Spermatogonial stem cell specific markers are expressed in the Piwil2 expressing

Till now, four cell surface markers, beta1-integrin (Itgb1), alpha6-integrin (Itga6), Thy-1 (CD90) and CD9 were identified on the surface of spermatogonial stem cells (Shinohara et al., 1999; Kubato et al., 2003; Kanatsu-shinohora et al., 2004). We found CD9 and Itga6 expression in Piwil2 expressing NIH3T3 cells (Fig. 3.38). Studies have begun to reveal the phenotype of mouse spermatogonialstem cells. Their surface phenotype is side scatter^low, α6-integrin⁺,ß1-integrin⁺, CD24⁺, thy-1⁺, α5-integrin^-, c-kit^- and MHC^- (Shinohara et al., 1999; Shinohara et al., 2000; Kubota et al., 2003). These stem cells also express Oct-4 and Stra8(Giuili et al., 2002; Tadokoro et al., 2002). Interestingly, Piwil2 and Stra8 protein expression was detected in about 30% of NIH3T3-pcDNA-Piwil2 cells. Some of side scatter^low, α6-integrin⁺,ß1-integrin⁺, CD24⁺, thy-1⁺, α5-integrin^-, c-kit^- and MHC^- are also expressed on other typesof stem cells. For example, Thy-1 and ß1-integrinare expressed on hematopoietic stem cells (Spangrude et al., 1988; Williams et al., 1991) and Oct-4and Itga6 are expressed on ES cells (Xu et al., 2001). However, spermatogonialstem cells are not identical with other stem cells. Hematopoietic stem cells express Itga4 and c-kit (Williams et al., 1991; Ikuta et al., 1992), which areabsent on spermatogonial stem cells (Shinohara et al., 1999). Oct-4, a marker of ES cells, is not expressed on hematopoietic stem cells (Pesce et al., 2001).Nonetheless, the search for common molecules for different stemcells has facilitated the identification of stem cell surfacemarkers (Shinohara et al., 1999; Kubota et al., 2003).

CD9 can now be added to the list of molecules that are expressed on several types of stem cells. CD9 is a type III membrane protein with four transmembrane domains and is involved in cell adhesion, migration, proliferation and fusion (Ikeyama et al., 1993;

Masellis-Smith et al., 1994; Hadjiargyrou et al., 1995). It is expressed on many types of

cells, including bone marrow,brain and muscle cells (Ikeyama et al., 1993; Masellis-Smith et al., 1994; Hadjiargyrou et al., 1995). It is also expressed on oocytes and plays an important role in fertilization; disruptionof the CD9 molecule by gene targeting results in female infertility(Miyado et al., 2000). CD9 associates withintegrins, including ß1- and α6-integrin (Park et la., 2000), and it may play a role in signal transduction and in regulatingcellular adhesion (Wright et al., 1994). As these integrin molecules areinvolved in the binding of cells to the basement membrane, this complex of molecules may be expressed on spermatogonial stemcells and may regulate cell adhesion. In a previous study, theaddition of leukemia inhibitory factor (LIF) or STAT3 was found to result in CD9 expression in ES cells, which indicates that the LIF/STAT3 pathway is critical for maintaining CD9 expression. The LIF/STAT3 pathway is essential for maintaining the undifferentiatedstate of ES cells (Niwa et al., 1998; Matsuda et al., 1999). A weak CD9 expression was detected in Piwil2 expressing NIH3T3 cells (Fig. 3.38).

An increase of Itga6 expression and a slight change in expression of Itgb1 was observed in NIH3T3-pcDNA-Piwil2 cells. Testicular germ cells selected by anti-α 6-integrinantibody appeared to be twice as effective in stem cell activityas those selected by anti-ß1-integrin antibody (Shinohara et al., 1999). Integrins are heterodimeric transmembrane receptors consisting of one α and one ß subunit. The two subunits collaborate to bind ligands, which are extracellular matrix proteins or counter-receptors of the Ig superfamily. It was reported that thestem cell or progenitor cell populations of other self-renewingtissues express also integrin molecules. For example, extracellular matrix receptors of the integrin family have been identified as important regulators of epidermal homeostasis, influencing the balance between stem cell renewal and differentiation (Watt, 2002).

