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Department of Urology,3 Mount Sinai Hospital, University of Toronto, Ontario, Canada, M5G 1X5
Division of Urology,4
Department of Molecular and Cellular Biology,5 Baylor College of Medicine, Houston, Texas 77030
Department of Urology,6 Eastern Virginia School of Medicine, Norfolk, Virginia 23454
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Efforts to identify SSCs among other testicular cells advanced in 1994 when a functional transplantation assay was developed that recolonized the seminiferous tubules of infertile recipients with donor SSCs [1, 2]. Since that landmark publication, different in vivo and in vitro techniques have been used to enrich the SSC population. Taking advantage of the detrimental effect of temperature on spermatogenesis, Shinohara et al. [3] reported a 25-fold increase in colonization of the recipient testes using donor cells from cryptorchid testes, in essence minimizing the haploid germ cell in the donor population. Flow-cytometric selection of 
6-integrinhi-Side Scatterlo-v-integrin– cells further enriched the SSC population to 1 in 30 testicular cells [4]. An alternative model for cell enrichment selected cells with an active Stra8 gene promoter that achieved a 700-fold enrichment of SSCs in a type of transgenic mice [5].
Several stem cells of different lineages share the ability to extrude Hoechst 33342, a nuclear-binding vital dye. The side population (SP) cells that exhibit low Hoechst staining are highly enriched for hematopoietic stem cells [6], skeletal muscle stem cells [7], and neural stem cells [8]. Members of the ATP-binding cassette (ABC) transporter family, such as MDR1 and BCRP1/ABCG2, are thought to contribute to the dye efflux of the SP cells [9, 10]. However, contradictory findings regarding the nature of the testicular SP cells have recently been reported. Lassalle et al. [11] and Falciatori et al. [12] reported an enrichment of SSCs in the SP cells isolated from adult and immature mouse testes, but Kubota et al. [13] could not colonize sterile testis with the Hoechst-sorted testicular SP cells. Our unpublished findings resemble the results of Kubota et al. and show that the SP may not contain SSCs or viable SSCs. One of the potential explanations for this discrepancy is the source of the donor cells (i.e., cryptorchid vs. noncryptorchid testis). The other possible cause is that because Hoechst dye is highly toxic to cells, the SSCs may not have survived the transplantation process.
To circumvent the toxicity of the Hoechst dye, we explored the use of a less toxic dye, rhodamine 123 (Rho), which is a mitochondrial dye that has been used both alone [14–16] and in combination with Hoechst dye to isolate hematopoietic stem cells [16–20]. The amount of fluorescence emitted by a cell stained with Rho dye reflects either its mitochondrial content or its kinetics. It is postulated that the quiescent nature of stem cells results in a low fluorescent emission detectable by the flow cytometer. This population that stains low with Rho dye, designated as Rholow, can be isolated and examined. Our aim was to compare the stem cell enrichment of the testicular Rholow population and that of Hoechst SP cells. We hypothesized that the Rholow cell population contains more viable cells and, therefore, may enrich the SSC population as demonstrated by the germ cell transplantation assay.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Donor Testicular Cell Isolation
Cells for transplantation were isolated from the testes of the transgenic mouse line, ROSA26, which are maintained on a C57BL/6 x 129/Sv genetic background and express the Escherichia coli LacZ gene. Many cell types, including all stages of germ cell differentiation, stain positively (blue) in a colorimetric reaction with 5-bromo-4-chloro-3-indolyl ß-galactoside, allowing accurate tracking of the transplanted cells. Two types of donors, cryptorchid and noncryptorchid mice, were studied. The testes of the cryptorchid donor mice were surgically sutured to the upper abdominal wall at 6–8 wk of age. These mice were killed and their testes harvested 6–8 wk postoperatively.
After harvest and decapsulation of the donor testes, the seminiferous tubules were minced mechanically and enzymatically digested using collagenase and trypsin as described elsewhere [21]. The single cells were washed and resuspended to a desired concentration in a medium supplemented with Dulbecco modified Eagle medium, 5% Hepes, and 10% fetal bovine serum.
Hoechst 33342 and Rho Dye Staining
The donor testicular cells were stained with Hoechst and/or Rho dye. For the Hoechst dye staining, cells were resuspended at 106 cells/ml and stained with the dye (bis-benzimide H 33342; Sigma-Aldrich, St. Louis, MO) at a concentration of 5 µg/ml for 90 min at 37°C. After the incubation, cells were kept in serum media at 4°C to prohibit leakage of Hoechst dye from the cells. For the Rho staining, 106 cells/ml were exposed to 0.1 µg/ml of Rho (Sigma-Aldrich) for 20 min at 37°C in the dark. The cells were then washed and resuspended with the serum-enriched media and kept on ice. For combined staining, the same method was used as described for the Hoechst staining, but Rho was added during the last 20 min of the incubation. Propidium iodide (2 µg/ml; Sigma-Aldrich) was added to the final suspension before the flow-cytometric sorting to exclude dead cells from the flow-cytometric profile.
