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Genes to Cells (2008) 13, 365-374. doi:10.1111/j.1365-2443.2008.01171.x
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GDNF-mediated signaling via RET tyrosine 1062 is essential for maintenance of spermatogonial stem cells

 Mayumi Jijiwa, Kumi Kawai, Jun Fukihara, Akari Nakamura, Masaki Hasegawa, Chikage Suzuki, Tomoko Sato, Atsushi Enomoto, Naoya Asai, Yoshiki Murakumo and Masahide Takahashi*

Department of Pathology, Nagoya University Graduate School of Medicine 65 Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Well-organized spermatogenesis, including the maintenance of spermatogonial stem cells (SSCs), is indispensable for continuous male fertility. Signaling by glial cell line-derived neurotrophic factor (GDNF) via the RET/GDNF family receptor {alpha}1 (GFR{alpha}1) receptor complex is essential for self-renewal of murine SSCs and may also regulate their differentiation. When phosphorylated, tyrosine 1062 in RET presents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for activation of a variety of intracellular signaling pathways. In this study, we investigated the role of signaling via RET tyrosine 1062 in spermatogenesis using RET Y1062F knockin mice (Y1062F mice), in which tyrosine 1062 was replaced with phenylalanine. Homozygous Y1062F mice showed marked atrophy of testes due to reduced production of germ cells. RET-expressing spermatogonia in seminiferous tubules of homozygous Y1062F mice decreased after postnatal day (P) 7 and germ cells were almost undetectable by P21. These phenomena appeared to be due to a lack of SSC self-renewal and inability to maintain the undifferentiated state. Our findings suggest that RET signaling via tyrosine 1062 is essential for self-renewal of SSCs and regulation of their differentiation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Mammalian spermatogenesis requires a continuous supply of differentiating germ cells. To achieve this, spermatogonial stem cells (SSCs) must have the capacities for both self-renewal and maintenance of the undifferentiated state. In addition, well-regulated asymmetric differentiation is crucial to retain some stem cells throughout the reproductive period and to continuously generate germ cells. The balance between these processes is controlled by the molecules expressed by germ cells and Sertoli cells (Meng et al. 2000; Naughton et al. 2006), which must interact appropriately for normal spermatogenesis. One mechanism for modulating spermatogenesis includes the glial cell line-derived neurotrophic factor (GDNF)/RET signaling system. RET is a receptor tyrosine kinase that possesses several binding sites for signal-transducing molecules. GDNF binding to GDNF family receptor {alpha}1 (GFR{alpha}1) induces RET activation that leads to binding of various molecules to the RET intracellular domain (Jing et al. 1996; Treanor et al. 1996). These include SHC, Enigma, FRS2, DOK family proteins, IRS1/2, PLC{gamma}, PKC{alpha}, GRB2 and GRB7/GRB10 (Takahashi 2001; Ichihara et al. 2004). Some molecules are involved in activation of the phosphatidylinositol-3 kinase (PI3K)–AKT and/or RAS–MAPK pathways (Takahashi 2001; Arighi et al. 2005; Asai et al. 2006).

In the testes, RET and its co-receptor GFR{alpha}1 are expressed by the gonocytes which are immature spermatogonia, and a subset of type A spermatogonia which are considered SSCs (Dettin et al. 2003; von Schonfeldt et al. 2004; Naughton et al. 2006). The ligand GDNF is produced by Sertoli cells (Golden et al. 1999; Meng et al. 2000; Viglietto et al. 2000).

GDNF is the first molecule that was found to be involved in regulation of SSC self-renewal (Meng et al. 2000). The availability of the transplantation assay made it possible to study SSCs in culture (Nagano et al. 1998), and led to development of systems supporting continuous replication of murine SSCs in vitro (Kanatsu-Shinohara et al. 2003). Studies using serum-free culture medium demonstrated that GDNF is the essential growth factor supporting mouse SSC self-renewal (Kubota et al. 2004). Gene-targeted mice carrying one Gdnf-null allele exhibited depletion of stem cell reserves, whereas mice over-expressing GDNF accumulated undifferentiated spermatogonia. These mice were unable to properly regulate the differentiation signals in germ cells (Meng et al. 2000). GDNF also supports self-renewal of rat SSCs in vitro (Ryu et al. 2005).

