A novel mouse model of testicular granulosa cell tumors

A novel mouse model of testicular granulosa cell tumors Abstract STUDY QUESTION What is the role of dysregulated transforming growth factor beta (TGFB) signaling in the development of sex cord-stromal tumors in the testis? SUMMARY ANSWER Overactivation of TGFB signaling results in the development of testicular tumors resembling granulosa cell tumors (GrCTs). WHAT IS KNOWN ALREADY In an earlier study, we demonstrated that constitutively active TGFB receptor 1 (TGFBR1) in ovarian somatic cells promotes the development of ovarian GrCTs. However, the consequence of dysregulation of TGFB signaling in the pathobiology of the testis, remains poorly defined. STUDY DESIGN, SIZE, DURATION To identify the impact of dysregulation of TGFB signaling on the testis, we generated mice with constitutive activation of TGFBR1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase. The effect of constitutively active TGFBR1 on testis development and the timeline of testicular tumor formation were examined. We further investigated the molecular features of testicular tumors and determined the expression of beta-catenin (CTNNB1) known to be involved in testicular GrCT development. PARTICIPANTS/MATERIALS, SETTING, METHODS Male mice with constitutive activation of TGFBR1 were examined at various developmental stages (i.e. from 1 week up to 6 months) along with controls. Testis samples were collected and processed for histological and molecular analyses, including haematoxylin and eosin (H and E) staining, real-time PCR, immunohistochemistry, immunofluorescence and western blotting. Immunostaining/immunoblotting and real-time PCR experiments were performed using at least three animals per genotype. Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tail t-test and reported when P value is <0.05. MAIN RESULTS AND THE ROLE OF CHANCE Mice harboring constitutively active TGFBR1 in the testes developed tumors resembling testicular GrCTs, a rare type of tumors in the testis. The formation of testicular tumors led to altered cell proliferation, loss of germ cells and defective spermatogenesis. Immunohistochemically, these tumors were positive for inhibin alpha (INHA), forkhead box O1 (FOXO1), and more importantly, forkhead box L2 (FOXL2), a protein specifically expressed in the ovary and required for normal granulosa cell differentiation and function. Consistent with the immunohistochemical findings, FOXL2 proteins were only detectable in testes of TGFBR1-CAAcre mice but not those of controls by western blotting, suggesting potential alteration of Sertoli cell fate. To explore mechanisms underlying the tumor-promoting effect of TGFBR1 overactivation, we examined the expression of CTNNB1. The results revealed increased expression of CTNNB1 in testicular tumors in TGFBR1-CAAcre mice. Collectively, this study uncovered tumorigenic function of enhanced TGFB signaling in the testis. LARGE-SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This study was performed using mice, and the direct relevance of the experimental paradigm and findings to human testicular GrCTs awaits further investigation. Of note, constitutive activation of TGFBR1 was employed to enhance TGFB/SMAD signaling activity and may not be interpreted as the genetic cause of the disease. WIDER IMPLICATIONS OF THE FINDINGS This mouse model may prove to be a useful addition to the mouse genetics toolkit for GrCT research. Our finding that dysregulation of TGFB signaling results in the development of testicular GrCTs supports a common origin between Sertoli cells and granulosa cells, and highlights the paramount importance of balanced TGFB signaling in reproduction and development. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University awarded to Q.L. The authors declare no competing interest. testicular granulosa cell tumor, gonad, TGFBR1, SMAD2/3, mouse model Introduction Transforming growth factor beta (TGFB) superfamily members, which are structurally related proteins, regulate a broad range of cellular properties, including, but not limited to growth, differentiation, migration and apoptosis (Massague, 1998; Massague et al., 2000). Because of their fundamental roles in these cellular events, TGFB superfamily members exhibit diverse physiological and pathological functions in multiple organ systems. In the reproductive system, recent studies, particularly those using genetics and functional genomics approaches uncovered many previously unrecognized roles of this growth factor family in reproductive development and cancer (Massague et al., 2000; Padua and Massague, 2009; Li, 2014; Fang et al., 2016). TGFB signaling is known to be a double-edged sword in tumor development, exhibiting tumorigenic or tumor suppressive effects at different tumor stages (Massague, 2008). To initiate signal transduction, TGFB ligands recruit type II and type I receptor complexes and activate canonical and/or non-canonical pathways depending on the signaling context (Akhurst and Hata, 2012). In general, TGFB ligands signal through TGFBR1/TGFBR2-SMAD2/3 axis, whereas bone morphogenetic proteins (BMPs) activate BMP type II and type I receptors and the SMAD1/5/9 signaling branch. SMAD4 is a common SMAD utilized by both TGFB and BMP signaling to access gene regulatory apparatus within the nuclear compartment (Fang et al., 2016). Approximately 95% of malignant testicular tumors belong to germ cell tumors, comprising seminomas and nonseminomas (Motzer et al., 2012). Sex cord-stromal tumors (3–5%) of the testis include Leydig cell tumor, Sertoli cell tumor, granulosa cell tumor (GrCT), mixed sex cord-stromal tumor and unclassified sex cord-stromal tumor (Moch et al., 2016). Testicular GrCTs are rare tumors (Mohapatra et al., 2016). The formation of testicular GrCTs has been reported in several mouse models (Matzuk et al., 1992; Boyer et al., 2009; Richards et al., 2012). The pathogenesis of GrCTs is poorly understood in both genders, due in part to the limited access to human GrCT specimens. A breakthrough has been recently made toward understanding the etiology of ovarian GrCTs. Forkhead box L2 (FOXL2) is an ovary-expressed gene essential for granulosa cell differentiation (Uhlenhaut et al., 2009). It was discovered that the vast majority of adult type GrCTs in the ovary bear a missense mutation of FOXL2 (402 C→G; C134W) (Shah et al., 2009). This mutation was also found in testicular GrCTs of males (Lima et al., 2012), indicating the importance of FOXL2 mutation in the pathogenesis of GrCTs. However, how FOXL2 mutation activates tumorigenic cascade within granulosa cells is not known. Interestingly, experimental evidence supports a potential link between FOXL2 mutation and alteration of TGFB/activin signaling in GrCT development (Rosario et al., 2012; Cheng et al., 2014; Fang et al., 2016). TGFB superfamily signaling is involved in the development of GrCTs (Middlebrook et al., 2009). In an earlier study, we created a mouse model harboring constitutively active TGFB receptor 1 (TGFBR1) using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase (Gao et al., 2016). Amhr2-Cre has been widely used to target genes in granulosa and theca cells of the ovary, myometrial and endometrial cells of the uterus (Li et al., 2008, 2011; Ren et al., 2009; Wang et al., 2016), and Sertoli cells and Leydig cells of the testis (Boyer et al., 2008). Expression of Amhr2-Cre has been detected as early as 11.5 days post-coitum in the urogenital ridges of both males and females, and the Cre activity is present in the somatic cells of gonads and the mesenchyme of Mullerian ducts (Jamin et al., 2002). To generate constitutively active TGFBR1, three missense mutations in the GS domain were made to activate the TGFBR1 kinase (T204D) and block the binding of the TGFBR1 inhibitor, FKBP12 (L193A/P194A) (Bartholin et al., 2008). A LoxP-flanked stop sequence was engineered to direct the expression of constitutively active TGFBR1 to Cre-expressing cells/tissues. Overactivation of TGFBR1 in the ovary results in formation of ovarian tumors reminiscent of GrCTs (Gao et al., 2016). Here, we report that male mice with constitutive activation of TGFBR1 in the testes developed tumors resembling testicular GrCTs. This study developed a useful mouse model for testicular GrCTs. Materials and Methods Ethics statement Animal use protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University. All animals were handled in accordance with the guidelines by the IACUC of Texas A&M University. Animals and tissue collection Mice harboring a latent constitutively active TGFBR1 (TGFBR1CA) were created as described (Bartholin et al., 2008; Vincent et al., 2010). The TGFBR1CA flox/flox mice were crossed to Amhr2-Cre line (Jamin et al., 2002) to generate mice containing constitutively active TGFBR1 in the testes (TGFBR1CA flox/+; Amhr2-Cre). These mice were termed TGFBR1-CAAcre. Mice were on a mixed C57BL/6/129 background and maintained by controlled light/dark cycles with free access to food and water. Testis samples were collected from both control and TGFBR1-CAAcre males during developmental stages for histological and immunohistochemical analyses described below. Genotyping was performed using DNA extracted from mouse tails described previously (Bartholin et al., 2008; Gao et al., 2016). PCR products were separated on 1% agarose gels containing ethidium bromide and digital images captured using a VWR Gel Imager. Histology Testis samples from control and experimental mice were fixed in 10% (v/v) neutral buffered formalin and/or Bouin’s solution for histology and immunostaining. Tissue samples were processed and embedded using the histology core lab in the Department of Veterinary Integrative Biosciences at Texas A&M University. Haematoxylin and eosin (H and E) staining was performed using standard procedures. Immunohistochemistry and immunofluorescence Immunohistochemistry (IHC) and Immunofluorescence (IF) were performed as described elsewhere (Gao et al., 2015, 2016). In brief, paraffin sections (5 μm) were deparaffinized in xylene, rehydrated in graded alcohol and boiled in 10 mM citrate buffer (pH 6.0) for 20 min to retrieve the antigen. After antigen retrieval, sections were treated with 0.3% H2O2 for 30 min to quench endogenous peroxidase activity. This step was only needed for IHC. Then, sections were sequentially incubated with non-immune serum (for IHC) or bovine serum albumin (for IF), primary antibodies with indicated dilutions (Table 1) overnight at 4°C and secondary antibodies conjugated with horseradish peroxidase (HRP; 1:300; for IHC) or Alexa Fluor 488/594 (1:400; for IF). Intervening washes were performed using Tris-buffered saline (TBS; pH 7.4). Following secondary antibody incubation, an avidin-biotin complex (ABC; Vector Laboratories, USA) was applied to amplify immunoreactive signals, which were developed using NovaRed substrate (Vector Laboratories; USA; for IHC). The sections were counterstained using haematoxylin, mounted with Permount and examined under BX43F microscope (Olympus; USA). For IF, sections were directly mounted with ProLong Gold Slowfade media containing 4′, 6-diamidino-2-phenylindole (DAPI; Catalog number: P36931; Invitrogen, USA) after incubation with secondary antibody. Fluorescence signals were visualized using a IX73 microscope (Olympus, USA) interfaced with an XM10 CCD camera and cellSens Software. Isotype-matched IgG controls were included to determine the staining background of the experiment. Table I Primary antibodies for immunostaining and western blotting analyses. Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Table I Primary antibodies for immunostaining and western blotting analyses. Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Western blotting Western blotting was conducted as described (Li et al., 2008). Protein lysates from mouse testis tissues were prepared using radioimmunoprecipitation assay buffer containing both proteinase (Catalog number: 04 693 116 001; MilliporeSigma, USA) and phosphatase inhibitors (Catalog number: 4 906 837 001; MilliporeSigma, USA). Protein quantification was performed using bicinchoninic acid reagent purchased from Thermo Scientific (USA). Approximately 30 μg of proteins were loaded on each lane and separated by electrophoresis using 12% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, USA). