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Keywords:

  • microarrays;
  • Sertoli cells;
  • Sox8;
  • Sox9;
  • spermatogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

The SOX8 and SOX9 transcription factors are involved in, among others, sex differentiation, male gonad development and adult maintenance of spermatogenesis. Sox8−/− mice lacking Sox9 in Sertoli cells fail to form testis cords and cannot establish spermatogenesis. Although genetic and histological data show an important role for these transcription factors in regulating spermatogenesis, it is not clear which genes depend upon them at a genome-wide level. To identify transcripts that respond to the absence of Sox8 in all cells and Sox9 in Sertoli cells we measured mRNA concentrations in testicular samples from mice at 0, 6 and 18 days post-partum. In total, 621 and 629 transcripts were found at decreased or increased levels, respectively, at different time points in the mutant as compared to the control samples. These mRNAs were categorized as preferentially expressed in Sertoli cells or germ cells using data obtained with male and female gonad samples and enriched testicular cell populations. Five candidate genes were validated at the protein level. Furthermore, we identified putative direct SOX8 and SOX9 target genes by integrating predicted SOX-binding sites present in potential regulatory regions upstream of the transcription start site. Finally, we used protein network data to gain insight into the effects on regulatory interactions that occur when Sox8 and Sox9 are absent in developing Sertoli cells. The integration of testicular samples with enriched Sertoli cells, germ cells and female gonads enabled us to broadly distinguish transcripts directly affected in Sertoli cells from others that respond to secondary events in testicular cell types. Thus, combined RNA profiling signals, motif predictions and network data identified putative SOX8/SOX9 target genes in Sertoli cells and yielded insight into regulatory interactions that depend upon these transcription factors. In addition, our results will facilitate the interpretation of genome-wide in vivo SOX8 and SOX9 DNA binding data.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

High-mobility-group (HMG) transcription factors that bind to the A/TACAAT/A motif control sex determination in mammals. The process is initiated in the mouse by the expression of Sex-determining region Y (Sry) in a cell population destined to become testicular Sertoli nurse cells. These specialized cells are essential for testicular architecture, germ cell development and fertility (Jegou, 1993; Griswold, 1998; Maclean & Wilkinson, 2005; Petersen & Soder, 2006). Sry's sole known function is to transcriptionally activate Sex-determining region Y box 9 (Sox9), encoding one of 20 Sox proteins known in human and mice to date (Schepers et al., 2002). Sox9 activation in the gonad is initiated by Sry and Steroidogenic Factor 1 (Sf1) and maintained by Sf1 in cooperation with SOX9 protein itself (Foster et al., 1994; Harley et al., 2003; Wissmuller et al., 2006). Genetic evidence from human patients diagnosed with campomelic dysplasia, a skeletal malformation syndrome that includes male-to-female sex reversal (Foster et al., 1994; Wagner et al., 1994), and mouse Sertoli cell-type specific deletion experiments (Chaboissier et al., 2004; Barrionuevo et al., 2006) marked out Sox9 as a key factor for Sertoli cell differentiation and sex determination. More recent work focusing on Sox9's role in Sertoli cells after foetal sex determination revealed an unexpected role in adult testicular maintenance and fertility, but the molecular mechanism of this late function remains elusive (Barrionuevo et al., 2009).

SOX8, SOX9 and SOX10 were classified as SoxE proteins because they share highly conserved HMG and C-terminal transactivation domains and their genomic loci are organized similarly (Schepers et al., 2002). Moreover, Sox8 shows a broadly overlapping pattern of foetal and adult testicular expression with Sox9 (Schepers et al., 2000; Takada & Koopman, 2003) and it was postulated to partially compensate for Sox9 functions during sexual development (Schepers et al., 2003; Chaboissier et al., 2004). Initially, no essential role in male gonad development was observed for Sox8 (Sock et al., 2001), but subsequent work in a different strain background revealed late-onset infertility (O'Bryan et al., 2008) reminiscent of the phenotype found when Sox9 was deleted in Sertoli cells after the onset of sex differentiation (Barrionuevo et al., 2009). Mutant Sox8 mice lacking Sox9 in Sertoli cells prior to the sex determination stage fail to form testis cords and are unable to establish the first wave of spermatogenesis (Barrionuevo et al., 2009). These results imply that Sox8 and Sox9 are redundant during foetal sex determination and post-natal establishment of spermatogenesis, but not during maintenance of adult spermatogenesis and fertility. Recent work reported altered gene expression in adult testicular samples from heterozygous vs. homozygous Sox8 mice (Singh et al., 2009), and an intermediate expression signature as compared to normal males and females for embryonic testes at E13.5 in mice lacking Sox9 in Sertoli cells during early gonadogenesis (Lavery et al., 2011).

High-density oligonucleotide microarrays (GeneChips) covering mammalian genes yield robust signals that are comparable between laboratories and across species which allows for large-scale integration of novel data with published information available via certified repositories (Irizarry et al., 2005). Consequently, microarrays have been employed to gain insight into the rodent and human testicular expression programs yielding data on both somatic and germ cells (Schultz et al., 2003; Schlecht et al., 2004; Shima et al., 2004; Chalmel et al., 2007b, 2012). The challenging task of interpreting microarray data has been facilitated by functional annotation of genes using controlled vocabularies (ontologies) (Consortium, 2008). Furthermore, integrating expression profiling data with predictions of evolutionarily conserved transcription factor binding sites [reviewed in (Chalmel et al., 2007a; Hannenhalli, 2008)] as well as regulatory-, and protein-network data is feasible (Chatr-aryamontri et al., 2007; Kerrien et al., 2007; Breitkreutz et al., 2008).

