogenesis exceeding nontransplanted host control levels was recorded. A more plausible explanation for the partly normal features of prepubertal transgenic testes could lie in the first wave of spermatogenesis, which has been suggested to deviate from subsequent spermatogenic cycles in life for several reasons [42, 43]. As the transplanted transgene-containing spermatogonial cells can only have been derived from subsequent ‘waves,’ they would not be expected to yield normal spermatogenesis in the host.
In the transgenic mouse, spermatogonial clusters were found to disappear around the time of maturation of the blood-testis barrier, leaving only tubules with a rim of spermatogonia lining the basal membrane [17]. It was suggested that at a later stage, the formation of a mature blood-testis barrier had prevented new clusters. In the present study, clusters were formed by GDNF-overexpressing germ cells transplanted to adult host mice. Whether the transplanted cells were inside or outside of the blood-testis barrier was not entirely clear. However, Sertoli cell nuclei were often noted at the boundaries of the clusters or lying over rims of spermatogonial cells, suggesting that the transplanted cells were actually at the basal side of the blood-testis barrier. This would suggest that Sertoli cells had attempted to move the cluster cells in the direction of the basal lamina, finally resulting in the formation of rims. More extensive time point analyses showing an increase in this phenomenon with time would be supportive of this idea. As clusters had still been able to form underneath, the presence of the intact blood-testis barrier in adult host mice in itself apparently does not have an inhibitory effect. Interestingly, in the central nervous system, GDNF has been shown to enhance the formation of the blood-brain barrier (BBB), decreasing the permeability of in vitro layers of BBB-forming endothelial cells to a tracer dye [44]. In this light, it cannot be excluded that long-term exposure of Sertoli cells to hGDNF at least partially contributed to the formation of rim-containing tubules in both the GDNF-overexpressing transgenic mice and the mice transplanted with transgenic germ cells. Whether Sertoli cells actually express one or more of the receptors involved in GDNF signaling is not clear. Ret and GFR have been found to be expressed only on germ cells [17]; on the other hand, a role of GDNF in Sertoli cell proliferation has been found [16], which would suggest the presence of a functional receptor.
At any rate, overexpression of GDNF appears to strongly affect germ cell behavior in vivo. Cells in the clusters in transgenic animals and host animals transplanted with GDNF-overexpressing spermatogonial cells are generally unable to initiate differentiation, displaying morphologic characteristics of spermatogonia. It will now be worthwhile to study the expression and role of GDNF and its receptors in overexpressing cells in vitro and to investigate the capability of overexpressors to reproduce the transgenic phenotype in host mice after culturing.
In conclusion, we have shown that local fractionated irradiation is an efficient method for depleting endogenous spermatogenesis in host animals. Moreover, germ cells overexpressing GDNF reproduce the abnormal features observed in donor transgenic mice on transplantation into irradiated host mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grant HD 36476-02. 
2 Correspondence: Laura B. Creemers, Department of Cell Biology, HP G02.525, UMCU, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. FAX: 31 30 2541797; l.b.creemers{at}lab.azu.nl
Accepted: December 19, 2001.
Received: August 15, 2001.
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