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Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis¹

ILARIA FALCIATORI^*, GIOVANNA BORSELLINO, NIKOLAOS HALIASSOS^*, CARLA BOITANI^*, SERENA CORALLINI^*, LUCA BATTISTINI, GIORGIO BERNARDI, MARIO STEFANINI^* and ELENA VICINI^*,2

^* Department of Histology and Medical Embryology, University of Rome "La Sapienza," Rome, Italy,
Neuroimmunology and Flow Cytometry Unit, Santa Lucia Foundation Scientific Institute (IRCCS), Rome, Italy; and
Department of Neuroscience, University of Rome "Tor Vergata", Rome, Italy

² Correspondence: Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via Antonio Scarpa 14, Rome 00161, Italy. E-mail: elena.vicini{at}uniroma1.it

SPECIFIC AIMS

Spermatogenesis is maintained by spermatogonial stem cells (SSC) that can self-renew and generate spermatogonia committed to differentiation. Although the morphology and kinetics of spermatogonia have been characterized, there is a lack of SSC specific morphological and biochemical markers for their identification and isolation. One molecular feature common to several stem cells is the so-called ‘side population’ (SP) phenotype, which can be assessed after staining with fluorescent DNA binding dyes and FACS analysis. In the present study, we tested the hypothesis that SSC might exhibit the SP phenotype.

PRINCIPAL FINDINGS

1. Identification of a SP population in immature murine testis
SP phenotype conferred by ABC transporter expression has been shown to be suitable to isolate stem cells from a variety of tissues such as those of bone marrow, muscle and mammary gland. To assess whether a cell population with SP phenotype is present in the immature testis, cells from 7- and 20-day-old mice were enzymatically isolated and stained with Hoechst 33342. FACS analysis identified, at both ages analyzed, a small population of Hoechst-effluxing SP cells that was termed Testis-SP (T-SP). Verapamil and reserpine treatments greatly reduced the T-SP fraction. FACS analysis of PI-stained sorted T-SP cells showed that these cells were mostly diploid.

2. T-SP cells are not of hematopoietic origin
To determine whether T-SP cells derive from hematopoietic SP cells circulating in the blood, we analyzed the expression of CD45, CD34 and Sca-1. FACS analysis demonstrated that T-SP cells were CD45^neg, CD34^neg and brightly positive for Sca-1 expression. These results indicate that even though T-SP cells are not derived from blood contamination, they express Sca-1 similarly to other SP cells.

3. T-SP cell surface antigen expression
We investigated the distribution of surface antigens known to be expressed in SP stem cells of other tissues and/or spermatogonial cells. Several populations were identified and gated by means of Hoechst staining and antibody labeling (Fig. 1 ). We found that cells in the T-SP region were located in the R1 and R2 gates, while non-SP cells were located in the R3-R6 gates. To determine the germ cell identity of T-SP cells, we first analyzed the expression of EE2, Ep-CAM and c-kit, all known to be markers of spermatogonial cells. The majority of the cells in the T-SP (R1+R2 gates) were positive for EE2 and Ep-CAM, thus indicating that they belong to the spermatogonial lineage. EE2 and Ep-CAM double-positive spermatogonial cells were also located in the R3 gate but were absent in the other gates. Spermatogonial cells in the distal tip of the T-SP region (R1 gate) were c-kit^neg, whereas those in the R2 gate were c-kit^pos. c-kit^pos spermatogonial cells were also located in the R3 gate, though not in other gates. These data indicate that T-SP is not a pure stem cell population since it contains c-kit^pos differentiating spermatogonial cells. T-SP cells were highly 6-integrin^pos, v-integrin^neg and brightly positive for Sca-1. Sca-1 expression gradually decreased from the R1 gate, in which the fluorescence intensity values were highest, to the R6 gate. These results indicate that T-SP cells in the R1 gate and adult SSC share a common expression profile of surface antigens (c-kit^neg, v-integrin^neg and 6-integrin^pos).

View larger version (52K): [in this window] [in a new window]   Figure 1. Immunophenotype of T-SP cells by multicolor flow cytometric analysis. Cells from 20-day-old mouse seminiferous tubules were stained with 5 µg/ml Hoechst and antibodies against indicated surface antigens. A)6 individual gates (R1-R6) were identified and represented with different colors. T-SP region comprised R1 and R2 gates. B) Expression analysis of the indicated antigens in individual gated populations. Solid lines, staining with specific antibodies; dotted lines, staining with isotype control antibodies.