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories; USA), which were subsequently incubated with primary antibodies at 4°C overnight (Table 1). After being washed thoroughly, membranes were further incubated with HRP-conjugated anti-rabbit/rat/goat secondary antibodies (1:20 000; Jackson ImmunoResearch, USA). Immunoreactive signals were developed using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, USA). Signals were visualized and captured using Kodak Image Station 4000 mm PRO digital scanner or Bio-Rad ChemiDoc MP Imaging System. Membranes were stripped and reprobed with antibody directed to beta-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). Quantification of western blotting results was performed using NIH image J (version 1.51e). RNA preparation and reverse transcription-real-time PCR RNA samples were isolated from testes of control and TGFBR1-CAAcre males using RNeasy Mini Kit (Qiagen, USA) based on instructions provided by the supplier. Potential genomic DNA contamination was eliminated by inclusion of an additional on-column DNase digestion step. Concentrations of the RNA preparations were determined by using NanoDrop Spectrophotometer ND 1000 (NanoDrop Technologies, USA). RNA samples were then subjected to reverse transcription for cDNA synthesis (Gao et al., 2014). Real-time PCR was performed using a CFX Connect Real-time PCR Detection System (Bio-Rad Laboratories; USA). The 10 μl reaction system contained iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories; USA) or TaqMan Universal PCR Master Mix, cDNA and oligo primers (Gao et al., 2016) or the Taqman probe. Assays were performed in duplicates for each sample and a mean cycle threshold (CT) value was calculated. Primers for Ccnd2 were 5′-GAGTGGGAACTGGTAGTGTTG-3′ (forward) and 5′-CGCACAGAGCGATGAAGGT-3′ (reverse) (PrimerBank ID 6 753 310a1) (Spandidos et al., 2008). Gja1 expression was analyzed using Taqman probe (Assay ID: Mm01179639_s1). Ribosomal protein L19 (Rpl19) was used as an internal control to normalize gene expression (Livak and Schmittgen, 2001; Gao et al., 2014). A Taqman probe for Rpl19 (Assay ID: Mm02601633_g1) was included in Taqman gene expression assays. Statistical analysis Comparisons of means between two groups were made using unpaired two-tail t-test. Data are presented as mean ± SEM. Statistical significance was defined when P value is <0.05 and marked as *P < 0.05, **P < 0.01 and ***P < 0.001. Results Generation of mice with constitutively active TGFBR1 in testes using Amhr2-Cre recombinase We previously reported that constitutive activation of TGFBR1 in the ovary using Amhr2-Cre recombinase leads to the development of ovarian sex cord-stromal tumors reminiscent of GrCTs (Gao et al., 2016). Because Amhr2-Cre is also expressed in Sertoli and Leydig cells of the testis (Boyer et al., 2008), we examined potential effects of overactivation of TGFBR1 on the development and function of the testis by generating a mouse model harboring constitutively active TGFBR1 in the testis (Fig. 1A). As expected, TGFBR1CA proteins were readily detectable in the testes of TGFBR1-CAAcre males but not controls by western blotting using anti-hemagglutinin (HA) antibody (Fig. 1B). These findings support that Amhr2-Cre drives the expression of TGFBR1CA in mouse testes. TGFBR1 phosphorylates and activates SMAD2/3 signal transducers. To verify activation of TGFB signaling in TGFBR1-CAAcre testes, we performed western blotting to determine the levels of phospho-SMAD2/3 in the testes of TGFBR1-CAAcre males. Results showed that the levels of both phospho-SMAD2 and phospho-SMAD3 were elevated in TGFBR1-CAAcre testes compared with controls (Fig. 1B). SMAD2, SMAD3 and beta-actin (ACTB) were included as controls (Fig. 1B). Figure 1 View largeDownload slide Constitutive activation of TGFBR1 in mouse testes. (A) A diagram depicting the generation of mice with constitutive activation of TGFBR1 using Amhr2-Cre recombinase. (B) Western blotting analysis of hemagglutinin (HA)-tagged TGFBR1CA, phospho-SMAD2 and phospho-SMAD3 in the testes of control and TGFBR1-CA male mice at the age of 2 weeks. Each lane represents an independent testis sample. n = 4. SMAD2, SMAD3 and ACTB were included as controls. Figure 1 View largeDownload slide Constitutive activation of TGFBR1 in mouse testes. (A) A diagram depicting the generation of mice with constitutive activation of TGFBR1 using Amhr2-Cre recombinase. (B) Western blotting analysis of hemagglutinin (HA)-tagged TGFBR1CA, phospho-SMAD2 and phospho-SMAD3 in the testes of control and TGFBR1-CA male mice at the age of 2 weeks. Each lane represents an independent testis sample. n = 4. SMAD2, SMAD3 and ACTB were included as controls. TGFBR1-CAAcre males develop testicular tumors with impaired spermatogenesis Sterility was observed in TGFBR1-CAAcre male mice when they were used as breeders. To determine the cause of sterility, we performed macroscopic and histological analyses of testes and epididymides of control and TGFBR1-CAAcre males. Gross testicular tumors with hemorrhagic foci were observed in TGFBR1-CAAcre mice around 2 months of age with full penetrance (Fig. 2A). In contrast, no tumors were observed in control mice (Fig. 2A). Testicular tumors progressed with age (Fig. 2B). Histological analysis of 2-month-old testes showed that while control mice had organized seminiferous tubules (Fig. 2C and E), TGFBR1-CAAcre males developed testicular tumors resembling GrCTs (Fig. 2D and F), which were observed in the ovary of TGFBR1-CAAcre female mice (Gao et al., 2016). Microscopically, seminiferous tubules were displaced by circumscribed tumor nests containing poorly differentiated cells or granulosa-like cells (Fig. 2D and F and Supplementary Fig. 1B and D). Remnant seminiferous tubules lacking mature germ cells which were observed in control testes could be found near the edge of TGFBR1-CAAcre testes (Supplementary Fig. 1A–D). Both histological results and IF analyses using antibody directed to smooth muscle actin alpha (ACTA2) to mark the structure of epididymis demonstrated lack of sperm in the epididymis of TGFBR1-CAAcre males (Fig. 2H and J), and this was in sharp contrast to wild type controls where abundant sperm/sperm DNA could be found within the epididymis (Fig. 2G and I). Analysis of testes from 5–6-month-old TGFBR1-CAAcre males demonstrated development of more hemorrhagic tumors (Supplementary Fig. 1F and H), in contrast to control testes containing morphologically normal seminiferous tubules (Supplementary Fig. 1E and G). Thus, testicular tumors resulting from constitutive activation of TGFBR1 are detrimental to normal testis development and spermatogenesis. Figure 2 View largeDownload slide Sustained activation of TGFBR1 in the testis leads to sex cord-stromal tumor formation. (A, B) Development of testicular tumors in TGFBR1-CAAcre males at the age of 2 and 4 months. Scale bars in panels (A and B) equal 500 μm. Images of paired testes from two controls and two TGFBR1-CAAcre mice are shown. Testes from control and TGFBR1-CAAcre mice around 2 months of age were examined for testicular tumor development. Macroscopic and microscopic tumors were found in all TGFBR1-CAAcre males (n = 9) but not controls (n = 10). (C–F) H and E staining of testes from control and TGFBR1-CAAcre males at 2 months of age. Panels (E and F) represent higher magnification images for panels (C and D), respectively. (G–J) Loss of sperm in the epididymis of TGFBR1-CAAcre mice revealed by haematoxylin and eosin staining and immunofluorescence. Antibody directed to ACTA2 (red) was used to label seminiferous tubule structures. Note that controls had abundant sperm (G) and DNA (I) within the epididymis (blue arrows), while TGFBR1-CAAcre males showed a lack of sperm/DNA in the epididymis (H and J). Scale bar is representatively shown in (C) and equals 25 μm (E–H), 50 μm (I and J) and 100 μm (C and D). Figure 2 View largeDownload slide Sustained activation of TGFBR1 in the testis leads to sex cord-stromal tumor formation. (A, B) Development of testicular tumors in TGFBR1-CAAcre males at the age of 2 and 4 months. Scale bars in panels (A and B) equal 500 μm. Images of paired testes from two controls and two TGFBR1-CAAcre mice are shown. Testes from control and TGFBR1-CAAcre mice around 2 months of age were examined for testicular tumor development. Macroscopic and microscopic tumors were found in all TGFBR1-CAAcre males (n = 9) but not controls (n = 10). (C–F) H and E staining of testes from control and TGFBR1-CAAcre males at 2 months of age. Panels (E and F) represent higher magnification images for panels (C and D), respectively. (G–J) Loss of sperm in the epididymis of TGFBR1-CAAcre mice revealed by haematoxylin and eosin staining and immunofluorescence. Antibody directed to ACTA2 (red) was used to label seminiferous tubule structures. Note that controls had abundant sperm (G) and DNA (I) within the epididymis (blue arrows), while TGFBR1-CAAcre males showed a lack of sperm/DNA in the epididymis (H and J). Scale bar is representatively shown in (C) and equals 25 μm (E–H), 50 μm (I and J) and 100 μm (C and D). Disruption of testis development in postnatal TGFBR1-CAAcre males To define a timeline of testicular tumor development in the TGFBR1-CAAcre males, we first performed histological analyses using testes from both control and TGFBR1-CAAcre mice at the age of 1 week, 2 weeks and 1 month. No histological difference was observed in the testes of TGFBR1-CAAcre mice versus controls at 1 week of age (Supplementary Fig. 2A and B). Although the basic histological structure of seminiferous tubules appeared to be maintained in the TGFBR1-CAAcre testes by 2 weeks of age (Supplementary Fig. 2C and D), microscopic lesions were evident within the testes of TGFBR1-CAAcre mice at 1 month of age (Supplementary Fig. 2F and H). Compared with control testes consisting of highly organized seminiferous tubules (Supplementary Fig. 2E and G), loss of germ cells occurred within the seminiferous tubules of TGFBR1-CAAcre testes (Supplementary Fig. 2F and H). Enrichment of Sertoli-like cells was observed in abnormal tubules of TGFBR1-CAAcre mice (Supplementary Fig. 2F). Tumor nodules were readily discernible in TGFBR1-CAAcre testes at this stage (Supplementary Fig. 2H). The histological findings were confirmed by immunohistochemical staining of Y box protein 2 (YBX2, also known as MSY2), a marker for germ cells (Gu et al., 1998) and Ki67 (a marker for cell proliferation). YBX2 was abundantly expressed in germ cells within seminiferous tubules in control testes (Fig. 3A). However, loss of YBX2-positive cells was pronounced in the seminiferous tubules of TGFBR1-CAAcre males at 1 month of age (Fig. 3B). The adverse effect of constitutive activation of TGFBR1 on germ cell production could be observed at 2 weeks (Supplementary Fig. 2I and J). Moreover, the neoplastic tumor foci contained proliferative cells, evidenced by intense staining of Ki67 versus controls (Fig. 3C and D). Enhanced cell proliferation of TGFBR1-CAAcre testes was further supported by upregulation of cyclin D2 (CCND2) protein and transcripts (Fig. 3E and F). Interestingly, double IF demonstrated colocalization of Ki67 and SOX9, a Sertoli cell marker, in 1-month-old TGFBR1-CAAcre testes (yellow/orange labeling; Fig. 3J–L), in sharp contrast to control testes where SOX9 and Ki67 signals did not overlap (Fig. 3G–I). A similar result supporting Sertoli cell proliferation in TGFBR1-CAAcre testes was found using 2-week-old mice (Supplementary Fig. 3). Since Sertoli cells stop proliferating around 2 weeks after birth in mice (Vergouwen et al., 1991), our results indicate that sustained activation of TGFBR1 extends the proliferation of Sertoli cells. As gap junction protein alpha 1 (GJA1), also known as Connexin 43, is implicated in Sertoli cell development and conditional loss of GJA1 in Sertoli cells impedes the differentiation but extends the proliferation of Sertoli cells (Sridharan et al., 2007), we examined the expression of GJA1 in TGFBR1-CAAcre testes to determine whether altered Sertoli cell proliferation was associated with reduced expression of GJA1. A significant change of Gja1 mRNA abundance was not found in the testes of TGFBR1-CAAcre mice at 2 weeks of age (Supplementary Fig. 4A), although localization of GJA1 proteins appeared to be increased in cells within the basal region of the seminiferous tubules (Supplementary Fig. 4B and C). At 1 month of age, mRNA levels of Gja1 were markedly elevated and abundant immunoreactive signals of GJA1 detected in TGFBR1-CAAcre testes compared with controls (Supplementary Fig. 4D–F). These results are in concordance with reports that TGFB upregulates GJA1 expression in human granulosa cells and trophoblast cells (Chen et al., 2015; Cheng et al., 2015) and suggest that altered Sertoli cell proliferation in TGFBR1-CAAcre mice is not a result of deficient production of GJA1. Figure 3 View largeDownload slide Depletion of germ cells and alteration of cell proliferation in the testes of TGFBR1-CAAcre males. (A–D) Immunostaining of YBX2 and Ki67 in the testes of control and TGFBR1-CAAcre mice at 1 month of age. Note the presence of abundant YBX2-positive germ cells within the seminiferous tubules in controls (A). However, loss of YBX2-positive cells was observed in TGFBR1-CAAcre testes (B). Meanwhile, tumor foci were positively stained for Ki67 (D), indicative of cell proliferation. Three mice per group were examined. (E) Western blotting analysis of CCND2 expression in testes of 1-month-old control and TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. (F) Increased expression of Ccnd2 transcripts in TGFBR1-CAAcre testes versus controls using real-time PCR. Data are presented as mean ± SEM. n = 5. ***P < 0.001. (G–L) Immunofluorescence staining of SOX9 and Ki67 using testes from control and TGFBR1-CAAcre males at 1 month of age. Note the presence of double positive cells (yellow/orange) in TGFBR1-CAAcre testes (L) but not controls (I). Scale bar is representatively shown in (A) and equals 25 μm (C, D and G–L) and 50 μm (A and B). Figure 3 View largeDownload slide Depletion of germ cells and alteration of cell proliferation in the testes of TGFBR1-CAAcre males. (A–D) Immunostaining of YBX2 and Ki67 in the testes of control and TGFBR1-CAAcre mice at 1 month of age. Note the presence of abundant YBX2-positive germ cells within the seminiferous tubules in controls (A). However, loss of YBX2-positive cells was observed in TGFBR1-CAAcre testes (B). Meanwhile, tumor