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Glycobiology. 2011 February; 21(2): 152–161.
Published online 2010 September 20. doi:  10.1093/glycob/cwq133
PMCID: PMC3031337

Expression regulation and function of heparan sulfate 6-O-endosulfatases in the spermatogonial stem cell niche

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a heparan sulfate (HS)-binding factor. GDNF is produced by somatic Sertoli cells, where it signals to maintain spermatogonial stem cells (SSCs) and reproduction. Here, we investigate the roles of extracellular HS 6-O-endosulfatases (Sulfs), Sulf1 and Sulf2, in the matrix transmission of GDNF from Sertoli cells to SSCs. Although Sulfs are not required for testis formation, Sulf deficiency leads to the accelerated depletion of SSCs, a testis phenotype similar to that of GDNF+/− mice. Mechanistically, we show that Sulfs are expressed in GDNF-producing Sertoli cells. In addition, reduced Sulf activity profoundly worsens haplo-deficient GDNF phenotypes in our genetic studies. These findings establish a critical role of Sulfs in promoting GDNF signaling and support a model in which Sulfs regulate the bioavailability of GDNF by enzymatically remodeling HS 6-O-desulfation to release GDNF from matrix sequestration. Further, Sertoli cell-specific transcriptional factor Wilm's tumor 1 (WT1) directly activates the transcription of both Sulf1 and Sulf2 genes. Together, our studies not only identify Sulfs as essential regulators of GDNF signaling in the SSC niche, but also as direct downstream targets of WT1, thus establishing a physiological role of WT1 in Sertoli cells.

Keywords: glial cell line-derived neurotrophic factor, heparan sulfate, sertoli cell, spermatogonial stem cell, Sulfs

Introduction

The extracellular heparan sulfate (HS) 6-O-endosulfatases (Sulfs), Sulf1 and Sulf2, are unique regulators of extracellular signaling (Ai et al. 2005). Sulfs specifically act on HS, a complex, negatively charged polysaccharide chain that plays diverse roles in embryogenesis, regeneration and tumor progression (Esko and Lindahl 2001; Nybakken and Perrimon 2002; Sasisekharan et al. 2002). The HS chain is composed of tandem repeats of uronic acid linked to glucosamine. During its biosynthesis in the Golgi, this disaccharide unit undergoes several modifications, including sulfation at the N-, 2-O- and 3-O- positions of uronic acid and the 6-O position of glucosamine (Esko and Lindahl 2001). These modifications allow HS to interact with a variety of extracellular signals and receptors. Both Sulf1 and Sulf2 selectively remove 6-O-sulfate groups from HS after biosynthesis, thereby differentially affecting multiple HS-dependent signaling pathways (Morimoto-Tomita et al. 2002; Ai et al. 2003, 2005). For those signaling pathways that require HS 6-O-sulfate groups to form functional signal–HS–receptor ternary complexes, such as the fibroblast growth factor (FGF) pathway (Schlessinger et al. 2000), Sulfs act as repressors (Lai et al. 2003, 2004; Wang et al. 2004). In contrast, for Wnts and glial cell line-derived neurotrophic factor (GDNF), Sulfs enzymatically reduce ligand binding to HS and increase their bioavailability, thereby promoting their signaling activities (Ai et al. 2003, 2007; Nawroth et al. 2007; Lemjabbar-Alaoui et al. 2010).

Sulf-enhanced GDNF signaling is essential for esophageal innervation (Ai et al. 2007). Sulf1 is co-expressed with GDNF by esophageal smooth muscle, whereas Sulf2 is expressed by innervating neural progenitors located within smooth muscle layers (Ai et al. 2007). Although the disruption of both Sulf genes has no effect on GDNF expression, over-6-O-sulfated HS in Sulf1−/−;Sulf2−/− embryos sequesters GDNF, leading to reduced GDNF signaling in innervating neurons. As a result, Sulf1−/−;Sulf2−/− mice have defective esophageal smooth muscle innervation and exhibit growth defects and partial postnatal death (Lamanna et al. 2006; Ai et al. 2007). Reduced neurite outgrowth from embryonic Sulf1−/−;Sulf2−/− esophagi can be fully rescued by excess GDNF, establishing that Sulfs play a quantitative role, rather than a qualitative role, in the GDNF pathway (Ai et al. 2007). Approximately 50% adult Sulf1−/−;Sulf2−/− mice survive into adulthood with reduced body weight and subfertility (Lamanna et al. 2006; Ai et al. 2007).