4. T-SP population is enriched in functional SSC
To evaluate the stem cell enrichment of T-SP we used the germ cell transplantation assay. Seminiferous tubule cell suspensions and sorted T-SP cells (R1+R2 gates) were obtained from 20-day-old ROSA 26 mice (which constitutively express the lacZ-gene) and transplanted into adult busulfan-treated, germ cell-depleted mouse testes. Two months after transplantation, the colonization of the recipient testes by donor stem cell-derived spermatogenesis was evaluated both quantitatively and qualitatively. Our results show a 13-fold increase in colonization efficiency of the T-SP population compared with the total Hoechst-unstained cell suspension. To investigate the pattern of donor-derived spermatogenesis, we next performed a histological analysis on serially sectioned T-SP- (n=6) and total cell-derived colonies (n=6). T-SP-derived colonies were organized in cell associations qualitatively similar to those of normal adult mouse, though with a lower number of haploid germ cells. Cell associations were regularly arranged along segments of repopulated tubules in co-ordinated successions of stages (e.g., from II to XII, from I to VIII, from VIII to XII). The length of each stage was shorter than that observed in normal mouse spermatogenesis. In two colonies, spermatogenesis was complete and spermiation was observed. By contrast, in total cell-derived colonies only one or two adjacent germ cell associations (most frequently between stages IX and XII) were observed, even when the colony length was comparable to that of T-SP-derived colonies.

CONCLUSIONS AND SIGNIFICANCE

In this study, we present data on the identification of a SP population in immature mouse testis (Fig. 2 ). By means of FACS analysis, we found that this population is CD45^neg, CD34^neg and Sca-1^pos; moreover, germ cell transplantation experiments indicate that the SP population is enriched in SSC activity if compared with total unsorted cells. A testis SP population has recently been identified in cryptorchid mouse testis. When assayed by means of germ cell transplantation experiments, these cells do not generate donor-derived spermatogenesis, which indicates that they do not display spermatogonial stem cell activity. These results are apparently in contrast to the data presented here. However, since immature and cryptorchid testes represent two different experimental models, different subsets of cells may constitute the testis SP populations in these models, a hypothesis supported by surface antigen analysis and transplantation experiments. Here T-SP cells were characterised in immature testis, hence during the first wave of spermatogenesis. Experiments designed to investigate whether the SP phenotype of spermatogonia and/or SSC is lost during testis development and/or after experimental cryptorchidism are in progress.

View larger version (45K): [in this window] [in a new window]   Figure 2. Schematic diagram.

The tyrosine kinase receptor c-kit and its ligand Steel (SCF) are essential for spermatogonial proliferation and differentiation; moreover, it has been demonstrated that spermatogonial stem cells are c-kit^neg whereas differentiating spermatogonia are c-kit^pos. We found that T-SP is not a pure stem cell population since it contains c-kit^pos differentiating spermatogonial cells. This is consistent with the observation that hematopoietic SP cells not only display highly enriched stem cell activity, but also contain differentiating cells. In the T-SP region, c-kit^neg spermatogonial cells are located in the distal tip (R1 gate). Interestingly, the distal tip of the SP region in bone marrow contains the highest concentration of stem cells able to repopulate recipient animals. Since SSC are c-kit^neg, these data suggest that it may be possible to obtain further enrichment in SSC activity by selecting cells in the R1 gate after Hoechst and c-kit staining.

Sca-1 expression levels in the R1-3 gates were found to inversely correlate with c-kit expression levels. c-kit^neg spermatogonia in the R1 gate were brightly Sca-1^pos, whereas the Sca-1 expression level was lower in the c-kit^pos cells. These data indicate that Sca-1 is downregulated during spermatogonial differentiation.

One of the most interesting observations in our study is that T-SP cells were able to give rise to an almost complete seminiferous epithelium wave as early as two months after transplantation. By contrast, we found that in total cell-derived colonies only one or two adjacent germ cell associations were present at the same time-point, in agreement with previous observations by other authors. It is noteworthy that the appearance of different stages, and therefore the presence of an epithelium wave in total cell-derived colonies, has previously been reported to occur six months after transplantation. Although the regulative mechanisms underlying the establishment of the seminiferous epithelium wave have not yet been identified, it is reasonable to assume that the start of spermatogenesis requires not only co-ordination but also adequate quantities of SSC in each of the tubular segments, where a specific cell association subsequently develops. The presence of numerous adjacent cellular associations in the T-SP-derived colonies two months after transplantation suggests a higher density of SSC in these colonies than in the total cell-derived colonies. A higher density of SSC in T-SP-derived colonies may mirror the concentration of stem cells in the T-SP cell suspension. If this is the case, a single colony would derive not from one, but from several stem cells. An alternative explanation is that T-SP cells represent a subset of SSC whose kinetics of proliferation and/or differentiation during recolonization differs from those of SSC present in the total cell suspension. Experiments on the dynamics of early events in seminiferous tubule repopulation after transplantation may clarify these aspects. Germ cell transplantation seems to be a promising approach to the study of the mechanisms which regulate the initiation and co-ordination of spermatogenesis within the testicular parenchyma.

It has recently been demonstrated that mouse embryonic, neural and hematopoietic stem cells share a number of gene products. Interestingly, all the afore-mentioned stem cells express high levels of ABC transporters. Here we demonstrate the presence in the immature testis of a Sca-1-positive cell population which displays SP phenotype and spermatogonial stem cell activity. SSC global gene expression analysis may shed more light on the possible existence of an overlapping set of gene products between male germ and somatic stem cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0744fje

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