Because Gfr{alpha}1- and Ret-deficient mice die early in life, there is only limited understanding of the mechanisms by which RET signaling influences spermatogenesis. Recently, mice expressing a dominant-negative RET mutant have been shown to manifest abnormalities in germ cell maturation (Jain et al. 2004). In addition, whole-testis transplantation techniques showed that Gdnf-, Gfr{alpha}1- and Ret-deficient testes led to severe SSC depletion by P7, which was due to lack of SSC proliferation and an inability to maintain an undifferentiated state (Naughton et al. 2006).

We recently generated Ret Y1062F knockin mice (Y1062F mice) in which tyrosine 1062 in mouse genomic Ret was replaced with phenylalanine (Jijiwa et al. 2004). Upon phosphorylation, tyrosine 1062 in RET presents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins such as SHC, FRS2 and Dok family proteins (Takahashi 2001; Ichihara et al. 2004). Thus, a mutation of tyrosine 1062 resulted in the impairment of activation of intracellular signaling pathways including RAS–ERK and PI-3K–AKT pathways (Besset et al. 2000; Hayashi et al. 2000). Homozygous Y1062F mice exhibited growth retardation and hypoplasia of the enteric nervous system and the kidneys, and died within 4 weeks of birth (Jijiwa et al. 2004). The expression level of the mutant RET protein in homozygous mice was comparable to that in wild-type and heterozygous mice, although GDNF-mediated phosphorylation of AKT and ERK was significantly impaired in homozygous mice.

In this study, we examined the histology of Y1062F mouse testes from neonate up to postnatal day 28 (P28). Homozygous Y1062F mice revealed SSC reduction probably because of poor self-renewal and an inability to maintain the undifferentiated state of SSCs. Our findings suggested that signaling via RET tyrosine 1062 plays an essential role in maintenance of the biological properties of SSCs.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Testis phenotype in Y1062F knockin mice

As previously reported, no significant difference in gross appearance and body weight was observed at birth between wild-type, heterozygous and homozygous Y1062F mice (Jijiwa et al. 2004). However, homozygous mutant mice displayed growth retardation soon after birth, whereas no abnormalities were detected in heterozygous mice (Fig. 1b). Although testes of homozygous mutant mice were comparable in size to those of wild-type and heterozygous mice at birth (data not shown), the former became smaller than the latter at P7 and showed progressive atrophy after P14 (Fig. 1a,c). To evaluate the degree of testis atrophy, the testis weight : body weight ratio of Y1062F mice was analyzed. While testes of homozygous Y1062F mice grew proportionately to their body weight until P14, the ratios in homozygous mice were about one-third to one-fourth of those in wild-type and heterozygous mice at P21 and P28 (Fig. 1d). In contrast, no significant difference in the heart weight : body weight ratio was observed between the three genotypes (Fig. 1e). These findings indicate that testes of homozygous Y1062F mice grew until around P14 and then presented progressive atrophy beyond growth retardation.


Figure 1
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  		Figure 1  Testicular hypoplasia in Ret Y1062F knockin mice. (a) Macroscopic findings of testes of wild-type (Wt) and homozygous (Ho) Y1062F mice. The testis of the homozygous Y1062F mouse at P14 was approximately one-third that of the wild type mouse. (b) Body weight of Y1062F mice at P0, P7, P14, P21 and P28. Although no significant differences were observed between the three genotypes at birth, severe growth retardation was seen in homozygous Y1062F mice after birth. "n" indicates numbers of examined mice in this study. (c) Testicular weight of Y1062F mice. In addition to small size, testes in homozygous mice showed atrophy after P14. (d) Testicular weight : body weight ratios of Y1062F mice. (e) Heart weight : body weight ratios of Y1062F mice. Bars indicate standard deviations. Wt, wild-type mouse; He, heterozygote; Ho, homozygote.

 
Histological findings of Y1062F mouse testis

Next, we investigated testicular histology using hematoxylin–eosin stained cross-sections. At P0 and P7, histological findings of testes were not significantly different between the three genotypes, although the size of seminiferous tubules in homozygous mutant mice at P7 was slightly smaller than that in wild-type and heterozygous mice (Fig. 2a). At P14, germ cell differentiation towards spermatocytes was impaired in some tubules of homozygous mice, and at P21, germ cells were remarkably reduced in all tubules. By P28, germ cells had almost disappeared from their tubules, revealing only Sertoli cells. The number of seminiferous tubules per section (the greatest sagittal section of each testis) in homozygous mutant mice appeared to be slightly less than that in wild-type and heterozygous mice, although it was not significantly different between the three genotypes (Fig. 2b).