In addition to its essential role in enteric neurogenesis, GDNF is also required for postnatal spermatogenesis and self-renewal of spermatogonial stem cells (SSCs) (Meng et al. 2000; Naughton et al. 2006; Oatley and Brinster 2008). SSCs are a subset of spermatogonia that are located at the basal layer of the seminiferous tubules. The only other cell type in the basal layer that directly contacts SSCs is the supporting somatic Sertoli cells. Sertoli cells express GDNF that signals in a paracrine manner to SSCs. GDNF functions by binding to GDNF receptor GFRα1, and the GDNF–GFRα1 complex recruits tyrosine kinase receptor Ret for signaling (Baloh et al. 2000). During the first postnatal week, GDNF, GFRα1 and Ret are expressed at high levels to promote the proliferation of SSCs and maintain their undifferentiated state (Jain et al. 2004; Naughton et al. 2006). Reduced GDNF levels in GDNF+/− mice cause abnormal differentiation and accelerated depletion of SSCs, whereas the overexpression of GDNF leads to the accumulation of undifferentiated SSCs and the formation of seminomatous tumors (Meng et al. 2000, 2001). These findings establish that the level of GDNF is critically regulated in the testis. We speculated that Sulfs may play a role in regulating the GDNF signaling pathway in the testes to affect fertility because of abundant Sulf mRNA expression (Morimoto et al. 2002).

Wilm's tumor 1 (WT1) is a zinc finger protein that is persistently expressed in Sertoli cells in the adult and essential for spermatogenesis. Functions of WT1 are complex and dependent on alternative splicing and expression ratios of various splicing isoforms (Discenza and Pelletier 2004; Yang et al. 2007). Four major WT1 protein isoforms are produced after two major alternative splicing events that involve exon 5 and nine nucleotides encoding for lysine, threonine and serine (KTS) at the 3′ end of exon 9. These include two isoforms without KTS(−KTS) that bind to DNA and function as transcription factors and two WT1(+KTS) isoforms that are involved in RNA and protein interactions (Discenza and Pelletier 2004; Yang et al. 2007). Embryonic disruption of Wt1 leads to severe defects in urogenital development (Kreidberg et al. 1993; ,Hammes et al. 2001; Gao et al. 2006). WT1 is also required to maintain the SSCs in adult testes based on previous studies using Sertoli cell-specific Wt1 knockdown transgenic mice (Rao et al. 2006) and mice that overexpress a WT1(−KTS) isoform in a Wt1+/− background (Lahiri et al. 2007). Direct targets of WT1 in Sertoli cells that engage the paracrine nature of WT1 function in SSCs remain to be identified.

Here, we investigate the function and regulation of Sulf1 and Sulf2 in the adult testes. We demonstrate that Sulfs are required for GDNF-dependent maintenance of SSCs, thus providing a mechanism for the observed subfertility of Sulf1−/−;Sulf2−/− male mice. In addition, WT1 is identified as a direct transcriptional regulator of both Sulf1 and Sulf2 genes in Sertoli cells. Together, our results reveal a functional link between WT1, Sulfs and GDNF signaling regulation in the SSC niche.

