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ABSTRACT: Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-Agar-Culture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfrα-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.
The physiologic conditions needed to maintain and differentiate cultured SSCs were previously analyzed in conventional culture systems by addition of testicular cells (Lee et al, 1997; Nagano et al, 2003) and/or certain factors (eg, leukemia inhibitory factor [LIF], de Miguel et al, 1996; Kanatsu-Shinohara et al, 2003; glia cell line—derived neurotrophic factor [GDNF], Meng et al, 2000; Kanatsu-Shinohara et al, 2003; Kubota et al, 2004b; basic fibroblast growth factor, Kanatsu-Shinohara et al, 2003; Kubota et al, 2004b; or stem cell factor [SCF], Allard et al, 1996; Blanchard et al, 1998; de Rooij et al, 1998) that had been identified for spermatogonial propagation. All of these factors have been proposed to be crucial for premeiotic germ cell development.
The conditions allowing male germ cells to enter meiosis are unknown. However, entry into meiosis relies on the integrity of the testicular microenvironment, as it is easily achieved in organ culture (Schlatt et al, 1999) but rarely observed in cell culture. Therefore, we and others assume that testicular somatic cells create unique physical and paracrine support for the developing germ cells, allowing them to enter meiosis (Hofmann et al, 1992; Lee et al, 1997; Nagano et al, 2003). In vivo, the seminiferous tubule offers three compartments for germ cells. 1) The basal compartment, offering physical contacts with the basement membrane, peritubular cells, Sertoli cells, and other premeiotic germ cells. Here, the germ cell receives paracrine and endocrine signals from the interstitium. 2) The intraepithelial compartment, offering only contact with the Sertoli cells and other meiotic and postmeiotic germ cells. 3) The adluminal compartment, allowing contact with Sertoli cells and postmeiotic germ cells, as well as signal molecules from the luminal fluid. The stem cell niches are part of the basal compartment, which offers the most versatile compartment within the seminiferous tubules. Stem cell niches could be established through specific extracellular matrix—specific contacts or specific signaling cascades and will provide specific physical support and environmental features allowing recognition and settlement of SSCs. They also might provide crucial factors needed for maintenance of pluripotent abilities of SSCs (Spradling et al, 2001).
In general, mammalian SSC culture experiments have been performed in conventional “two-dimensional” cell culture approaches using culture dishes or flasks (eg, Dirami et al, 1999; Feng et al, 2002; Hasthorpe, 2003; Nagano et al, 2003; Kanatsu-Shinohara et al, 2004a, 2005a,b). The physical support for SSCs in a conventional culture is different from the natural niche environment zof the seminiferous epithelium, and it remains conjectural whether stem cell niches can be reestablished in a monolayer culture of Sertoli cells. Hence, a three-dimensional culture approach might offer more appropriate opportunities for cell growth.
The Soft-Agar-Culture-System (SACS), a three-dimensional cell culture approach, was first established to characterize clonal expansion of bone marrow cells and to identify factors involved in the regulation of their proliferation and differentiation (Lin et al, 1975; Quaroni et al, 1979; Huleihel et al, 1993; Horowitz et al, 2000). Applied to testicular stem cells, it might also provide an improved structural environment for clonal expansion of germ cells. Here, we are testing this hypothesis to explore whether SACS can be used as an innovative methodology for analysis of germ cell development. Previously published studies demonstrated the importance of a three-dimensional structure for the differentiation of mouse and human testicular cells and the support of in vitro spermatogenesis (Lee et al, 2006, 2007).
To isolate spermatogonia from testicular tissues, particularly from immature animals, several approaches are available, such as gravity sedimentation to separate cells of different size in percoll (Koh et al, 2004) or with the STAPUT technique (Dirami et al, 1999), fluorescence-activated cell sorting (Shinohara et al, 2000; Fujita et al, 2005; Guan et al, 2006), or magnetic-activated cell sorting (MACS; von Schönfeldt et al, 1999; Buageaw et al, 2005). The MACS system is fast and causes minimal stress to the spermatogonial cells during isolation and enrichment. One of the most crucial steps to enrich SSCs is the availability of highly specific markers. Signaling pathway proteins or receptors exclusively expressed on the surface of spermatogonia can be specifically utilized for cell separation by MACS (von Schönfeldt et al, 1999; Buageaw et al, 2005). To isolate the population of undifferentiated spermatogonia in mice, marker proteins such as GDNF family receptor-alpha-1 (Gfrα-1; Meng et al, 2000; von Schönfeldt et al, 2004), Cd-9 (tetraspanin transmembrane protein; Kanatsu-Shinohara et al, 2004b), and Thy-1 (glycosyl phosphatidylinositol—anchored surface antigen; Kubota et al, 2004a; Oatley et al, 2007) have been suggested to show prevalence for this cell type. MAC-sorted cells have previously been cultured using standard procedures. These studies showed the possibility of maintaining proliferating SSCs in vitro for up to 6 months (Kubota et al, 2004b). However, the in vitro production of meiotic and postmeiotic germ cells, which would indicate an optimal culture condition not only for SSCs, but also for survival and differentiation of their progeny, turned out to be extremely difficult. Thus far, no culture system was able to maintain the viability of differentiating spermatogonia and to support the meiotic and postmeiotic spermatogenic progress. In this study, we aimed to characterize a novel three-dimensional culture system and determine the survival, expansion, and differentiation of germ cells.
