Gene Ontology (GO) analysis with Molecular Function, Cellular Component and Biological Processes categories.

<p>Pie chart shows the distribution of the DEG’s in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice that were matched to A) a Molecular Function, B) a Cellular Component, and C) a Biological Process, using GO.</p

Most highly significant canonical pathways identified in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT identified using QIAGEN’S Ingenuity Pathway Analysis.

<p>Most highly significant canonical pathways identified in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT identified using QIAGEN’S Ingenuity Pathway Analysis.</p

Principal component analysis (PCA) of the microarray-derived transcriptomal results for oviducts collected from ESR1KO mice and WT littermates at 23 days of age, or treated with 5 IU PMSG at 23 days of age and collected 48 h later.

<p>Red: ESR1KO, Blue: WT, Green: PMSG-treated ESR1KO, Purple: PMSG-treated WT.</p

Smad3 physically interacts with Nur77.

<p>(<b>A</b>) mKG_N-MC-NLS-Smad3 and mKG_C-MC-Nur77 were transfected into HeLa cells for 24 hours. Interaction between Nur77 and NLS-Smad3 yielded fluorescent green signals in the nucleus. The single fusion protein alone (mKG_N-MC-NLS-Smad3 or mKG_C-MC-Nur77) and another pair (mKG_N-MC-NLS-Smad3 and mKG_C-MN-Nur77) gave no fluorescent signal. The scale bars represent 25 µm. (<b>B</b>) [³⁵S] methionine-labeled Smad3 produced by <i>in vitro</i> translation was incubated with the GST-Nur77 fusion protein and its deletion mutants. Coomassie blue staining shows the protein level of the purified GST, GST-Nur77 and GST-Nur77 deletion mutant (bottom). (<b>C</b>) [³⁵S] methionine-labeled Smad3 deletion mutants were incubated with GST-Nur77 fusion protein. The data are representative of three independent experiments.</p

TGF-β1 signaling interferes with Nur77 binding to DNA.

<p>(<b>A and B</b>) TGF-β1 inhibits the recruitment of Nur77 to the P450c17 promoter. ChIP assays were performed using purified primary Leydig cells treated with 300 µM of 8-Br-cAMP and 10 ng/ml of TGF-β1 for 2 hours (A) and R2C cells treated with 10 ng/ml of TGF-β1 for the indicated time (B). Anti-Nur77 antibody was used for immunoprecipitation. The immunoprecipitates were analyzed by PCR using a pair of specific primers spanning a region containing the Nur77 binding site of the P450c17 promoter. A negative control PCR for nonspecific immunoprecipitation was performed using primers specific to the GAPDH coding region. (<b>C</b>) The interference with Nur77 binding to NBRE by Smad3. The GST-Nur77 fusion protein was incubated with α-³²P-labeled NBRE oligonucleotide, along with increasing amounts of purified GST-Smad3 (lanes 7 and 8) proteins. A 100-fold excess of cold NBRE oligomer (lane 9) or nonspecific oligomer (ARE, lane 10) was added. Positions of the specific protein-DNA complex and the free probe are indicated. The data are representative of three independent experiments.</p

TGF-β1 signaling regulates steroidogenic gene expression, affecting testicular testosterone levels in mice.

<p>(<b>A</b>) Decreased Tgfbr2^fl°^x allele in purified primary Leydig cells isolated from mice harboring the Cyp17iCre transgene. The genomic DNA isolated from primary Leydig cells of Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was amplified for Tgfbr2 intron region containing the LoxP site. A pair of β-actin primers was used as the control for the amount of genomic DNA. (<b>B</b>) Decreased TGF-β1-mediated repression of steroidogenic gene expression with Tgfbr2 silencing. Purified primary Leydig cells from the testes of 12-week-old Tgfbr2^flox/flox (n = 6) and Tgfbr2^flox/flox Cyp17iCre (n = 6) mice were treated with 300 µM of 8-Br-cAMP and 2 ng/ml of TGF-β1 for 24 hours, and mRNA expression levels were measured using qRT-PCR. β-actin expression was used as a loading control. The data are presented as the mean ± SEM. **, P<0.01; ***, P<0.01. (<b>C</b>) Testicular testosterone levels were measured by RIA in the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice. (<b>D</b>) Total protein (100 µg) from the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was subjected to western blot analysis for protein levels of steroidogenic genes. The relative level of each protein/GAPDH was quantified by densitometric analysis using Image J software. In panels C and D, the data are presented as the mean ± SD (n = 10). **, P<0.01.</p