We report a whole-genome mRNA profiling analysis of post-natal testes from Sox8−/− mice harbouring a Sertoli-cell specific Sox9 deletion from E13.5 onwards. Global transcript concentrations were measured in newborn mice (0 days post-partum, dpp) and compared to mice at the age of 6 and 18 dpp covering developmental stages prior to or at the onset of spermatogenesis. We included total testes and ovary samples, enriched Sertoli cells and germ cells at mitotic, meiotic and post-meiotic stages as controls to help determine the predominant cellular origin of target transcripts. Finally, we integrated expression data with information on predicted DNA binding motifs and protein network data to establish a comprehensive picture of the regulatory processes that depend on testicular expression of Sox8 and Sox9 during early post-natal male gonad development.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

Mouse strains

To obtain homozygous Sox8 mutants bearing Sox9 flanked by Cre recombinase binding sites (flox) for inactivation in Sertoli cells, females harbouring Cre under the control of the Anti-Müllerian Hormone (AMH) gene's promoter and a floxed allele of Sox9 (AMH-Cre;Sox9flox/flox) were crossed with Sox8−/− males of a mixed inbred line consisting of two widely used genetic backgrounds termed C57BL/6 and 129/Sv (Sock et al., 2001). This procedure yielded mice with the desired AMH-Cre;Sox9flox/flox;Sox8−/− genotype. To mark mutant Sertoli cells a Cre reporter allele encoding R26E-EYFP, a version of the Enhanced Yellow Fluorescent Protein kindly supplied by F. Constantini (Srinivas et al., 2001), was crossed in to obtain AMH-Cre;Sox9flox/flox;Sox8−/− EYFP/+ testes. Genotyping was carried out using the Polymerase Chain Reaction (PCR) as described in reference (Barrionuevo et al., 2009).

Testicular sample preparation

Total testis samples were prepared from control and AMH-Cre; Sox9flox/flox;Sox8−/− double mutant animals at 0, 6 and 18 days post-partum (dpp). Testes of at least three animals for each time points were decapsulated and combined into duplicate pools using standard laboratory practice.

Target synthesis, GeneChip hybridization and raw data production

Total RNA preparation, cRNA target synthesis and raw data production using MG430 2.0 GeneChips (Affymetrix, Santa Clara, CA, USA) were essentially executed as previously published (Chalmel et al., 2007b).

GeneChip data analysis

Data analysis was carried out using the Annotation, Mapping, Expression and Network (AMEN) analysis software (Chalmel & Primig, 2008). Probe sets yielding a signal higher than the detection threshold (median of the normalized dataset, cutoff 5.3) and a fold-change ≥2.0 between Sox8/Sox9 mutant testes and control samples at 0, 6 and 18 dpp were selected. A Linear Model for Microarray Data (LIMMA) statistical test (F-value adjusted with the False Discovery Rate method: ≤ 0.01) was employed to identify significantly differentially expressed probe sets. For further classification we divided the differentially expressed probe-sets into groups being significantly decreased or increased in Sox8/Sox9 mutant testes testis either at 0, 6 dpp or both as compared to wild-type samples. Five probe-sets were identified that showed opposite patterns at 0 and 6 dpp.

Minimum information about a microarray experiment compliance

Raw data CEL files (containing information at the GeneChip probe cell level) corresponding to total-testis samples are available via the European Bioinformatics Institute's ArrayExpress public repository (Parkinson et al., 2009). E-TABM-531 corresponds to 12 duplicate samples collected at 0, 6 and 18 dpp from C57BL/6 control and Sox8/Sox9 mutant mice respectively. Normalized data for all loci represented on the GeneChip are available for viewing at www.germonline.org/ (Gattiker et al., 2007).

Conserved regulatory motif prediction and protein network analysis

The prediction of transcription factor binding sites conserved across species was performed using the Promoter Analysis Protocol with the same parameters as previously described (Lardenois et al., 2010). Binding site enrichment was estimated on the minSUM_good profile from the TRANSFAC Professional database release 2010.1 (Matys et al., 2006). The protein interaction data were integrated with RNA profiling and phenotype information using an approach described (Matys et al., 2006).

Immunohistochemistry and immunofluorescence analysis

We collected mouse gonads in 1× PBS (phosphate buffered saline) and fixed them in FEAA (37% formaldehyde:ethanol:acetic acid, 3:6:1) before embedding them in paraffin. Histological sections were 7 μm thick. Alternatively, a number of gonads were fixed in PFS (4% paraformaldehyde in PBS buffer) and cryosectioned at 6 μm. Immunostaining was performed using the standard protocol for Peroxidase Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) and samples were counterstained with haematoxylin as previously described (Barrionuevo et al., 2009).