Figure 2
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  		Figure 2  Histological findings of testes of Ret Y1062F knockin mice. (a) Histology of testes of Ret Y1062F knockin mice. At P0 and P7, testes of homozygous Y1062F mice showed similar histological findings to those of wild-type mice. Gonocytes and spermatogonia are indicated by red and black arrow heads, respectively. At P14, germ cell differentiation was impaired in some tubules (red asterisks) of homozygous mice. At P21 and P28, germ cells had almost disappeared in tubules of homozygous mice. Bars indicate 50 µm. (b) Numbers of seminiferous tubules per section. Numbers of tubules were counted in the greatest sagittal sections of testes. They were not statistically significant between wild-type and homozygous mice. Statistical analysis was not performed at P28, because only two homozygous mice were available. (c) Immunohistochemistry of testis with anti-GATA4 antibody. Sertoli cells were positively stained (brown color). A broken line indicates the peripheral margin of a seminiferous tubule. Cells with blue nuclei indicated by red dots were counted as germ cells. (d) Quantification of germ cells. The numbers of germ cells in 20 seminiferous tubules per testis were counted in two to five mice of each genotype. The numbers of germ cells per tubule were statistically significant between wild-type (or heterozygous mice) and homozygous mice at P7, P14 and P21 (asterisks, P < 0.05). Statistical analysis was not performed at P28, because only two homozygous mice were available. (e) Testis of heterozygous Y1062F mice at P14. Focal lesions, which showed the defect of germ cell differentiation in tubules (red asterisks), were observed in three of five Y1062F heterozygous mice examined. (f) Testis of heterozygous Y1062F mice at P21. Two of the five examined heterozygous mice exhibited focal Sertoli cell-only tubules.

 
To quantify the number of germ cells, immunohistochemistry using anti-GATA4 antibody with hematoxylin counter staining was performed (Fig. 2c). GATA4 is a transcription factor known to be stably expressed in postnatal murine Sertoli cells and Leydig cells (Ketola et al. 1999; Imai et al. 2004). Thus, GATA4-negative cells in seminiferous tubules were recognized as germ cells (Fig. 2c). Although germ cells increased in homozygous Y1062F mice until P14, they markedly decreased in number after P14 (Fig. 2d). These findings indicate that the atrophy seen in homozygous testes was due to the reduction of germ cells.

Testes of heterozygous mutant mice revealed grossly normal histology at all observed time points. However, we found focal lesions in seminiferous tubules in three of five heterozygous mice at P14 and two of five heterozygous mice at P21 in which differentiation of germ cells was impaired (Fig. 2e,f), whereas such changes were not detected at P28. At P21, the affected tubules contained only Sertoli cells (Fig. 2f). This finding may suggest a dosage effect of the tyrosine 1062 RET signal, which impacts differentiation of germ cells during a restricted period in neonatal mice.

Localization of RET-positive spermatogonia in Y1062F testis

RET is expressed with its co-receptor GFR{alpha}1 in two germ cell types (Dettin et al. 2003; von Schonfeldt et al. 2004; Naughton et al. 2006). One is the gonocyte which is the initial germ cell of spermatogenesis, and the other is the undifferentiated spermatogonium. To investigate the expression of RET protein in germ cells of RET Y1062F mice, immunohistochemistry using anti-RET antibody was performed (Fig. 3a). In the three genotypes, RET-expressing cells were located mainly in the inner area of seminiferous tubules at P0 and moved to the periphery by P7. The intensity of RET staining was almost the same between the three genotypes at P0, whereas it appeared to decrease in homozygous Y1062F mice at P7 (Fig. 3a). At P14, RET-positive cells in testes of homozygous mice markedly decreased compared with those in testes of wild-type and heterozygous mice (Fig. 3a,b). At P21 and P28, no RET-positive cells were identified in testes of homozygous mutant mice.


Figure 3
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  		Figure 3  RET expression in gonocytes and spermatogonia. (a) Immunohistochemistry of testes with anti-RET antibody. RET staining was detected in the gonocytes in the inner area of tubules at P0. RET-positive cells moved to the periphery at P7. They were difficult to detect in homozygous Y1062F mice after P14. Bars indicate 50 µm. (b) Quantitative analysis of RET-expressing cells. Twenty seminiferous tubules in two to five mouse testes of each genotype were randomly selected. The average numbers of RET-positive cells per tubule of each mouse were calculated at each time point. Statistical significance of RET-positive cells was observed between wild-type (or heterozygous mice) and homozygous mice at P14 and P21 (asterisks, P < 0.05). (c) Expression levels of RET protein in testes. Total cell lysates of testes of each genotype at P7, P14 and P28 were subjected to Western blotting with anti-RET antibody (detected at 175 and 155 kDa). AKT was also immunoblotted as an internal control (detected below 62 kDa).