Results

Sulf1 and Sulf2 are co-expressed by Sertoli cells, but differentially expressed by spermatogenic cells in adult testes

We analyzed the expression of Sulf1 and Sulf2 in adult testes by in situ hybridization, reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemistry. Sulf1 mRNA was detected in almost all spermatogenic cells except spermatogonia in the basal layer of seminiferous tubules (Figure 1A). A few cells in the basal layer also express Sulf1 mRNA and were confirmed to be Sertoli cells by RT–PCR using primary cultures of isolated Sertoli cells (Figure 1B) (Li et al. 2001) and immunostaining (Figure 1C). In contrast, Sulf2 mRNA was detected in all cells in the basal layer, including both Sertoli cells and SSCs, and a subset of spermatocytes and spermatids (Figure 1A). Consistently, Sulf-specific antibodies detected both Sulfs in Sertoli cells, which are distinguishable from spermatogonia by their distinct punctate heterochromatin staining by Hoechst dye and by the expression of WT1 (Figure 1C; Supplementary data, Figure S1) (Ai et al. 2007). However, only Sulf2 is expressed in all spermatogonia in the basal layer, including SSCs, which can be distinguished from Sertoli cells by their uniformly Hoechst-labeled round nuclei and by the expression of Ki67 and Oct4 (Figure 1C, Supplementary data, Figure S1). Although Sulf1 is not present in spermatogonia, it exhibits a broader expression in spermatocytes and spermatids than Sulf2 (Figure 1A and C). Interestingly, Sulf1 and Sulf2 are both localized in the acrosome of spermatids (Figure 1C). In summary, Sulf1 and Sulf2 exhibit partially overlapping expression in adult testes. In the basal layer of seminiferous tubules, where SSCs depend on GDNF secreted by neighboring Sertoli cells for self-renewal, both Sulfs are expressed by Sertoli cells, whereas only Sulf2 is expressed by SSCs.

Fig. 1.
Sulf1 and Sulf2 exhibit partially overlapping expression in the adult testes. (A) In situ hybridization detects Sulf1 and Sulf2 mRNA expression in seminiferous tubules of 3-month-old mice. Sulf1 mRNA is detected in all spermatocytes, but is largely excluded ...

Sulf1−/−;Sulf2−/− male mice exhibit accelerated depletion of SSCs

To test whether Sulf deficiency leads to testis phenotypes, cross-sections of testes from Sulf1+/−;Sulf2+/− control and Sulf1−/−;Sulf2−/− mice were compared by histology and immunohistochemistry. At postnatal day 10 (P10) and at 3 months of age, the seminiferous tubules of Sulf1−/−;Sulf2−/− mice were indistinguishable from controls, as shown by hematoxylin and eosin (H&E) staining, Ki67 immunolabeling of spermatogonia and a mosaic pattern of WT1+ Sertoli cells in the basal layer (Figure 2A). However, at 8 months, half of Sulf1−/−;Sulf2−/− male mice (7 of 12) exhibited significant percentages of atrophic seminiferous tubules (Figure 2). These atrophic tubules are characterized by a reduced diameter, lack of sperm in the lumen and selective loss of Ki67+ germ cells with remaining WT1+ Sertoli cells in the basal layer (Figure 2A). Sulf1−/−;Sulf2−/− and control mice have comparable levels of apoptosis in the testes at 4 months (Supplementary data, Figure S2). Thus, Sulf1−/−;Sulf2−/− mice are defective in the maintenance of SSCs, a testis phenotype similar to that of GDNF+/− mice (Meng et al. 2000; Naughton et al. 2006). Mice with only one Sulf wild-type allele also exhibit testicular atrophy at 8 months of age at a lower frequency than Sulf1−/−;Sulf2−/− mice (6 of 23, Figure 2B). We did not observe any significant difference in the frequency of testicular atrophy between Sulf1−/−;Sulf2+/− mice (2 of 11) and Sulf1+/−;Sulf2−/− mice (4 of 12), suggesting that Sulf1 and Sulf2 have redundant roles in the maintenance of SSCs.

Fig. 2.
Sulf mutant mice exhibit accelerated depletion of SSCs at 8 months. (A) Cross-sections of the testes from Sulf1+/−;Sulf2+/− and Sulf1−/−;Sulf2−/− mice at P10 and 3 and 8 months of age were stained with H&E ...