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Many studies have analyzed in vitro culture effects on gonocytes or SSCs (Hasthorpe et al, 2000; Hasthorpe 2003; Izadyar et al, 2002, 2003; Kanatsu-Shinohara et al, 2003, 2004a, 2005a; Nagano et al, 2003; Kubota et al, 2004b). In general, conventional culture approaches were employed. These did not provide structural conditions that would closely resemble the natural testicular environment. Therefore, our study aimed to establish and validate a novel method for spermatogonial cell culture to improve propagation and differentiation of these undifferentiated germline cells. In contrast to conventional culture performed in dishes or flasks, the three-dimensional agar structure of SACS offers conditions mimicking some structural features of the in vivo situation. SACS was used to examine supporting and limiting effects of somatic testicular cells cocultured in the solid phase of the system. As previously published, the use of different supporter cell types revealed aspects of SSC culture (single spermatogonia; Asingle; de Rooij et al, 2000) in terms of SSC line establishment (Shinohara et al, 2000; Nagano et al, 2003; Kubota et al, 2004b; Kanatsu-Shinohara et al, 2005a) and differentiation into more developed spermatogenic stages (Tres et al, 1983; Gerton et al, 1984; Hue et al, 1998; Tesarik et al, 1998a,b; Feng et al, 2002; Sousa et al, 2002). However, a culture system that allows complete spermatogenesis to occur is still far from routine methodology.
Successful enrichment and separation of isolated testicular cells from mouse tissue using MACS has previously been described (von Schönfeldt et al, 1999; Kubota et al, 2004b; Buageaw et al, 2005; Oatley et al, 2007). Supplementation with somatic cells resulted in a stabilized and more differentiated in vitro population of germ cells. The experimental setting combining MACS separation of the testicular cell fraction and SACS allowed the use of the various fractions achieved by the MAC sorting. A successful MACS separation depends on the use of cell surface markers expressed exclusively on undifferentiated SSCs (Sofikitis et al, 2005). Therefore, Gfrα-1, Cd-9, and Thy-1 were analyzed as putative mouse SSC markers (Meng et al, 2000; Kanatsu-Shinohara et al, 2004b; von Schönfeldt et al, 2004; Oatley et al, 2007; He et al, 2007). The fact that all 3 markers detect the same cell population (same size and granularity in flow cytometric analysis of the enriched fractions) and that we localized exclusively single spermatogonia and small chains/groups of spermatogonia indicated that Cd-9 is a marker for Asingle and Aaligned spermatogonia. In contrast, Gfrα-1 appeared to be expressed exclusively in single spermatogonia, rendering out our favorite marker for SSC separation. Previous studies using MACS confirmed that Gfrα-1 is an excellent marker for SSCs as a co-enrichment of Oct-3/4, which is considered a specific marker for pluripotent cells and germline stem cells (primordial germ cell, embryonic germ cells, and embryonic stem cells), was observed in the enriched fraction (Ohbo et al, 2003; Buageaw et al, 2005). Furthermore, our results are strongly supported by a recently published study by He et al (2007), who showed the double expression of Oct-3/4 and Gfrα-1 in type A spermatogonia in 6 dpp murine testes. Taken together, these and our results indicate an expression of Gfrα-1 in SSCs before the initial differentiation and expansion into pairs and chains starts, which is also indicated by expression of Cd-9.
To explore the effect of more sensitive separation approaches, we compared indirect approaches with magnetically labeled secondary antibodies to strategies using biotin-labeled secondary antibodies and antibiotin magnetic MicroBeads. The better result in cell numbers but similar outcome in the degree of enrichment using the latter approach let us conclude that the efficiency of isolation depends on the enhancement of a rather weak cellular labeling. This indicates that even low expression of Gfrα-1 on the cell surface could be detected. This finding also confirms previous observations that sub-populations of SSCs exist which are characterized by different levels of Gfrα-1 expression (Buagaew et al, 2005).