Goat polyclonal antibodies against DHH (1:50, sc-1193; Santa Cruz Biotechnology, Santa Cruz, CA, USA), FST (1:50, AF669; R&D Systems, Minneapolis, MN, USA) and GFP (1:400, NB100-1770; Novus Biologicals, Littleton, CO, USA), as well as rabbit polyclonal antibodies against GDNF (1:400, ab18956; Abcam, Cambridge, UK), FOXL2 [1:600, provided by R. Veitia (Cocquet et al., 2005)] and PTGDS (1:500, kindly provided by F. Poulat [Moniot et al., 2009)] were employed. GDNF and PTGDS immunofluorescence signals were revealed with an anti-rabbit antibody conjugated with FITC (1:200, F0382; Sigma, St. Louis, MO, USA); for FOXL2 we used a donkey anti-rabbit FITC-conjugated reagent (1:200, ab6798; Abcam). GFP was revealed using a donkey anti-goat antibody conjugated with Texas-Red (1:200, ab6883; Abcam). Images were captured using a Zeiss CCD camera at the default settings. Fluorescent signals were recorded with a Zeiss Axioplan 2 fluorescence microscope equipped with the ISIS imaging system and appropriate fluorescent filter sets (MetaSystems, Altlussheim, Baden-Wuerttemberg, Germany). Sections of control and mutant gonads were always mounted on the same slide. At least three pairs of control and mutant gonads were analysed for each time point.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

Sample description, experimental design and quality control

In this report, Sox8−/− mice lacking Sox9 in Sertoli cells after Cre induction at E13.5 (AMH-Cre; Sox9flox/flox; Sox8−/−) are referred to as Sox8/Sox9 mutant whereas Sox9flox/flox; Sox8+/+ mice that are phenotypically normal are used as controls. The sample choice was guided by previous results showing that mutant testes at 0 dpp contain fewer albeit morphologically normal cords than the controls, whereas at 6 dpp a clear effect on cord structure becomes apparent. This phenotype is exacerbated at 18 dpp where pathologically small testes contain only a few remaining abnormal cords whose meiotic germ cell population undergoes widespread apoptosis [(Barrionuevo et al., 2009); Fig. S1 A–C].

Expression profiling complex organs such as male wild-type and mutant testes which consist of multiple somatic and germ cell types at different stages of growth and development yields data that are difficult to interpret because both transcriptional effects and changing cell ratios are at the origin of distinct transcript concentrations. However, standard cell purification procedures, although in principle suitable for profiling studies (Wrobel & Primig, 2005), likely trigger rapid transcriptional responses which may mask the effect caused by deleting the Sox8 and Sox9 regulatory proteins. Because of this concern and practical issues such as sample size and cost effectiveness and given that mutant testes displayed a clear phenotype at 6 dpp (Barrionuevo et al., 2009) we analysed the mRNA profiles of duplicate total testicular samples using Mouse Genome 240 2.0 GeneChips at 0, 6 and 18 dpp.

Total RNAs and pooled cRNAs appeared homogenous across all samples analyzed (Fig. S2 A and B). Plotting data on probe intensities revealed no hybridization artefacts (C), and showed normal 5′-3′ probe set intensities (which is a measure for mRNA length; D) as well as typical signal intensity distributions (E). Comparing samples based on their overall expression signals using a distance matrix together with a dendrogram yielded the expected results: replicates were juxtaposed, the control sample and the mutant showing the most severe phenotype at 18 dpp were set apart whereas the 0 dpp/6 dpp controls and mutant samples were grouped together respectively (F).

The global testicular RNA signature of early postnatal Sox8/Sox9 mutant mice

We next sought to identify genes for which reproducible signal differences were obtained at 0 and 6 dpp and, as a control for the onset of spermatogenesis, 18 dpp. First, we selected loci showing a statistically significant change between control and Sox8/Sox9 mutant samples (see 'Materials and methods'). Finally, the resulting genes were grouped into two clusters showing increased or decreased signals in the mutant testis as compared to the control sample [Fig. 1(A)]. Among 506 genes displaying differential transcript concentrations at 0 dpp 202 were scored as showing significantly increased-, and 304 as showing decreased mRNA levels. As expected, we identified a larger number of transcripts at 6 dpp where the phenotype is more severe: among 1014 genes we found 539 to be increased, and 475 to be decreased in the mutant samples. For 72 (C1) and 422 genes (C3) we found signals to be increased only at 0 or 6 dpp, respectively, whereas for 135 genes (C2) signals were stronger in both 0 and 6 dpp samples [Fig. 1(B)]. Likewise, mRNA concentrations for 133 (C4) and 313 genes (C6) were decreased only at 0 or 6 dpp, respectively, whereas 175 (C5) displayed weaker signals at both 0 and 6 dpp [Fig. 2(B)]. Finally, Actn3 and Ccl17 (C7) yielded weaker signals at 0 dpp, but stronger ones at 6 dpp. As expected, the largest differences were found at 18 dpp where mutant mice showed phenotypes severely affecting most, if not all testicular cell types: 4 468 loci showed distinct mRNA concentrations including 2 286 (C8) that were stronger, and 2 182 (C9) that were weaker [Fig. 1(A)], see also filtering options in Data S1.)

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Figure 1. Gene filtration and gene categorization procedure. (A) A flow chart outlines the procedure we employed to filter and cluster genes that display statistically significant signal changes when controls and mutant samples at 0, 6 or 18 dpp were compared. The numbers of genes and probe-sets are given. (B) A colour-coded Venn diagram summarizes the distribution of genes (represented as Entrez Gene IDs) into categories where mRNA concentrations are increased (red) and decreased (blue) at the 0 and 6 dpp time points in the Sox8/Sox9 mutant vs. the wild-type control. The genes are organized as classes C1–C9. Note that the numbers of increased and decreased transcripts in the Venn diagram do not precisely add up with the numbers indicated in (A) because several probe-sets can belong to one gene and genes can therefore be in different classes depending on the signals obtained with their probe-sets.