Sulfs promote GDNF signaling in the embryos and SSC niche

Our previous studies show that Sulfs reduce GDNF-HS binding by enzymatically removing 6-O-sulfate groups from HS chains, thereby releasing GDNF from esophageal smooth muscle to signal in enteric neurons (Ai et al. 2007). Here, Sulfs are expressed in GDNF-producing Sertoli cells (Figure 1) and may play a similar role in regulating GDNF signaling in the SSC niche. To test this hypothesis, we assessed the effect of Sulf deficiency on GDNF function when the GDNF level is reduced in GDNF+/− mice (,Meng et al. 2000; Gianino et al. 2003). After crossing Sulf mutant mice with GDNF+/− mice, we found that GDNF;Sulf compound mice exhibit postnatal death and testes atrophy more pronounced than those caused individually by GDNF haplodeficiency and Sulf deficiency combined. Among 117 pups from 14 crosses between Sulf1−/−;Sulf2+/− and GDNF+/−;Sulf1+/−;Sulf2+/− mice, no GDNF+/−;Sulf1−/−;Sulf2−/− pups survived up to postnatal day 21 (zero survived, predicted 7.3), compared with 100% survival of GDNF+/− pups and ~60% survival of Sulf1−/−;Sulf2−/− pups (five survived, predicted 7.3) (Table I) (Ai et al. 2007). The complete lethality of GDNF+/−;Sulf1−/−;Sulf2−/− mice is caused by defects in postnatal survival, not prenatal survival, since embryonic day 18.5 GDNF+/−;Sulf1−/−;Sulf2−/− embryos were identified (6 of 89 embryos, predicted 5.6) at almost equal frequency as Sulf1−/−;Sulf2−/− embryos (7 of 89, predicted 5.6) (Table I). We suspect that the esophageal innervation of GDNF+/−;Sulf1−/−;Sulf2−/− mice is affected to a greater extent than Sulf1−/−;Sulf2−/− and GDNF+/− mice. As suspected, although the number of enteric neurons in the esophagus is not affected by Sulf deficiency and is decreased to ~50% by GDNF haplodeficiency (Ai et al. 2007), it is reduced by more than 3-fold in GDNF+/−;Sulf1−/−;Sulf2−/− esophagi (Supplementary data, Figure S3). The resulting severe reduction of esophageal innervation in GDNF+/−;Sulf1−/−;Sulf2−/− embryos likely causes the complete postnatal death observed in these pups.

Table I.
Genetic crosses between Sulf mutant mice and GDNF+/− mice and Ret+/− mice

Testicular atrophy in mice with one Sulf wild-type allele is also profoundly accelerated in the GDNF+/− background. At 3 months, these Sulf mutant mice do not exhibit atrophy (Figures 2 and and3),3), and only 15% of GDNF+/−;Sulf1+/−;Sulf2+/− mice (4 of 25) showed low levels of testicular atrophy (Figure 3). However, 90% of GDNF+/−;Sulf1−/−;Sulf2+/− and GDNF+/−;Sulf1+/−;Sulf2−/− mice (10 of 11) developed testicular atrophy at 3 months (Figure 3), establishing a synergic effect of reduced Sulf activity and decreased GDNF level on the maintenance of SSCs.

Fig. 3.
Sulfs have a gene dose-dependent effect on testicular atrophy in GDNF+/− background, but not in Ret+/− background. Testicular atrophy was quantified at 3 months after crossing Sulf mutant mice with GDNF+/− mice and Ret+/− ...

To rule out the possibility that phenotypes of GDNF;Sulf compound mice were affected by strain (Sulf mutant mice were in a mixed 129/BL6 background), we crossed Sulf1−/−;Sulf2+/− mice with Ret+/− mice, which are in the same C57BL/6 background as GDNF+/− mice (,Sánchez et al. 1996; Enomoto et al. 2001). Ret+/− mice have no reported haplo-deficient phenotypes in the esophagi, and we did not observe any testicular atrophy in Ret+/−;Sulf1+/−;Sulf2+/− mice (Figure 3), indicating that Ret is not rate limiting. In addition, Ret does not directly bind to GDNF and functions independently of HS, and therefore Sulfs (Baloh et al. 2000; Parkash et al. 2008). We found that Ret+/− background has no effect on postnatal survival or testis phenotype in Sulf1−/−;Sulf2−/− mice (Table I, Figure 3). Collectively, these studies establish that Sulfs are required to promote the signaling activities of limited GDNF in vivo both in the embryo and in the adult.