On day 10 pp in the juvenile immature mouse testis, the SSC proportion is up to 100-fold higher compared with adult tissue (de Rooij et al, 2000; McLean et al, 2003; Aponte et al, 2005). We determined a proportion of 21%–24% Gfrα-1-positive cells in immature preparations prior to sorting. In addition, isolated spermatogonia from immature mice showed better viability (Creemers et al, 2002) and differentiation potential (Nagano et al, 2003). Therefore, the use of juvenile male germ cells seems to be beneficial for spermatogonial in vitro development.
The SACS we used consists of 2 phases of different agar concentrations forming a gel and a solid phase according to Huleihel et al, 1993. This arrangement allows addition of different supplemental factors or supporter cell lines (eg, Sertoli cells) to the solid agar phase without contaminating the gel phase containing the enriched SSCs.
Colony morphology was different when the cultured spermatogonia were grown in different SACS approaches; once established, it did not change during continued culture. During the first 24 hours of SACS spermatogonial cell number decreased independently of the presence of tubular cells and was shown to occur via apoptosis due to abundant TUNEL-positive cells. However, cell survival was enhanced when germ cells are cocultured with cells of the tubular and interstitial fraction, and this early effect was sustained throughout the culture period up to 16 days. Better survival of spermatogonia in the presence of somatic cells confirms findings from conventional in vitro experiments (Dirami et al, 1999; Izadyar et al, 2003).
During murine male germ cell development, a first wave of apoptosis occurs at day 16 pp (Zheng et al, 2006). We also observed an apoptotic wave in 16-dayold germ cells (isolated at day 10 pp and maintained for 6 days in vitro). This response could reflect the first wave of apoptosis found in vivo. However, when a somatic cell supported the germ cell, this apoptotic wave was not seen. It can be speculated that factors produced by Sertoli cells and/or Leydig cells have a positive effect on the cultured spermatogonia.
During the development of the immature testis, Sertoli cells differentiate terminally (eg, Tarulli et al, 2006). Sertoli cells produce 2 isoforms of SCF, a paracrine growth factor, which has inhibiting effects on apoptosis in early spermatogenesis (Print et al, 2000; Huleihel et al, 2004). The soluble form is predominantly expressed and important in the juvenile testis; the membrane-bound isoform is crucial for adult spermato-genesis (Blanchard et al, 1998; de Rooij et al, 1998). Considering the antiapoptotic effect of SCF, our data obtained from SACS suggest an effect on apoptotic inhibition during spermatogonial differentiation.
If optimal culture conditions exist, meiosis should be initiated and completed in vitro. Boule is a meiosis marker highly expressed in mice in late pachytene or diplotene stage spermatocytes (Xu et al, 2001). We confirmed here that Boule protein in immature mouse testis is not detected before day 16 pp. In SACS-cultured germ cells, we found Boule expression at day 13 of culture when the spermatogonia were cocultured with all other somatic testicular cells in the gel phase of the agar, but not when spermatogonia were cultured alone or with the tubular somatic fraction. As an additional marker to determine meiotic processes in vitro, we analyzed Crem expression indicating for postmeiotic/round spermatid stages. Crem is known to be expressed in round spermatids (Delmas et al, 1993; Wistuba et al, 2002), and is therefore the optimal marker to analyze meiosis completion. In the SACS approach using all testicular cells (intratubular and interstitial; Table 1), we show that Crem-positive cells appear at least at day 21 of culture. This might be an effect of testosterone as a product of Leydig cells located in the interstitium, which is considered to be a crucial factor inhibiting apoptotic events during meiosis (Print et al. 2000).
The observed mRNA expression profile supports the results obtained by immunohistochemistry. In the approach containing all testicular cells, almost all meiotic genes were expressed at the mRNA level already after 1 day of culture. This can be explained by the well-known shift between transcription and translation of genes during spermatogenesis (Kleene et al, 1984; Kleene, 1996; Iguchi et al, 2006). Although only the germ cells that were supported by all other testicular cells progress up to meiosis, the early expression of these mRNAs might be a necessary step preparing the later differentiation. Hence, in the other experiments, we did not find this expression pattern, and maybe for this reason we also failed to detect meiosis. Therefore, these results indicate that our coculture approach allowed germ cells to enter meiosis in vitro without any addition of growth factors.
The hypothesis that in vitro meiosis is even possible without a direct cell-cell contact has to be investigated further. Therefore, additional experiments combining the supporting factors (eg, LIF, GDNF, SCF, and/or hormones like testosterone) with a three-dimensional environment might result in completed spermatogenesis in vitro.