 
RET expression in testes was investigated by Western blotting. Whereas the levels of RET protein expression were comparable between wild-type and heterozygous mice at each stage (P7, P14 and P28), it significantly decreased at P7 and almost disappeared at P21 and P28 in homozygous mutant mice (Fig. 3c). This result correlated with the numbers of RET-positive cells in seminiferous tubules of each genotype.

The number of PLZF-positive cells decreases in parallel to that of RET-positive cells in Y1062F testis

To confirm that SSCs in testes of homozygous Y1062F mice actually decreased, immunohistochemistry using anti-PLZF antibody, a stem cell marker, was performed. PLZF is a transcriptional repressor that regulates the epigenetic state of undifferentiated cells (Buaas et al. 2004). Its expression was also restricted to gonocytes and undifferentiated spermatogonia, a subset of which is considered to be SSCs (Costoya et al. 2004). PLZF-positive staining was detected in the nuclei of inner (P0, P7) and peripheral (P7, P14 and P28) cells of wild-type and heterozygous mutant mice like RET staining (Fig. 4a and data not shown). Immunohistochemistry of serial sections using anti-RET and anti-PLZF antibody revealed that the two proteins were expressed in the same cells (Fig. 4b). PLZF-positive cells markedly decreased at P14 and were almost undetectable at P21 and P28 in homozygous Y1062F mice (Fig. 4a). Quantitative analysis revealed that changes in the numbers of PLZF-positive cells (Fig. 4c) were similar to those of RET-positive cells (Fig. 3b) in the three genotypes.


Figure 4
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  		Figure 4  Number of spermatogonial stem cells. (a) Immunohistochemistry of testes with anti-PLZF antibody. Nuclei of gonocytes and spermatogonia were positively-stained with anti-PLZF antibody. Bars indicate 50 µm. (b) Immunohistochemistry using serial section demonstrated that RET and PLZF were expressed in the same cells in tubules of a homozygous Y1062F mouse at P3 (black arrows). (c) Quantitative analysis of PLZF-expressing spermatogonial stem cells. Twenty seminiferous tubules in two to five mice testes of each genotype were randomly selected. The average numbers of PLZF-positive cells per tubule of each mouse were calculated at each time point. Statistical significance of PLZF-positive cells was observed between wild-type (or heterozygous mice) and homozygous mice at P14 and P21 (asterisks, P < 0.05).

 
Analysis of germ cell apoptosis

To evaluate whether the reduction of SSCs in testes of homozygous mutant mice was due to apoptosis, we used TUNEL staining to examine apoptotic cells in testes at P7 and P14 when RET-expressing germ cells were rapidly disappearing. Apoptotic cells were detected in the inner area rather than in the periphery of seminiferous tubules where SSCs are present (Fig. 5a). The ratios of apoptotic cells per germ cell were similar between the three genotypes (Fig. 5b), suggesting that apoptosis does not contribute to the reduction of SSCs in homozygous Y1062F mice.


Figure 5
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  		Figure 5  Apoptosis of germ cells in Y1062F mice. (a) TUNEL-positive apoptotic cells in seminiferous tubules at P14. TUNEL-positive apoptotic cells are indicated by arrows. Bars indicate 100 µm. (b) The ratio of apoptotic cells per germ cells. No significant difference in the numbers of apoptotic cells was observed in tubules of the three genotypes.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Spermatogenesis is a strictly regulated process initiated shortly after birth. In neonatal mouse testes, spermatogonial precursor cells, called gonocytes, begin to migrate from the inner area of the seminiferous tubules to the periphery (Orth et al. 2000; McLean et al. 2003). By P3, gonocytes reach the basement membrane, followed by differentiation into type A spermatogonia, generally regarded as SSCs (McLean et al. 2003). P2 to P4 is a critical period for SSCs to establish stem cell niches and interact with Sertoli cells (McLean et al. 2003), and type A spermatogonia are observed around P6 (Bellve et al. 1977). Subsequently, primary spermatocytes appear around P10, and pachytene or secondary spermatocytes differentiate by P18 (McLean et al. 2003). According to our observations, spermatids can be seen at P21, and immature sperm are found in some tubules at P28.

