Journal: Sensors (Basel, Switzerland)

Article Title: Electrochemical Immunoassay Using Open Circuit Potential Detection Labeled by Platinum Nanoparticles

doi: 10.3390/s18020444

Figure Lengend Snippet: Schematic illustration of the immobilization of human chorionic gonadotropin hormone (hCG) antigens and platinum nanoparticle (PtNP)-Labeled hCG Antibody (Pt-Mab-hCG) onto Mab-FSH-immobilized immunosensor.

Article Snippet: Monoclonal anti-human α-subunit of follicle-stimulating hormone (Mab-FSH) with an affinity constant of 2.8 × 10 −9 M −1 , and monoclonal anti-human chorionic gonadotropin (Mab-hCG) with an affinity constant of 4.9 × 10 −9 M −1 , were purchased from Medix Biochemica (Kauniainen, Finland).

Techniques: Labeling

Journal: Biochemical and biophysical research communications

Article Title: Blocking FSH Action Attenuates Osteoclastogenesis

doi: 10.1016/j.bbrc.2012.04.104

Figure Lengend Snippet: A. Blocking antibodies for FSH reverse the effect of FSH on osteoclast formation. Bone marrow cells from 6 month old mice were isolated and osteoclast differentiation was induced with murine RANKL and murine CSF-1 [1]. This increased osteoclast formation by ~20%; duplicate experiments are shown. In the first experiment monoclonal anti-FSHβ was added in excess and this eliminated eliminating the effect of FSH (p = 0.001). In the second experiment polyclonal anti-FSHβ also returned osteoclast formation to background (p = 0.003). B. The effect of FSHR deletion on osteoclast formation is shown. Multinucleated TRAP-expressing osteoclasts 5-days after RANK-L treatment were attenuated in FSHR−/− cultures compared with FSHR+/− or wild type (p < 0.001 FSHR−/− relative to WT littermates). The size of the effect was similar to that seen when FSH is added during osteoclast formation in wild type cells (A). C. Photomicrographs of multinucleated TRAP-expressing cells in wild type, FSHR+/−, and FSHR−/− marrow cell cultures with 30 ng/ml of FSH during differentiation in RANKL and CSF-1. The knockout cells produce fewer multinucleated cells.

Article Snippet: We used a highly specific blocking monoclonal antibody against human FSHβ (IgG1) (MedixMab cat. #6602, BiosPacific, Emeryville, CA).

Techniques: Blocking Assay, Isolation, Expressing, Knock-Out

Journal: Biochemical and biophysical research communications

Article Title: Blocking FSH Action Attenuates Osteoclastogenesis

doi: 10.1016/j.bbrc.2012.04.104

Figure Lengend Snippet: (A) FSHR isoform 1 is expressed in the ovary, but a truncated form is the main form found in monocytes and osteoclasts. Primer set 1 (Methods), amplifying across exon 9, shows the full length FSHR fragment from distal exon 8 to early exon 10, and the smaller isoform missing exon 9. The smaller, type 2 isoform, 140 bp, is barely visible in ovary, but is the major product in osteoclasts made from CD14 cells with 14 day incubation in RANKL and CSF-1. Exon 9 is a short extracellular exon just distal to the FSH-binding sequence and proximal to the invariant transmembrane signaling region, exon 10. Results from three reactions in a temperature gradient PCR are shown. Further reactions were annealed at 54°C. (B) FSHR isoform 2 with exon 9 omitted. Primer set 2, the forward primer of which extends across the exon 8–10 boundary, was used, and the products of 30 cycles of amplification were then re-amplified with an internal nested primer set (131 bp) for a further 20 cycles. Transcript of this isoform was seen in fractions of peripheral blood mononuclear cells (unselected, CD14-selected, CD14-depleted, and osteoclasts from CD14 cells), and ovarian control (COV) cells. Presence of the smaller isoform, at low levels, in ovarian cells was previously described (see text).

Article Snippet: We used a highly specific blocking monoclonal antibody against human FSHβ (IgG1) (MedixMab cat. #6602, BiosPacific, Emeryville, CA).

Techniques: Incubation, Binding Assay, Sequencing, Amplification, Control

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF

doi: 10.1186/s12958-015-0017-0

Figure Lengend Snippet: Staining for Granulosa cells and Immunofluorescence staining for FSH receptor. (A) Aggregated granulosa cells stained with Papanicolaou (20x magnification). Bar = 20 μm. Localisation of FSH receptor (FSHR) in human granulosa cells (GC) after six days in culture, Immunofluorescence FSHR staining with monoclonal mouse anti-human FSHR (primary), chicken anti-mouse (Alexa, secondary), counterstained with DAPI for nuclei. (B) GCs in control culture (without FSH) showing cytoplasmic vesicles; (C) GC culture stimulated with FSH 0.1 IU/mL showing outgrowth with fibroblast-like morphology; (D) GC culture stimulated with FSH 1 IU/mL showing an intracellular signal for FSHR which could indicate de novo production (40x magnification). Bar = 40 μm.

