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Testis |
Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3A 1A1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of the spermatogonial transplantation technique allows detection of stem cells in the male germ line even though at present no definitive markers exist for these cells. Following transplantation of donor testis cells, donor-derived spermatogenesis is reconstituted and maintained in recipient testes, and the recipient can regain fertility [2, 3]. Since the complete and long-term regeneration of spermatogenesis can be accomplished only by stem cells, this technique represents an unequivocal biological assay system to detect SSCs. Upon transplantation, donor-derived spermatogenesis is established in the form of distinct segments (colonies) along recipient seminiferous tubules, allowing a straightforward quantification of functional SSCs by counting the number of colonies [4]. Therefore, spermatogonial transplantation provides not only qualitative but also quantitative analyses of SSCs.
This SSC quantification analysis based on the colony number is legitimate only when one colony arises from one stem cell. On the basis of the one colony-one stem cell hypothesis, the biology of SSCs has been quantitatively studied under various physiological and experimental conditions. For example, the number of SSCs increases during postnatal development, and SSCs are enriched 
25-fold in experimental cryptorchid testes [5–7]. However, the validity of the one colony-one stem cell hypothesis has not been intensively examined. Dobrinski et al. [8] have shown that the number of colonies established in a recipient testis linearly correlates with the number of donor cells injected into the testis when the colony number ranges from 0.2 to 20 colonies per testis. As demonstrated in the studies of hematopoietic progenitor cells [9, 10], this linear relationship implies that one colony arises from one stem cell, supporting the one colony-one stem cell hypothesis. However, the indirect approach taken in this study did not provide evidence on the clonality of each individual colony. In addition, no information is available on the clonality of colonies when more than 20 colonies are formed in a recipient testis.
To evaluate the clonal origin of somatic cell lineages or tissues, various approaches have previously been taken, including cytological analyses for hematopoietic progenitors [11–14] and the use of chimeric or transgenic mice for intestinal crypts [15–17]. The clonality of hematopoietic colonies formed in the spleen following bone marrow transplantation (colony-forming unit-spleen) was intensively examined using cytological analyses [11–14]. Becker et al. [11] transplanted donor marrow cells derived from heavily irradiated mice and, following excision of colonies formed in the spleen of recipient mice, examined the karyotype of the cells contained in one colony. They demonstrated that at least 95% of the cells in one donor-derived colony contained a unique abnormal karyotype. Although the experiments were designed in such a way that the number of colonies formed in one spleen was very small (1.1 colonies per spleen), the results directly revealed the clonal origin of colony-forming unit-spleen. To evaluate the clonality of intestinal crypts, Ponder et al. [15] and Schmidt et al. [16, 17] used chimeric mice to analyze the distribution of cells that expressed a marker gene unique to each of two mouse strains used to generate aggregation chimeras. The results showed that intestinal crypts contained the cells of two genotypes in the neonatal period but contained those of only one genotype in adults, demonstrating a transition to clonality in the intestinal crypt during postnatal development.
To evaluate the one colony-one stem cell hypothesis for SSCs in the present study, we undertook an approach that conceptually combines cytological analyses and the use of chimeric animals described above. We obtained donor testis cells from two transgenic mouse lines and transplanted a mixture of these two cell populations into recipient testes. We genotyped the resulting colonies of donor-derived spermatogenesis based on the detection of a transgene unique to each of the two transgenic lines. A colony was evaluated to have arisen from one stem cell (clonal) when only one genotype was found in the colony, whereas a colony was determined to be multiclonal when two genotypes were detected. We further analyzed the relationship between the clonality and the density of colonies and also between the clonality and the colony length.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of McGill University. Homozygous B6;129S-Gtrosa26Sor transgenic mice (Jackson Laboratory, Bar Harbor, ME; designated ROSA herein), which express ß-galactosidase in all types of male germ cells [4, 18], were maintained in our animal facility. Two transgenic mouse strains were generous gifts from Dr. A. Peterson [19]. One strain contained a transgene with a 9-kilobase (kb) region of myelin basic protein (MBP) promoter (number 17 in Forghani et al. [19], herein designated MBP mice), which includes the Schwann cell enhancer (SCE) sequence (0.6 kb), conjugated with the 3-kb lacZ sequence (see Fig. 1A). The other strain contained a transgene with the SCE sequence and the minimal 0.3-kb heat shock protein (HSP) 68 promoter sequence conjugated with lacZ (number 49 in Forghani et al. [19], herein designated HSP mice) (Fig. 1A). Both strains were hemizygous on the C57BL/6 (B6) genetic background and do not show defects in development and reproduction [19]. The MBP strain has been estimated to carry three copies of the transgene, and the HSP strain has been estimated to carry two copies [19] (A. Peterson, personal communication).
