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a Institute of Reproductive Medicine of the University Münster, 48149 Münster, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Magnetic cell separation has become a widely used method for separating many different types of cells. Methods have been described for the isolation of human lymphocytes [11], dendritic cells [12], fetal cells from maternal blood [13], granulocytes [14], and megakaryotic cells [15], as well as natural killer cells, T cells, tumor cells, and epithelial cells. Blood stem cell technology has profited significantly from the introduction of magnetic cell sorting [16]. Negative and positive selection strategies using depletion columns or positive selection columns have been implemented; and for many applications, multi-sort options are available from the manufacturer of the magnetic beads and separation columns (Miltenyi Biotech, Auburn, CA).
In this study we applied commercially available IgGs directed against the c-kit receptor protein to the immunomagnetic isolation of spermatogonia. Binding of the antibody to c-kit was characterized by immunohistochemical procedures on testicular tissue sections. The percentage of c-kit/fluorescein isothiocyanate (FITC)-positive cells and the DNA content of the sorted cell fractions were analyzed by flow cytometry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fully regressed testes from photoinibited hamsters (n = 27) that had been kept in short photoperiods (16L:8D) for 8–14 wk were used for initial experiments to develop the basic principles of the digestion protocol and isolation procedure, since these testes present a physiologically enriched source of spermatogonia [17, 18]. In subsequent experiments, normal testes showing the full germ cell complement from photostimulated adult hamsters (n = 10), adult mice (n = 9), and adult marmoset monkeys (n = 8) were used. All animals were derived from colonies at our institute and were treated in accordance with the Federal German Law on the handling of experimental animals.
Cell Isolation from the Seminiferous Epithelium
The testes were excised and decapsulated. Testicular tissue was minced using fine scissors and transferred into culture medium (Gibco, Gaithersburg, MD; Dulbecco's modified Eagle's medium [DMEM]/F12) containing collagenase type I (Sigma Chemical Co., St. Louis, MO; 1 mg/ml) and DNase (Sigma; 0.5 mg/ml). Digestion was performed at 37°C for 10 min in a shaking water bath operated at 110 cycles/min. Interstitial cells were separated by sedimentation at unit gravity for 10 min and washed in DMEM/F12. A final digestion of the basal lamina components was carried out in a mixture of collagenase type I (Sigma; 1 mg/ml), DNase (Sigma; 0.5 mg/ml), and hyaluronidase (Sigma; 0.5 mg/ml) under the same conditions as for the first digestion step. The single-cell suspension obtained was washed successively with medium and PBS containing 2 mM EDTA (Sigma) and 0.5% fetal calf serum (FCS; Gibco). Undigested remains of the tunica albuginea were eliminated by filtering the cell suspension through a 50-µm nylon mesh. No visible amounts of the single-cell suspension were retained in the filter. Cell number and concentration were established microscopically using a Thoma chamber (Hecht, Sondheim, Germany) and were later confirmed by flow cytometric analysis.
Magnetic Labeling and Separation of Cells
Aliquots of cell preparations with a final concentration of 107 cells/100 µl were labeled using an indirect labeling technique with the magnetic beads attached to a secondary antibody in order to amplify binding. The separation procedure was carried out according to the manufacturer's instructions (Miltenyi Biotech). In brief, the cells were incubated with polyclonal rabbit anti-c-kit IgGs (Santa Cruz Biotechnology, Santa Cruz, CA; C-19, catalogue no. sc-168, diluted 1:16) for 15 min at 6–10°C. After two washes with PBS (supplemented with EDTA and FCS as described above), the cells were labeled with goat anti-rabbit IgG microbeads (Miltenyi; order no. 486–02) diluted 1:5. The cells were washed again and stained with FITC-conjugated swine anti-rabbit F(ab')2 IgG fragments (Dako, Glostrup, Denmark; no. F 0054, diluted 1:10) for 20 min at 6–10°C to allow subsequent flow cytometric analysis. Alternatively, fluorescent labeling was performed after the separation, yielding comparable results. Control samples were processed as above, but incubation with the c-kit antibody was omitted. Prior to flow cytometric analysis the cells were protected from strong light.