Thy-1 (CD90) is a glycoprotein of the immunoglobin superfamily, which is expressed in a variety of stem cells, including mouse SSCs (Goldschneider et al., 1978; Beech et al., 1983;

Sprangrude et al., 1988; Baum et al., 1992; Ling and Neben, 1997; Jiang et al., 2002;

Kubota et al., 2003). By flow cytometric analysis and the testicular transplantation assay, it was demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal pup and in adult testes of wild type mouse (Kubota et al., 2004). Increased expression of two stem cell surface markers, Thy-1 and Itga6, supports again the hypothesis that Piwil2 protein is able to induce normal NIHT3T cells towards cells with stem cell-like characters.

Taken together, our data suggest a crucial role of stem cell protein Piwil2 in regulation or modulation of other stem cell proteins which are involved in proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion (Intga6) and cell signaling (Thy-1). These findings suggest that Piwil2 modulates cells towards cells with spermatogonial stem cell phenotype.

Therefore, immunological separationusing surface antigenic properties (anti-Thy-1, CD-9, Stra8, Hsp90 antibody) is a major approach for enrichmentof SSCs. These cells selected by using anti-piwil2 and anti-Itga6 antibody can be useful to determine the degree of enrichment by using flow cytometric analysis.

In our future works, to obtain a pure or highly enriched stem cellpopulation, it is critical to identify unique markersthat are expressed on SSCs because the antigenic profileof SSCs establishes the basis for selective separation. Particularly, identification of markers that are expresseduniquely on SSCs, but not on other somatic cells or differentiatedspermatogenic cells, facilitates enrichment of SSCs. It is also important to notice that expression of spermatogonial stem cell markers is conserved during development, indicating possible associationwith biological properties of the SSCs.

In conclusion, the present data report for the first time, that the expression of Piwil2 is specifically restricted to testes of mouse and human. However, Piwil2 expression was highly detected in various cancer cell lines and cancer tissues of mouse and human. The correlation between Piwil2 expression and the germline property is also supported by the finding that Piwil2 is only overexpressed in seminomas, but not in nonseminomas or in somatic tumors of the adult testis in human. Nonseminomas do not show enhanced Piwil2 expression because these tumors have lost their germline properties, even though they also originate from the same precursor cells as seminomas. The expression of Piwil2 ectopically in fibroblast mouse cells supports the hypothesis that Piwil2 expression is highly upregulated by the Stat3/Bcl-XL signaling pathway. Furthermore, we investigated the role of Piwil2 in cell metabolism using siRNA of Piwil2 in mouse spermatogonia tumor cells and human breast cancer cells. Candidate Piwil2-regulated genes were found to be involved in preventing apoptosis of mouse tumor germ cells and human breast cancer cells. Among the genes of which expression changed on silencing Piwil2 is Stat3/Bcl-XL, an antiapoptotic genes. Stat3 proteins are persistently activated in breast cancer and promote tumor cell survival (Gritsko et al., 2006).

By contrast, dramatic overexpression of Stat3 has been shown in antiapoptotic cells, in which Piwil2 was not expressed. Our results provide first finding that Piwil2 directly regulates the Stat3 expression. Notably, Stat3 expression in antiapoptotic cells is significantly correlated with decreased Piwil2 expression in GC-1 and MDA-MB-231 cells and our findings presented here indicate that Piwil2 expression in NIH3T3-pcDNA-Piwil2 cells induces Stat3/Bcl-XL expression and increases proliferation. Thereby apoptosis is prevented.

Piwil2 transgenic mice that are homozygous for this targeted allele are viable, normal in size and do not display any severe physical abnormalities. Homozygous males are subfertile and have smaller testes than wild type controls. Decreased numbers of sperms are produced in response to degenerated spematocytes. The testes of the Piwil2 transgenic mice exhibit round spermatid arrest and spermatogenesis looks apparently normal up till round spermatids. Histological analysis and TUNEL assay for apoptotic cells in Piwil2 testis showed an increase of apoptosis compared with wild type.

We have confirmed Piwil2 expression in SSCs and shownthat alpha6-integrin (Itga6), Thy-1 (CD90), Stra8, Hsp90 and CD9 antibodies can be used to obtain enriched fractionsof SSCs by MACS. Our results indicate the existence of a distinctsubpopulation of SSCs in the mouse pup testis that expressesa higher level of alpha6-integrin (Itga6), Thy-1 (CD90), Stra8, Hsp90 and CD9. This subpopulation is also positive for the pluripotencymarker Piwil2. It will be interesting in future studiesto confirm the existence of subpopulations of SSCs that maintaindifferent degrees of developmental potential.