Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration
- Universidad de Granada, Spain
- University of Freiburg, Germany
- Facultad de Ciencias, Universidad de Granada, Spain
- University of California, San Diego, United States
Figures
Figure 1 with 7 supplements
Sox9 and Sox8 maintain the function and integrity of the adult mouse testis.
(A) Diagram illustrating the time course of TX administration. Mice were fed with a TX-supplemented diet during one month, between P60 (0 datx) and P90 (30 datx). After this period, mice were fed …
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Figure 1—source data 1
Comparison of the number of SOX9+ cells per testis tubule in both SC-DKO mutants and TX-treated controls (Sox9f/f;Sox8-/-).
- https://doi.org/10.7554/eLife.15635.004
Figure 1—figure supplement 1
Analysis of the CRE-recombination efficiency in SC-DKO mice.
(A) Immunostaining of the EYFP protein in mutant mice at different times-points after the initiation of the TX treatment. The number of cells expressing EYFP increases with time (from a to e and …
Figure 1—figure supplement 2
Redundant role for Sox9 and Sox8 in the maintenance of adult testis cord architecture.
Haematoxilin-eosin stained transversal sections of P120 testes (left column) and epididymides (right column) from control mice (a–b) and four different types of Sox9/8 DKO (Wt1) mice differing in …
Figure 1—figure supplement 3
Relative abundance of the most relevant morphological features observed in the testes of P120 control and Sox9/8 mutant mice differing in the number of Sox9/8 mutant alleles 60 days after TX administration during 5 days with a feeding-gauge needle.
Categories of relative abundance: (−) not present, (+) sporadic, (++) scarce, (+++) abundant, (++++) generalized.
Figure 1—figure supplement 4
Time course of the testis phenotype of control and SC-DKO (Wt1) mice.
Representative micrographs of H&E stained sections of non-treated Sox8-/- (a–f), TX-treated Sox8-/- (g–l) and TX-treated Sox8/9 DKO (Wt1) (m–r) mouse testis at different time-points. T, normal …
Figure 1—figure supplement 5
Relative abundance of the most relevant morphological features observed throughout the timecourse of testis regression in double Sox8/Sox9 mutant mice.
Categories of relative abundance: (−) not present, (+) sporadic, (++) scarce, (+++) abundant, (++++) generalized.
Figure 1—figure supplement 6
Functional status of the BTB in Sox9/8 DKO testes.
The location of a biotin tracer (red fluorescence) shows that the BTB is impermeable in the control (Sox9f/f;Sox8-/-) testis (a) as it did not enter into the adlumninal compartment of the …
Figure 1—figure supplement 7
Expression bar plots of two adult Leydig cell markers.
Hsd3b1: 3β-hydroxysteroid dehydrogenase; Insl3: Insulin-Like 3. Data obtained from our transcriptome analysis.
Figure 2 with 4 supplements
Genetic reprogramming in somatic cells of adult Sox9/8 SC-DKO (Sox9).
(A) Expression of FOXL2 (green fluorescence) in P150 (90 datx) TX-treated control (Sox9f/f;Sox8-/-) (a) and in Sox9/8 SC-DKO (Sox9) mouse testes analyzed at P90 (30 datx) (b), P105 (45 datx) (c), …
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Figure 2—source data 1
Genes with significant differential expression among untreated controls, TX-treated controls, Sox8/9 SC-DKO mutants and control ovary at P150 (90 datx) identified from the bioinformatic analysis of our transcriptome.
- https://doi.org/10.7554/eLife.15635.013
Figure 2—figure supplement 1
Expression of Foxl2 in somatic cells of adult Sox9/8 DKO (Wt1).
Immunofluorescence for FOXL2 (green fluorescence) in P150 (90 datx) TX-treated control (Sox9f/f;Sox8-/-) (a) and Sox9/8 DKO (Wt1) mouse testes analyzed at P90 (30 datx) (b), P105 (45 datx) (c), and …
Figure 2—figure supplement 2
Heatmaps showing the expression of genes involved in 8 selected pathways (A–H), relative to their expression in control testes.
Gene sets where obtained from the Pathway Unification Database except for the Myosin genes Family that where obtained from the list of differentially expressed genes. The pathways, URL, and number …
Figure 2—figure supplement 3
Cluster analysis of (A) replicates and (B) conditions.
The global gene expression patterns of mutants are closer to the expression in ovaries than to that in normal testes. Both controls with and without Tamoxifen treatment also group together as …
Figure 2—figure supplement 4
Quantification of genes with differential expression and Jensen–Shannon (JS) distances between conditions.
(A) Number of genes showing differential expression in pairwise comparisons of the different conditions.
(B) Jensen–Shannon (JS) distances heatmap between conditions.
Figure 3
Identification of the somatic cells expressing FOXL2.