For the subsequent magnetic separation, ice-cold degassed PBS buffer containing EDTA and FCS was utilized. An RS+ separation column (Miltenyi) was flushed with 500 µl of degassed buffer. The cell suspension was also resuspended in degassed buffer (500 µl/107 cells) and poured into the column reservoir. C-kit-positive, and thus magnetically labeled, cells were retained within the magnetized matrix of the column, whereas nonlabeled cells passed through and were collected as the nonmagnetic fraction. To increase the purity of the magnetic fraction, the column was rinsed three times with 500 µl of buffer. The effluent of the washing steps was also collected as nonmagnetic fraction. In order to retrieve the magnetic fraction, the column was removed from the separator, and 1 ml of degassed buffer was added to the reservoir. The cells were flushed out of the column with the aid of a plunger.
Quantification of FITC-Positive Cells and DNA Content by Single-and Dual-Parameter Flow Cytometry
In order to determine the percentage of FITC-positive cells, the cells were analyzed on a Coulter (Krefeld, Germany) flow cytometer EPICS XL equipped with a 15-mW argon-ion laser at an excitation wavelength of 488 nm. The green signals of FITC on log scale of each of the unsorted/sorted cell fractions were collected using a 520-band pass filter (505–545 nm). A marker was set in the FITC histogram as the cut-off between background signals and positive staining, which was determined by comparison with the control sample. A minimum of 10 000 cells was analyzed in each run.
Aliquots of the cells were stained with propidium iodide (PI, 1 mg/ml; Sigma) to check the viability of the cells. The red signals of PI on log scale were quantified using a 620-band pass filter (605–635 nm). The PI-positive cells were considered dead cells and the PI-negative cells considered alive. The viability was also tested by trypan blue dye exclusion test.
PI staining was also carried out to discriminate the different testicular germ cell populations based on the nuclear DNA content. Cells were recognized as elongated and round haploid spermatids (HC and 1C), diploid spermatogonia and somatic cells (2C), S-phase cells that synthesize DNA (S-ph), and tetraploid primary spermatocytes and G2 spermatogonia (4C) [19]. Dual-parameter flow cytometry was performed to quantify the number of FITC-positive cells among the various testicular somatic and germ cell populations. Freshly isolated cells from the various fractions of the FITC-labeled cells were fixed for 12 h in 0.1% buffered paraformaldehyde (pH 7.4) in the dark at 4°C. The cells were washed and counterstained with DNase-free staining solution containing RNase (40 µg/ml; Sigma) and PI (25 µg/ml; Sigma) in PBS for 20 min at room temperature in the dark. Forward scatter was used as triggering parameter. The green fluorescence signals of the FITC-labeled cells on log scale in each of the unsorted/sorted fractions was quantified using a 520-band pass filter (505–545 nm). Red fluorescence signals of the PI staining were displayed on linear scale using a 620-band pass filter (605–635 nm). In order to avoid signal spillover from FITC emission, a color compensation of 20% was applied. Cell debris and aggregates were eliminated from the analysis by gating.