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Figure 2. Gene clustering and GO term analysis. (A) A false-colour heat map provides an overview of increased or decreased mRNA signal intensities obtained with replicate control (Sox9flox/flox;Sox8+/+) and mutant (AMH-Cre; Sox9flox/flox;Sox8−/−) total testis samples at the time points given at the bottom. The testicular samples are compared to wild-type enriched testicular cell types as indicated and two external wild-type controls (Sox8+/+; Sox9+/+; Testis, Ovary). Genes are grouped into classes (C1–C9) according to their pattern as defined in Fig. 1 and grouped within each class by hierarchical clustering. A colour scale indicates log2 transformed signal values. (B) Significantly enriched GO terms and their identification numbers are given followed by the total number of genes associated with the term and the numbers of genes observed vs. expected by chance for the increased (clusters C1–C3) and decreased (clusters C4–C6) classes. The total number of genes associated with a biological process term and the genes in the classes are shown at the top. A colour scale of p-values for enriched (red) and depleted (blue) terms is shown at the bottom.

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We subsequently combined the data set obtained with testis samples from control and mutant mice with expression data from purified Sertoli cells, spermatogonia, spermatocytes and spermatids (Chalmel et al., 2007b) as well as total testis and ovary samples (the last two were obtained from the NCBI's Gene Expression Omnibus repository) to determine which cell types predominantly expressed fluctuating transcripts. Pair-wise hierarchical clustering was employed to organize genes within each of the nine classes as defined in [Fig. 2(A) C1–C9]. The outcome of this analysis yielded two broad conclusions: first, genes for which transcripts display increased or decreased signals at 0 and 6 dpp typically show peak expression in enriched Sertoli cells and spermatogonia [Fig. 2(A), classes C1–C7]. Second, transcripts that increase in the mutant at 18 dpp are predominantly expressed in enriched Sertoli cells and spermatogonia whereas those that decrease typically peak in spermatocytes (including many transcripts that appear to be present also in spermatids) [Fig. 2(A), classes C8–C9]. We note that the signals which increase at 18 dpp do so only moderately whereas the signal decrease observed is substantial. Our interpretation is that stronger signals are likely ascribable to proportional Sertoli cell enrichment whereas weaker ones are referable to a failure to build up germ cell populations in the mutant testicular tissue.

Differential mRNA profiles correlate with biological processes known to require Sox8 and Sox9

We then analysed the functional relevance of expression signals changing at 0 and 6 dpp by searching for enriched Gene Ontology (GO) terms (using the Biological Process ontology) among genes showing increased or decreased signals (genes associated with GO terms are available via the filtering options in Data S1, Fig. 2(B). Among genes showing stronger signals we found enrichment for kidney development (GO:0001822; 16 observed/4 expected by chance, p-value 2.4 × 10−5) and prostate gland development (GO:0030850; 6/1, 2.5 × 10−3), which is interesting in the context of abnormal renal functions in a patient with mutated Sox9 (Cost et al., 2009) and Sox9's role in prostate cancer (Thomsen et al., 2010). Other GO terms enriched in that category reflect the known induction in Sox8/Sox9 mutant testes of genes involved in female gonad development (GO:0008585; 9/2, 4.0 × 10−4) and female sex differentiation (GO:0046660; 11/2, 6.9 × 10−5) (Chaboissier et al., 2004; Barrionuevo et al., 2009). Importantly, we found functions related to the progressive degeneration of testicular tissue in the mutant that triggers a response to stress (GO:0006950; 66/41, 7.7 × 10−4) and signaling pathway (0023033; 73/39, 1.9 × 10−6) and that perturbs cell proliferation (GO:0008283; 25/9, 2.5 × 10−5) and cell migration (GO:0016477; 18/8, 6.0 × 10−3) processes. Furthermore, we identified GO terms related to hormone signalling including lipid-, (GO:0006629; 36/21, 8.2 × 10−3), steroid-, (GO:0008202; 21/5, 3.6 × 10−7) and hormone metabolic process (GO:0042445; 18/3, 5.3 × 10−9), and regulation of hormone levels (GO:0010817; 20/4, 7.5 × 10−8).

Furthermore, the category showing decreased signals is enriched for genes bearing reproduction (GO:0000003; 56/19, 5.7 × 10−12), male sex differentiation (GO:0046661; 8/2, 4.0 × 10−3), male gonad development (GO:0008584; 8/1, 4.0 × 10−4) as well as meiosis (GO:0007126; 13/3, 1.5 × 10−5) and spermatid differentiation (GO:0048515; 9/2, 4.9 × 10−4) [Fig. 3(B) and Data S1.]. This is consistent with spermatogenesis not being able to become established normally in the mutant testes (Fig. S1 A–C).

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Figure 3. Detection of ovary-specific markers in mutant testes. (A) Shown are immunohistochemical (IHC) cytoplasmic staining patterns (brown) obtained with an anti-Fst antibody on an ovarian control sample (XX, left part), and testicular sections (XY, middle and right part) from three different genetic backgrounds as indicated on top. Scale bar = 20 μm. (B) Immunofluorescence signals (green) obtained with an anti-FOXL2 antibody are given for ovary and testis samples as in (A). DAPI was used to stain DNA (blue) and Sertoli cells are marked in the mutant by EYFP (red) to confirm FOXL2 accumulation in the mutant. Samples were isolated from newborn mice. Scale bar = 20 μm.