Sulf1 and Sulf2 promoters contain a functional WT1 binding site

To fully understand the Sulf function in the SSC niche, we investigated the transcriptional regulation of Sulf genes in Sertoli cells. By searching conserved mammalian Sulf promoter sequences, we identified a single, conserved WT1-binding site at ~100 and ~125 bp upstream of transcription initiation sites of Sulf1 and Sulf2 genes, respectively (Figure 4A) (,Nakagama et al. 1995; Klattig et al. 2007).

Fig. 4.
WT1 binds to promoters of the Sulf1 and Sulf2 genes to regulate their transcription. (A) Alignment of promoter sequences of the Sulf1 and Sulf2 genes from mouse (M), rat (R) and human (H) identifies a conserved WT1-binding site (in red) at ~100 ...

To test whether WT1 controls Sulf gene expression, we first performed chromatin immunoprecipitation to assess WT1 binding to Sulf promoters using a mouse TM4 Sertoli cell line. The TM4 cell line expresses WT1 and Sulfs (Supplementary data, Figure S4). Both Sulf1 and Sulf2 promoter sequences were enriched in a pulldown by the WT1 antibody, compared with control immunoglobin G (IgG), as demonstrated by a greater abundance of PCR product in WT1-treated samples (Ab) than IgG-treated samples (IgG) using two different primer sets (A and B) for each promoter (Figure 4B). Quantification by real-time PCR showed that the WT1 antibody enriched Sulf1 promoter by 4.2+/−0.9-fold and Sulf2 promoter by 3.1+/−0.7-fold. Therefore, WT1 binds to both Sulf1 and Sulf2 promoters.

Second, we tested the activities of candidate WT1-binding sites in transcriptional activities Sulf promoters. Wild-type Sulf promoters, including sequences from positions −156 to +10 of Sulf1 and −250 to + 156 of Sulf2, were cloned into a pGL3-firefly luciferase vector. Candidate WT1-binding site was disrupted by converting essential G/C nucleotides into A/T (Figure 4C). The transcriptional activities of wild-type and mutated Sulf promoters were compared by a dual luciferase assay after transfecting TM4 cells with the Sulf–firefly luciferase construct, a TK–renilla luciferase construct (as a transfection control) and an expression vector of green fluorescent protein (GFP) or WT1(−KTS). Transfection of the GFP expression vector serves to test the trans-activities of endogenous WT1. We found that the Sulf1 promoter was activated 6-fold above the basal level by overexpressed WT1(−KTS), and the wild-type WT1-binding site is required for this activation (Figure 4D). In contrast, the Sulf2 promoter was activated ~3-fold above the basal level through the WT1-binding site in GFP-transfected cells, indicating that endogenous WT1 is sufficient to activate the Sulf2 promoter, although this activity was diminished by overexpressed WT1(−KTS) (Figure 4D). Further, WT1(+KTS), a predominant RNA-binding form, had no effect on either the Sulf1 promoter or the Sulf2 promoter (Figure 4D). Therefore, WT1 directly regulates Sulf1 and Sulf2 gene transcription.

After establishing a direct WT1 regulation of Sulf expression in a Sertoli cell line, we investigated the physiological roles of WT1 in the expression of Sulfs in the SSC niche. Wt1+/− mice express approximately 50% of the wild-type level of WT1 isoforms (Supplementary data, Figure S5), thus providing an animal model to assess the dose-dependent transcriptional activities of WT1 on Sulf promoters. Both Sulf1 and Sulf2 mRNA levels were found to be reduced by ~3-fold and ~2-fold, respectively, in primary Wt1+/− Sertoli cells, compared with wild-type Sertoli cells (Figure 4E). Collectively, WT1 acts as an upstream activator of Sulf gene expression in Sertoli cells.