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These three strains of transgenic mice were used to produce two donor mouse lines (Fig. 1A). ROSA mice were mated with either MBP or HSP mice, and the genotype of resulting F1 generations (MBP x ROSA and HSP x ROSA) was confirmed by the polymerase chain reaction (PCR) using genomic DNA obtained from a tail tip under the amplification conditions described below (Fig. 1B). These double-transgenic mice were used as donors at 73.2 ± 4.6 days of age (17 preparations in total).
Since ROSA mice were on the B6 x 129 genetic background, immunologically compatible B6 x 129 F1 mice were used as recipients. Recipient mice were prepared by an i.p. injection of 50 mg/kg of busulfan at 4 wk of age to destroy endogenous spermatogenesis [4] and used for transplantation 4 or more weeks later.
Transplantation
Donor cells were prepared from the testes of MBP x ROSA and HSP x ROSA mice using a two-step enzymatic digestion and injected into the recipient seminiferous tubules through the rete testis [20]. In preliminary experiments, donor testis cells derived from each transgenic line were individually injected into recipient testes at the concentration of 100 x 106 cells/ml. As described in Results, the ratio of colony numbers after transplantation of HSP x ROSA and MBP x ROSA donor cells was 1:1.7. In subsequent experiments, donor testis cells from MBP x ROSA mice were prepared at 400 x 106 cells/ml and those from HSP x ROSA mice at 260 x 106 cells/ml for a technical readiness. These two donor cell populations were mixed at a 1:1 ratio, and the cell mixture was further diluted serially to 1/2 and 1/5 before transplantation to obtain different numbers of colonies per recipient testis. Approximately 7 µl of donor cell preparation was injected into a recipient testis.
Analysis
Two months after transplantation, recipient testes were collected and stained with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) as described previously [20]. Donor-derived spermatogenesis was visualized as blue colonies, owing to the lacZ activity of ROSA genotype [4]. Following X-gal staining, recipient testes were rinsed in Dulbecco phosphate-buffered saline and immediately analyzed without poststaining fixation. The length of each colony was measured with an eyepiece micrometer using a stereomicroscope [4]. Then, each colony was individually excised and stored in a microcentrifuge tube at -80°C until DNA preparation. The entire length of a blue-stained colony was obtained, excluding nonstained fragments of seminiferous tubules. At least 72% of all colonies in a recipient testis were excised and analyzed. Genomic DNA was extracted from each colony using the DNeasy Tissue Kit 250 (Qiagen GmbH, Hilden, Germany).