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Transcript fluctuations detected by microarray analysis of testicular samples correlate with changing protein levels within gonads

Among the genes displaying a strong and early signal increase we found Fst encoding Follistatin. The protein exists in different isoforms including one that is important for follicle formation and female fertility (Lin et al., 2008; Kimura et al., 2010a,2010b). Our microarray-based observation is in keeping with the presence of Fst mRNA in cultured embryonic gonads (E11.5) from mice lacking Sox9 in Sertoli cells (Chaboissier et al., 2004). We then asked if the abnormal presence of Fst mRNA would result in the accumulation of the Follistatin protein in testicular tissue. Immunohistochemical analysis of newborn gonadal sections using a polyclonal antibody showed the protein to be present in control ovarian tissue, whereas it was undetectable in testes from AMH-Cre; Sox9flox/flox; Sox8+/− mice and clearly present in the cytoplasm of Sertoli cells in gonads from Sox8/Sox9 mutant mice [Fig. 3(A)]. Similarly, we found a moderate and late signal increase at 18 dpp for Foxl2, a gene involved in ovarian development (Uda et al., 2004). This is in keeping with the finding that Sox8 and Sox9 down-regulate Foxl2 during embryonic male gonad development and the accumulation of the protein in mutant testis samples (Georg et al., 2012). Our mRNA signal and immunohistochemical data by Georg et al. was unambiguously confirmed for Sertoli cells and shown to be specific for them by immunofluorescence using antibodies against FOXL2 and EYFP that in our experimental system is a highly specific marker for mutated Sertoli cells [Fig. 3(B)].

We next investigated the known Sertoli cell marker proteins GDNF (Glial cell line derived neurotrophic factor; Fig. 4(A), PTGDS [Prostaglandin D2 synthase; Fig. 4(B)] and DHH [Desert hedgehog; Fig. 4(C)], where decreasing mRNA concentrations in the absence of Sox8 and Sox9 as determined by our microarray study confirmed previously published RT-PCR and whole-mount RNA in situ hybridization assays (Wilhelm et al., 2007; Barrionuevo et al., 2009). GDNF is important for Sertoli-dependent germ stem cell differentiation (Meng et al., 2000), DHH is involved in the formation of seminiferous tubules (Clark et al., 2000), and PTGDS expression was reported to depend upon Sox9 induction (Moniot et al., 2009). The results show strongly reduced protein levels in Sox8/Sox9 mutant testes as compared to controls in all three cases. These data indicate that Sox9-dependent changes in mRNA concentrations correlate well with the corresponding protein levels in testicular Sertoli cells.

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Figure 4. Down-regulation of Sertoli cells marker proteins in mutant testes. (A) Immunofluorescence signals in the cytoplasm of Sertoli cells obtained with an antibody against GDNF (green) are given for sections that represent the typical wild-type (left half) and mutant (right half) testicular morphology. DNA was visualized using DAPI (blue). Dark circular structures within the Sertoli cell areas are immature germ cells associated with them. Scale bar = 40 μm. (B) A similar experiment using an anti-PTGDS antibody (green) and DAPI (blue) is shown. Scale bar = 60 μm. (C) IHC patterns are given for DHH in the cytoplasm of Sertoli cells present in controls (left) and mutant (right) male gonads. Samples were isolated from newborn mice. Scale bar = 40 μm.

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Genome-wide prediction of SOX8 and SOX9 regulatory motifs in the proximal promoters of potential testicular target genes

Predicting evolutionary conserved regulatory motifs in conjunction with high-throughput expression data is helpful for identifying direct transcription factor target genes (Kellis et al., 2003; Schlecht et al., 2008; Van Loo & Marynen, 2009). Although the approach has its limitations because not all conserved motifs are functional and not all functional motifs are conserved (Birney et al., 2007), genes such as Sox4 and Sox10 have been investigated successfully using this method (Lee et al., 2008; Liao et al., 2008). In an initial exploratory approach, we predicted at the genome-wide level conserved matches to two SOX9 motifs and one generic SOX site within the proximal promoters defined as the region −1 kb upstream of the transcription start site [see (Lardenois et al., 2010) for methods]. We then validated the approach by asking if it correctly identified known SOX9 target genes and found that loci falling into the increased (Hapln1, Mia1, Sox5) or decreased (Cbln4, Ptgds, Sox6, Vnn1) categories were identified as having at least one match to a SOX9 motif in their proximal promoter regions. SOX9 is known to regulate Amh (De Santa Barbara et al., 1998; Arango et al., 1999) and shows substantially weaker expression in the mutant, but the promoter element recognized by Transfac motif M01284 was not selected because it was not sufficiently conserved. Another result was that 15 known target genes showed no significant difference between controls and mutant testicular samples at 0 and 6 dpp (Fig. 5). This either was attributable to extremely moderate signal changes, which were below our threshold level of detection (Acan, Cldn7, Col2a, Col9a) or to a lack of expression altogether in pre-pubertal or adult testes (Col11a2, Comp, Foxl2, Matn1, Nkx3-2). In six cases, we observed invariably weak (Cd3e) or strong (Col4a2, Creb1, Nfya, Sp1, Stat1) signals across the samples, which indicates that SOX8 and SOX9 do not contribute to the regulation of these genes in testicular tissue to a level accessible by GeneChip analysis (Fig. 5).

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Figure 5. Identification of potential testicular Sox8/Sox9 target genes. A heat map is given for samples as described in Fig. 2(A). Genes were organized within the increased, decreased and no change classes by hierarchical clustering. Genes bearing highly conserved SOX9 motifs in their proximal promoters are shown in green. The scale is as shown in Fig. 2(A).