foci were positively stained for Ki67 (D), indicative of cell proliferation. Three mice per group were examined. (E) Western blotting analysis of CCND2 expression in testes of 1-month-old control and TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. (F) Increased expression of Ccnd2 transcripts in TGFBR1-CAAcre testes versus controls using real-time PCR. Data are presented as mean ± SEM. n = 5. ***P < 0.001. (G–L) Immunofluorescence staining of SOX9 and Ki67 using testes from control and TGFBR1-CAAcre males at 1 month of age. Note the presence of double positive cells (yellow/orange) in TGFBR1-CAAcre testes (L) but not controls (I). Scale bar is representatively shown in (A) and equals 25 μm (C, D and G–L) and 50 μm (A and B). To further probe the cellular and molecular alterations resulting from constitutive activation of TGFBR1 in the testis, we performed IF using antibodies directed to SOX9 and ACTA2 (Fig. 4). Results showed that ACTA2-labeled seminiferous tubules (green) in the testes of TGFBR1-CAAcre were structurally comparable to those of controls at 2 weeks of age, except for disorganized arrangement of Sertoli cells labeled by SOX9 (red; Fig. 4A–F). In accordance with the histological observation of enrichment of Sertoli-like cells in the dysplastic seminiferous tubules in 1-month-old TGFBR1-CAAcre males (Supplementary Fig. 2F), markedly disorganized arrangement of SOX9-positive cells was found within these abnormal tubules (Fig. 4G–I). This result also provided indirect support for the aforementioned observation of germ cell depletion in TGFBR1-CAAcre testes. In addition, SOX9 staining was low to undetectable in tumor foci/nodules, in contrast to the strong immunoreactive signals of SOX9 observed in adjacent dysplastic tubules (Fig. 4J–L). Therefore, these studies suggest potential loss of SOX9 expression in tubule-like structures/neoplastic nodules during tumor development and progression. Figure 4 View largeDownload slide Immunofluorescence analysis of testicular tumor development in TGFBR1-CAAcre males. (A–L) Immunofluorescence staining of SOX9 and ACTA2 using testes from control and TGFBR1-CAAcre males at the age of 2 weeks, 1 month and 2 months. Note that seminiferous tubules in the testes of TGFBR1-CAAcre males were structurally comparable to those of controls at 2 weeks of age (A–F), except for disorganized arrangement of Sertoli cells. However, at 1 month of age, abnormal seminiferous tubules containing dramatically disorganized arrangement of SOX9-positive cells were found in TGFBR1-CAAcre testes (G–I). Minimal expression of SOX9 was observed in testicular tumor foci/nodules of TGFBR1-CAAcre mice, in sharp contrast to the adjacent abnormal tubules consisting of SOX9-positive cells. Dotted blue lines mark small and large tumor foci/nodules (sTu/lTu). Results of immunofluorescence were verified by immunohistochemical staining of SOX9, in which three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–I) and 80 μm (J–L). Figure 4 View largeDownload slide Immunofluorescence analysis of testicular tumor development in TGFBR1-CAAcre males. (A–L) Immunofluorescence staining of SOX9 and ACTA2 using testes from control and TGFBR1-CAAcre males at the age of 2 weeks, 1 month and 2 months. Note that seminiferous tubules in the testes of TGFBR1-CAAcre males were structurally comparable to those of controls at 2 weeks of age (A–F), except for disorganized arrangement of Sertoli cells. However, at 1 month of age, abnormal seminiferous tubules containing dramatically disorganized arrangement of SOX9-positive cells were found in TGFBR1-CAAcre testes (G–I). Minimal expression of SOX9 was observed in testicular tumor foci/nodules of TGFBR1-CAAcre mice, in sharp contrast to the adjacent abnormal tubules consisting of SOX9-positive cells. Dotted blue lines mark small and large tumor foci/nodules (sTu/lTu). Results of immunofluorescence were verified by immunohistochemical staining of SOX9, in which three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–I) and 80 μm (J–L). Testicular tumors in TGFBR1-CAAcre males express markers of GrCTs As testicular tumors in TGFBR1-CAAcre mice histologically resembled ovarian GrCTs, we examined the expression of several granulosa cell markers that are expressed by GrCTs including inhibin alpha (INHA), forkhead box O1 (FOXO1) and FOXL2 within the testes of TGFBR1-CAAcre males. IHC revealed that testicular tumor nodules in 2-month-old TGFBR1-CAAcre mice were positively stained for INHA (Fig. 5B and C), FOXO1 (Fig. 5E and F) and FOXL2 (Fig. 5H and I) compared with controls (Fig. 5A, D and G). Notably, FOXL2 is a granulosa cell lineage marker expressed in the ovary but not the testis (Wilhelm et al., 2009). To determine the temporal expression of FOXL2 in TGFBR1-CAAcre testes, we examined mice at 1 month of age, when microscopic tumor foci were detectable (Supplementary Fig. 2H). Interestingly, FOXL2 staining was found in some abnormal seminiferous tubules or tumor nodules in TGFBR1-CAAcre testes (Fig. 5K and L), in stark contrast to control testes which did not express FOXL2 (Fig. 5J). Representative negative controls using isotype-matched IgGs are depicted in Supplementary Fig. 5. Supporting the IHC findings, protein bands of FOXL2 were only detected in the testes of TGFBR1-CAAcre mice but not those of controls by western blotting (Fig. 5M). These results indicate that constitutive activation of TGFBR1 in the testis may alter Sertoli cell fate. Figure 5 View largeDownload slide Testicular tumors of TGFBR1-CAAcre males express granulosa cell tumor markers. (A–I) Immunostaining of INHA, FOXO1 and FOXL2 in control and TGFBR1-CAAcre testes at 2 months of age. Note that intensive immunoreactive signals for INHA, FOXO1 and FOXL2 were detected in testicular tumors of TGFBR1-CAAcre males compared with controls (A–I). (J–L) Immunostaining of FOXL2 in control and TGFBR1-CAAcre testes at 1 month of age. Note that FOXL2 was not expressed in control testis (J) but localized to some tumor foci (K and L; red arrows). Panels (C, F, I and L) represent higher magnification images for corresponding panels (B, E, H and K). Three mice per group were examined. Scale bar is representatively shown in (A), and equals 25 μm (A, C, D, F, G, I, J and L) and 100 μm (B, E, H and K). (M) Western blotting analysis of FOXL2 in 1-month-old control and TGFBR1-CAAcre testes. Note that the FOXL2 proteins were only detected in the testes of TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. Figure 5 View largeDownload slide Testicular tumors of TGFBR1-CAAcre males express granulosa cell tumor markers. (A–I) Immunostaining of INHA, FOXO1 and FOXL2 in control and TGFBR1-CAAcre testes at 2 months of age. Note that intensive immunoreactive signals for INHA, FOXO1 and FOXL2 were detected in testicular tumors of TGFBR1-CAAcre males compared with controls (A–I). (J–L) Immunostaining of FOXL2 in control and TGFBR1-CAAcre testes at 1 month of age. Note that FOXL2 was not expressed in control testis (J) but localized to some tumor foci (K and L; red arrows). Panels (C, F, I and L) represent higher magnification images for corresponding panels (B, E, H and K). Three mice per group were examined. Scale bar is representatively shown in (A), and equals 25 μm (A, C, D, F, G, I, J and L) and 100 μm (B, E, H and K). (M) Western blotting analysis of FOXL2 in 1-month-old control and TGFBR1-CAAcre testes. Note that the FOXL2 proteins were only detected in the testes of TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. It has been well established that doublesex and mab-3 related transcription factor 1 (DMRT1) maintains testis identity; and Sertoli cells could be reprogramed to granulosa cells in the testis upon deletion of Dmrt1 due to activation of Foxl2 and other female-promoting genes (Raymond et al., 2000; Matson et al., 2011). In postnatal mouse testis, DMRT1 is expressed in mitotic spermatogonia and Sertoli cells (Matson et al., 2011). To explore whether the aberrant expression of FOXL2 in our mouse model was associated with loss of DMRT1 expression, we performed immunohistochemical analysis of DMRT1 and IF of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre mice. DMRT1 was expressed in the testes from both control and TGFBR1-CAAcre mice at 2 weeks of age (Supplementary Fig. 6). At 1 month of age, expression of FOXL2 in TGFBR1-CAAcre testes could be detected in a subset of DMRT1-negative cells within disorganized tubules (Fig. 6B, E, H and K) or in majority of cells within some tumor nodules containing scattered DMRT1-expressing cells (Fig. 6C, F, I and L). In tumor foci where FOXL2-positive cells prevailed, no DMRT1 was detected (data not shown). In contrast, immunoreactive signals for FOXL2 were not detected in control testes (Fig. 6A, D, G and J). These results provide circumstantial evidence supporting a potential link between loss of DMRT1 and altered Sertoli cell fate in TGFBR1-CAAcre testes. Figure 6 View largeDownload slide Immunofluorescence analysis of DMRT1 and FOXL2 distribution during testicular tumor development. (A–L) Immunofluorescence staining of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre males at the age of 1 month. Note that while DMRT1 was readily detectable in control testes, FOXL2 specific signals were not observed as expected (A, D, G and J). Scattered FOXL2-postive cells were found within some abnormal tubules in TGFBR1-CAAcre testes (B, E, H and K). Panels (C, F, I and L) show a small tumor nodule in which many tumor cells were positive for FOXL2 but lacked DMRT1 expression. Three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–L). Figure 6 View largeDownload slide Immunofluorescence analysis of DMRT1 and FOXL2 distribution during testicular tumor development. (A–L) Immunofluorescence staining of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre males at the age of 1 month. Note that while DMRT1 was readily detectable in control testes, FOXL2 specific signals were not observed as expected (A, D, G and J). Scattered FOXL2-postive cells were found within some abnormal tubules in TGFBR1-CAAcre testes (B, E, H and K). Panels (C, F, I and L) show a small tumor nodule in which many tumor cells were positive for FOXL2 but lacked DMRT1 expression. Three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–L). As mentioned above, tumor nodules in TGFBR1-CAAcre testes expressed minimal SOX9. However, loss of SOX9 may also occur during the development of Sertoli cell tumors (Chang et al., 2009). To further determine whether tumor cells expressed other Sertoli cell markers, we examined Wilms tumor 1 (WT1), a Sertoli cell expressed protein that is detectable in mouse Sertoli cell tumors (Chang et al., 2009). The results showed that WT1 was expressed in Sertoli cells of control testes and abnormal seminiferous tubules (Fig. 7A-D), but not the parenchyma of the tumors at 2 months of age (Fig. 7C and D). In addition, to elucidate whether the tumors expressed Leydig cell markers, we analyzed the expression of CYP17A1 (Liu et al., 2016). Results showed that CYP17A1 was localized to Leydig cells in control testes (Fig. 7E and F). However, only scattered CYP17A1-postive cells were observed in testicular tumors of TGFBR1-CAAcre mice (Fig. 7G and H), supporting the finding that tumors were not derived from Leydig cells. These results collectively indicate that constitutive activation of TGFBR1 in the testis leads to development of sex cord-stromal tumors resembling GrCTs. Figure 7 View largeDownload slide Analysis of Sertoli cell and Leydig cell markers in tumors from TGFBR1-CAAcre males. (A–H) Immunostaining of WT1 (A–D) and CYP17A1 (E–H) in control and TGFBR1-CAAcre testes at 2 months of age. Panels (B, D, F and H) represent higher magnification images for corresponding panels (A, C, E and G). Note that tumor nodules (Tu) were not major sites for the expression of WT1 (C and D) or CYP17A1 (G and H) in TGFBR1-CAAcre mice. Strong immunoreactive signals of WT1 were observed in Sertoli cells of controls (A and B) and abnormal seminiferous tubules of TGFBR1-CAAcre mice (C and D; blue arrows). In contrast, expression of CYP17A1 was mainly found in Leydig cells of control testes (E and F) and inter-nodular areas of testicular tumors (blue arrows; G and H). Dotted yellow lines in panels (G and H) mark the borders of tumor nodules. Testis samples from three mice per group were examined. Scale bar is representatively shown in (A) and equals 25 μm (B, D, F and H) and 100 μm (A, C, E and G). Figure 7 View largeDownload slide Analysis of Sertoli cell and Leydig cell markers in tumors from TGFBR1-CAAcre males. (A–H) Immunostaining of WT1 (A–D) and CYP17A1 (E–H) in control and TGFBR1-CAAcre testes at 2 months of age. Panels (B, D, F and H) represent higher magnification images for corresponding panels (A, C, E and G). Note that tumor nodules (Tu) were not major sites for the expression of WT1 (C and D) or CYP17A1 (G and H) in TGFBR1-CAAcre mice. Strong immunoreactive signals of WT1 were observed in Sertoli cells of controls (A and B) and abnormal seminiferous tubules of TGFBR1-CAAcre mice (C and D; blue arrows). In contrast, expression of CYP17A1 was mainly found in Leydig cells of control testes (E and F) and inter-nodular areas of testicular tumors (blue arrows; G and H). Dotted yellow lines in panels (G and H) mark the borders of tumor nodules. Testis samples from three mice per group were examined. Scale bar is representatively shown in (A) and equals 25 μm (B, D, F and H) and 100 μm (A, C, E and G). WNT/CTNNB1 signaling is active in GrCTs resulting from TGFBR1 overactivation Misregulation of WNT/beta-catenin (CTNNB1) signaling is implicated in tumorigenesis of GrCTs in multiple species including humans, horses and mice (Boerboom et al., 2005; Boyer et al., 2009). To understand how constitutive activation of TGFBR1 promoted the development of testicular tumors in TGFBR1-CAAcre mice, we examined the expression of CTNNB1 known to be involved in GrCT development (Boerboom et al., 2005; Boyer et al., 2009; Richards et al., 2012). Herein, we demonstrated that the levels of CTNNB1 were increased in testicular tumor tissues of TGFBR1-CAAcre mice at 1 month of age compared with controls (Fig. 8A and B). IHC confirmed the results of western blotting by revealing extensive immunoreactive signals of CTNNB1 in testicular tumors of TGFBR1-CAAcre males versus controls (Fig. 8C–E). Moreover, variable degrees of nuclear/cytoplasmic staining of CTNNB1 were observed in testicular tumor tissues (Fig. 8E). These results suggest that dysregulation of WNT/CTNNB1 signaling is potentially linked to GrCT development in TGFBR1-CAAcre mice. Figure 8 View largeDownload slide WNT/CTNNB1 signaling is active in GrCTs resulting from overactivation of TGFBR1. (A) Western blotting analysis of CTNNB1 expression in control and TGFBR1-CAAcre testes of 1-month-old males. ACTB was included as an internal control. n = 4. (B) Quantification of the western blotting result shown in panel (A). Image J was used to calculate the ratio of CTNNB1 to ACTB for each sample. Results are presented as percentage, in which relative protein levels of control group are set to 100%. n = 4. Data are presented as mean ± SEM. ***P < 0.001. (C–E) Immunostaining of CTNNB1 in tumor nodules of testes from TGFBR1-CAAcre mice along with controls. Panel (E) represents a higher magnification image of panel (D). Three mice per group were examined. Scale bar is representatively shown in (C), and equals 10 μm (E) and 50 μm (C and D). Figure 8 View largeDownload slide WNT/CTNNB1 signaling is active in GrCTs resulting from overactivation of TGFBR1. (A) Western blotting analysis of CTNNB1 expression in control and TGFBR1-CAAcre testes of 1-month-old males. ACTB was included as an internal control. n = 4. (B) Quantification of the western blotting result shown in panel (A). Image J was used to calculate the ratio of CTNNB1 to ACTB for each sample. Results are presented as percentage, in which relative protein levels of control group are set to 100%. n = 4. Data are presented as mean ± SEM. ***P < 0.001. (C–E) Immunostaining of CTNNB1 in tumor nodules of testes from TGFBR1-CAAcre mice along with controls. Panel (E) represents a higher magnification image of panel (D). Three mice per group were examined. Scale bar is representatively shown in (C), and equals 10 μm (E) and 50 μm (C and D). Discussion GrCTs may arise from the testis with low incidence (Jimenez-Quintero et al., 1993; Wang et al., 2002). The adult type GrCTs of the testis are extremely rare and occur after puberty, whereas the juvenile type GrCTs account for approximately 1–4% of total prepubertal testicular tumors and the majority of them develop within the first 6 months (Lawrence et al., 1985; Shukla et al., 2004; Dudani et al., 2008). While adult GrCTs are associated with somatic mutation (Lima et al., 2012), the juvenile type GrCTs may be associated with chromosomal abnormalities and gonadal dysgenesis (Young et al., 1985; Kos et al., 2005). Animal models are useful to investigate the pathogenesis of this disease. Several mouse models are available for sex cord-stromal tumors, including but not limited to, mice with targeted deletion of Inha (Matzuk et al., 1992), Smad1/5 (Pangas et al., 2008) and BMP type 1 receptors (Edson et al., 2010), mice with overexpression of R-spondin 1 (Rspo1) (De Cian et al., 2017), mice containing KRAS activation and dominant-stable CTNNB1 (Richards et al., 2012), and mice with dominant-stable CTNNB1 or both dominant-stable CTNNB1 and Pten inactivation (Boerboom et al., 2005; Boyer et al., 2009; Richards et al., 2012). Although it is challenging to faithfully model human GrCTs, mouse models have helped to gain significant insights into the pathobiology and signaling cascades of GrCT development. We previously reported that overactivation of TGFBR1 using Amhr2-Cre to target mouse granulosa cells provokes the development of ovarian GrCTs (Gao et al., 2016). Since Amhr2-Cre has been successfully used to conditionally target/overexpress genes in somatic cells of the gonad in both sexes (Jamin et al., 2002; Jeyasuria et al., 2004; Boyer et al., 2008, 2009; Pangas et al., 2008; Papaioannou et al., 2009; Tanwar et al., 2010; Kyronlahti et al., 2011), we determined the phenotypic consequence of constitutive activation of TGFBR1 using Amhr2-Cre in the testis. Formation of microscopic abnormalities observed in testicular tubules of TGFBR1-CAAcre males before puberty suggests balanced TGFB signaling is essential for testis development. Development of testicular tumors was accompanied by increased cell proliferation and depletion of germ cells. It was reported that TGFB superfamily signaling is active in the mouse testis, and the dosage of activin A maintains balanced numbers of Sertoli cells and germ cells (Mendis et al., 2011). As activin and TGFB signaling shares the same downstream SMADs, SMAD2/3, it appears possible that loss of germ cells in TGFBR1-CAAcre testes may be associated with altered SMAD2/3 signaling activity. To strengthen the conclusion that testicular tumors resembled GrCTs, we examined the spatiotemporal localization of several protein markers including SOX9, CYP17A1 and FOXL2 for the respective Sertoli cells, Leydig cells and granulosa cells. During the development of testicular GrCTs, we observed accumulation of Sertoli cells within seminiferous tubules, expression of Ki67 in Sertoli cells and reduction of SOX9 expression but gain of FOXL2 expression in tumor nodules. The origin of testicular GrCT is poorly defined. However, based on the aforementioned findings and the observation that testicular GrCTs arose within seminiferous tubules, it is tempting to postulate that Sertoli cells might transdifferentiate into granulosa tumor cells. Moreover, neither the histological feature nor the expression pattern of CYP17A1 supported that these tumors were Leydig cell tumors. Of note, the most common cell type of Leydig cell tumors is medium-sized hexagonal cell that has eosinophilic/vacuolar cytoplasm, irregular round/oval shaped nucleus and recognizable cell border (Mostofi, 1973). It is unclear whether gain of FOXL2 expression in TGFBR1-CAAcre testes is a cause or consequence of testicular GrCT development. It has been suggested that both granulosa cells and Sertoli cells share the same progenitors (Albrecht and Eicher, 2001), and granulosa cells can transdifferentiate into Sertoli cells in adult female mice lacking FOXL2 (Uhlenhaut et al., 2009). Conversely, it is logic to speculate that the fate of Sertoli cells may be altered to resemble granulosa cells when FOXL2 is misregulated. Indeed, Sertoli cells could be reprogramed to granulosa cells upon conditional loss of DMRT1 due to activation of Foxl2 and other female-promoting genes (Matson et al., 2011). Another study showed that constitutive activation of CTNNB1 in Sertoli cells promotes FOXL2 expression ectopically in the testis, resulting in the transformation of Sertoli cells into granulosa-like cells (Li et al., 2017). Moreover, upregulation of FOXL2 in testicular GrCTs was found in an independent mouse model, where CTNNB1 and AKT signaling was genetically enhanced (Boyer et al., 2009). Our result is also in agreement with the discovery that aberrant expression of FOXL2 links to the development of human juvenile GrCTs of the testis (Kalfa et al., 2008). The finding of gain of FOXL2-expressing cell population but loss of cells positive for DMRT1, a key protein that maintains Sertoli cell identify (Raymond et al., 2000; Matson et al., 2011) in testicular tubules of TGFBR1-CAAcre males during tumor development highlights the importance of TGFB pathway in maintaining the balance of ‘male-female’ characteristics in the gonad. In view of the early onset of tumorigenesis, the potential of TGFBR1-CAAcre mice as a model for the juvenile GrCTs should be further explored. Our current findings support an equally important role of TGFB signaling in the development of GrCTs in both male and female gonads (Gao et al., 2016). Somatic mutation of FOXL2 has been identified as a hallmark of ovarian adult GrCTs (Shah et al., 2009). FOXL2 mutation was also revealed in testicular GrCTs (Lima et al., 2012). Moreover, activation of TGFB signaling appears to be associated with GrCT development (Pangas et al., 2008; Middlebrook et al., 2009; Edson et al., 2010; Liu et al., 2015; Kim et al., 2016). Interestingly, FOXL2 mutation may impact activin/TGFB signaling (Rosario et al., 2012; Cheng et al., 2014; Fang et al., 2016). Previous studies indicated that unopposed activin signaling resulting from loss of inhibin promotes testicular tumor development partially through SMAD3, a critical regulator of sex cord-stromal tumor development (Li et al., 2007). Thus, SMAD mediated signaling likely contributes to testicular tumor development in our model system, as is supported by increased phospho-SMAD2/3 levels in the testes of TGFBR1-CAAcre males. TGFB signaling is complex and known to interact with other pathways. WNT pathway plays fundamental roles in normal cell functions and the development of multiple types of cancers (Giles et al., 2003). Activation of the WNT/CTNNB1 pathway is involved in the pathogenesis of GrCTs; mice expressing a dominant-stable CTNNB1 mutant in ovarian granulosa cells develop GrCTs (Boerboom et al., 2005). Simultaneous dysregulation of CTNNB1 and phosphatidylinositol 3-kinase (PI3K)/AKT pathways causes testicular GrCT formation (Boyer et al., 2009). Increased expression of phospho-AKT was found in both ovarian tumors (Gao et al., 2016) and testicular tumors (unpublished observation) from TGFBR1-CAAcre mice. Moreover, the R-Spondin family proteins activate WNT signaling pathway (Kim et al., 2008). Recent studies discovered that amplification of RSPO1 signaling leads to GrCT formation through altering the fate and properties of granulosa cells (De Cian et al., 2017). These studies collectively indicate that enhanced WNT signaling promotes GrCT development. Supporting interactions between TGFB and WNT signaling (Akhmetshina et al., 2012), we demonstrated that CTNNB1 expression was upregulated in testicular tumors. Moreover, WNT signaling appears to be active in gonadal tumors resulting from constitutive activation of TGFBR1. Of note, CTNNB1 signaling is suppressed during normal testis development, and WT1 negatively regulates CTNNB1 signaling (Chang et al., 2008). Thus, lack of WT1 in tumor nodules of TGFBR1-CAAcre testes may facilitate the expression of CTNNB1. While our studies uncovered a role of TGFBR1 activation in GrCT development, a potential regulatory mechanism of SMAD2/3-CTNNB1 activation in the pathogenesis of human GrCTs warrants further investigation. Illuminating the signaling network that underpins GrCT development will provide opportunities for designing new therapeutic interventions. In summary, the current study created a new mouse model resembling testicular GrCTs. This mouse model may prove to be a useful addition to the mouse genetics toolkit for testicular GrCT research. Supplementary data Supplementary data are available at Molecular Human Reproduction Online. Acknowledgements The authors thank Dr David Zarkower for kindly providing the DMRT1 antibody. We also thank the staff from comparative medicine program for animal care. Authors’ roles X.F., N.N. and Y.G. conducted the experiments, collected the data and analyzed the results. D.F.V. and L.B. created and provided mice containing the latent constitutively active TGFBR1. Q.L. contributed to study conception and supervised the project. X.F. and Q.L. drafted the manuscript. All authors critically revised and/or commented on the manuscript and approved the final version of the manuscript and its submission. Conflict of interest None declared. Funding National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University (to Q.L.). References Akhmetshina A , Palumbo K , Dees C , Bergmann C , Venalis P , Zerr P , Horn A , Kireva T , Beyer C , Zwerina J et al. . Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis . Nat Commun 2012 ; 3 : 735 . Akhurst RJ , Hata A . Targeting the TGF beta signalling pathway in disease . Nat Rev Drug Discov 2012 ; 11 : 790 – 811 . Albrecht KH , Eicher EM . Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor . Dev Biol 2001 ; 240 : 92 – 107 . Bartholin L , Cyprian FS , Vincent D , Garcia CN , Martel S , Horvat B , Berthet C , Goddard-Leon S , Treilleux I , Rimokh R et al. . 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Human Reproduction Oxford University Press

A novel mouse model of testicular granulosa cell tumors

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Oxford University Press
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© The Author 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Abstract