Discussion

Our study investigates the function and gene regulation of Sulfs in the SSC niche. We demonstrate that Sulfs are required for the maintenance of SSCs, a process known to be critically dependent upon the dose of GDNF produced by neighboring Sertoli cells (Meng et al. 2000; Naughton et al. 2006; Oatley and Brinster 2008). Genetic studies of Sulf mutant mice crossed with GDNF+/− mice show that loss and reduction of Sulf activities synergize with GDNF haplo-insufficiency to profoundly impact GDNF-dependent postnatal survival, esophageal innervation and maintenance of SSCs, thus establishing a critical role of Sulfs in promoting the signaling activities of GDNF. In addition, WT1 is shown to be a direct transcriptional activator of Sulf1 and Sulf2 genes in Sertoli cells. This finding not only identifies a physiological role for WT1 in adult testis, but also provides a mechanism underlying the paracrine nature of WT1 function. In our model (Figure 4F), WT1 activates the expression of Sulf1 and Sulf2 in Sertoli cells. Sulfs enzymatically remove HS 6-O-sulfate groups from heparan sulfate proteoglycans in the extracellular matrix of Sertoli cells and neighboring SSCs by secretion, whereby GDNF sequestration by the matrix is reduced (,Morimoto-Tomita et al. 2002; Ai et al. 2007). As a result, Sulfs promote GDNF interaction with GDNF receptor for signaling in SSCs. This, together with additional WT1-regualted factors (Rao et al. 2006), functions to maintain SSCs in adult testis (Figure 4F). Loss of Sulf activity leads to over-6-O-sulfated HS that sequesters GDNF in the extracellular matrix, resulting in reduced GDNF bioavailability and accelerated depletion of SSCs.

Sulfs quantitatively regulate GDNF signaling activity rather than functioning as essential components of the GDNF pathway. Disruption of two, three and all four Sulf wild-type alleles revealed a Sulf gene dosage effect on testicular atrophy in the adult, as well as esophageal hypo-innervation in the embryos. Both Sulf1+/−;Sulf2+/− mice and Sulf single mutant mice show marginal changes in HS 6-O-sulfation in tissues where both Sulf genes are expressed, and they do not exhibit any testis or esophageal phenotypes (Figure 2) (Ai et al. 2007), whereas mice with only one wild-type allele exhibit phenotypes at low frequencies, indicating that two Sulf wild-type alleles are sufficient and required for GDNF activity. We did not observe any significant difference in the penetration of testis and esophageal phenotypes between Sulf1−/−;Sulf2+/− mice and Sulf1+/−;Sulf2−/− mice, further supporting the functional redundancy of Sulf1 and Sulf2 in remodeling HS 6-O-desulfation in the extracellular matrix (,Morimoto-Tomita et al. 2002; Ai et al. 2006).

In addition to the role of Sulfs in Sertoli cells, we also detected the presence of both Sulf enzymes in the acrosome of spermatids. It remains to be tested whether Sulfs are released from acrosome and function with digestive enzymes to facilitate the breakdown of the zona pellucida during fertilization. If true, Sulfs possess the capacities of HS degradation, and observed subfertility in Sulf1−/−;Sulf2−/− males may be due to the combined defects in the maintenance of SSCs and in HS degradation during fertilization.

The identification of functional WT1-binding sequences within Sulf promoters provides molecular mechanisms underlying WT1-dependent Sulf expression in both Sertoli cells and podocytes (Hartwig et al. 2010; Ratelade et al. 2010). In primary Sertoli cells, WT1 is critically required to activate both Sulf1 and Sulf2 expression (Figure 4E). In addition to the WT1-binding site identified in our study, WT1 may also regulate Sulf2 expression in vivo via a second putative binding site downstream of the transcription initiation site of Sulf2 (Hartwig et al. 2010). Consistently, in a Sertoli cell line, the Sulf1 promoter is activated by overexpressed WT1, and the basal Sulf2 promoter activity requires the WT1-binding site. However, the overexpression of WT1 in this Sertoli cell line appears to diminish the basal transcriptional activity of the Sulf2 promoter. One possibility is that WT1, when expressed at high levels, recruits low-affinity inhibitory cofactors that bind to the Sulf2 promoter and block transcription. Alternatively, overexpressed WT1 activates the expression of inhibitory transcriptional cofactors that selectively bind to the Sulf2 promoter as a feedback mechanism. The mechanism underlying the WT1 regulation of Sulf2 expression is likely complex and requires further investigation.