The presence of the second transgene (HSP and/or MBP) in a colony was examined by PCR using genomic DNA as a template and primers specific to each gene. The sequence of the forward primer for HSP was 5'-CCATCCAGAGACAAGCGAAGAC-3', which is located within the 3'-end of the HSP sequence, and that of the reverse primer was 5'- TTGAGGGGACGACGACAGTATC-3', which is found near the 5' end of lacZ (Fig. 1A). The sequence of the forward primer for MBP was 5'- GAGGACAACACCTTCAAAGACAGG-3', which is located within the 3' end of MBP promoter sequence, and that of the reverse primer was 5'- AATGTGAGCGAGTAACAACCCG-3', which is found near the 5' end of lacZ (Fig. 1A). These primer sets give rise to 441-base pair (bp) amplicons for the HSP gene and 610-bp amplicons for the MBP gene. The amplification was performed for each transgene in a separate reaction tube using the same DNA sample. Because the ratio of copy numbers of HSP and MBP constructs was 2:3 (see above), 450 and 300 ng of genomic DNA were used in one reaction for the detection of HSP and MBP sequences, respectively, to provide the same amount of specific template sequences. As a negative control for PCR, genomic DNA was prepared as above from the liver of homozygous ROSA mice. The amplification reaction mixture (25 µl per reaction) was composed of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleotides mix, 0.5 µM each of forward and reverse primers, and 1 U of Taq DNA polymerase (Taq DNA polymerase kit; Roche Diagnostics GmbH, Mannheim, Germany). Following incubation at 95°C for 2 min, the amplification was performed for 35 cycles at 95°C for 1 min, 59°C for 1 min, and 72°C for 1 min and completed with an additional incubation at 72°C for 10 min. After amplification, the reaction mixture (10 µl) was loaded to a 1.5% agarose gel and the PCR products stained with ethidium bromide. The presence of each transgene in a colony was determined by visual detection of PCR products corresponding to each target sequence.
Two preliminary experiments were performed to determine the sensitivity of the PCR-based assay described above (see Fig. 2). The testes of ROSA x HSP and ROSA x MBP mice were stained with X-gal, small fragments of stained seminiferous tubules (20 mg) were excised, and genomic DNA was extracted. Serial dilutions of genomic DNA were prepared (700–0.07 ng for one reaction) and used as a template for PCR under the same conditions described above. The PCR products were stained with ethidium bromide as above, and the intensity of the bands was measured using a densitometric analysis system of a gel documentation apparatus (FluorChem 8800; Alpha Innotech Co., Los Angeles, CA).
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All data were expressed as mean ± SEM. Statistical analyses were performed using SYSTAT 10.2 (SYSTAT Software Inc., Richmond, CA) for ANOVA followed by Tukey multiple comparison (see Table 1), Student t-test (two-group comparisons of colony length), or linear regression (see Fig. 3). The logarithmic transformation was used for the analyses of colony length to stabilize variances and normalize data distribution [21].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used three transgenic mouse strains (ROSA, MBP, and HSP mice) to establish two lines of donor mice that have a set of two transgenes (ROSA and MBP or ROSA and HSP) in their genome (Fig. 1A and B). Although MBP and HSP mice carry the lacZ sequence in their transgene constructs, the marker gene was not efficiently expressed in the testes of these mice, probably due to the neuron-specific promoters in the constructs (Fig. 1A and C). These two strains were thus not suitable donors to visualize donor-derived spermatogenesis after transplantation. Since ROSA mice express lacZ in all types of male germ cells even when they are hemizygous [4, 22], these mice were mated with either MBP or HSP mice (Fig. 1A). The seminiferous tubules of the resulting male offspring (ROSA x MBP F1 and ROSA x HSP F1) were strongly stained with X-gal, as a result of the common marker gene derived from the ROSA strain (Fig. 1D). The use of these two lines of double-transgenic mice as donors allowed clear visualization of spermatogenic colonies after transplantation regardless of the donor origin, whereas the second transgenes (MBP and HSP) provided the markers to evaluate the genetic composition of cells in each colony (Fig. 1B).
