Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 1. Immunofluorescence showing membrane, cytoplasmic, and nuclear staining of ESR1 in MA10 (A), TM4 (B), 15P-1 (C), MSC-1 (D), and PSCs (E) cells. To differentiate ESR1-positive Sertoli cells from germ cells in isolated PSCs (G) and the testicular section (H), double labeling of ESR1 (E, green) and a Sertoli cell marker vimentin (F, red) was performed. Arrow heads in panel (H) indicate Sertoli cells in a somniferous tubule, and an arrow indicates Leydig cells in the interstitial space that is stained positively for ESR1. Panel (I) is isolated PSCs stained with hematoxylin and eosin. Panel (J) shows that the nuclei of isolated PSCs are immunostained for a Sertoli cell-specific marker GATA-2. Panel (K) is a negative control, in which GATA-2 antibody was omitted and panel (L) is a positive control, in which only the nuclei of Sertoli cells in the seminiferous tubules were immunostained for GATA-2. The immunostaining procedure was performed by an avidin–biotin immunoperoxidase method as described previously.42 Mag (A) to (H) and (L) ¼ 350 and (I) to (K) ¼ 500. ESR indicates estrogen receptor; mag, magnification; PSC, primary mouse Sertoli cell. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Immunofluorescence, Membrane, Staining, Isolation, Labeling, Marker, Negative Control, Positive Control, Immunostaining, Avidin-Biotin Assay

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 2. The RT-PCR (A) and Western blot (B) analyses demon- strate that similar to mouse Leydig cells (MA10), all Sertoli cell lines (TM4, 15P-1, and MAC-1) and primary Sertoli cells (PSC) express both Esr1 and Esr2. MA10 serves as a positive control cell line. Ribo- somal protein large subunit 19 (Rpl19) and b-actin serve as internal controls for RT-PCR and Western blotting, respectively. ESR indicates estrogen receptor; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 6. Induction of Greb1 expression by 17b-estradiol (A) and the selective ESR1 agonist PPT in TM4 cells is attenuated by coincubation with either the selective ESR1 antagonist MPP or an ESR inhibitor ICI 182,780 but not selective ESR2 antagonist PHTPP. Ribosomal protein large subunit 19 (Rpl19) serves as an internal control for reverse transcription-polymerase chain reaction (RT-PCR). Data are presented as mean + standard error of the mean (SEM; n ¼ 3). aP < .01 compared to dimethyl sulfoxide (DMSO) and bP < .05 compared to PPT. ESR indicates estrogen receptor.

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Expressing, Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 7. Western blot demonstrating overexpression of ESR1 pro- tein levels in MSC-1 cells (A). Overexpression of Esr1 in MSC-1 cells enhances 17b-estradiol (B) and the ESR1 agonist PPT-induced Greb1 expression, which can be counteracted by coincubation with an ESR inhibitor ICI 182,780 (B) or the selective ESR1 antagonist MPP (C), respectively. The empty vector pcDNA3 is transfected in parallel with Esr1 expression vector as a negative control for overexpression of ESR1. Ribosomal protein large subunit 19 (Rpl19) and b-actin serve as internal controls for reverse transcription-polymerase chain reac- tion (RT-PCR) and Western blotting, respectively. Data are presented as mean + standard error of the mean (SEM; n ¼ 3). aP < .05 and bP < .01 compared to dimethyl sulfoxide (DMSO), cP < .01 compared to E2 (B) and PPT (C) in PCDNA3. ESR indicates estrogen receptor.

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Western Blot, Over Expression, Expressing, Plasmid Preparation, Transfection, Negative Control, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 8. The RT-PCR and Western blot analyses show that Esr1 siRNA transfection markedly reduces Esr1 messenger RNA (mRNA) and protein levels in TM4 cells (A). Knockdown of Esr1 in TM4 cells abolishes 17b-estradiol (E2)-induced Greb1 expression (B). A scrambled RNA sequence (control siRNA) is transfected in parallel with the Esr1 siRNA as a specificity control for Esr1 knockdown. Ribosomal protein large subunit 19 (Rpl19) and b-actin serve as internal con- trols for RT-PCR and Western blotting, respectively. Data are pre- sented as mean + standard error of the mean (SEM; n ¼ 3).aP < .01 compared to dimethyl sulfoxide (DMSO) and bP < .01 compared to E2 in control siRNA. ESR indicates estrogen receptor; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small inter- fering RNA.

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Knockdown, Expressing, Sequencing, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 9. Panel (A) is a representative genotyping picture, which demonstrates that a deleted form of Esr1 (Esr1D) is only detected in Amh-Creþ/Esr1flox/flox mice (Lines 4 and 6) but not in Amh-Cre/Esr1- flox/flox mice (Lines 3 and 5). Panel (B) shows a dramatic decrease in Esr1 messenger RNA (mRNA) levels in primary Sertoli cells isolated from the Amh-Creþ/Esr1flox/flox testes. Panel (C) demonstrates that Sertoli cell-specific ablation of Esr1 significantly curtails 17b-estradiol- induced Greb1 expression. Ribosomal protein large subunit 19 (Rpl19) serves as an internal control for reverse transcription-polymerase chain reaction (RT-PCR). Data are presented as means + standard error of the mean (SEM; n ¼ 3). aP < .01 and bP < .05 compared to dimethyl sulfoxide (DMSO).

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Isolation, Expressing, Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: Expression of genomic functional estrogen receptor 1 in mouse sertoli cells.

doi: 10.1177/1933719114527355

Figure Lengend Snippet: Figure 10. A schematic drawing illustrating the relative positions of 2 consensus estrogen responsive elements (ERE1 and ERE2) in the mouse Greb1 upstream regulatory regions (A). Chromatin immuno- precipitation (ChIP) assays reveal that ESR1 but not ESR2 binding to both EREs in the Greb1 promoter in mouse Sertoli cell line TM4 (B). Genomic DNA input and rabbit immunologublin G (IgG) serve as a positive and a negative control, respectively. ESR indicates estrogen receptor; ERE, estrogen response element.

Article Snippet: On the day of hormonal treatments, the medium was changed to phenol red- and serum-free DMEM and cells were then incubated with 17b-estradiol (E2, 10 10 mol/L; Sigma), ESR inhibitor ICI 182,780 (10 8 mol/L), ESR1 agonist PPT (4,40,400- (4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol) (10 7 mol/L), ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) (10 7 mol/L), ESR2 agonist Diarylpropionitrile (DPN) (10 7 mol/L), ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) (10 7 mol/L; Tocris, Minneapolis, Minnesota), or vehicle dimethyl sulfoxide (Sigma) for 16 hours unless indicated elsewhere.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Negative Control