ALK5 signaling inhibits Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A and B</b>) MA-10 cells were transiently transfected with the ALK5 WT (wild type), ALK5 mutant (TD; constitutively active form or KR; inactive form), and Nur77 expression plasmids, along with the indicated reporter. The CMVβ expression plasmid was used as a control for transfection efficiency. (<b>C</b>) Whole cell extracts and subcellular fractions of primary Leydig cells, which were treated with 300 µM of 8-Br-cAMP and 2.5 ng/ml of TGF-β1 for 4 hours, were analyzed by western blot analysis with anti-Nur77, anti-pSmad3, anti-α-Tubulin (cytoplasmic marker) and anti-Lamin B (nuclear marker) antibodies. (<b>D</b>) MA-10 cells were transiently transfected with scrambled or Nur77 siRNA, ALK5 (TD) expression plasmid and P450c17 promoter reporter (top). Silenced Nur77 protein levels in HEK293T cells, which were transiently transfected with scrambled or Nur77 siRNA, Flag-Nur77 and CMVβ expression vector for 48 hours, were determined by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

ALK5-activated Smad3 represses Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A</b>) MA-10 cells were transiently transfected with siRNA, Nur77, ALK5 (TD) and an indicated reporter for 48 hours and were harvested for luciferase assay. The CMVβ expression plasmid was used as a control for transfection efficiency (bottom). The silencing efficiencies of Smad2 and Smad3 siRNA were determined by western blot analysis (top). (<b>B</b>) MA-10 cells were transiently transfected with Nur77, increasing amounts of Smad (60 and 150 ng) expression plasmids and the NBRE reporter construct. (<b>C–E</b>) MA-10 cells were transiently transfected with ALK5 WT, ALK5 mutant (TD or KR), Smad3 and Nur77 expression plasmids, along with the indicated reporter construct. (<b>F</b>) MA-10 cells were transiently transfected with expression plasmids of Nur77, ALK5 (TD), Flag-Smad3 (WT) or a phosphorylation mutant (S3A or S3D), and NurRE-luc reporter construct (top). A similar amount of expressed protein was confirmed by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. *, P<0.5; **, P<0.01; ***, P<0.001; ns, not significant.</p

TGF-β1/ALK5 signaling represses cAMP-induced steroidogenic gene expression in Leydig cells.

<p>(<b>A and B</b>) The culture medium of purified mouse primary Leydig cells treated with 300 µM of 8-Br-cAMP and 5 ng/ml of TGF-β1 (A) and R2C cells treated with vehicle or 5 ng/ml of TGF-β1 (B) for 24 hours was collected for the measurement of testosterone levels by RIA. (<b>C and D</b>) The expression levels of steroidogenic genes in primary Leydig cells (C), which were treated with 300 µM of 8-Br-cAMP, 2.5 ng/ml of TGF-β1 and 10 µM SB431542 for 24 hours, and R2C cells (D), which were treated with 5 ng/ml of TGF-β1 for 24 hours, were analyzed by qRT-PCR. (<b>E</b>) The expression level of Tgfbr2 and Tgfbr1 was analyzed using total RNAs from primary Leydig, R2C and MA-10 cells by RT-PCR. (<b>F</b>) MA-10 cells were transiently transfected with the ALK5 (TD; constitutively active form) expression plasmid, along with an indicated reporter of the natural promoter, in medium containing 5% charcoal stripped FBS. Twenty four hours after transfection, the cells were treated with 300 µM of 8-Br-cAMP for 24 hours and harvested for luciferase assay. The pSV-β-gal expression plasmid was used as a control for transfection efficiency. The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

Table <b>1.</b> Ovarian function of heterozygous Edn2+/? mice and WT mice.

<p>Table <b>1.</b> Ovarian function of heterozygous Edn2+/? mice and WT mice.</p