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To find putative target genes under strong negative SOX8 and SOX9 control we identified 157 transcripts whose proximal promoters contained at least one predicted SOX motif and for which mRNA was reliably detectable only in the mutant, but not the control samples at either 0 or 6 dpp or both. The control vs. mutant data were compared to results obtained with purified Sertoli and germ cells as well as total testis and ovary samples [Fig. 6(A)]. One subgroup contains loci strongly expressed in ovary, but not (or much less) in Sertoli-, and germ cells such as Apoa1 (metabolism and corticosteroid synthesis), Bmpr1b [limb skeleton development and female fertility, (Edson et al., 2010)], Cyp19a1 (male and female fertility), Esr2 (development, female fertility), Foxp2 (post-natal development) and Ptger2 (ovulation). Another group comprises loci that show elevated transcript concentrations in both ovary and purified Sertoli cells including some involved in reproductive functions like Nr5a2 and Tbx3 and stress-, or inflammatory response such as Bcl2a1a, Cxcl14, Thbs1 and Tnfrsf1b.

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Figure 6. Transcription factor DNA binding site enrichment (A) Two heat maps represent genes for which mRNAs are detected only in mutant testicular samples (increased) or for which signals in the mutant were weaker than in the controls and above background in purified Sertoli cells (decreased). Samples are as in Fig. 2(A). Known Sox8/Sox9 target genes are marked by a red bar. (B) The names and binding site matrix identifiers (left), the total number of promoter sequences containing a hit (middle), and the numbers of promoters observed vs. expected by chance (right) for genes falling into increased and decreased categories are shown. The total numbers of promoters investigated and those containing at least one predicted motif in each category are given at the top of the columns. Transcription factors falling into the decreased category are shown in blue. Data are displayed for each matrix associated with a given regulator. A colour scale showing depletion and enrichment is shown at the bottom.

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We next selected 372 putative target transcripts under positive control by SOX8 and SOX9 using three criteria: they contained SOX motifs in their promoters, we found significantly weaker signals in 0 and 6 dpp Sox8/Sox9 mutants than in control testis and they displayed peak mRNA concentrations in purified Sertoli cells [Fig. 6(A)]. This group contained, as expected, Sox8 and Sox9 themselves and known Sox9 targets such as Ptgds (Wilhelm et al., 2007) and Cbln4 (Bradford et al., 2009). Unexpectedly, we also found Sox6 that has not previously been associated with Sertoli cell functions. Other potential target genes include Ttyh1 (chlorine channel involved in cell adhesion), Ctse (peptidase), Dusp15 (phosphatase), Zdhhc14 (zinc-finger protein), Rnf208 (ring finger protein) and uncharacterized genes not yet identified as Sox8/Sox9-dependent in a testicular context such as D3Bwg0562e, 3110035E14Rik, 4933402E13Rik and Zfp945, possibly encoding a zinc finger protein.

Identification of enriched regulatory motifs in the promoters of putative Sox8/Sox9-dependent genes

Only a few of the putative Sox8/Sox9 target genes for which we find changing signals in the Sox8/Sox9 mutant show a strong effect, perhaps indicating that SOX8 and SOX9 mostly act on promoters in combination with other regulators. We therefore investigated which DNA binding motifs and their cognate transcription factors might co-regulate Sox-dependent testicular genes. To address this question, we searched a 1 kb fragment upstream of the transcription start site for enriched binding sites and found 27 regulatory motifs associated with 15 transcription factors [Fig. 6(B)]. Both SOX9 sites fell into the class of decreased mRNAs, which is consistent with the protein being a transcriptional activator. Additional target motifs found in the decreased class are bound by VDR [encoding the Vitamin D receptor essential for male and female fertility, (Yoshizawa et al., 1997)], MXI1/MYCN (organ function), TCF7/TCFL1 (TCF7 is critical for T-cell development), JUN (hepatogenesis), IKZF1 (blood cell maturation), NR2F2 [angiogenesis and heart development, male and female fertility (Kurihara et al., 2007; Petit et al., 2007; Qin et al., 2008)], TBCLD30 (Rab GTPase activator) and NFIA [brain development, male and female fertility, (das Neves et al., 1999)]. In addition, we found enrichment in the increased class of regulatory elements bound by HMGA1 [cardiovascular system, germline transmission in a heterozygous mouse is not possible, but homozygous mice are fertile; (Fedele et al., 2006)], TCF7L1 (embryogenesis), FOXO1 (embryonic vascularization), DOCK9 (guanine nucleotide exchange) and FOXA1 (post-natal metabolism). Finally, the motifs of SOX9, JUN, IKZF1, ZIC3 (embryonic development) were enriched in both groups; one possible explanation might be that they act as both positive and negative regulators depending upon the promoter context (Fig. 6, see Mouse Genome Database for references concerning phenotypes not related to reproduction).

Protein network analysis of regulatory interactions in the testis

To gain insight into the level at which genes deregulated in the absence of Sox8 and Sox9 interact with each other, we combined transcript profiling data and gene annotation data with information on protein–protein interactions. This analysis identified a large complex of 67 proteins (Fig. 7). It consists of 13 proteins annotated as transcription factors and 29 interacting proteins falling into the increased class together with 10 transcription factors and 14 interacting proteins belonging to the decreased category. CCND1 shows distinct signals for two probe-sets and thus falls into both classes. The class showing lower signals in the mutant obviously not only includes SOX8 and SOX9 but also FOXA1, HHEX (embryonic development), JUN, NOTCH1 (embryonic organogenesis), REL (cytokine production), RORA (brain development), SOX6 (cardiogenesis and growth) and VDR.

image

Figure 7. Regulatory network analysis. The network is colour coded with nodes given in red and blue for transcription factors showing stronger (increased TF) or weaker (decreased TF) signals in the mutant respectively. Green edges represent interactions with target genes showing stronger or weaker signals in the mutant. Targets are given in dark blue (increased) and light green (decreased) respectively. The thickness of the edge represents the number of published protein-DNA interactions. A legend for the colour code is shown.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