(A) Double immunofluorescence for FOXL2 (green, nuclear) and EYFP (red, cytoplasmic) (B) Double immunofluorescence for SOX9 and WT1 in P150 (90 datx) TX-treated control (Sox9f/f;Sox8-/-) testes (a) …
Figure 4 with 1 supplement
Role of Dmrt1 in Sertoli-to-granulosa cell transdifferentiation.
(A) Double immunofluorescence for SOX9 and FOXL2 in TX-treated control (Sox9f/f;Sox8-/-) testis (a), Sox9/8 SC-DKO mutant testes analyzed at P105 (45 datx) and P150 (90 datx) (b,c) and a control …
Figure 4—figure supplement 1
Role of Dmrt1 in Sertoli-to-granulosa cell transdifferentiation.
Double immunofluorescence for SOX9 and FOXL2 (A), and for FOXL2 and DMRT1 (C) as shown in Figure 4 but showing separated the single color channels (a', c' and e' are higher magnifications of the …
Figure 5
Sox9 and Dmrt1 act in the same pathway during Sertoli-to-granulosa transdifferentiation.
(A) Log2 fold change scatterplot comparing the microarray data from the P28 SC-Dmrt1 KO testes reported by Matson et al., 2011b; GEO accession: GSE27261) with the RNA-seq data from our P150 (90 …
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Figure 5—source data 1
List of 8910 genes showing significant differential expression respect to normal testis in both Dmrt1 and Sox9/8 mutants.
- https://doi.org/10.7554/eLife.15635.022
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Figure 5—source data 2
Log2 fold change of expression of both Dmrt1 and Sox9/8 mutants respect to controls in a set of genes belonging to the all-trans-retinoic acid-mediated apoptosis path and RA receptors-mediated signaling from the PathCards database.
- https://doi.org/10.7554/eLife.15635.023
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Figure 5—source data 3
Comparison of the number of FOXL2+ cells per transversal testis cord section in Sox9/8 DKO (Sox9) WIN 18,446-treated mice and vehicle-injected controls
- https://doi.org/10.7554/eLife.15635.024
Figure 6
Incidence of apoptosis in Sox9/8 SC-DKO testes.
(A) TUNEL staining in testis sections of TX-treated control (Sox9f/f;Sox8-/-) at P120 (60 datx) (a) and Sox9/8 SC-DKO at different time-points (b–d). (B) Double immunofluorescence for TUNEL and …
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Figure 6—source data 1
Comparison of the number of TUNEL-positive cells per section area unit in both SC-DKO mutants and TX-treated controls.
- https://doi.org/10.7554/eLife.15635.026
Figure 7
Model for the regulation of mammalian sex maintenance.
Positive regulation is indicated by arrows. Negative regulation is indicated by perpendicular lines. See text for a detailed explanation.
Figure 8
Pairwise scatterplots comparing log10 FPKM between different conditions.
Dispersion is lower when comparing similar conditions (controls, mutants) and higher when comparing mutant with control conditions. Notice that dispersion observed when mutants are compared with …
Figure 9
Sashimi plots of the Sox9 and Sox8 genes in mutant and control conditions.
Vertical lines indicate coverage and curved lines indicate splicing. The mm10 row shows the positions of the exons and introns and the translated region as annotated in the mm10 UCSC mouse genome. …
Tables
Table 1
Antibodies used in this study.
| Gene product | Raised in | Working dilution | References |
|---|---|---|---|
| Laminin | rabbit | 1:100 | Sigma L9393 |
| ACTA2 | mouse | 1:200 | Sigma A2547 |
| Claudin11 | rabbit | 1:100 | Santa Cruz Biotechnology, CA sc-25711 |
| DMC1 | goat | 1:100 | Santa Cruz Biotechnology, CA sc-8973 |
| PCNA | mouse | 1:100 | Santa Cruz Biotechnology, CA sc-56 |
| CYP14A1 (P450scc) | goat | 1:200 | Santa Cruz Biotechnology, CA sc-18043 |
| SOX9 | rabbit | 1:100 | Santa Cruz Biotechnology, CA sc-20095 |
| SOX9 | goat | 1:10 | Santa Cruz Biotechnology, CA sc-17341 |
| WT1 | rabbit | 1:100 | Santa Cruz Biotechnology, CA sc-192 |
| FOXL2 | goat | 1:100 | Abcam ab5096 |
| GFP | rabbit | 1:100 | Novus Biologicals NB600-308 |
| WT1 | mouse | 1:30 | DAKO M3561 (clone 6F-H2) |
| CYP19A1 (Aromatase) | mouse | 1:10 | GeneTex GTX41561 |
| DMRT1 | rabbit | 1:400 | Gift from Dr. Silvana Guioli |
Additional files
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Supplementary file 1
Complete lists of genes included in the 8 molecular pathways mentioned in Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.15635.031