In testis, RET and its co-receptor GFR{alpha}1 are mainly expressed by a subset of spermatogonia including SSCs (Dettin et al. 2003; von Schonfeldt et al. 2004; Naughton et al. 2006; He et al. 2007), and the ligand GDNF is secreted by Sertoli cells (Golden et al. 1999; Meng et al. 2000; Viglietto et al. 2000). In the present study, we analyzed the role of signaling via RET tyrosine 1062 in spermatogenesis. Ret Y1062F knockin mice, in which tyrosine 1062 in RET was replaced with phenylalanine, enabled us to investigate the effect of RET signaling on early spermatogenesis. Homozygous Y1062F mice showed remarkable testicular atrophy due to the reduction of germ cells. Growth retardation of homozygous Y1062F mice may affect some hormone/growth factor conditions which lead to the decrease of germ cells.

We observed with immunohistochemical analysis that both RET and PLZF-positive spermatogonia were markedly reduced in number after P7 and testes exhibited Sertoli cell-only tubules at P28. Because SSCs are the source of germ cells, it is likely that the decrease of SSCs leads to a reduction of germ cells and testicular atrophy. Many reports have provided evidence that GDNF is the primary growth factor supporting mouse SSC self-renewal. Thus, the reduction of SSC in homozygous Y1062F mouse testes could be due to the decrease of GDNF-mediated RET signaling that plays a crucial role in SSC self-renewal. Alternatively, it is possible that the blockage/reduction in SSC/spermatogonial differentiation leads to similar results.

The numbers of RET- and PLZF-positive cells at birth, which are considered to be gonocytes, are comparable between the three genotypes. Although their numbers in homozygous mutant mice at P7 increased, they were less numerous than those in the other two genotypes, suggesting that self-renewal of SSCs was impaired at this stage. A study using a whole-testis transplantation technique demonstrated a normal number of gonocytes at birth in RET-deficient testes followed by deficient SSC self-renewal and severe depletion of germ cells at P7. Lee et al. (2007) reported that the PI3K–AKT signaling pathway plays a central role in the self-renewal of SSCs. They examined the effects of inhibitors of PI3K and MAPK pathways using the SSC culture system. When SSCs were cultured in the presence of LY294002 (a PI3K-specific inhibitor), the growth of SSCs was significantly inhibited and the number of cells did not increase after 6 days of culture. In contrast, when SSCs were cultured in the presence of PD98059 (a MEK-specific inhibitor) at inhibitory concentrations (Burdon et al. 1999), SSCs continued to proliferate, and their growth rate and morphologies were comparable to untreated cells (Lee et al. 2007). Interestingly, the activation of PI3K plays a crucial role in the proliferation and maintenance of undifferentiated ES cells (Jirmanova et al. 2002; Paling et al. 2004; Watanabe et al. 2006). The fact that the PI3K–AKT pathway was significantly impaired in Y1062F mutant mice (Jijiwa et al. 2004) supports the importance of this pathway in the self-renewal of SSCs.

In addition, it has been noted that RET activation regulates its own expression (Kodama et al. 2004; Peterson et al. 2004). Because RET expression decreased in homozygous Y1062F mice at P7, it is possible that the impairment of signaling via tyrosine 1062 down-regulates its expression, leading to the SSC reduction.

Despite the SSC reduction, spermatogonia in testes of homozygous Y1062F mice initially proceeded to differentiate. Testes of homozygous mice contained SSCs and spermatocytes at P14, although spermatocytes as well as SSCs had almost disappeared by P21. This finding suggests that not only self-renewal of SSCs but also maintenance of their undifferentiated state may be impaired in homozygous Y1062F mice. Previous investigation using Gdnf+/– and GDNF over-expressing mice revealed that the dosage of GDNF regulates the fate and lineage determination of undifferentiated spermatogonia (Meng et al. 2000). At a low-level of GDNF, spermatogonia favor differentiation, and at a high level, they favor self-renewal. Decreased GDNF signaling through RET Y1062 may have results similar to those seen during a shortage of GDNF. Thus, it is possible that SSCs were unable to maintain the undifferentiated state and proceeded to differentiate despite the insufficiency of the SSC pool (Paling et al. 2004; Lee et al. 2007; Li et al. 2007). As for GFR{alpha}1, He et al. (2007) reported very recently that gene silencing of Gfr{alpha}1 in SSCs leads to their differentiation via inactivation of RET.