Flow-Cytometric Analysis and Cell Sorting
The procedure employed for flow-cytometric cell analysis of the Hoechst SP is described elsewhere [6]. Briefly, the Hoechst dye-stained cell solution was excited with the ultraviolet laser at 350 nm (DAKOCYTOMATION, Carpinteria, CA). Forward and side light scatter of the cells was used to eliminate debris. Cells with low side scatter were gated for further analysis with dual-wavelength filters, 405 nM (blue) and 630 nM (red), to detect the resulting fluorescence. For the analysis of Rho staining, an argon laser was used to excite the cells, and a single filter at 570–40 nM detected the resulting fluorescence.
As a negative control for the Hoechst staining, verapamil (25 and 75 µg/ml; Sigma-Aldrich), which blocks the ABC membrane transporter from extruding the Hoechst dye, was added to an aliquot of cell solution before the Hoechst dye incubation. The same procedure was applied to an aliquot of cells to be stained with Rho to examine whether the ABC transporters also are involved in the differential staining of Rho.
For germ cell transplantation, the Rho-stained cell populations gated for low (Rholow) and high (Rhohi) fluorescence were sorted and resuspended in the serum media.
Donor Cell Transplantation and Recipients
Donor cells were transplanted into the seminiferous tubules of recipient mice (age, 3–4 mo) according to techniques described previously [21]. Briefly, the recipient mice were anesthetized in accordance with the guidelines for animal handling approved by the AIRB. Trypan blue dye (0.4% Gibco BRL, Carlsbad, CA) was added to the cell solution to assist in visualizing the injected tubules. The cell solution was injected via the efferent duct or rete testis into the seminiferous tubules using a micropipette. Transjector 5246 (Eppendorf, Hamburg, Germany) was used to regulate the rate of injection. The sterile transgenic mouse line WBB6F1/J-Kitw/Kitw-v (designated W/Wv) from the Jackson Laboratory (Bar Harbor, ME) served as the recipients. The W/Wv mice are sterile with a Sertoli cell-only testicular phenotype.
Staining of Testicular Tissue to Assess Spermatogenesis and Transplantation Success
The recipients were killed 3 mo posttransplantation, and testes were harvested as per the animal protocol. With the tunica albuginea removed, the seminiferous tubules were dispersed and fixed in CHO fixative (3% paraformaldehyde, 0.2% glutaraldehyde, and 2% sucrose in PBS; pH 7.4– 7.5) for 90 min at 4°C. The tubules were then rinsed in PBS and washed with buffer Solution A (60 min) and Solution B (twice for 30 min each time) from Specialty Media (Phillipsburg, NJ). The tubules were then incubated in COMPLETE B-Gal Tissue Stain Solution (Specialty Media) for 2–3 h at 37°C. Successful donor cell colonization with spermatogenesis is clearly visible as blue seminiferous tubules, and each blue segment originates from one SCC [22, 23]. The number of colonies was counted under magnification with a dissecting microscope. The testes were then refixed with CHO fixative for 12 h (or overnight) before being embedded in paraffin. Sections of the paraffinized blocks were cut (thickness 5 µm) and counterstained with nuclear fast red solution for further analysis.
Statistical Analysis
SigmaStat version 3.1 software (Systat Software, Inc., Point Richmond, CA) was used for data analysis. For Table 1, the Mann-Whitney rank sum test was used to compare the cryptorchid and noncryptorchid donor cells. For Table 2, we used the Kruskal-Wallis one-way ANOVA on ranks with three degrees of freedom to compare the transplant results from Rholow cells with those of the other groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Hoechst staining patterns of the testicular cells from cryptorchid (Fig. 1A) and noncryptorchid (Fig. 1C) testes were quite different. From the cryptorchid donor cells, three distinct regions (R1, R2, and R3, representing haploid, diploid, and tetraploid cell populations, respectively) were detected in addition to the SP region. In contrast, the noncryptorchid testicular cells exhibited a more diverse pattern that can be gated into five regions (R4 to R8). The SP cell population represented approximately 1.6% of the total cell population in cryptorchid testes but only 0.45% in the noncryptorchid testes.
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To examine the Rho staining of the cells in each distinct region, the percentage of cells that fell in the Rholow region after argon laser excitation was measured (Table 1). The SP regions of both cryptorchid and noncryptorchid testes had the highest portion of cells that were also Rholow. No significant difference was observed between cryptorchid and noncryptorchid SP cells (Fig. 1, B and C). The Rholow cells were almost undetectable in the other regions (R1 to R8), ranging from 0% to 5.5%. These data suggest that the SP and Rholow cells share similar characteristics in the flow-cytometric analysis.