Abstract STUDY QUESTION What is the role of dysregulated transforming growth factor beta (TGFB) signaling in the development of sex cord-stromal tumors in the testis? SUMMARY ANSWER Overactivation of TGFB signaling results in the development of testicular tumors resembling granulosa cell tumors (GrCTs). WHAT IS KNOWN ALREADY In an earlier study, we demonstrated that constitutively active TGFB receptor 1 (TGFBR1) in ovarian somatic cells promotes the development of ovarian GrCTs. However, the consequence of dysregulation of TGFB signaling in the pathobiology of the testis, remains poorly defined. STUDY DESIGN, SIZE, DURATION To identify the impact of dysregulation of TGFB signaling on the testis, we generated mice with constitutive activation of TGFBR1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase. The effect of constitutively active TGFBR1 on testis development and the timeline of testicular tumor formation were examined. We further investigated the molecular features of testicular tumors and determined the expression of beta-catenin (CTNNB1) known to be involved in testicular GrCT development. PARTICIPANTS/MATERIALS, SETTING, METHODS Male mice with constitutive activation of TGFBR1 were examined at various developmental stages (i.e. from 1 week up to 6 months) along with controls. Testis samples were collected and processed for histological and molecular analyses, including haematoxylin and eosin (H and E) staining, real-time PCR, immunohistochemistry, immunofluorescence and western blotting. Immunostaining/immunoblotting and real-time PCR experiments were performed using at least three animals per genotype. Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tail t-test and reported when P value is <0.05. MAIN RESULTS AND THE ROLE OF CHANCE Mice harboring constitutively active TGFBR1 in the testes developed tumors resembling testicular GrCTs, a rare type of tumors in the testis. The formation of testicular tumors led to altered cell proliferation, loss of germ cells and defective spermatogenesis. Immunohistochemically, these tumors were positive for inhibin alpha (INHA), forkhead box O1 (FOXO1), and more importantly, forkhead box L2 (FOXL2), a protein specifically expressed in the ovary and required for normal granulosa cell differentiation and function. Consistent with the immunohistochemical findings, FOXL2 proteins were only detectable in testes of TGFBR1-CAAcre mice but not those of controls by western blotting, suggesting potential alteration of Sertoli cell fate. To explore mechanisms underlying the tumor-promoting effect of TGFBR1 overactivation, we examined the expression of CTNNB1. The results revealed increased expression of CTNNB1 in testicular tumors in TGFBR1-CAAcre mice. Collectively, this study uncovered tumorigenic function of enhanced TGFB signaling in the testis. LARGE-SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This study was performed using mice, and the direct relevance of the experimental paradigm and findings to human testicular GrCTs awaits further investigation. Of note, constitutive activation of TGFBR1 was employed to enhance TGFB/SMAD signaling activity and may not be interpreted as the genetic cause of the disease. WIDER IMPLICATIONS OF THE FINDINGS This mouse model may prove to be a useful addition to the mouse genetics toolkit for GrCT research. Our finding that dysregulation of TGFB signaling results in the development of testicular GrCTs supports a common origin between Sertoli cells and granulosa cells, and highlights the paramount importance of balanced TGFB signaling in reproduction and development. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University awarded to Q.L. The authors declare no competing interest. testicular granulosa cell tumor, gonad, TGFBR1, SMAD2/3, mouse model Introduction Transforming growth factor beta (TGFB) superfamily members, which are structurally related proteins, regulate a broad range of cellular properties, including, but not limited to growth, differentiation, migration and apoptosis (Massague, 1998; Massague et al., 2000). Because of their fundamental roles in these cellular events, TGFB superfamily members exhibit diverse physiological and pathological functions in multiple organ systems. In the reproductive system, recent studies, particularly those using genetics and functional genomics approaches uncovered many previously unrecognized roles of this growth factor family in reproductive development and cancer (Massague et al., 2000; Padua and Massague, 2009; Li, 2014; Fang et al., 2016). TGFB signaling is known to be a double-edged sword in tumor development, exhibiting tumorigenic or tumor suppressive effects at different tumor stages (Massague, 2008). To initiate signal transduction, TGFB ligands recruit type II and type I receptor complexes and activate canonical and/or non-canonical pathways depending on the signaling context (Akhurst and Hata, 2012). In general, TGFB ligands signal through TGFBR1/TGFBR2-SMAD2/3 axis, whereas bone morphogenetic proteins (BMPs) activate BMP type II and type I receptors and the SMAD1/5/9 signaling branch. SMAD4 is a common SMAD utilized by both TGFB and BMP signaling to access gene regulatory apparatus within the nuclear compartment (Fang et al., 2016). Approximately 95% of malignant testicular tumors belong to germ cell tumors, comprising seminomas and nonseminomas (Motzer et al., 2012). Sex cord-stromal tumors (3–5%) of the testis include Leydig cell tumor, Sertoli cell tumor, granulosa cell tumor (GrCT), mixed sex cord-stromal tumor and unclassified sex cord-stromal tumor (Moch et al., 2016). Testicular GrCTs are rare tumors (Mohapatra et al., 2016). The formation of testicular GrCTs has been reported in several mouse models (Matzuk et al., 1992; Boyer et al., 2009; Richards et al., 2012). The pathogenesis of GrCTs is poorly understood in both genders, due in part to the limited access to human GrCT specimens. A breakthrough has been recently made toward understanding the etiology of ovarian GrCTs. Forkhead box L2 (FOXL2) is an ovary-expressed gene essential for granulosa cell differentiation (Uhlenhaut et al., 2009). It was discovered that the vast majority of adult type GrCTs in the ovary bear a missense mutation of FOXL2 (402 C→G; C134W) (Shah et al., 2009). This mutation was also found in testicular GrCTs of males (Lima et al., 2012), indicating the importance of FOXL2 mutation in the pathogenesis of GrCTs. However, how FOXL2 mutation activates tumorigenic cascade within granulosa cells is not known. Interestingly, experimental evidence supports a potential link between FOXL2 mutation and alteration of TGFB/activin signaling in GrCT development (Rosario et al., 2012; Cheng et al., 2014; Fang et al., 2016). TGFB superfamily signaling is involved in the development of GrCTs (Middlebrook et al., 2009). In an earlier study, we created a mouse model harboring constitutively active TGFB receptor 1 (TGFBR1) using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase (Gao et al., 2016). Amhr2-Cre has been widely used to target genes in granulosa and theca cells of the ovary, myometrial and endometrial cells of the uterus (Li et al., 2008, 2011; Ren et al., 2009; Wang et al., 2016), and Sertoli cells and Leydig cells of the testis (Boyer et al., 2008). Expression of Amhr2-Cre has been detected as early as 11.5 days post-coitum in the urogenital ridges of both males and females, and the Cre activity is present in the somatic cells of gonads and the mesenchyme of Mullerian ducts (Jamin et al., 2002). To generate constitutively active TGFBR1, three missense mutations in the GS domain were made to activate the TGFBR1 kinase (T204D) and block the binding of the TGFBR1 inhibitor, FKBP12 (L193A/P194A) (Bartholin et al., 2008). A LoxP-flanked stop sequence was engineered to direct the expression of constitutively active TGFBR1 to Cre-expressing cells/tissues. Overactivation of TGFBR1 in the ovary results in formation of ovarian tumors reminiscent of GrCTs (Gao et al., 2016). Here, we report that male mice with constitutive activation of TGFBR1 in the testes developed tumors resembling testicular GrCTs. This study developed a useful mouse model for testicular GrCTs. Materials and Methods Ethics statement Animal use protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University. All animals were handled in accordance with the guidelines by the IACUC of Texas A&M University. Animals and tissue collection Mice harboring a latent constitutively active TGFBR1 (TGFBR1CA) were created as described (Bartholin et al., 2008; Vincent et al., 2010). The TGFBR1CA flox/flox mice were crossed to Amhr2-Cre line (Jamin et al., 2002) to generate mice containing constitutively active TGFBR1 in the testes (TGFBR1CA flox/+; Amhr2-Cre). These mice were termed TGFBR1-CAAcre. Mice were on a mixed C57BL/6/129 background and maintained by controlled light/dark cycles with free access to food and water. Testis samples were collected from both control and TGFBR1-CAAcre males during developmental stages for histological and immunohistochemical analyses described below. Genotyping was performed using DNA extracted from mouse tails described previously (Bartholin et al., 2008; Gao et al., 2016). PCR products were separated on 1% agarose gels containing ethidium bromide and digital images captured using a VWR Gel Imager. Histology Testis samples from control and experimental mice were fixed in 10% (v/v) neutral buffered formalin and/or Bouin’s solution for histology and immunostaining. Tissue samples were processed and embedded using the histology core lab in the Department of Veterinary Integrative Biosciences at Texas A&M University. Haematoxylin and eosin (H and E) staining was performed using standard procedures. Immunohistochemistry and immunofluorescence Immunohistochemistry (IHC) and Immunofluorescence (IF) were performed as described elsewhere (Gao et al., 2015, 2016). In brief, paraffin sections (5 μm) were deparaffinized in xylene, rehydrated in graded alcohol and boiled in 10 mM citrate buffer (pH 6.0) for 20 min to retrieve the antigen. After antigen retrieval, sections were treated with 0.3% H2O2 for 30 min to quench endogenous peroxidase activity. This step was only needed for IHC. Then, sections were sequentially incubated with non-immune serum (for IHC) or bovine serum albumin (for IF), primary antibodies with indicated dilutions (Table 1) overnight at 4°C and secondary antibodies conjugated with horseradish peroxidase (HRP; 1:300; for IHC) or Alexa Fluor 488/594 (1:400; for IF). Intervening washes were performed using Tris-buffered saline (TBS; pH 7.4). Following secondary antibody incubation, an avidin-biotin complex (ABC; Vector Laboratories, USA) was applied to amplify immunoreactive signals, which were developed using NovaRed substrate (Vector Laboratories; USA; for IHC). The sections were counterstained using haematoxylin, mounted with Permount and examined under BX43F microscope (Olympus; USA). For IF, sections were directly mounted with ProLong Gold Slowfade media containing 4′, 6-diamidino-2-phenylindole (DAPI; Catalog number: P36931; Invitrogen, USA) after incubation with secondary antibody. Fluorescence signals were visualized using a IX73 microscope (Olympus, USA) interfaced with an XM10 CCD camera and cellSens Software. Isotype-matched IgG controls were included to determine the staining background of the experiment. Table I Primary antibodies for immunostaining and western blotting analyses. Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Table I Primary antibodies for immunostaining and western blotting analyses. Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Name Manufacturer Catalog no. Host IHC IF WB ACTA2 Abcam, USA Ab76549 mouse 1:2000 YBX2 Abcam, USA Ab33164 rabbit 1:500 Ki67 Abcam, USA Ab16667 rabbit 1:200 Ki67 BD Biosciences 550 609 mouse 1:20 DMRT1 Obtained from Dr David Zarkower N/A rabbit 1:400 GJA1 Cell Signaling Technology, USA 3512 rabbit 1:100 SOX9 EMD Millipore, USA Ab5535 rabbit 1:2000 1:2000 WT1 Abcam, USA Ab89901 rabbit 1:200 INHA Bio-Rad Laboratories, USA MCA951ST mouse 1:300 FOXL2 Abcam, USA Ab5096 goat 1:1500 1:1000 FOXO1 Cell Signaling Technology, USA 2880 rabbit 1:800 CTNNB1 Cell Signaling Technology, USA 8480 rabbit 1:100 1:1000 CTNNB1 BD, USA 610 153 Mouse 1:100 CYP17A1 Santa Cruz Biotechnology, USA SC-46 081 Goat 1:1500 pSMAD2 Cell Signaling Technology, USA 3101s rabbit 1:1000 pSMAD2 EMD Millipore, USA Ab3849I rabbit 1:1000 pSMAD3 Abcam, USA Ab52903 rabbit 1:1000 SMAD2 Cell Signaling Technology, USA 5339 rabbit 1:1000 SMAD3 Abcam, USA Ab28379 rabbit 1:1000 HA Roche, Germany 12 013 819 001 rat 1:500 GAPDH Cell Signaling Technology, USA 2118 Rabbit 1:1000 ACTB Sigma-Aldrich, USA A3854 mouse 1:100 000 Western blotting Western blotting was conducted as described (Li et al., 2008). Protein lysates from mouse testis tissues were prepared using radioimmunoprecipitation assay buffer containing both proteinase (Catalog number: 04 693 116 001; MilliporeSigma, USA) and phosphatase inhibitors (Catalog number: 4 906 837 001; MilliporeSigma, USA). Protein quantification was performed using bicinchoninic acid reagent purchased from Thermo Scientific (USA). Approximately 30 μg of proteins were loaded on each lane and separated by electrophoresis using 12% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, USA). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories; USA), which were subsequently incubated with primary antibodies at 4°C overnight (Table 1). After being washed thoroughly, membranes were further incubated with HRP-conjugated anti-rabbit/rat/goat secondary antibodies (1:20 000; Jackson ImmunoResearch, USA). Immunoreactive signals were developed using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, USA). Signals were visualized and captured using Kodak Image Station 4000 mm PRO digital scanner or Bio-Rad ChemiDoc MP Imaging System. Membranes were stripped and reprobed with antibody directed to beta-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). Quantification of western blotting results was performed using NIH image J (version 1.51e). RNA preparation and reverse transcription-real-time PCR RNA samples were isolated from testes of control and TGFBR1-CAAcre males using RNeasy Mini Kit (Qiagen, USA) based on instructions provided by the supplier. Potential genomic DNA contamination was eliminated by inclusion of an additional on-column DNase digestion step. Concentrations of the RNA preparations were determined by using NanoDrop Spectrophotometer ND 1000 (NanoDrop Technologies, USA). RNA samples were then subjected to reverse transcription for cDNA synthesis (Gao et al., 2014). Real-time PCR was performed using a CFX Connect Real-time PCR Detection System (Bio-Rad Laboratories; USA). The 10 μl reaction system contained iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories; USA) or TaqMan Universal PCR Master Mix, cDNA and oligo primers (Gao et al., 2016) or the Taqman probe. Assays were performed in duplicates for each sample and a mean cycle threshold (CT) value was calculated. Primers for Ccnd2 were 5′-GAGTGGGAACTGGTAGTGTTG-3′ (forward) and 5′-CGCACAGAGCGATGAAGGT-3′ (reverse) (PrimerBank ID 6 753 310a1) (Spandidos et al., 2008). Gja1 expression was analyzed using Taqman probe (Assay ID: Mm01179639_s1). Ribosomal protein L19 (Rpl19) was used as an internal control to normalize gene expression (Livak and Schmittgen, 2001; Gao et al., 2014). A Taqman probe for Rpl19 (Assay ID: Mm02601633_g1) was included in Taqman gene expression assays. Statistical analysis Comparisons of means between two groups were made using unpaired two-tail t-test. Data are presented as mean ± SEM. Statistical significance was defined when P value is <0.05 and marked as *P < 0.05, **P < 0.01 and ***P < 0.001. Results Generation of mice with constitutively active TGFBR1 in testes using Amhr2-Cre recombinase We previously reported that constitutive activation of TGFBR1 in the ovary using Amhr2-Cre recombinase leads to the development of ovarian sex cord-stromal tumors reminiscent of GrCTs (Gao et al., 2016). Because Amhr2-Cre is also expressed in Sertoli and Leydig cells of the testis (Boyer et al., 2008), we examined potential effects of overactivation of TGFBR1 on the development and function of the testis by generating a mouse model harboring constitutively active TGFBR1 in the testis (Fig. 1A). As expected, TGFBR1CA proteins were readily detectable in the testes of TGFBR1-CAAcre males but not controls by western blotting using anti-hemagglutinin (HA) antibody (Fig. 1B). These findings support that Amhr2-Cre drives the expression of TGFBR1CA in mouse testes. TGFBR1 phosphorylates and activates SMAD2/3 signal transducers. To verify activation of TGFB signaling in TGFBR1-CAAcre testes, we performed western blotting to determine the levels of phospho-SMAD2/3 in the testes of TGFBR1-CAAcre males. Results showed that the levels of both phospho-SMAD2 and phospho-SMAD3 were elevated in TGFBR1-CAAcre testes compared with controls (Fig. 1B). SMAD2, SMAD3 and beta-actin (ACTB) were included as controls (Fig. 1B). Figure 1 View largeDownload slide Constitutive activation of TGFBR1 in mouse testes. (A) A diagram depicting the generation of mice with constitutive activation of TGFBR1 using Amhr2-Cre recombinase. (B) Western blotting analysis of hemagglutinin (HA)-tagged TGFBR1CA, phospho-SMAD2 and phospho-SMAD3 in the testes of control and TGFBR1-CA male mice at the age of 2 weeks. Each lane represents an independent testis sample. n = 4. SMAD2, SMAD3 and ACTB were included as controls. Figure 1 View largeDownload slide Constitutive activation of TGFBR1 in mouse testes. (A) A diagram depicting the generation of mice with constitutive activation of TGFBR1 using Amhr2-Cre recombinase. (B) Western blotting analysis of hemagglutinin (HA)-tagged TGFBR1CA, phospho-SMAD2 and phospho-SMAD3 in the testes of control and TGFBR1-CA male mice at the age of 2 weeks. Each lane represents an independent testis sample. n = 4. SMAD2, SMAD3 and ACTB were included as controls. TGFBR1-CAAcre males develop testicular tumors with impaired spermatogenesis Sterility was observed in TGFBR1-CAAcre male mice when they were used as breeders. To determine the cause of sterility, we performed macroscopic and histological analyses of testes and epididymides of control and TGFBR1-CAAcre males. Gross testicular tumors with hemorrhagic foci were observed in TGFBR1-CAAcre mice around 2 months of age with full penetrance (Fig. 2A). In contrast, no tumors were observed in control mice (Fig. 2A). Testicular tumors progressed with age (Fig. 2B). Histological analysis of 2-month-old testes showed that while control mice had organized seminiferous tubules (Fig. 2C and E), TGFBR1-CAAcre males developed testicular tumors resembling GrCTs (Fig. 2D and F), which were observed in the ovary of TGFBR1-CAAcre female mice (Gao et al., 2016). Microscopically, seminiferous tubules were displaced by circumscribed tumor nests containing poorly differentiated cells or granulosa-like cells (Fig. 2D and F and Supplementary Fig. 1B and D). Remnant seminiferous tubules lacking mature germ cells which were observed in control testes could be found near the edge of TGFBR1-CAAcre testes (Supplementary Fig. 1A–D). Both histological results and IF analyses using antibody directed to smooth muscle actin alpha (ACTA2) to mark the structure of epididymis demonstrated lack of sperm in the epididymis of TGFBR1-CAAcre males (Fig. 2H and J), and this was in sharp contrast to wild type controls where abundant sperm/sperm DNA could be found within the epididymis (Fig. 2G and I). Analysis of testes from 5–6-month-old TGFBR1-CAAcre males demonstrated development of more hemorrhagic tumors (Supplementary Fig. 1F and H), in contrast to control testes containing morphologically normal seminiferous tubules (Supplementary Fig. 1E and G). Thus, testicular tumors resulting from constitutive activation of TGFBR1 are detrimental to normal testis development and spermatogenesis. Figure 2 View largeDownload slide Sustained activation of TGFBR1 in the testis leads to sex cord-stromal tumor formation. (A, B) Development of testicular tumors in TGFBR1-CAAcre males at the age of 2 and 4 months. Scale bars in panels (A and B) equal 500 μm. Images of paired testes from two controls and two TGFBR1-CAAcre mice are shown. Testes from control and TGFBR1-CAAcre mice around 2 months of age were examined for testicular tumor development. Macroscopic and microscopic tumors were found in all TGFBR1-CAAcre males (n = 9) but not controls (n = 10). (C–F) H and E staining of testes from control and TGFBR1-CAAcre males at 2 months of age. Panels (E and F) represent higher magnification images for panels (C and D), respectively. (G–J) Loss of sperm in the epididymis of TGFBR1-CAAcre mice revealed by haematoxylin and eosin staining and immunofluorescence. Antibody directed to ACTA2 (red) was used to label seminiferous tubule structures. Note that controls had abundant sperm (G) and DNA (I) within the epididymis (blue arrows), while TGFBR1-CAAcre males showed a lack of sperm/DNA in the epididymis (H and J). Scale bar is representatively shown in (C) and equals 25 μm (E–H), 50 μm (I and J) and 100 μm (C and D). Figure 2 View largeDownload slide Sustained activation of TGFBR1 in the testis leads to sex cord-stromal tumor formation. (A, B) Development of testicular tumors in TGFBR1-CAAcre males at the age of 2 and 4 months. Scale bars in panels (A and B) equal 500 μm. Images of paired testes from two controls and two TGFBR1-CAAcre mice are shown. Testes from control and TGFBR1-CAAcre mice around 2 months of age were examined for testicular tumor development. Macroscopic and microscopic tumors were found in all TGFBR1-CAAcre males (n = 9) but not controls (n = 10). (C–F) H and E staining of testes from control and TGFBR1-CAAcre males at 2 months of age. Panels (E and F) represent higher magnification images for panels (C and D), respectively. (G–J) Loss of sperm in the epididymis of TGFBR1-CAAcre mice revealed by haematoxylin and eosin staining and immunofluorescence. Antibody directed to ACTA2 (red) was used to label seminiferous tubule structures. Note that controls had abundant sperm (G) and DNA (I) within the epididymis (blue arrows), while TGFBR1-CAAcre males showed a lack of sperm/DNA in the epididymis (H and J). Scale bar is representatively shown in (C) and equals 25 μm (E–H), 50 μm (I and J) and 100 μm (C and D). Disruption of testis development in postnatal TGFBR1-CAAcre males To define a timeline of testicular tumor development in the TGFBR1-CAAcre males, we first performed histological analyses using testes from both control and TGFBR1-CAAcre mice at the age of 1 week, 2 weeks and 1 month. No histological difference was observed in the testes of TGFBR1-CAAcre mice versus controls at 1 week of age (Supplementary Fig. 2A and B). Although the basic histological structure of seminiferous tubules appeared to be maintained in the TGFBR1-CAAcre testes by 2 weeks of age (Supplementary Fig. 2C and D), microscopic lesions were evident within the testes of TGFBR1-CAAcre mice at 1 month of age (Supplementary Fig. 2F and H). Compared with control testes consisting of highly organized seminiferous tubules (Supplementary Fig. 2E and G), loss of germ cells occurred within the seminiferous tubules of TGFBR1-CAAcre testes (Supplementary Fig. 2F and H). Enrichment of Sertoli-like cells was observed in abnormal tubules of TGFBR1-CAAcre mice (Supplementary Fig. 2F). Tumor nodules were readily discernible in TGFBR1-CAAcre testes at this stage (Supplementary Fig. 2H). The histological findings were confirmed by immunohistochemical staining of Y box protein 2 (YBX2, also known as MSY2), a marker for germ cells (Gu et al., 1998) and Ki67 (a marker for cell proliferation). YBX2 was abundantly expressed in germ cells within seminiferous tubules in control testes (Fig. 3A). However, loss of YBX2-positive cells was pronounced in the seminiferous tubules of TGFBR1-CAAcre males at 1 month of age (Fig. 3B). The adverse effect of constitutive activation of TGFBR1 on germ cell production could be observed at 2 weeks (Supplementary Fig. 2I and J). Moreover, the neoplastic tumor foci contained proliferative cells, evidenced by intense staining of Ki67 versus controls (Fig. 3C and D). Enhanced cell proliferation of TGFBR1-CAAcre testes was further supported by upregulation of cyclin D2 (CCND2) protein and transcripts (Fig. 3E and F). Interestingly, double IF demonstrated colocalization of Ki67 and SOX9, a Sertoli cell marker, in 1-month-old TGFBR1-CAAcre testes (yellow/orange labeling; Fig. 3J–L), in sharp contrast to control testes where SOX9 and Ki67 signals did not overlap (Fig. 3G–I). A similar result supporting Sertoli cell proliferation in TGFBR1-CAAcre testes was found using 2-week-old mice (Supplementary Fig. 3). Since Sertoli cells stop proliferating around 2 weeks after birth in mice (Vergouwen et al., 1991), our results indicate that sustained activation of TGFBR1 extends the proliferation of Sertoli cells. As gap junction protein alpha 1 (GJA1), also known as Connexin 43, is implicated in Sertoli cell development and conditional loss of GJA1 in Sertoli cells impedes the differentiation but extends the proliferation of Sertoli cells (Sridharan et al., 2007), we examined the expression of GJA1 in TGFBR1-CAAcre testes to determine whether altered Sertoli cell proliferation was associated with reduced expression of GJA1. A significant change of Gja1 mRNA abundance was not found in the testes of TGFBR1-CAAcre mice at 2 weeks of age (Supplementary Fig. 4A), although localization of GJA1 proteins appeared to be increased in cells within the basal region of the seminiferous tubules (Supplementary Fig. 4B and C). At 1 month of age, mRNA levels of Gja1 were markedly elevated and abundant immunoreactive signals of GJA1 detected in TGFBR1-CAAcre testes compared with controls (Supplementary Fig. 4D–F). These results are in concordance with reports that TGFB upregulates GJA1 expression in human granulosa cells and trophoblast cells (Chen et al., 2015; Cheng et al., 2015) and suggest that altered Sertoli cell proliferation in TGFBR1-CAAcre mice is not a result of deficient production of GJA1. Figure 3 View largeDownload slide Depletion of germ cells and alteration of cell proliferation in the testes of TGFBR1-CAAcre males. (A–D) Immunostaining of YBX2 and Ki67 in the testes of control and TGFBR1-CAAcre mice at 1 month of age. Note the presence of abundant YBX2-positive germ cells within the seminiferous tubules in controls (A). However, loss of YBX2-positive cells was observed in TGFBR1-CAAcre testes (B). Meanwhile, tumor foci were positively stained for Ki67 (D), indicative of cell proliferation. Three mice per group were examined. (E) Western blotting analysis of CCND2 expression in testes of 1-month-old control and TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. (F) Increased expression of Ccnd2 transcripts