Donor cells prepared individually from the testes of ROSA x HSP and ROSA x MBP mice were mixed at a 1:1 ratio for transplantation. Here, it is important that the 1:1 mixture ratio of two donor cell populations be based on the ratio of the number of colonies formed after transplantation and not the ratio of the number of donor testis cells in the cell mixture, because the two transgenic mouse lines could have different concentrations of colony-forming cells (i.e., SSCs) in their testes. In preliminary experiments, therefore, we individually transplanted donor testis cells derived from ROSA x MBP and ROSA x HSP mice to identify a transplantation condition under which the 1:1 ratio of colonies can be accomplished. When donor testis cells derived from the ROSA x MBP mice alone were transplanted, we observed 4.6 ± 1.0 colonies/106 cells injected per testis (n = 15 testes, 3 experiments). When donor testis cells derived from ROSA x HSP mice alone were transplanted, we observed 7.6 ± 1.5 colonies/106 cells injected per testis (n = 12, 2 exp.). These results indicated that when the same number of donor cells were injected, the ROSA x HSP donor cells produced 1.7-fold more colonies than the ROSA x MBP donor cells. In subsequent experiments, we prepared the donor cell mixture at the ratio of 1:1.5 in the number of ROSA x HSP vs. ROSA x MBP cells for practical reasons and serially diluted the cell mixture to obtain varying colony numbers per recipient testis after transplantation.
Evaluation of PCR Assay Sensitivity
The presence or absence of the second marker genes (HSP and MBP) in individual colonies was detected using PCR (Fig. 1A and B). In preliminary experiments, we examined the sensitivity of this PCR-based detection method. Testes of ROSA x HSP and ROSA x MBP mice were stained with X-gal, and genomic DNA was prepared from stained seminiferous tubules of each mouse line. PCR was performed using serial dilutions of the genomic DNA preparation, ranging from 0.07 to 700 ng per reaction. Following ethidium bromide staining of agarose gels, a linear relationship was observed for both marker genes between the staining intensity of PCR bands and the log10 values of the template amounts (Fig. 2, lower panel). The PCR products were clearly and consistently visible for both genes when 7 ng of genomic DNA was used as a template (Fig. 2, upper panel). The PCR products that resulted from the amplification of 1.75 ng of DNA were visible only inconsistently for both genes (not shown). Based on these results, in subsequent experiments, we used at least 300 ng of genomic DNA prepared from each colony as the PCR template in one assay reaction (see Materials and Methods). Thus, this PCR-based assay method should detect the second marker genes if they are present in at least 2% (7/300) of DNA preparations used for one reaction.
Evaluation of the Transplantation Condition
Following transplantation, we analyzed a total of 52 recipient testes and 695 colonies by X-gal staining and PCR (Table 1, columns 5 and 6). The number of colonies observed per testis ranged from 1 to 55 (Table 1, columns 2 and 3). Among the 695 colonies analyzed, we found 314 colonies with only the MBP gene, 277 colonies with only the HSP gene, and 104 colonies with both genes. Therefore, the overall ratio of MBP-only vs. HSP-only colonies was 1.13 (314/277). In addition, when the numbers of MBP-only and HSP-only colonies were compared in each individual testis, the ratio was 1.12 ± 0.16 (n = 25 testes with 10 or more colonies per testis). These results indicate that the transplantation condition used in this study allowed a near 1:1 ratio of colonies with each of the two genotypes, providing an even competition for both donor stem cell populations to colonize recipient testes.
Relationship Between Clonality of Colonies and Colony Density in a Recipient Testis
Since the clonality of a colony may vary, depending on the number of colonies formed in a testis (colony density), we first analyzed the relationship between these two parameters (Table 1, Fig. 3). The overall colony density was 15.7 ± 2.1 colonies per testis (n = 52). Among the recipient testes analyzed, a total of 12 testes contained 1–4 colonies, and all colonies in these testes showed a single genotype, only HSP or MBP (Table 1, line 1). The percentage of clonal colonies in one recipient testis decreased as the colony density increased (Table 1, column 10, and Fig. 3). When the colony density was 5–9 colonies per testis, 96.0% of colonies showed a single genotype (clonal). Although 93.8% of colonies were clonal at the colony density of 10–19 per testis, the percentage of clonal colonies decreased to 87.7% at 20–29 per testis and 76.1% at 
30 per testis. A linear correlation was detected between the clonality and the colony density (P < 0.001), by which 90% of colonies were estimated to be clonal when 19 colonies were formed in a recipient testis (Fig. 3). In accordance with these results, no testes had all colonies with only one genotype when 20 or more colonies were formed per testis (Table 1, column 9, lines 4 and 5). Thus, the fraction of colonies that are clonal is estimated to be 100% when the colony density is less than 5 and 90% when the colony density is less than 20 but decreases with increasing density of colonies. These results support the one colony-one stem cell hypothesis but demonstrate that the colony density needs to be less than 20 to achieve the one colony-one stem cell condition in 90% of the cases.