Immunohistochemistry
Bouin's-fixed and routinely embedded tissue was used. Paraffin sections (5 µm) from male Djungarian hamsters, mice, and marmoset monkeys were immunohistochemically stained for c-kit. An indirect method using peroxidase-labeled secondary antibodies was carried out, with reagent dilutions and incubation conditions selected according to manufacturer instructions. Briefly, the sections were deparaffinized and rehydrated in an ethanol series. To recover protein antigenicity, sections were heated in a microwave oven in glycin/HCl buffer (50 mM, pH 3.5) for 10 min at 100°C. Endogenous peroxidase activity was quenched by a 5-min treatment with 3% hydrogen peroxide in a humidified chamber, followed by blocking of nonspecific antibody binding with 5% normal goat serum supplemented with 0.1% BSA, for 20 min at room temperature. Slides were rinsed in Tris-buffered saline (TBS) three times, 5 min each, between each incubation, and all antibodies were diluted in TBS/0.1% w:v BSA. Sections were then incubated at room temperature for 1 h in a humidified chamber in TBS/BSA as a negative control or with rabbit anti-c-kit antibodies (1:20; Santa Cruz) raised against a peptide corresponding to an amino acid sequence within the carboxy-terminal domain of c-kit p 145 of human origin. For localization of the c-kit receptor, the sections were immunostained with horseradish peroxidase-conjugated goat anti-rabbit IgGs (at a 1:50 dilution for 1 h) followed by 3,3'-diaminobenzidine tetrahydrochloride in urea buffer (5–12 min; Sigma tablets). Positive staining appeared as a brown precipitate in the cells. The sections were counterstained with hematoxylin and examined by light microscopy; representative areas of the testis were photographed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Indirect immunohistochemical staining of c-kit using a polyclonal rabbit antibody was performed in order to characterize the distribution of c-kit protein in histological sections. Spermatogonia demonstrated positive staining in the basal region of the seminiferous tubules in the marmoset monkey (Fig. 1A), Djungarian hamster (Fig. 1, C and D), and mouse (not shown). C-kit immunoreactivity was also detected within the acrosomal granules and acrosomal regions of round and elongated spermatids. The Leydig cells of all species, including the photoinhibited Djungarian hamster, also demonstrated positive staining. All other germ cell types, Sertoli cells, and peritubular cells showed no staining for c-kit. Omitting the primary antibody resulted in the absence of any positive staining (Fig. 1B).
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Spermatogonial Cell Isolation
Sedimentation at unit gravity after the first digestion step allowed the efficient separation of seminiferous tubules from interstitial cells. Sequential enzymatic digestion of the decapsulated testis tissue resulted in a single-cell suspension. The final cell concentration after filtration of the cell suspension was in the range of 1–3 x 108/g testis tissue.
Cytological Analysis
In all species, obvious differences were observed between the unsorted and the nonmagnetic cell fractions in comparison to the magnetic fraction. As a typical example, Figure 2 illustrates the microscopic appearance of freshly isolated cells from photoregressed Djungarian hamster testes. A heterogenous single-cell fraction containing all cells of the regressed tubular compartment was obtained in the unsorted fraction (Fig. 2A). In contrast, the magnetic fraction consisted of a relatively homogenous population of cells (Fig. 2C, Fig. 3). This fraction contained small to large clusters of cells. In their cytological characteristics, many of these cells resembled A-spermatogonia (Fig. 3) as previously described [20]. The cytological picture of the freshly isolated nonmagnetic fraction was very similar to that of the unsorted cell fraction (Fig. 2B). Analysis of cytospins yielded similar results.
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Flow Cytometric Analysis
In terms of absolute cell numbers, the magnetic cell separation allowed sorting of fractions of testicular cells with a recovery of around 105 cells in the magnetic fraction/107 cells obtained after digestion. In the first experiments, flow cytometry was used to analyze the relative number of FITC-labeled versus nonlabeled cells in the unsorted nonmagnetic and magnetic fractions obtained. Since the FITC-labeled secondary antibodies are directed against the c-kit antibody, these data should reflect the relative number of c-kit-positive cells in each fraction. The relative numbers of FITC-positive cells in fractions from photoinhibited and fully active Djungarian hamster and mouse testes are shown in Figure 4. The unsorted cell fractions contained about 4–5% FITC-positive cells in rodents compared with approximately 10% in the monkey. After separation, the magnetic fraction showed a 4- to 7-fold enrichment of FITC-immunopositive cells. All nonmagnetic fractions were slightly depleted of FITC-positive cells. The maximum enrichment rate was 11-fold in long-photoperiod (LD) hamsters. Two independent attempts to separate marmoset monkey testes yielded increases in FITC-positive cells from 10.2% and 13.3% before the separation to 37.5% and 49.6% in the magnetic fraction. The nonmagnetic fraction was slightly depleted of FITC-positive cells (4.2% and 8.8%). The viability of the cells in the magnetic fractions as estimated by trypan blue test and confirmed by flow cytometric analysis ranged from 81% to 92% among the species examined.
