Intracellular cAMP levels in FSHRwt, FSHRmt, and FSHRwt/mt cells following treatment with FSH.

<p>Intracellular cAMP levels in 293T cells transfected with empty vector, FSHRwt, FSHRmt, or FSHRwt/mt followed by treatment without and with FSH at 10 mIU/mL (FSH 10) or 100 mIU/mL (FSH 100). Results were obtained with the cAMP-Glo Max assay kit and are shown as ∆relative light unit (∆RLU), the difference between treatment with and without FSH. Data expressed the mean ± SEM, obtained from three independent experiments. Single and double asterisks show the significant difference compared to MOCK cells treated with 10 mIU/mL and 100 mIU/mL of FSH, respectively (<i>P</i> <0.05). Two symbols, § and ¶, show the significant difference compared to FSHRmt cells treated with 10 mIU/mL and 100 mIU/mL of FSH, respectively (<i>P</i> <0.05).</p

Phosphorylation of ERK1/2 (p42/44 ERK) in FSHRwt, FSHRmt, and FSHRwt/mt cells following treatment with FSH.

<p>Phosphorylation of ERK1/2 in 293T cells transfected with empty vector, FSHRwt, FSHRmt, or FSHRwt/mt followed by treatment without (FSH 0) or with 100 mIU/mL FSH (FSH 100) were analyzed by immunoblotting using antibodies against phospho-ERK1/2 and total ERK1/2. Each band was quantified with ImageJ® software. Phosphorylation level of ERK1/2 was normalized to the the intensity of total ERK1/2 expression band. Data express the mean ± SEM, obtained from three independent experiments. Single, double and triple asterisks show the significant difference compared to non-stimulated FSHRwt, FSHRmt and FSHRwt/mt cells, respectively (<i>P</i><0.05).</p

Proliferation of FSHRwt, FSHRmt, and FSHRwt/mt cells following treatment with FSH.

<p>Proliferation activities of 293T cells transfected with the indicated vector followed by treatment with 100 mIU/mL FSH for the indicated days. Growth was measured using the CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit and described as relative light unit (RLU). Data show the mean ± SEM, obtained from three independent experiments. Asterisks show the significant difference compared to FSHRwt cells after two days of culture (<i>P</i><0.05).</p

Luciferase activities of FSHRwt, FSHRmt, and FSHRwt/mt cells following treatment with FSH in combination with the patient’s serum.

<p>MOCK, FSHRwt, FSHRmt, and FSHRwt/mt cells were treated without and with 100 mIU/mL FSH in combination with control or the patient’s serum and subjected to a luciferase assay. Firefly luciferase activities were normalized with the sea pansy luciferase activities, and described as relative light units (RLU). Each bar indicates the mean ± SEM, obtained from three different independent experiments. Asterisks, double asterisks and a triple asterisks show the significant difference compared to MOCK cells treated with control serum, MOCK cells treated with patient’s serum, and FSHRwt treated with patient’s serum, respectively (<i>P</i> <0.05).</p

<i>In vivo</i> human endometrial stem cell assay using ESP and EMP.

<p>(<b>A</b>) Summary of procedures for <i>in vivo</i> endometrial stem cell assay. E₂+P₄, treatment with estradiol in combination with progesterone. (<b>B</b>) Representative BLI of a ventrally positioned NOG mouse one week after xenotransplantation using suspensions harboring the endometrial constructs expressing RedFluc derived from <i>TdTom-ESP</i> (left panel) or <i>TdTom-EMP</i> (right panel). (<b>C</b>) Quantitative assessment of BLI signals derived from the reconstituted endometrial tissues containing <i>TdTom-ESP</i> (n = 5) or <i>TdTom-EMP</i> (n = 6). Each bar indicates the mean+SEM. (<b>D</b>) Representative BLI (upper two panels) and fluorescence images (lower two panels) of kidneys excised from NOG mice transplanted with <i>TdTom-ESP</i> (left two panels) or <i>TdTom-EMP</i> (right two panels).</p

Macroscopic and microscopic findings of human endometrium-like tissues reconstituted in the <i>in vivo</i> stem cell assay.