Another possible explanation for the disappearance of SSCs is that SSCs undergo apoptosis due to the loss of GDNF-mediated signaling. However, TUNEL staining analysis demonstrated that the number of apoptotic cells per germ cells was not significantly different between the three genotypes at key points (P7 and P14) showing SSC reduction. Apoptosis occurred randomly in the inner area of tubules regardless of genotypes. Thus, it is unlikely that the disappearance of SSCs in homozygous Y1062F mice resulted from apoptosis, although the possibility that Y1062F mutation affected the survival of SSCs is not completely excluded.

A number of spermatocytes were present at P14 in homozygous Y1062F mice. While meiotic spermatocytes and round spermatids were observed in wild-type and heterozygous mutant mice, they were hardly detected in homozygous mice at P21. This finding suggested that germ cell maturation may be arrested at the spermatocyte stage in homozygous mutant mice. Similar maturation impairment was observed in mice expressing a dominant-negative RET mutation (Jain et al. 2004). The mice exhibited severely diminished RET kinase activity, implying that RET signaling is required in early spermatogenesis (between P0 and P10) to establish normal germ cell numbers and spermatogonial maturation. Interestingly, the numbers of spermatogonia in those mutant mice were comparable to wild-type mice even at P28.

In conclusion, we elucidated the role of signaling via RET tyrosine 1062 in spermatogenesis using Ret Y1062F knockin mice. The signal appeared to be essential for SSC self-renewal and maintenance of the undifferentiated state of SSCs. Reduced self-renewal and maintenance of "stemness" did not allow the formation of the basal population of SSCs, thereby differentiating cells could not be maintained. In addition, our findings suggested that signaling via tyrosine 1062 may be involved in germ cell maturation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Generation of Ret Y1062F knockin mice

Ret Y1062F knockin mice were produced and genotyped as described previously (Jijiwa et al. 2004). For genotyping, genomic DNA was extracted from mouse tails and subjected to genomic PCR, followed by SspI digestion (Jijiwa et al. 2004). This study was approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine.

Preparation of tissue samples

Male mice were sacrificed under general anesthesia. Testes were isolated and their weight determined. One testis of each mouse was frozen for protein extraction, and the other was fixed in 10% neutral buffered formalin for histological analysis. Frozen testes were homogenized in SDS sample buffer (2% SDS, 20 Tris–HCl pH 6.8, 5 mM EDTA pH 8.0, 80 mM dithiothreitol) and boiled for 5 min. Testes fixed in formalin were dehydrated and embedded in paraffin, and sectioned to 5 µm thickness.

Antibodies

Anti-RET rabbit polyclonal antibody (R787) was described previously (Jijiwa et al. 2004). Anti-PLZF (H-300) and anti-GATA4 (H-112) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Histology and immunohistochemistry

Hematoxylin and eosin staining was performed by conventional methods. For immunohistochemistry, slides were deparaffinized in xylene and rehydrated in a graded series of ethanols (100%, 90% and 70%). Antigen retrieval (microwave 10 min, 0.01 M citrate buffer, pH 6.0) was performed for all antibodies. Endogenous peroxidase was inhibited with 0.3% hydrogen peroxide in methanol for 15 min at room temperature. Nonspecific binding sites were blocked with 10% normal goat serum for 60 min at room temperature. The sections were incubated with the primary antibodies (anti-RET and anti-GATA4) overnight at 4 °C or for 2 h at room temperature (anti-PLZF). The slides were incubated with the secondary antibody conjugated to horseradish peroxidase-labeled polymer (EnVision+, goat, anti-rabbit, Dako, Hamburg, Germany) for 60 min at room temperature. The reaction products were visualized with diaminobenzidine. Counterstaining was performed with hematoxylin.

Western blotting

Lysates containing 30 µg of total protein were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were blocked for 1 h at room temperature in 5% skim milk in TPBS (phosphate-buffered saline containing 0.05% Tween 20) with gentle shaking and incubated with the primary antibody overnight at 4 °C. After three TPBS washes, the membranes were incubated with the secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit immunoglobulin horseradish peroxidase; Dako) for 1 h at room temperature. The reaction was examined with an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions.

TUNEL staining

Apoptotic germ cells were detected by TUNEL staining using Apoptosis in situ Detection Kit Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Statistical analysis

Statistical significance was evaluated by Student's t-test. Because we obtained only two homozygous Y1062F mice at p28, statistical analysis between wild-type and homozygous mice was not performed at P28.