Mice bearing a homozygous deletion of Sox8 and a targeted deletion of Sox9 in testicular Sertoli cells from E13.5 onwards are unable to form testis cords and fail to establish spermatogenesis when they reach adulthood (Chaboissier et al., 2004; Barrionuevo et al., 2009). To uncover genes and biological processes that are deregulated in post-natal testis lacking both Sox8 and Sox9, we carried out a whole-genome protein-coding RNA profiling analysis of total testicular samples from Sox8/Sox9 mutant mice vs. phenotypically normal controls. To distinguish transcripts showing signal peaks in somatic cells from those for which we measured the highest concentration in germ cells, the output of this experiment was combined with our own profiling data obtained with enriched Sertoli cells, spermatogonia, spermatocytes and spermatids as well as wild-type testis and ovary samples from a certified repository (Chalmel et al., 2007b). Furthermore, we integrated motif predictions to identify DNA binding regulators that might directly or indirectly cooperate with or respond to Sox8 and Sox9 in the testis. Finally, protein network data were used to explore the level of direct protein–protein or protein-DNA interactions that might play a role in establishing the infertility phenotype reported.

Our experiment has yielded a number of reference loci for which we indeed detected the changes in transcript levels anticipated for bona fide SOX8/SOX9 target genes. However, we also identified loci for which no signal changes were found in spite of the presence of conserved binding motifs in their putative promoter regions [see Fig. 4(B)]. An obvious explanation for this result is that motif predictions are prone to yield false-positives, but it is also conceivable that some of the binding sites are functional in somatic cells, but not in testicular tissue, because transcripts for some of the loci are (practically) not detectable in testicular samples. It might also be that signal changes were too subtle to be revealed by GeneChips. This known issue notwithstanding, we have previously shown that it is useful to combine expression data and information about promoter motifs when interpreting GeneChip data from complex mammalian samples (Lardenois et al., 2010; Chalmel et al., 2012).

Transcript concentrations change in the absence of Sox8 and Sox9 in testicular Sertoli cells

The experimental design we chose enabled us to explore the GeneChip data in two major directions: first, we identified transcripts that were detectable only in mutant samples, but not in normal testis. In this group, one would expect genes involved in cell survival – given the deleterious effect of the Sox8 and Sox9 double deletion on cellular interactions and thus tissue architecture within the testis – and ovarian functions as Sox9 is important for maintaining Sertoli cell function and for preventing them from becoming granulosa-like cells (Chaboissier et al., 2004; Barrionuevo et al., 2009). Indeed, we found Xaf1 (apoptosis) as well as Nr5a2 [steroidogenesis; activated by FOXO1 in granulosa cells and required for female fertility; (Labelle-Dumais et al., 2007; Liu et al., 2009)], Esr2 [female fertility related to granulosa cell function; (Dupont et al., 2000)] and Ptger2 [fertility via optimal ovulation; (Hizaki et al., 1999; Kennedy et al., 1999)]. Intriguingly, we also identified Foxl2os, a putative long intergenic noncoding RNA (lincRNA) antisense to Foxl2 [eyelid development and adult ovary function; (Cocquet et al., 2005; Garcia-Ortiz et al., 2009), for review, see (Jagarlamudi & Rajkovic, 2012)], as being present at high levels in the mutant testes. To the best of our knowledge, this is the first evidence suggesting that the testicular level of a lincRNA may be dependent upon SOX8 and SOX9. The sense/antisense transcript configuration at the Foxl2 locus hints at a regulatory mechanism involving lincRNA; related phenomena are well established in budding yeast gametogenesis (Hongay et al., 2006; van Werven et al., 2012), and are being extensively studied in the fields of mammalian development and disease; for review, see (Esteller, 2011; Wang & Chang, 2011; Guttman & Rinn, 2012). In any case, it appears that the FOXL2 protein is very stable in Sertoli cells as it is clearly present in samples from Sox8/Sox9 mutant mice right after birth whereas its mRNA is low at 0 and 6 dpp and accumulates to unambiguously detectable levels in the 18 dpp sample (see Fig. 3; Data S1.).

In the case of three other regulatory proteins for which we measured increased transcript concentrations in the double mutant, we observed an interesting phenomenon in so far as their levels were also found to increase during testicular dysfunction in late adult mice lacking Sox9 in their Sertoli cells (Lardenois et al., 2010). It is therefore conceivable that Nfia (brain development, fertility), Nr2f2 (development) and Ppara [lipid metabolism in Sertoli cells, (Thomas et al., 2011)] are part of similar response mechanisms triggered by the lack of Sox8 and Sox9 during early male gonad development, and to the absence of Sox9 alone in Sertoli cells during the maintenance of spermatogenesis and testicular architecture in mature adults.

Identification of potential direct targets for SOX8 and SOX9 in Sertoli cells

The group of genes for which we detected an increase in mRNA is split into those that lack Sox motifs in their upstream regions and those that contain at least one predicted site within the region we investigated. The former group may reflect a broad tissular response to the phenotype and should therefore include loci related to stress that are not necessarily restricted to Sertoli cells as far as their transcription is concerned. Indeed, we identified genes such as Mpzl2 known to be induced in germ cells in a spermatogenesis-deficient mutant, (Nakata et al., 2012) and the co-activator CIDEA involved in lipid metabolism and cell death (Inohara et al., 1998; Wang et al., 2012). Intriguingly, we also found SCX, a bHLH transcription factor essential for gastrulation, previously reported to mediate SOX9- dependent gene expression during chondrogenesis as part of a multi-subunit complex (Furumatsu et al., 2010). SCX is actually expressed in Sertoli cells (Muir et al., 2008) and its lack of induction in the Sox8/Sox9 mutant points to a possible feedback loop whereby the SOX proteins are either indirectly required for SCX expression in Sertoli cells, or they directly activate the Scx promoter by binding to motifs outside of the region we examined.