in TGFBR1-CAAcre testes versus controls using real-time PCR. Data are presented as mean ± SEM. n = 5. ***P < 0.001. (G–L) Immunofluorescence staining of SOX9 and Ki67 using testes from control and TGFBR1-CAAcre males at 1 month of age. Note the presence of double positive cells (yellow/orange) in TGFBR1-CAAcre testes (L) but not controls (I). Scale bar is representatively shown in (A) and equals 25 μm (C, D and G–L) and 50 μm (A and B). Figure 3 View largeDownload slide Depletion of germ cells and alteration of cell proliferation in the testes of TGFBR1-CAAcre males. (A–D) Immunostaining of YBX2 and Ki67 in the testes of control and TGFBR1-CAAcre mice at 1 month of age. Note the presence of abundant YBX2-positive germ cells within the seminiferous tubules in controls (A). However, loss of YBX2-positive cells was observed in TGFBR1-CAAcre testes (B). Meanwhile, tumor foci were positively stained for Ki67 (D), indicative of cell proliferation. Three mice per group were examined. (E) Western blotting analysis of CCND2 expression in testes of 1-month-old control and TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. (F) Increased expression of Ccnd2 transcripts in TGFBR1-CAAcre testes versus controls using real-time PCR. Data are presented as mean ± SEM. n = 5. ***P < 0.001. (G–L) Immunofluorescence staining of SOX9 and Ki67 using testes from control and TGFBR1-CAAcre males at 1 month of age. Note the presence of double positive cells (yellow/orange) in TGFBR1-CAAcre testes (L) but not controls (I). Scale bar is representatively shown in (A) and equals 25 μm (C, D and G–L) and 50 μm (A and B). To further probe the cellular and molecular alterations resulting from constitutive activation of TGFBR1 in the testis, we performed IF using antibodies directed to SOX9 and ACTA2 (Fig. 4). Results showed that ACTA2-labeled seminiferous tubules (green) in the testes of TGFBR1-CAAcre were structurally comparable to those of controls at 2 weeks of age, except for disorganized arrangement of Sertoli cells labeled by SOX9 (red; Fig. 4A–F). In accordance with the histological observation of enrichment of Sertoli-like cells in the dysplastic seminiferous tubules in 1-month-old TGFBR1-CAAcre males (Supplementary Fig. 2F), markedly disorganized arrangement of SOX9-positive cells was found within these abnormal tubules (Fig. 4G–I). This result also provided indirect support for the aforementioned observation of germ cell depletion in TGFBR1-CAAcre testes. In addition, SOX9 staining was low to undetectable in tumor foci/nodules, in contrast to the strong immunoreactive signals of SOX9 observed in adjacent dysplastic tubules (Fig. 4J–L). Therefore, these studies suggest potential loss of SOX9 expression in tubule-like structures/neoplastic nodules during tumor development and progression. Figure 4 View largeDownload slide Immunofluorescence analysis of testicular tumor development in TGFBR1-CAAcre males. (A–L) Immunofluorescence staining of SOX9 and ACTA2 using testes from control and TGFBR1-CAAcre males at the age of 2 weeks, 1 month and 2 months. Note that seminiferous tubules in the testes of TGFBR1-CAAcre males were structurally comparable to those of controls at 2 weeks of age (A–F), except for disorganized arrangement of Sertoli cells. However, at 1 month of age, abnormal seminiferous tubules containing dramatically disorganized arrangement of SOX9-positive cells were found in TGFBR1-CAAcre testes (G–I). Minimal expression of SOX9 was observed in testicular tumor foci/nodules of TGFBR1-CAAcre mice, in sharp contrast to the adjacent abnormal tubules consisting of SOX9-positive cells. Dotted blue lines mark small and large tumor foci/nodules (sTu/lTu). Results of immunofluorescence were verified by immunohistochemical staining of SOX9, in which three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–I) and 80 μm (J–L). Figure 4 View largeDownload slide Immunofluorescence analysis of testicular tumor development in TGFBR1-CAAcre males. (A–L) Immunofluorescence staining of SOX9 and ACTA2 using testes from control and TGFBR1-CAAcre males at the age of 2 weeks, 1 month and 2 months. Note that seminiferous tubules in the testes of TGFBR1-CAAcre males were structurally comparable to those of controls at 2 weeks of age (A–F), except for disorganized arrangement of Sertoli cells. However, at 1 month of age, abnormal seminiferous tubules containing dramatically disorganized arrangement of SOX9-positive cells were found in TGFBR1-CAAcre testes (G–I). Minimal expression of SOX9 was observed in testicular tumor foci/nodules of TGFBR1-CAAcre mice, in sharp contrast to the adjacent abnormal tubules consisting of SOX9-positive cells. Dotted blue lines mark small and large tumor foci/nodules (sTu/lTu). Results of immunofluorescence were verified by immunohistochemical staining of SOX9, in which three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–I) and 80 μm (J–L). Testicular tumors in TGFBR1-CAAcre males express markers of GrCTs As testicular tumors in TGFBR1-CAAcre mice histologically resembled ovarian GrCTs, we examined the expression of several granulosa cell markers that are expressed by GrCTs including inhibin alpha (INHA), forkhead box O1 (FOXO1) and FOXL2 within the testes of TGFBR1-CAAcre males. IHC revealed that testicular tumor nodules in 2-month-old TGFBR1-CAAcre mice were positively stained for INHA (Fig. 5B and C), FOXO1 (Fig. 5E and F) and FOXL2 (Fig. 5H and I) compared with controls (Fig. 5A, D and G). Notably, FOXL2 is a granulosa cell lineage marker expressed in the ovary but not the testis (Wilhelm et al., 2009). To determine the temporal expression of FOXL2 in TGFBR1-CAAcre testes, we examined mice at 1 month of age, when microscopic tumor foci were detectable (Supplementary Fig. 2H). Interestingly, FOXL2 staining was found in some abnormal seminiferous tubules or tumor nodules in TGFBR1-CAAcre testes (Fig. 5K and L), in stark contrast to control testes which did not express FOXL2 (Fig. 5J). Representative negative controls using isotype-matched IgGs are depicted in Supplementary Fig. 5. Supporting the IHC findings, protein bands of FOXL2 were only detected in the testes of TGFBR1-CAAcre mice but not those of controls by western blotting (Fig. 5M). These results indicate that constitutive activation of TGFBR1 in the testis may alter Sertoli cell fate. Figure 5 View largeDownload slide Testicular tumors of TGFBR1-CAAcre males express granulosa cell tumor markers. (A–I) Immunostaining of INHA, FOXO1 and FOXL2 in control and TGFBR1-CAAcre testes at 2 months of age. Note that intensive immunoreactive signals for INHA, FOXO1 and FOXL2 were detected in testicular tumors of TGFBR1-CAAcre males compared with controls (A–I). (J–L) Immunostaining of FOXL2 in control and TGFBR1-CAAcre testes at 1 month of age. Note that FOXL2 was not expressed in control testis (J) but localized to some tumor foci (K and L; red arrows). Panels (C, F, I and L) represent higher magnification images for corresponding panels (B, E, H and K). Three mice per group were examined. Scale bar is representatively shown in (A), and equals 25 μm (A, C, D, F, G, I, J and L) and 100 μm (B, E, H and K). (M) Western blotting analysis of FOXL2 in 1-month-old control and TGFBR1-CAAcre testes. Note that the FOXL2 proteins were only detected in the testes of TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. Figure 5 View largeDownload slide Testicular tumors of TGFBR1-CAAcre males express granulosa cell tumor markers. (A–I) Immunostaining of INHA, FOXO1 and FOXL2 in control and TGFBR1-CAAcre testes at 2 months of age. Note that intensive immunoreactive signals for INHA, FOXO1 and FOXL2 were detected in testicular tumors of TGFBR1-CAAcre males compared with controls (A–I). (J–L) Immunostaining of FOXL2 in control and TGFBR1-CAAcre testes at 1 month of age. Note that FOXL2 was not expressed in control testis (J) but localized to some tumor foci (K and L; red arrows). Panels (C, F, I and L) represent higher magnification images for corresponding panels (B, E, H and K). Three mice per group were examined. Scale bar is representatively shown in (A), and equals 25 μm (A, C, D, F, G, I, J and L) and 100 μm (B, E, H and K). (M) Western blotting analysis of FOXL2 in 1-month-old control and TGFBR1-CAAcre testes. Note that the FOXL2 proteins were only detected in the testes of TGFBR1-CAAcre males. Each lane represents an independent testis sample. n = 4. It has been well established that doublesex and mab-3 related transcription factor 1 (DMRT1) maintains testis identity; and Sertoli cells could be reprogramed to granulosa cells in the testis upon deletion of Dmrt1 due to activation of Foxl2 and other female-promoting genes (Raymond et al., 2000; Matson et al., 2011). In postnatal mouse testis, DMRT1 is expressed in mitotic spermatogonia and Sertoli cells (Matson et al., 2011). To explore whether the aberrant expression of FOXL2 in our mouse model was associated with loss of DMRT1 expression, we performed immunohistochemical analysis of DMRT1 and IF of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre mice. DMRT1 was expressed in the testes from both control and TGFBR1-CAAcre mice at 2 weeks of age (Supplementary Fig. 6). At 1 month of age, expression of FOXL2 in TGFBR1-CAAcre testes could be detected in a subset of DMRT1-negative cells within disorganized tubules (Fig. 6B, E, H and K) or in majority of cells within some tumor nodules containing scattered DMRT1-expressing cells (Fig. 6C, F, I and L). In tumor foci where FOXL2-positive cells prevailed, no DMRT1 was detected (data not shown). In contrast, immunoreactive signals for FOXL2 were not detected in control testes (Fig. 6A, D, G and J). These results provide circumstantial evidence supporting a potential link between loss of DMRT1 and altered Sertoli cell fate in TGFBR1-CAAcre testes. Figure 6 View largeDownload slide Immunofluorescence analysis of DMRT1 and FOXL2 distribution during testicular tumor development. (A–L) Immunofluorescence staining of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre males at the age of 1 month. Note that while DMRT1 was readily detectable in control testes, FOXL2 specific signals were not observed as expected (A, D, G and J). Scattered FOXL2-postive cells were found within some abnormal tubules in TGFBR1-CAAcre testes (B, E, H and K). Panels (C, F, I and L) show a small tumor nodule in which many tumor cells were positive for FOXL2 but lacked DMRT1 expression. Three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–L). Figure 6 View largeDownload slide Immunofluorescence analysis of DMRT1 and FOXL2 distribution during testicular tumor development. (A–L) Immunofluorescence staining of DMRT1 and FOXL2 using testes from control and TGFBR1-CAAcre males at the age of 1 month. Note that while DMRT1 was readily detectable in control testes, FOXL2 specific signals were not observed as expected (A, D, G and J). Scattered FOXL2-postive cells were found within some abnormal tubules in TGFBR1-CAAcre testes (B, E, H and K). Panels (C, F, I and L) show a small tumor nodule in which many tumor cells were positive for FOXL2 but lacked DMRT1 expression. Three independent samples per group were used. Scale bar is representatively shown in (A) and equals 20 μm (A–L). As mentioned above, tumor nodules in TGFBR1-CAAcre testes expressed minimal SOX9. However, loss of SOX9 may also occur during the development of Sertoli cell tumors (Chang et al., 2009). To further determine whether tumor cells expressed other Sertoli cell markers, we examined Wilms tumor 1 (WT1), a Sertoli cell expressed protein that is detectable in mouse Sertoli cell tumors (Chang et al., 2009). The results showed that WT1 was expressed in Sertoli cells of control testes and abnormal seminiferous tubules (Fig. 7A-D), but not the parenchyma of the tumors at 2 months of age (Fig. 7C and D). In addition, to elucidate whether the tumors expressed Leydig cell markers, we analyzed the expression of CYP17A1 (Liu et al., 2016). Results showed that CYP17A1 was localized to Leydig cells in control testes (Fig. 7E and F). However, only scattered CYP17A1-postive cells were observed in testicular tumors of TGFBR1-CAAcre mice (Fig. 7G and H), supporting the finding that tumors were not derived from Leydig cells. These results collectively indicate that constitutive activation of TGFBR1 in the testis leads to development of sex cord-stromal tumors resembling GrCTs. Figure 7 View largeDownload slide Analysis of Sertoli cell and Leydig cell markers in tumors from TGFBR1-CAAcre males. (A–H) Immunostaining of WT1 (A–D) and CYP17A1 (E–H) in control and TGFBR1-CAAcre testes at 2 months of age. Panels (B, D, F and H) represent higher magnification images for corresponding panels (A, C, E and G). Note that tumor nodules (Tu) were not major sites for the expression of WT1 (C and D) or CYP17A1 (G and H) in TGFBR1-CAAcre mice. Strong immunoreactive signals of WT1 were observed in Sertoli cells of controls (A and B) and abnormal seminiferous tubules of TGFBR1-CAAcre mice (C and D; blue arrows). In contrast, expression of CYP17A1 was mainly found in Leydig cells of control testes (E and F) and inter-nodular areas of testicular tumors (blue arrows; G and H). Dotted yellow lines in panels (G and H) mark the borders of tumor nodules. Testis samples from three mice per group were examined. Scale bar is representatively shown in (A) and equals 25 μm (B, D, F and H) and 100 μm (A, C, E and G). Figure 7 View