Our study in the testes has identified the essential physiological roles of Sulfs in the maintenance of adult tissue function. In addition, it reveals important physiological functions of WT1 in Sulf expression regulation. Although WT1 was initially identified as a tumor suppressor in kidney tumors, it has been found to be expressed by a number of tumors and has oncogenic roles in lung cancer, breast cancer, ovarian cancer, head and neck squamous carcinoma and pancreatic cancer (Yang et al. 2007). Interestingly, these tumors also exhibit deregulated Sulf1 or Sulf2 expression, and the downregulation of Sulf expression promotes FGF2/hepatocyte growth factor-dependent tumor growth, whereas the upregulation of Sulf expression enhances Wnt-dependent tumor growth (,Lai et al. 2003, 2004; Dai et al. 2005; Li et al. 2005; Nawroth et al. 2007; Lemjabbar-Alaoui et al. 2010). It remains to be tested whether WT1 in a dose-dependent manner regulates Sulf gene expression in different tumors and whether the level of WT1 serves as a biomarker for the prediction of growth factor dependency during tumor progression.

Materials and methods

Mice

Sulf single- and double-mutant mice were backcrossed for six generations into C57BL/6 background (Ai et al. 2007). GDNF+/− mice and Ret+/− mice in C57BL/6 background (,Sánchez et al. 1996; Enomoto et al. 2001; Gianino et al. 2003) were crossed with mice with one Sulf wild-type allele to generate GDNF+/−;Sulf and Ret+/−;Sulf compound mutant mice. Wt1+/− mice were at the second backcross into FVB background from 129 background. All animal studies were approved by the Institutional Animal Care and Use Committee at BUMC.

Histology, immunohistology and in situ hybridization

Testes from adult mice were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) at 4°C overnight. After cryoprotection in 30% sucrose/PBS, 10 μm sections were collected. For general morphology, sections were washed in PBS and stained with H&E. For immunohistochemistry, sections were blocked for 1 h in an antibody dilution buffer (3% bovine serum albumin, 10% goat serum, 2% horse serum and 0.1% Triton X-100 in PBS) and a primary antibody applied for 2 h at room temperature or overnight at 4°C. Primary antibodies used include: rabbit anti-WT1 (1:100, Santa Cruz Biotechnologies), rabbit anti-Sulf1 and Sulf2 (1:100) (Ai et al. 2007), rabbit anti-Ki67 (1:100, Abcam). Antigen–antibody complexes were detected using fluorescence-conjugated secondary antibodies (Molecular Probes) and examined with a fluorescence microscope (Leica DMR). For in situ hybridization, riboprobes of Sulf1 and Sulf2 were generated by in vitro transcription with digoxigenin-UTP using the DIG RNA labeling mix (Roche). Cryosections (15 μm) of testes were processed for in situ hybridization as described previously (Ai et al. 2007). Images were taken using a digital camera (Leica DC300F).

Quantitative RT–PCR

Seminiferous tubules were collected from the testes of 3-month-old mice. Primary Sertoli cells were isolated and cultured at ~104 cells per well in a six-well plate (Li et al. 2001). After 3 days in culture, the total RNA was extracted from cultures using RNeasy Mini Kit (Qiagen). RNA was reverse-transcribed using Superscript III (Invitrogen) followed by real-time PCR using an ABI 7000 machine (Applied Biosystems). PCR reactions were performed with 50 μL volume using Power Sybergreen Master Mix (Applied Biosystems). The relative level of mRNA expression of genes of interest was calculated by normalizing to β-actin using ΔCt. Primers for Sulf1 and Sulf2 were described previously (Langsdorf et al. 2007), and their sequences follow:

  • Sulf1 forward: 1756: 5′-GCTGCTGGTGACATCAGGAATG-3′;
  • Sulf1 reverse: 2079: 5′-AAGGGGTGAAGGTGACTCTTTAGC-3′;
  • Sulf2#2 forward: 1062: 5′-TCTGAACCCCCACATTGTCCTC-3′;
  • Sulf2#2 reverse: 1263: 5′-CACTTTGTCACCCTCCCTCTTG-3′.