Article Snippet: The slides were incubated with the primary antibody against FSH receptor (monoclonal mouse anti-human FSHR, R&D Systems, England, dilution 1:50), 5% normal goat serum (NGS), 0.5% casein, in PBS for 90 min. at room temperature After three washes in PBST, the slides were incubated with the second antibody, chicken anti-mouse IgG (H + L) labelled with Alexa® Fluor 488 (Invitrogen, USA, dilution 1:200) for 1 h at room temperature.

Techniques: Staining, Immunofluorescence, Control

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF

doi: 10.1186/s12958-015-0017-0

Figure Lengend Snippet: Effect of FSH on the expression of FSHR- and of AMH- mRNA in GCs from NC-IVF and c-IVF. Gene expression of AMH mRNA (panel A) and FSHR mRNA (panel B) levels by qPCR in the granulosa cell lysate after six days of culture in 28 GC preparations (NC-IVF, N = 14, open bars; c-IVF, N = 14, closed bars).

Article Snippet: The slides were incubated with the primary antibody against FSH receptor (monoclonal mouse anti-human FSHR, R&D Systems, England, dilution 1:50), 5% normal goat serum (NGS), 0.5% casein, in PBS for 90 min. at room temperature After three washes in PBST, the slides were incubated with the second antibody, chicken anti-mouse IgG (H + L) labelled with Alexa® Fluor 488 (Invitrogen, USA, dilution 1:200) for 1 h at room temperature.

Techniques: Expressing, Gene Expression

Journal: BBA Clinical

Article Title: Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression

doi: 10.1016/j.bbacli.2016.11.002

Figure Lengend Snippet: Aberrant expression of the FSHR and the LHR in a broad ovarian-like differentiation in endometriosis. A and B. RT-qPCR analysis of FSHR, LHR, INSL3 and steroidogenic enzymes CYP11, CYP17, CYP19 transcripts from eutopic ( n = 16) (A) and ectopic ( n = 16) (B) endometriotic tissues. Y axis: mRNA expression relative to control. The values shown are means ± SEM (*P < 0.05). C. Left: pre-immune IgG were used as controls. Right: immunohistochemical staining of FSHR, LHR and INSL3 with specific antibodies in control (disease-free) endometria ( n = 10) and ectopic endometriotic tissues ( n = 10). The arrow indicates a vessel. Illustrative pictures shown are representative of the median level of staining for each protein studied except for the LHR for which an aberrant expression of proteins is detected in only 21% of patients. All scale bars indicate 100 μm.

Article Snippet: The following antibodies were used: monoclonal antibody against LHR (clone 29, dilution: 1/200) , monoclonal antibody against FSHR (clone 323, dilution: 1/300) , and polyclonal antibodies against aromatase (H4, dilution: 1/50) (Acris Antibodies, San Diego, USA), CYP 17 A 1 (M-80-SC/66850, dilution: 1/100) (Santa Cruz Biotechnology Inc., USA), CYP11A1 (13363-1-AP, dilution 1/100) (Proteintech, USA) , INSL3 (ab65981, dilution 1/100) (abcam, FRA), GATA6 (ab22600, dilution 1/100) (abcam, FRA), and GATA 4 (SC:25310, dilution: 1/50) (Santa Cruz Biotechnology Inc., USA).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: BBA Clinical

Article Title: Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression

doi: 10.1016/j.bbacli.2016.11.002

Figure Lengend Snippet: The FSHR and LHR are functional in endometriosis inducing the same steroidogenic cascade as in ovaries. A. Kinetics of phospho-ERK 1/2 induction in primary cultures of epithelial endometriotic cells (EECs) stimulated by rFSH ( n = 6) or hCG ( n = 6). ERK 1/2 phosphorylation was quantified by densitometry with the ImageJ software (pERK/ERK 1/2 fold induction) (*P < 0.05). B. RT-qPCR analysis of EECs stimulated with rFSH ( n = 6) or hCG ( n = 6). Y axis: mRNA expression relative to control. Values are means ± SEM (*P < 0.05). C. Proliferative response to hCG in EECs and control epithelial endometrial cells ( n = 6 for each cell type). Cells were treated with various concentrations of hCG (0, 2.5, 5, 10, 20 IU/ml) for 48 h and cell proliferation was assessed by measuring [ 3 H ]thymidine incorporation during the last 16 h of culture. Results are expressed as the ratio of cpm for treated cells versus cpm for untreated cells. Y axis: proliferation relative to untreated cells. The red dotted line corresponds to untreated cells. ( n = 3 independent experiments; * P < 0.05; ** P < 0.01).