largeDownload slide Analysis of Sertoli cell and Leydig cell markers in tumors from TGFBR1-CAAcre males. (A–H) Immunostaining of WT1 (A–D) and CYP17A1 (E–H) in control and TGFBR1-CAAcre testes at 2 months of age. Panels (B, D, F and H) represent higher magnification images for corresponding panels (A, C, E and G). Note that tumor nodules (Tu) were not major sites for the expression of WT1 (C and D) or CYP17A1 (G and H) in TGFBR1-CAAcre mice. Strong immunoreactive signals of WT1 were observed in Sertoli cells of controls (A and B) and abnormal seminiferous tubules of TGFBR1-CAAcre mice (C and D; blue arrows). In contrast, expression of CYP17A1 was mainly found in Leydig cells of control testes (E and F) and inter-nodular areas of testicular tumors (blue arrows; G and H). Dotted yellow lines in panels (G and H) mark the borders of tumor nodules. Testis samples from three mice per group were examined. Scale bar is representatively shown in (A) and equals 25 μm (B, D, F and H) and 100 μm (A, C, E and G). WNT/CTNNB1 signaling is active in GrCTs resulting from TGFBR1 overactivation Misregulation of WNT/beta-catenin (CTNNB1) signaling is implicated in tumorigenesis of GrCTs in multiple species including humans, horses and mice (Boerboom et al., 2005; Boyer et al., 2009). To understand how constitutive activation of TGFBR1 promoted the development of testicular tumors in TGFBR1-CAAcre mice, we examined the expression of CTNNB1 known to be involved in GrCT development (Boerboom et al., 2005; Boyer et al., 2009; Richards et al., 2012). Herein, we demonstrated that the levels of CTNNB1 were increased in testicular tumor tissues of TGFBR1-CAAcre mice at 1 month of age compared with controls (Fig. 8A and B). IHC confirmed the results of western blotting by revealing extensive immunoreactive signals of CTNNB1 in testicular tumors of TGFBR1-CAAcre males versus controls (Fig. 8C–E). Moreover, variable degrees of nuclear/cytoplasmic staining of CTNNB1 were observed in testicular tumor tissues (Fig. 8E). These results suggest that dysregulation of WNT/CTNNB1 signaling is potentially linked to GrCT development in TGFBR1-CAAcre mice. Figure 8 View largeDownload slide WNT/CTNNB1 signaling is active in GrCTs resulting from overactivation of TGFBR1. (A) Western blotting analysis of CTNNB1 expression in control and TGFBR1-CAAcre testes of 1-month-old males. ACTB was included as an internal control. n = 4. (B) Quantification of the western blotting result shown in panel (A). Image J was used to calculate the ratio of CTNNB1 to ACTB for each sample. Results are presented as percentage, in which relative protein levels of control group are set to 100%. n = 4. Data are presented as mean ± SEM. ***P < 0.001. (C–E) Immunostaining of CTNNB1 in tumor nodules of testes from TGFBR1-CAAcre mice along with controls. Panel (E) represents a higher magnification image of panel (D). Three mice per group were examined. Scale bar is representatively shown in (C), and equals 10 μm (E) and 50 μm (C and D). Figure 8 View largeDownload slide WNT/CTNNB1 signaling is active in GrCTs resulting from overactivation of TGFBR1. (A) Western blotting analysis of CTNNB1 expression in control and TGFBR1-CAAcre testes of 1-month-old males. ACTB was included as an internal control. n = 4. (B) Quantification of the western blotting result shown in panel (A). Image J was used to calculate the ratio of CTNNB1 to ACTB for each sample. Results are presented as percentage, in which relative protein levels of control group are set to 100%. n = 4. Data are presented as mean ± SEM. ***P < 0.001. (C–E) Immunostaining of CTNNB1 in tumor nodules of testes from TGFBR1-CAAcre mice along with controls. Panel (E) represents a higher magnification image of panel (D). Three mice per group were examined. Scale bar is representatively shown in (C), and equals 10 μm (E) and 50 μm (C and D). Discussion GrCTs may arise from the testis with low incidence (Jimenez-Quintero et al., 1993; Wang et al., 2002). The adult type GrCTs of the testis are extremely rare and occur after puberty, whereas the juvenile type GrCTs account for approximately 1–4% of total prepubertal testicular tumors and the majority of them develop within the first 6 months (Lawrence et al., 1985; Shukla et al., 2004; Dudani et al., 2008). While adult GrCTs are associated with somatic mutation (Lima et al., 2012), the juvenile type GrCTs may be associated with chromosomal abnormalities and gonadal dysgenesis (Young et al., 1985; Kos et al., 2005). Animal models are useful to investigate the pathogenesis of this disease. Several mouse models are available for sex cord-stromal tumors, including but not limited to, mice with targeted deletion of Inha (Matzuk et al., 1992), Smad1/5 (Pangas et al., 2008) and BMP type 1 receptors (Edson et al., 2010), mice with overexpression of R-spondin 1 (Rspo1) (De Cian et al., 2017), mice containing KRAS activation and dominant-stable CTNNB1 (Richards et al., 2012), and mice with dominant-stable CTNNB1 or both dominant-stable CTNNB1 and Pten inactivation (Boerboom et al., 2005; Boyer et al., 2009; Richards et al., 2012). Although it is challenging to faithfully model human GrCTs, mouse models have helped to gain significant insights into the pathobiology and signaling cascades of GrCT development. We previously reported that overactivation of TGFBR1 using Amhr2-Cre to target mouse granulosa cells provokes the development of ovarian GrCTs (Gao et al., 2016). Since Amhr2-Cre has been successfully used to conditionally target/overexpress genes in somatic cells of the gonad in both sexes (Jamin et al., 2002; Jeyasuria et al., 2004; Boyer et al., 2008, 2009; Pangas et al., 2008; Papaioannou et al., 2009; Tanwar et al., 2010; Kyronlahti et al., 2011), we determined the phenotypic consequence of constitutive activation of TGFBR1 using Amhr2-Cre in the testis. Formation of microscopic abnormalities observed in testicular tubules of TGFBR1-CAAcre males before puberty suggests balanced TGFB signaling is essential for testis development. Development of testicular tumors was accompanied by increased cell proliferation and depletion of germ cells. It was reported that TGFB superfamily signaling is active in the mouse testis, and the dosage of activin A maintains balanced numbers of Sertoli cells and germ cells (Mendis et al., 2011). As activin and TGFB signaling shares the same downstream SMADs, SMAD2/3, it appears possible that loss of germ cells in TGFBR1-CAAcre testes may be associated with altered SMAD2/3 signaling activity. To strengthen the conclusion that testicular tumors resembled GrCTs, we examined the spatiotemporal localization of several protein markers including SOX9, CYP17A1 and FOXL2 for the respective Sertoli cells, Leydig cells and granulosa cells. During the development of testicular GrCTs, we observed accumulation of Sertoli cells within seminiferous tubules, expression of Ki67 in Sertoli cells and reduction of SOX9 expression but gain of FOXL2 expression in tumor nodules. The origin of testicular GrCT is poorly defined. However, based on the aforementioned findings and the observation that testicular GrCTs arose within seminiferous tubules, it is tempting to postulate that Sertoli cells might transdifferentiate into granulosa tumor cells. Moreover, neither the histological feature nor the expression pattern of CYP17A1 supported that these tumors were Leydig cell tumors. Of note, the most common cell type of Leydig cell tumors is medium-sized hexagonal cell that has eosinophilic/vacuolar cytoplasm, irregular round/oval shaped nucleus and recognizable cell border (Mostofi, 1973). It is unclear whether gain of FOXL2 expression in TGFBR1-CAAcre testes is a cause or consequence of testicular GrCT development. It has been suggested that both granulosa cells and Sertoli cells share the same progenitors (Albrecht and Eicher, 2001), and granulosa cells can transdifferentiate into Sertoli cells in adult female mice lacking FOXL2 (Uhlenhaut et al., 2009). Conversely, it is logic to speculate that the fate of Sertoli cells may be altered to resemble granulosa cells when FOXL2 is misregulated. Indeed, Sertoli cells could be reprogramed to granulosa cells upon conditional loss of DMRT1 due to activation of Foxl2 and other female-promoting genes (Matson et al., 2011). Another study showed that constitutive activation of CTNNB1 in Sertoli cells promotes FOXL2 expression ectopically in the testis, resulting in the transformation of Sertoli cells into granulosa-like cells (Li et al., 2017). Moreover, upregulation of FOXL2 in testicular GrCTs was found in an independent mouse model, where CTNNB1 and AKT signaling was genetically enhanced (Boyer et al., 2009). Our result is also in agreement with the discovery that aberrant expression of FOXL2 links to the development of human juvenile GrCTs of the testis (Kalfa et al., 2008). The finding of gain of FOXL2-expressing cell population but loss of cells positive for DMRT1, a key protein that maintains Sertoli cell identify (Raymond et al., 2000; Matson et al., 2011) in testicular tubules of TGFBR1-CAAcre males during tumor development highlights the importance of TGFB pathway in maintaining the balance of ‘male-female’ characteristics in the gonad. In view of the early onset of tumorigenesis, the potential of TGFBR1-CAAcre mice as a model for the juvenile GrCTs should be further explored. Our current findings support an equally important role of TGFB signaling in the development of GrCTs in both male and female gonads (Gao et al., 2016). Somatic mutation of FOXL2 has been identified as a hallmark of ovarian adult GrCTs (Shah et al., 2009). FOXL2 mutation was also revealed in testicular GrCTs (Lima et al., 2012). Moreover, activation of TGFB signaling appears to be associated with GrCT development (Pangas et al., 2008; Middlebrook et al., 2009; Edson et al., 2010; Liu et al., 2015; Kim et al., 2016). Interestingly, FOXL2 mutation may impact activin/TGFB signaling (Rosario et al., 2012; Cheng et al., 2014; Fang et al., 2016). Previous studies indicated that unopposed activin signaling resulting from loss of inhibin promotes testicular tumor development partially through SMAD3, a critical regulator of sex cord-stromal tumor development (Li et al., 2007). Thus, SMAD mediated signaling likely contributes to testicular tumor development in our model system, as is supported by increased phospho-SMAD2/3 levels in the testes of TGFBR1-CAAcre males. TGFB signaling is complex and known to interact with other pathways. WNT pathway plays fundamental roles in normal cell functions and the development of multiple types of cancers (Giles et al., 2003). Activation of the WNT/CTNNB1 pathway is involved in the pathogenesis of GrCTs; mice expressing a dominant-stable CTNNB1 mutant in ovarian granulosa cells develop GrCTs (Boerboom et al., 2005). Simultaneous dysregulation of CTNNB1 and phosphatidylinositol 3-kinase (PI3K)/AKT pathways causes testicular GrCT formation (Boyer et al., 2009). Increased expression of phospho-AKT was found in both ovarian tumors (Gao et al., 2016) and testicular tumors (unpublished observation) from TGFBR1-CAAcre mice. Moreover, the R-Spondin family proteins activate WNT signaling pathway (Kim et al., 2008). Recent studies discovered that amplification of RSPO1 signaling leads to GrCT formation through altering the fate and properties of granulosa cells (De Cian et al., 2017). These studies collectively indicate that enhanced WNT signaling promotes GrCT development. Supporting interactions between TGFB and WNT signaling (Akhmetshina et al., 2012), we demonstrated that CTNNB1 expression was upregulated in testicular tumors. Moreover, WNT signaling appears to be active in gonadal tumors resulting from constitutive activation of TGFBR1. Of note, CTNNB1 signaling is suppressed during normal testis development, and WT1 negatively regulates CTNNB1 signaling (Chang et al., 2008). Thus, lack of WT1 in tumor nodules of TGFBR1-CAAcre testes may facilitate the expression of CTNNB1. While our studies uncovered a role of TGFBR1 activation in GrCT development, a potential regulatory mechanism of SMAD2/3-CTNNB1 activation in the pathogenesis of human GrCTs warrants further investigation. Illuminating the signaling network that underpins GrCT development will provide opportunities for designing new therapeutic interventions. In summary, the current study created a new mouse model resembling testicular GrCTs. This mouse model may prove to be a useful addition to the mouse genetics toolkit for testicular GrCT research. Supplementary data Supplementary data are available at Molecular Human Reproduction Online. Acknowledgements The authors thank Dr David Zarkower for kindly providing the DMRT1 antibody. We also thank the staff from comparative medicine program for animal care. Authors’ roles X.F., N.N. and Y.G. conducted the experiments, collected the data and analyzed the results. D.F.V. and L.B. created and provided mice containing the latent constitutively active TGFBR1. Q.L. contributed to study conception and supervised the project. X.F. and Q.L. drafted the manuscript. All authors critically revised and/or commented on the manuscript and approved the final version of the manuscript and its submission. Conflict of interest None declared. 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Molecular Human ReproductionOxford University Press

Published: May 21, 2018

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