Sertoli cell culture and chromatin-immuno precipitation

Murine Sertoli cell line TM4 (ATCC) was grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics (Invitrogen). Approximately 5 × 106 TM4 cells were treated for 1 h in 1% PFA/PBS at room temperature to cross-link chromatin and associated proteins. Sonication was used to break the DNA into 400–500 bp fragments, and the supernatant was collected by centrifugation. One-fifth of the supernatant (100 μL) was then incubated with 5 μg of the WT1 antibody (1:100, Santa Cruz Biotechnologies) or 5 μg of rabbit IgG isotype control in a final volume of 300 μL overnight at 4°C. Complexes were pulled down using protein A/G beads (Fisher). The DNA fragments were released by protease K treatment of the pulldown followed by incubation at 65°C overnight to reverse the cross-link. After purification, the DNA was used in PCRs using two different sets of primers located within Sulf promoters. Sulf1A primers and Sulf2B primers were used in real-time PCR to quantify the abundance of Sulf promoter sequences in the WT1 antibody pulldown relative to an IgG pulldown. Primer sequences for chromatin-IP are as follows:

  • Sulf1A forward: 5′-TGCTCCTCCTCTTCTTGGAA-3′;
  • Sulf1A reverse: 5′-GATAAAACTGCCCGACCTGA-3′;
  • Sulf1B forward: 5′-ATACTCCTATTGGGTTGCCTGTTG-3′;
  • Sulf1B reverse: 5′-CTTCCTTCCTCCTCCTGAATGG-3′;
  • Sulf2A forward: 5′-TTGGAAGACTTTCAGGGGTTACG-3′;
  • Sulf2A reverse: 5′-ACGCACACACTTGCCATTGG-3′;
  • Sulf2B forward: 5′-CGGCTCTGTCTTGATTTCAGTATC-3′;
  • Sulf2B reverse: 5′-TGACTCCAGGTGGTGCTGTC-3′.

Constructs, mutagenesis and dual luciferase assay

Wild-type Sulf1 and Sulf2 promoter sequences were cloned into pTEasy vector (Promega) by PCR. The predicted WT1-binding sites were mutated by converting G/C into A/T by PCR. After sequencing, both wild-type and mutated Sulf promoter sequences were inserted into pGL3-Basic vector (Promega) to generate pSulf–firefly luciferase constructs. To measure promoter activities, TM4 Sertoli cells in 24-well culture plates were transfected with two luciferase reporter constructs and an expression vector as follows: 100 ng of pSulf–firefly luciferase under the transcriptional control of either Sulf1 or Sulf2 promoter; 10 ng of pTK–renilla luciferase to control for transfection efficiency and 200 ng of the expression vector for either Wt1 or GFP control. Cell transfection was performed using the Fugene 6 reagent (Roche). After 48 h, cells were lyzed and subjected to dual luciferase assay using Gluomax Multi Detection System (Promega). The transcriptional activities of cloned promoters were quantified by dividing firefly luciferase activity by renilla luciferase activity. Experiments were repeated three times with individual samples in triplicates.

Funding

This work was supported by BUMC startup funds and a grant from National Institute of Aging (1R01AG034939) to X.A.

Acknowledgements

We thank Carrie Ng for assistance with Wt+/− mice and Drs. Cardoso and Kotton at BUMC for comments on the manuscript.

Conflict of interest statement

None declared.

Abbreviations

GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; H&E, hematoxylin and eosin; HS, heparan sulfate; IgG, immunoglobin G; IP, immunoprecipitation; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RT–PCR, reverse transcriptase–polymerase chain reaction; SSC, spermatogonial stem cell; Sulf, heparan sulfate 6-O-endosulfatase; WT1, Wilm's tumor 1.

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