Relationship Between Clonality of Colonies and Colony Length
It has previously been shown that the length of colonies varies significantly among recipient testes [4]. A long colony may develop as a result of multiple SSCs colonizing in close proximity in the recipient seminiferous epithelium, which would cause merging of multiple colonies. Consistent with this, a trend was observed that colonies were longer as the colony density increased (Table 1, column 7). Therefore, we next evaluated the relationship of the clonality and the length of colonies (Table 2, Fig. 4).
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The average length of colonies was 2.56 ± 0.05 mm in the 695 colonies analyzed (Table 2, line 3). Among the clonal colonies, the colony length ranged from 0.7 to 10.5 mm, with an average of 2.42 mm (n = 591). Among the multiclonal colonies, the colony length ranged from 1.0 to 11.2 mm, with an average of 3.36 mm (n = 104). The average colony lengths were significantly different (P < 0.001) between clonal and multiclonal colonies, indicating that although long colonies can be clonal, they are more likely to be of multiclonal origin. As such, the percentage of clonal colonies decreased as the length of a colony increased (Fig. 4A). Although 100% and 92.8% of colonies were clonal when the length was <1 mm and 1–2 mm, respectively, 83.0% of colonies were clonal when the length was 2–3 mm. When the colony length exceeded 5 mm, only 54.5% of the colonies were clonal. Taken together, these results indicate that more than 90% of colonies were clonal when the length was less than 2 mm, but the probability for clonal origin of colonies decreased as the length of colonies increased.
We next analyzed the distribution of colony lengths in clonal and multiclonal colonies (Fig. 4B). Among the clonal colonies, 89.0% were in the range of 1–4-mm long, and the largest population (37.1%) was found in the 1–2-mm range. Only 3% of clonal colonies were longer than 5 mm. Among the multiclonal colonies, 75.0% were in the range of 1–4-mm long, and the largest population (37.5%) was found in the 2–3-mm range. Colonies longer than 5 mm represented 14% of multiclonal colonies. These results indicated that although the minimum and maximum values of colony length did not appear significantly different between clonal and multiclonal colonies (Table 2), greater numbers of longer colonies were found in the multiclonal group compared with the clonal group, confirming that longer colonies were more likely to be multiclonal. The results also indicated that a large fraction (75%) of colonies were 1–4 mm in length regardless of the clonality of colonies.
Finally, we analyzed the relationship between colony density and colony length. As described above, most colonies (90%) were clonal when colony density was less than 20 per testis. The length of colonies found in recipient testes with this range of colony density was 2.35 ± 0.08 mm (n = 226 colonies, clonal and multiclonal combined). The length of colonies in testes with higher densities was 2.66 ± 0.06 mm (n = 469). A significant difference was detected between these two groups (P 
0.002), indicating that a higher colony density resulted in more frequent formation of longer colonies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results obtained in this study support the hypothesis and indicate that spermatogonial transplantation is a highly quantitative assay for SSCs. These conclusions, however, are applicable under two transplantation conditions. First, the clonality is affected by the colony density (Fig. 3). The colony density needs to be less than 20 per testis to obtain a majority (90%) of clonal colonies. Second, although one SSC is apparently capable of producing a long colony (Table 2), longer colonies more frequently have a multiclonal origin (Fig. 4). Therefore, it is necessary to adjust the density and length of colonies for the accurate quantification of SSCs.