Article Snippet: The following antibodies were used: monoclonal antibody against LHR (clone 29, dilution: 1/200) , monoclonal antibody against FSHR (clone 323, dilution: 1/300) , and polyclonal antibodies against aromatase (H4, dilution: 1/50) (Acris Antibodies, San Diego, USA), CYP 17 A 1 (M-80-SC/66850, dilution: 1/100) (Santa Cruz Biotechnology Inc., USA), CYP11A1 (13363-1-AP, dilution 1/100) (Proteintech, USA) , INSL3 (ab65981, dilution 1/100) (abcam, FRA), GATA6 (ab22600, dilution 1/100) (abcam, FRA), and GATA 4 (SC:25310, dilution: 1/50) (Santa Cruz Biotechnology Inc., USA).

Techniques: Functional Assay, Software, Quantitative RT-PCR, Expressing

Journal: BBA Clinical

Article Title: Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression

doi: 10.1016/j.bbacli.2016.11.002

Figure Lengend Snippet: FSHR induces vascular and LHR induces inflammatory factors in endometriotic cells. A and B. RT-qPCR analysis from eutopic ( n = 16) (A) and ectopic ( n = 16) (B) endometric tissues. Y axis: mRNA expression relative to control. The values shown are means ± SEM (*P < 0.05). C. RT-qPCR analysis in EECs stimulated with rFSH ( n = 6) or hCG ( n = 6). Y axis: mRNA expression relative to control. Values are means ± SEM (*P < 0.05).

Article Snippet: The following antibodies were used: monoclonal antibody against LHR (clone 29, dilution: 1/200) , monoclonal antibody against FSHR (clone 323, dilution: 1/300) , and polyclonal antibodies against aromatase (H4, dilution: 1/50) (Acris Antibodies, San Diego, USA), CYP 17 A 1 (M-80-SC/66850, dilution: 1/100) (Santa Cruz Biotechnology Inc., USA), CYP11A1 (13363-1-AP, dilution 1/100) (Proteintech, USA) , INSL3 (ab65981, dilution 1/100) (abcam, FRA), GATA6 (ab22600, dilution 1/100) (abcam, FRA), and GATA 4 (SC:25310, dilution: 1/50) (Santa Cruz Biotechnology Inc., USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: BBA Clinical

Article Title: Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression

doi: 10.1016/j.bbacli.2016.11.002

Figure Lengend Snippet: Model of ovarian-like differentiation in endometriosis. In red, genes upregulated in eutopic endometriosis; in blue, additional genes upregulated in ectopic endometriosis. The aberrant production of GATA4/6 factors may trigger FSHR expression at the start of the disease in eutopic tissues, in a subset of patients (group A), and during disease progression in all ectopic lesions (groups A and B). Together with INSL3, these molecules may induce partial steroidogenesis in situ especially through aromatase induction. In a subset of ectopic lesions, strong GATA4/6 expression also induces aberrant LHR upregulation (group C), resulting in the strongest induction of the complete steroidogenic cascade and the highest levels of INSL3 transcripts. The upregulation of GATA4/6 factors by FSHR and LHR in endometriotic cells may result in an autoregulatory loop favoring estrogen-dependent disease progression. FSHR and LHR also have angiogenic and inflammatory properties, respectively.

Article Snippet: The following antibodies were used: monoclonal antibody against LHR (clone 29, dilution: 1/200) , monoclonal antibody against FSHR (clone 323, dilution: 1/300) , and polyclonal antibodies against aromatase (H4, dilution: 1/50) (Acris Antibodies, San Diego, USA), CYP 17 A 1 (M-80-SC/66850, dilution: 1/100) (Santa Cruz Biotechnology Inc., USA), CYP11A1 (13363-1-AP, dilution 1/100) (Proteintech, USA) , INSL3 (ab65981, dilution 1/100) (abcam, FRA), GATA6 (ab22600, dilution 1/100) (abcam, FRA), and GATA 4 (SC:25310, dilution: 1/50) (Santa Cruz Biotechnology Inc., USA).

Techniques: Expressing, In Situ