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The DNA content of the various cell fractions was analyzed by PI staining followed by flow cytometric analysis to determine the content of haploid, diploid, S-phase, and tetraploid cells in each fraction. The relative numbers of FITC-positive and diploid cells correlated well in each fraction. In a representative run using cells from fully active (LD) Djungarian hamster testes, there were 3.7% FITC-positive and 4.2% diploid cells before separation. The magnetic fraction was enriched to 14.9% FITC-positive cells and 14.2% diploid cells after separation. Similar grades of enrichment were achieved in all samples when analyzed for ploidy.
In three independent isolation attempts, this procedure was applied to marmoset monkey testes using dual-parameter flow cytometry to determine simultaneously the composition of the cell fractions with respect to FITC staining and ploidity. A representative example of the flow cytometric histograms is presented in Figure 5. A comparison of the DNA histograms (Fig. 5, a and f) shows an enrichment of diploid cells and a depletion of the haploid and tetraploid cells in the magnetic fraction. In the three isolation experiments, FITC-positive diploid cells were enriched 8.1-fold, 16.3-fold, and 10.9-fold, respectively, in the magnetic fraction compared with the unsorted fraction. Very few FITC-positive cells were detected among the haploid cells before separation, and only a minor enrichment was observed after separation. The results of the three experiments are summarized in Table 1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The c-kit receptor was chosen as the marker for the cell sorting since a number of findings support the presence of this receptor in the outer membrane of spermatogonia. 1) Spermatogonial proliferation and differentiation are dependent on the interaction between stem cell factor and c-kit [23]. 2) Spermatogonia express functional c-kit receptors [22]. 3) Naturally occurring c-kit mutants lack spermatogonia due to their failure to migrate from the hindgut into the gonadal ridge [24]. 4) Antibodies against c-kit block the proliferation of differentiating spermatogonia [25]. 5) Spermatogonia from wild-type mice transplanted into c-kit mutants reinduce normal spermatogenesis in focal areas of seminiferous tubules [3].
A commercially available polyclonal rabbit anti-c-kit antibody was chosen for the detection of c-kit. To examine the testicular binding characteristics of this antibody, immunohistochemical localization of c-kit was performed in the three animal models used for the experiments. All testes showed the same types of labeled cells: spermatogonia and spermatids in the seminiferous epithelium and Leydig cells in the interstitium. Interestingly, c-kit-positive interstitial cells were also observed in the photoinhibited Djungarian hamster testis. Although Leydig cells in the photoinhibited hamster do not express 3-beta hydroxysteroid dehydrogenase [18], which by functional definition precludes their identification as Leydig cells, they still express c-kit, thus suggesting that they do in fact have some functional characteristics of Leydig cells during the regression phase.
The successful binding of the anti-c-kit antibody in cell suspensions indicates that the c-kit receptor is localized on the outer cell membrane. This supports the findings that c-kit mRNA is expressed at high levels in isolated mouse and rat spermatogonia [22, 26] and that the proliferation of differentiating mouse spermatogonia can be blocked by anti-c-kit antibodies [25]. The possibility cannot be excluded that the enzymatic digestion during preparation of the single-cell suspension changes epitopes at the outer plasma membrane. However, the application of a similar digestion protocol revealed the immunohistochemical and functional presence of c-kit in isolated spermatogonia, indicating that the digestion did not alter the c-kit protein in the plasma membrane [22]. Since no haploid cells were enriched, the primary antibody seemed unable to detect c-kit receptor protein on the surface of spermatids, although spermatids were labeled by the immunohistochemical technique. The fact that the same antibody stained permeabilized spermatids on fixed sections but failed to label intact spermatids supports the report of the presence of a truncated form of the c-kit protein consisting only of parts of the intracellular domain [27]. As the truncated form is not localized on the external cell membrane, intact spermatids do not offer epitopes for c-kit antibody binding on the cell surface. Thus spermatids are excluded from the magnetic cell fraction, allowing efficient enrichment of spermatogonial cells.