These two parameters (colony density and length) can be modified by changing the concentration of donor cells and, thus, the number of SSCs injected into a recipient testis. Transplantation of donor cells at a lower concentration results in a lower colony density [8], and the colony density of <20 per testis gives rise to more clonal colonies (Fig. 3). For accurate SSC quantification, therefore, it is ideal to perform preliminary experiments to determine the donor cell concentration that provides this range of colony density. The adjustment of donor cell concentration may also be effective in manipulating the colony length because colony length and density are correlated. In this study, when the density of colonies was higher than 20 per testis, the colonies were significantly longer than those in the lower-density condition (<20 per testis). These results suggest that when more cells are injected into a confined space of the seminiferous tubules the probability that SSCs seed in close proximity may increase. In such a circumstance, multiple units of regenerating spermatogenesis arising from different SSCs would merge during the development and expansion of each unit of donor-derived spermatogenesis, resulting in the formation of a long colony. Therefore, transplantation of donor cells at a lower concentration reduces such a probability and leads to the formation of shorter and clonal colonies.
If an adjustment of donor cell concentration is necessary to obtain clonal colonies for SSC quantification, such a modification inevitably results in a difference in the number of testicular somatic cells and nonstem germ cells injected together with SSCs into recipient testes. It is possible that these cells affect the colonization efficiency of SSCs after transplantation and that the fidelity of SSC quantification could be influenced, depending on the concentration of donor cells. Although further studies are necessary to address this issue, such a possibility seems less likely. A previous study has shown that the number of colonies established after transplantation linearly correlates with the number of donor testis cells injected [8]. This linearity was obtained by injecting increasing concentrations of donor cells. Therefore, the concentration of donor cells and the presence of testis cells other than SSCs are apparently not a significant factor that affects SSC colonization efficiency and the accuracy of SSC quantification by transplantation. Together with the results of the present study, it is suggested that the most important factor that affects the clonal origin of colonies is the colony density and, therefore, the absolute number of SSCs injected into a recipient testis.
Although the experimental approach taken in this study allowed a highly efficient and sensitive evaluation of the colonality of colonies, it involves two limitations. First, even if two stem cells are present in one colony, this multiclonality cannot be detected with our assay system if one of the two stem cells undergoes mitotic arrest after transplantation or if one stem cell dominates the regeneration of spermatogenesis and produces more than 98% of daughter germ cells in a colony. It may be difficult, however, to detect the genotype of such a small population of germ cells in a colony, particularly a mitotically arrested single stem cell, using assay methods currently available. Second, we cannot rule out the possibility that the dissociation of donor cells was not complete and that these cells contained aggregates of two or more stem cells of one transgenic line, which formed the units that gave rise to colonies after transplantation. This limitation could partially be overcome by using a mixture of donor cells derived from more than two transgenic mouse lines. Another approach would be to transfer a marker gene into the genome of donor SSCs using a retroviral vector before transplantation [23, 24]. A transgene delivered by a retroviral vector randomly integrates into the host cell genome, providing each stem cell in an aggregate with a unique marker. However, these approaches are practically demanding or do not provide the versatility to produce different experimental conditions (e.g., a wide range of colony density as examined in this study). In addition, considering that SSCs comprise only 0.01% of the cells in a testis [7, 25], it appears rather unlikely that such a stem cell aggregation would occur frequently enough to significantly affect the sensitivity of our assay method. Therefore, although caution is necessary, we believe that the experimental approach taken in the present study is satisfactory to test the one colony-one stem cell hypothesis.
In conclusion, the results obtained in this study support the one colony-one stem cell hypothesis and suggest that adjustment of donor cell concentration is necessary to obtain a majority of clonal colonies in a recipient testis for accurate quantitative analyses of SSCs (<20 colonies per recipient testis). The present study thus provides an important platform to further investigate the biology of this powerful but elusive stem cell population in a quantitative manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Makoto Nagano, Royal Victoria Hospital, Room F3.07, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1. FAX: 514 843 1662; makoto.nagano{at}muhc.mcgill.ca
Received: 9 May 2003.
First decision: 27 May 2003.
Accepted: 10 July 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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