Role of ABC Transporter in Hoechst and Rho Cell Staining
It is postulated that the ABC transporter family is responsible for the extrusion of the Hoechst dye from the stem cells, but the mechanism of the differential staining of the Rho dye is less evident. Verapamil blocks the ABC transporters and inhibits the cells from "pumping out" the Hoechst dye. This is shown in Fig. 2, A and B, which indicates that the number of SP cells was markedly reduced when verapamil was added. The same result was not observed in the Rho-stained cells. No change was seen in the proportion of cells that were Rholow when 25 or 75 µg/ml of verapamil were added (Fig. 2, C and D). This result suggests that a different mechanism determines Rho cell staining.
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Enrichment of SSCs in the Rholow Cell Population
Approximately 5–7% of the total testicular cells were Rholow (Fig. 3). Overall, nine testes from the W/Wv were injected with the Rholow cells, five testes with Rhohi cells, and five testes with nonsorted cells. The mean number of Rholow cells injected was 5000 per testis (range, 3000–7500 cells), whereas the average number of cells transplanted in the Rhohi group was 8 x 104 (Table 2). The nonsorted group was transplanted at a higher concentration, and each testis received an average of 9.1 x 105 cells.
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The recipients' testes were processed and analyzed as described in the Materials and Methods. Successful colonization of the donor SSCs is shown in Fig. 4A, and the negative control testis is shown in Fig. 4B. Histologic sections confirm the presence of spermatogenesis (Fig. 4C) from the successfully transplanted testis and the Sertoli cell-only pattern (Fig. 4D) from the negative control W/Wv testis.
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Successful transplantation of the donor cells was observed in all three groups but at different frequencies. When the colony number was normalized to 105 cells injected per testis, we observed a 17-fold increase in the Rholow group over the Rhohi group, and a 20-fold enrichment with injected Rholow cells over the nonsorted cells (Table 2). In contrast, no colonies were observed in the eight recipient testes transplanted with the Hoechst SP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Can these results be extrapolated to explain the discrepancy in the reported characteristics of the testicular SP isolated using the Hoechst dye? Lassalle et al. [11] suggested that the testicular SP cells isolated from noncryptorchid adult mice versus those from cryptorchid donor mice reported by Kubota et al. [13] may account for the difference in their germ cell transplantation success. Our results do not support this explanation, because the testicular SP cells from both donor types have similar Rho staining.
The alternative explanation that the Hoechst dye toxicity is responsible for the failure of SSC colonization may be more plausible. Although Kubota et al. [13] showed that viability of the SP population immediately after cell sorting is adequate, the long-term survival of these cells is not defined. In light of the relatively low efficiency of transplantation, it is possible that the number of surviving cells injected simply was too low. Using the less toxic Rho dye, we show that a better chance of successful colonization and subsequent spermatogenesis exists following flow-cytometric enrichment.
We used cryptorchid testes as the donors for the transplantation. Therefore, the 20-fold increase in the Rholow cell population in comparison to that of the nonsorted cells represents an additional enrichment over the in vivo manipulation. Our results also show a 17-fold difference between the colonization from Rholow and Rhohi cells. This indicates that not all SCCs stain low with the Rho dye. Because Rho staining reflects the mitochondrial kinetics, it is conceivable that whereas majorities of the SSCs are quiescent (Rholow), some SSCs may be in the process of proliferation and, hence, have higher mitochondrial staining (Rhohi). The degree of enrichment may reflect the ratio between the dormant and proliferating SCCs in the donor testes.
Interestingly, the verapamil results suggest that although the ABC transporters most likely are responsible for the Hoechst dye exclusion in the SP cells, a different mechanism governs the degree of Rho staining by the stem cells.
The value of obtaining a purified population of SSCs cannot be overstated. In addition to the study of reproductive biology, the therapeutic potential in the treatment of male infertility with the germ cell transplantation technique also relies on the ability to exclude any malignant cells from the isolated testicular cell population. Progress using both in vitro (surface antibodies) and in vivo (cryptorchidism, thermo therapy) methods to enrich the SCC population are being made in many laboratories, and we believe that use of the differential staining properties of stem cells with Rho dye can add to the techniques available to overcome these challenges.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Dolores J. Lamb, Scott Department of Urology, Baylor College of Medicine, One Baylor Plaza, Alkek N 730, Houston, TX 77030. FAX 713 798 5577; dlamb{at}bcm.tmc.edu
Received: 26 June 2004.
First decision: 20 July 2004.
Accepted: 18 October 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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