A novel mouse model of testicular granulosa cell tumors.

study questionWhat 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,

methodsMale 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

findingsThis 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.