   Acknowledgements
 
We are grateful to N. Misawa, Y. Hayashi and Division of Experimental Animals, Nagoya University for excellent technical supports. We give special thanks to K. Uchiyama, K. Imaizumi and S. Kawai for kind assistance. This work was supported in part by Grants-in-Aid for the 21st Century Center of Excellence Research, Scientific Research (A), and Scientific Research on Priority Areas Cancer (to M.T.) and for Young Scientists (B) (to M.J.) from Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: Email: mtakaha{at}med.nagoya-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Arighi, E., Borrello, M.G. & Sariola, H. (2005) RET tyrosine kinase signaling in development and cancer. Cytokine Growth Factor Rev. 16, 441–467.[CrossRef][Medline]

Asai, N., Jijiwa, M., Enomoto, A., Kawai, K., Maeda, K., Ichiahara, M., Murakumo, Y. & Takahashi, M. (2006) RET receptor signaling: dysfunction in thyroid cancer and Hirschsprung's disease. Pathol. Int. 56, 164–172.[CrossRef][Medline]

Bellve, A.R., Cavicchia, J.C., Millette, C.F., O’Brien, D.A., Bhatnagar, Y.M. & Dym, M. (1977) Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J. Cell Biol. 74, 68–85.[Abstract/Free Full Text]

Besset, V., Scott, R.P. & Ibanez, C.F. (2000) Signaling complexes and protein–protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase. J. Biol. Chem. 275, 39159–39166.[Abstract/Free Full Text]

Buaas, F.W., Kirsh, A.L., Sharma, M., McLean, D.J., Morris, J.L., Griswold, M.D., de Rooij, D.G. & Braun, R.E. (2004) Plzf is required in adult male germ cells for stem cell self-renewal. Nat. Genet. 36, 647–652.[CrossRef][Medline]

Burdon, T., Stracey, C., Chambers, I., Nichols, J. & Smith, A. (1999) Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev. Biol. 210, 30–43.[CrossRef][Medline]

Costoya, J.A., Hobbs, R.M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., Orwig, K.E., Wolgemuth, D.J. & Pandolfi, P.P. (2004) Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet. 36, 653–659.[CrossRef][Medline]

Dettin, L., Ravindranath, N., Hofmann, M.C. & Dym, M. (2003) Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis. Biol. Reprod. 69, 1565–1571.[Abstract/Free Full Text]

Golden, J.P., DeMaro, J.A., Osborne, P.A., Milbrandt, J. & Johnson, E.M., Jr. (1999) Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp. Neurol. 158, 504–528.[CrossRef][Medline]

Hayashi, H., Ichihara, M., Iwashita, T., Murakami, H., Shimono, Y., Kawai, K., Kurokawa, K., Murakumo, Y., Imai, T., Funahashi, H., Nakao, A. & Takahashi, M. (2000) Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene 19, 4469–4475.[CrossRef][Medline]

He, Z., Jiang, J., Hofmann, M.C. & Dym, M. (2007) Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase. Biol. Reprod. 77, 723–733.[Abstract/Free Full Text]

Ichihara, M., Murakumo, Y. & Takahashi, M. (2004) RET and neuroendocrine tumors. Cancer Lett. 204, 197–211.[CrossRef][Medline]

Imai, T., Kawai, Y., Tadokoro, Y., Yamamoto, M., Nishimune, Y. & Yomogida, K. (2004) In vivo and in vitro constant expression of GATA-4 in mouse postnatal Sertoli cells. Mol. Cell. Endocrinol. 214, 107–115.[CrossRef][Medline]

Jain, S., Naughton, C.K., Yang, M., Strickland, A., Vij, K., Encinas, M., Golden, J., Gupta, A., Heuckeroth, R., Johnson, E.M. Jr. & Milbrandt, J. (2004) Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis. Development 131, 5503–5513.[Abstract/Free Full Text]

Jijiwa, M., Fukuda, T., Kawai, K., Nakamura, A., Kurokawa, K., Murakumo, Y., Ichihara, M. & Takahashi, M. (2004) A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia. Mol. Cell. Biol. 24, 8026–8036.[Abstract/Free Full Text]

Jing, S., Wen, D., Yu, Y., Holst, P.L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J.C., Hu, S., Altrock, B.W. & Fox, G.M. (1996) GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-{alpha}, a novel receptor for GDNF. Cell 85, 1113–1124.[CrossRef][Medline]