Contrary to the genes lacking a SOX motif in their proximal promoter, the latter group did contain at least one match to at least one of the three motifs we searched for. It included cases that reunited three features marking them out as potential direct targets for Sox8/Sox9-dependent gene activation: first, the presence of motifs, second, transcripts were detected in enriched Sertoli cells, and third, mRNA is found in normal testes at 0 and 6 dpp (decreasing at 18 dpp when the germ cell population increases in proportion to the Sertoli cells), but not in mutant testes. Coherently, one gene with this pattern is Cbln4 (brain signalling pathways) that was previously reported as being a direct target of SRY and SOX9 (Bradford et al., 2009). Another case is Gas7 [motor neuron function, (Huang et al., 2012)] the human orthologue of which was shown to depend upon SOX9 during chondrogenic differentiation of human mesenchymal stem cells (Chang et al., 2008). We have earlier made the observation that Sox6 (cardiac development, chondrogenesis) expression appears to be down-regulated in adult testis lacking Sox9 in Sertoli cells (Lardenois et al., 2010), and we now fail to detect Sox6 transcripts in early post-natal Sox8/Sox9 mutant testis as well. Although Sox6 has no known function in male gonads – perhaps because partially redundant SOX proteins are expressed in testicular cells – our findings are consistent with a previous observation that the protein is present not only in germ cells but also in Sertoli cells (Ohe et al., 2009). Finally, the data suggest novel Sox8/Sox9-dependent roles in testicular functions for as yet poorly characterized genes such as Rnf208 (Ring finger protein) and 8030411F24Rik (endopeptidase inhibitor) that may fulfil specific functions related to RNA and protein stability in male gonads.

Our motif prediction and protein network analysis have yielded a number of transcription factors that might co-regulate genes in Sertoli cells such as NFIA, a protein marked out by the network analysis as being potentially up-regulated in early post-natal Sox8/Sox9 mutant testes and in late Sox9 mutant male gonads [see Fig. 6 and (Lardenois et al., 2010)]. This notion is in keeping with the recent finding that SOX9 and NFIA cooperate during the onset of gliogenesis (Kang et al., 2012). As opposed to Nr2f2 and Ppara that show similar patterns of signal induction, Jun's transcripts were decreased in the double mutant and increased in the single mutant [Fig. 6 and (Lardenois et al., 2010)]. This may be a consequence of Jun-related immunological processes unfolding at late, but not early stages of testicular breakdown.

Among potentially interesting interaction partners identified in the network analysis, published experimental data mark out AFP [female fertility, inhibition of spermatogenesis upon heat shock in undescended testes; (Yazama & Tai, 2011)], DHH [formation of seminiferous tubules; (Barrionuevo et al., 2009; Clark et al., 2000)], see Fig. 3(D), EDN1 [cardiovascular and craniofacial development; (Chan et al., 2008)], GDNF (control of spermatogonial stem cell number; (Savitt et al., 2012); see Fig. 3(B), and RBP4 [maintenance of testis function during vitamin A starvation; (Ghyselinck et al., 2006)], as potentially relevant for the phenotype observed in the Sox8/Sox9 mutant.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

This analysis identified Sox8/Sox9-dependent testicular genes on the basis of whole-genome transcript profiles in conjunction with motif prediction data and information about protein networks. This genome-wide approach enabled us to identify genes likely regulated by SOX8 and SOX9 in Sertoli cells, and to distinguish them from others that indirectly respond to the double mutant phenotype (or for which signal changes are ascribable to altered cell populations in the mutant testis samples). This study, therefore, constitutes a rich source of information for subsequent hypothesis-driven work to elucidate the genetics of early male gonad development. Last not least, our results will be very helpful for the interpretation of ongoing efforts in the field to map the genome-wide in vivo DNA binding pattern of SOX9 in testicular cells using chromatin-immunoprecipitation and ultra-high throughput DNA sequencing.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

We thank J. Moore for stimulating discussions, and F. Petit and S. Jamin for critical reading of the manuscript and O. Collin (IRISA, Genouest) for MIMAS and GermOnline system administration. A. Lardenois received an INSERM Young Investigator fellowship and support from grant INERIS-STORM 10028NN awarded to B. Jégou. Deutsche Forschungsgemeinschaft grants Sche 195/15 and GRK 1104 awarded to G. Scherer and INSERM Avenir grant R07216NS awarded to M. Primig funded this work.

Author's contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information

F.C. and A.L. analyzed and interpreted microarray data and contributed to the manuscript, IG validated expression data, F.B. prepared the testicular samples, P.D. produced and quality controlled raw microarray data, B.J. interpreted data, G.S. designed research, and M.P. interpreted data and wrote the manuscript. All authors have read and approved the manuscript.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Author's contributions
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
andr49-sup-0001-DataS1.xlsxapplication/msexcel4912K

Data S1. Profiling and motif prediction output.

andr49-sup-0002-FigS1.tifimage/tif2301K

Figure S1. The AMH-Cre; Sox9 flox/flox;Sox8−/− phenotype.

andr49-sup-0003-FigS2.tifimage/tif920K

Figure S2. Total RNA, cRNA and data quality controls.

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