Disruption of postnatal folliculogenesis and development of ovarian tumor in a mouse model with aberrant transforming growth factor beta signaling

Abstract

Background: Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

Methods: A mouse model containing constitutively active TGFBR1 (TGFBR1^CA) using Gdf9-iCre (termed TGFBR1-CA^G9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CA^G9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3'-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues.

Results: TGFBR1-CA^G9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CA^G9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

Conclusions: These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.

Keywords: Gdf9-iCre; Ovary; TGFB; Tumor.

Fig. 1

Development of sex cord-stromal tumors in TGFBR1-CA^G9Cre mice. a-f Histological analysis of 8-week-old control and TGFBR1-CA^G9Cre mice. Note the presence of hemorrhagic cysts (c; blue arrows) and hemorrhage (d; blue arrows) and neoplastic regions containing mitotic figures (e and f; red arrows) in TGFBR1-CA^G9Cre ovaries compared with control ovaries (a and b). Panel f is a higher magnification image for the boxed region in panel (e). g and h Immunohistochemical analysis of Ki67 using 8-week-old TGFBR1-CA^G9Cre (g) and control (h) ovaries. Experiment was performed using ABC method, and signals were developed using NovaRED Peroxidase Substrate Kit. Sections were counterstained with hematoxylin. Scale bar is representatively depicted in (a) and equals 12.5 μm (f), 25 μm (e, g, and h), 50 μm (b), and 250 μm (a, c, and d). H & E staining and immunohistochemistry were conducted using 3-5 independent samples per group. i Gross ovarian tumor morphology of a 7-month-old mouse. Yellow arrows denote the ovary and ovarian tumors in the control and TGFBR1-CA^G9Cre mice, respectively

Fig. 2

Altered follicular development in TGFBR1-CA^G9Cre mice. a and b Follicle counts of control and TGFBR1-CA^G9Cre ovaries at PD5 (a) and PD7 (b). Data are mean ± s.e.m. n = 3. ^* P < 0.05. Ns, not significant. c and d Immunohistochemical localization of INHA in PD7 control and TGFBR1-CA^G9Cre ovaries. Arrows indicate abnormal follicle structures. Experiment was performed using ABC method, and signals were developed using NovaRED Peroxidase Substrate Kit. Sections were counterstained with hematoxylin. Four independent samples per group were used for immunohistochemical analyses. Scale bar is representatively depicted in (c) and equals 50 μm (c and d)

Fig. 3

Immunofluorescence analysis of follicular defects in TGFBR1-CA^G9Cre mice. a-f Double immunofluorescence of DDX4 (red) and ACTA2 (green) using PD12 control (a-c) and TGFBR1-CA^G9Cre (d-f) mice. Three independent samples per group were analyzed using immunohistochemistry and/or immunofluorescence. Note the presence of large follicles or abnormal follicle-like structures (dotted yellow lines) in the ovaries of TGFBR1-CA^G9Cre mice in comparison with age-matched controls. Scale bar is representatively depicted in (a), and equals 100 μm (a-f)

Fig. 4

Immunohistochemical analysis of ovarian tumor markers. a-j Expression of granulosa cell and germ cell markers in 8-week-old control and TGFBR1-CA^G9Cre ovaries. Representative images from immunohistochemical analysis of FOXL2 (a and b), INHA (c and d), FOXO1 (e and f), AMH (g and h), and DDX4 (i and j) are shown. k and l Negative controls using isotype-matched rabbit and goat IgGs. Experiment was performed using ABC method, and signals were developed using NovaRED Peroxidase Substrate Kit. Sections were counterstained with hematoxylin. Five independent samples per group were utilized in this analysis. Scale bar is representatively depicted in (a) and equals 50 μm (a-l)

Fig. 5

Immunofluorescence of SOX9 in control and TGFBR1-CA^G9Cre ovaries. a-c SOX9 expression in control ovaries. d-i SOX9 expression in TGFBR1-CA^G9Cre ovaries. DAPI (blue) was used to counterstain the nucleus. Arrows indicate abnormal expression of SOX9 proteins (red) in ovarian tumors. At least three control and TGFBR1-CA^G9Cre mice at the age of ~2 months were analyzed. The results of immunofluorescence were confirmed by immunohistochemistry (not shown). Tu, tumor. Scale bar is depicted in (a) and equals 50 μm (a-i)

Fig. 6

Apoptosis analysis of ovaries from control and TGFBR1-CA^G9Cre mice. a PD3 ovarian sections treated with DNase I as positive control. b Negative control where TdT was replaced with water. c-f Representative images of apoptosis analysis using PD3 (n = 4) and PD21 (n = 5) ovarian sections. Apoptotic cells were labeled with TdT and signals developed using DAB. Sections were counterstained with Methyl Green. Scale bar is representatively depicted in (a) and equals 20 μm (a-f)

Fig. 7

Evidence of TGFBR1 activation in ovarian granulosa cells of TGFBR1-CA^G9Cre mice. a-d Real-time PCR analysis of expression of TGFBR1 ^CA, Smad7, Inha, and Zp3 in ovaries from control and TGFBR1-CA^G9Cre mice at PD3 and PD7. Real-time PCR was performed using ΔΔCT method. Data are mean ± s.e.m. n = 4-5. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001. Ns, not significant. e Western blotting analysis of TGFBR1^CA, phospho-SMAD2/3, and HSD3B using ovaries from 2-month-old control and TGFBR1-CA^G9Cre mice. TGFBR1^CA was detected using an anti-HA antibody. n = 3. Each lane represents an independent sample. f-i X-gal staining using ovaries from Rosa26/Gdf9-iCre mice (f-h) and Rosa26 control mice (i). Panels g and h are higher magnification images of two different fields of panel (f). At least 3 independent samples per group were used. j Real-time PCR analysis of the expression of Gli1, Gli2, and Tgfbr3 using ovaries from 8-week-old control and TGFBR1-CA^G9Cre mice. Data are mean ± s.e.m. n = 4-5. ^* P < 0.05 and ^*** P < 0.001. k-n RNAscope in situ hybridization analysis of Gli1 mRNA distribution using 8-week-old control (k) and TGFBR1-CA^G9Cre ovaries (l). n = 4. Positive and negative controls using TGFBR1 ^{CA flox/+} ovaries were shown in (m) and (n), respectively. Sections were counterstained with hematoxylin. Scale bar is representatively shown in (f) and equals 25 μm (g, h, and k-n) and 100 μm (f and i)