As was seen after analysis of the composition of the cell fractions by successive flow cytometric measurement of FITC and PI staining or by dual-parameter flow cytometry, the positively selected cell fraction consisted of diploid FITC-positive cells, indicating that the great majority of these cells must be either somatic c-kit-positive cells or c-kit-positive spermatogonia. The cell preparations were depleted of Leydig cells by sedimentation during the initial digestion step. Differential sedimentation at unit gravity after enzymatic digestion of testicular tissue is a simple and efficient procedure for the separation of seminiferous tubules and interstitial tissue, and it has been widely used in the past in many laboratories to generate Sertoli cell cultures (for review see [28]). Since Leydig cells were shown to be the only immunopositive somatic cell type expressing c-kit, the magnetic cell fractions were expected to consist mainly of spermatogonia.
Initially, photoinhibited Djungarian hamster testes were used to establish the separation procedure, as they present a physiological source of enriched spermatogonia. In the fully regressed period, testis volume is reduced to less than 5% as compared with the active period, and spermatogonia and very few early spermatocytes are the only germ cell types present in the seminiferous epithelium [18]. In subsequent experiments, testes from adult mice, photostimulated hamsters, and marmoset monkeys were utilized. The application of a polyclonal anti-c-kit antibody for magnetic cell separation enabled us to label and sort fractions of testicular cells with a recovery of around 105 cells in the magnetic fraction/107 cells sorted. Depending upon the preparation, up to 55% of these cells were recognized as c-kit positive by flow cytometry. The viability of the cells ranged from 82% to 91%. The efficiency of our spermatogonial separation procedure was slightly lower than that of the elutriation procedure described for immature rats [21]. Specific fractions of the elutriation procedure consisted of 51% A-spermatogonia and 74% B-spermatogonia/preleptotene spermatocytes, which were identified by morphological criteria [21]. Using the fully photoregressed hamster testis (which is comparable to that in the immature rat), we achieved an enrichment up to 25% of spermatogonia that were functionally identified by diploidy and the presence of c-kit receptors.
The superiority of magnetic cell sorting became clear when normal testes showing the full germ cell complement were used for the separation procedure. Immature animals are a prerequisite for both centrifugal elutriation [21] and sedimentation velocity in a BSA gradient [20, 22]. The size, shape, and density of the cells determine the separation efficiency using these two approaches, so the high number of spermatocytes and round spermatids that are similar to spermatogonia in these respects make it impossible to select and isolate the comparatively small number of spermatogonial cells. Furthermore, spermatogonia are connected by cytoplasmic bridges, and thus the majority are possibly eluted in small aggregates. This limits the isolation efficiency, since their elution pattern in BSA gradients and centrifugal elutriation depends on particle size. The presence of small fragments, however, does not interfere with the magnetic enrichment procedure, highlighting another advantage of our new approach. On the other hand, isolation of small cell clumps may be limiting the purity of the magnetic fractions as c-kit-negative cells located there could be coseparated. Therefore, a careful digestion of the tissue is mandatory to detach all cell types not interlinked by cytoplasmic bridges.
In this study we have shown that spermatogonial cells were enriched up to 25–54% when normal testes from Djungarian hamsters, mice, and marmoset monkeys were used. Since the enrichment efficiency was similar in regressed and nonregressed testes, it seems to be independent of the number of nontarget cells eluted from the column in the nonmagnetic fraction. However, the efficiency of separation is probably determined by the binding affinity of the primary antibody. Magnetic cell sorting is specifically useful for separation of a few cells from a larger number of unwanted cells in the cell preparation [11–15]. According to the supplier (Milteniy Biotech), an enrichment of up to 10 000-fold of the target cell population can be achieved using a positive selection strategy. Magnetic cell sorting allows the separation of rare target cells with frequencies down to 1 in 1 x 108. These methodological features render this approach particularly appropriate for the isolation of spermatogonia from fully active testes. It should also be useful for any other testicular cell type for which specific antibodies are available.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stefan Schlatt, Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48149 Münster, Germany. FAX: 49 251 835 6093; schlats{at}uni-muenster.de
Accepted: March 31, 1999.
Received: December 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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