Jirmanova, L., Afanassieff, M., Gobert-Gosse, S., Markossian, S. & Savatier, P. (2002) Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells. Oncogene 21, 5515–5528.[CrossRef][Medline]

Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S. & Shinohara, T. (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616.[Abstract/Free Full Text]

Ketola, I., Rahman, N., Toppari, J., Bielinska, M., Porter-Tinge, S.B., Tapanainen, J.S., Huhtaniemi, I.T., Wilson, D.B. & Heikinheimo, M. (1999) Expression and regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis. Endocrinology 140, 1470–1480.[Abstract/Free Full Text]

Kodama, Y., Murakumo, Y., Ichihara, M., Kawai, K., Shimono, Y. & Takahashi, M. (2004) Induction of CRMP-2 by GDNF and analysis of the CRMP-2 promoter region. Biochem. Biophys. Res. Commun. 320, 108–115.[CrossRef][Medline]

Kubota, H., Avarbock, M.R. & Brinster, R.L. (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 101, 16489–16494.[Abstract/Free Full Text]

Lee, J., Kanatsu-Shinohara, M., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Kimura, T., Nakano, T., Ogura, A. & Shinohara, T. (2007) Akt mediates self-renewal division of mouse spermatogonial stem cells. Development 134, 1853–1859.[Abstract/Free Full Text]

Li, J., Wang, G., Wang, C., Zhao, Y., Zhang, H., Tan, Z., Song, Z., Ding, M. & Deng, H. (2007) MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation 75, 299–307.[CrossRef][Medline]

McLean, D.J., Friel, P.J., Johnston, D.S. & Griswold, M.D. (2003) Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol. Reprod. 69, 2085–2091.[Abstract/Free Full Text]

Meng, X., Lindahl, M., Hyvonen, M.E., Parvinen, M., de Rooij, D.G., Hess, M.W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H., Lakso, M., Pichel, J.G., Westphal, H., Saarma, M. & Sariola, H. (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 1489–1493.[Abstract/Free Full Text]

Nagano, M., Avarbock, M.R., Leonida, E.B., Brinster, C.J. & Brinster, R.L. (1998) Culture of mouse spermatogonial stem cells. Tissue Cell 30, 389–397.[CrossRef][Medline]

Naughton, C.K., Jain, S., Strickland, A.M., Gupta, A. & Milbrandt, J. (2006) Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol. Reprod. 74, 314–321.[Abstract/Free Full Text]

Orth, J.M., Jester, W.F., Li, L.H. & Laslett, A.L. (2000) Gonocyte–Sertoli cell interactions during development of the neonatal rodent testis. Curr. Top Dev. Biol. 50, 103–124.[CrossRef][Medline]

Paling, N.R., Wheadon, H., Bone, H.K. & Welham, M.J. (2004) Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J. Biol. Chem. 279, 48063–48070.[Abstract/Free Full Text]

Peterson, S. & Bogenmann, E. (2004) The RET and TRKA pathways collaborate to regulate neuroblastoma differentiation. Oncogene 23, 213–225.[CrossRef][Medline]

Ryu, B.Y., Kubota, H., Avarbock, M.R. & Brinster, R.L. (2005) Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc. Natl. Acad. Sci. USA 102, 14302–14307.[Abstract/Free Full Text]

von Schonfeldt, V., Wistuba, J. & Schlatt, S. (2004) Notch-1, c-kit and GFR{alpha}-1 are developmentally regulated markers for premeiotic germ cells. Cytogenet. Genome Res. 105, 235–239.[CrossRef][Medline]

Takahashi, M. (2001) The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev. 12, 361–373.[CrossRef][Medline]

Treanor, J.J., Goodman, L., de Sauvage, F., et al. (1996) Characterization of a multicomponent receptor for GDNF. Nature 382, 80–83.[CrossRef][Medline]

Viglietto, G., Dolci, S., Bruni, P., Baldassarre, G., Chiariotti, L., Melillo, R.M., Salvatore, G., Chiappetta, G., Sferratore, F., Fusco, A. & Santoro, M. (2000) Glial cell line-derived neutrotrophic factor and neurturin can act as paracrine growth factors stimulating DNA synthesis of Ret-expressing spermatogonia. Int. J. Oncol. 16, 689–694.[Medline]

Watanabe, S., Umehara, H., Murayama, K., Okabe, M., Kimura, T. & Nakano, T. (2006) Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 25, 2697–2707.[CrossRef][Medline]

Received: 2 October 2007
Accepted: 3 January 2008




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