<p>(<b>A</b>) Representative macroscopic images (left two panels) of the transplanted site (arrowheads) of NOG mice eight weeks after xenotransplantation of <i>TdTomato-ESP</i>. H&E staining was performed on the transplanted lesion (right; scale bar, 100 µm). (<b>B</b>) Representative immunofluorescence staining of the endometrial constructs derived from <i>TdTomato-ESP</i> (upper panels) and <i>TdTomato-EMP</i> (lower panels) using antibodies against vimentin (Vm), and TdTom followed by Hoechst staining. Note that localization of TdTom-expressing cells was focal as surrounded by a dotted line in the endometrial constructs derived from <i>TdTomato-ESP</i>, whereas much less TdTom-expressing cells were sporadically distributed in <i>TdTomato-EMP</i>-derived constructs. Scale bars, 100 µm.</p

Co-expression of each endometrial lineage marker and TdTom in <i>TdTom-ESP</i> and <i>TdTom-EMP</i> kidneys.

<p>Representative immunofluorescence images of the <i>TdTom-ESP</i>- and <i>TdTom-EMP</i>-derived reconstituted endometrial tissues immunostained with anti-TdTom antibody together with antibodies against vimentin (Vm) (<b>A</b>), cytokeratin (Ck) (<b>D</b>), α-smooth muscle actin (α-SMA) (<b>G</b>), and CD31 (<b>J</b>). Nuclei were stained with Hoechst dye. Yellow arrowheads indicate cells co-expressing TdTom and the corresponding endometrial lineage marker (<b>A</b>, <b>D</b>, <b>G</b>, and <b>J</b>). Typical scattergrams from microscopic analyses of co-localization of TdTom and an endometrial lineage marker such as Vm (<b>B</b>), Ck (<b>E</b>), α-SMA (<b>H</b>), or CD31 (<b>K</b>) in reconstituted tissues derived from <i>TdTomato-ESP</i> or <i>TdTomato-EMP</i>. The horizontal and vertical axes of the scattergrams indicate the intensities of Alexa 488 (green, lineage marker) and Alexa 555 (red, TdTom). We set the cutoff point referring intensity of mouse kidney parenchyma in each section as controls. Black and white bar graphs – <i>TdTomato-ESP</i> and <i>TdTomato-EMP</i>, respectively – indicate the mean+SEM of the percentage of cells doubly positive for TdTom and Vm (<b>C</b>), Ck (<b>F</b>), α-SMA (<b>I</b>), or CD31 (<b>L</b>) among whole TdTom⁺ cells, as determined by image analysis using TissueQuest software (n = 36 visual fields per group). Bars, 20 µm. * <i>P</i><0.0005, ** <i>P</i><0.01. Note that TdTom-positive epithelial cells, which are surrounded by dotted lines, localize adjacent to TdTom positive stromal cells in (A).</p

New FSHR mutation and family pedigree.

<p>(A) Result of nucleotide-sequencing of exon 10 of the hFSHR of the patient in comparison to the respective wild-type sequence. The transversion G>A at position c.1536 is indicated by an arrow on the wild-type sequence. In-frame amino acids are indicated above each sequence. (B) The location of the amino acid substitution in the FSHR protein. The previously reported activating mutations linked to sOHSS are also indicated [42]. (C) The sequences around the target region of FSHR in other species. The relevant amino acid sequences of human luteinizing hormone receptor (LH) and thyroid-stimulated hormone receptor (TSHR), which are highly homologous to human FSHR as analyzed by Blastp (<a href="http://blast.ncbi.nlm.nih.gov/blast.cg" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cg</a>), are also listed. (D) Family pedigree. The arrow indicates the patient. The black symbol indicates the occurrence of the sOHSS symptoms. Half-shaded symbols indicate unaffected heterozygotes. Circles represent females and squares male family members.</p

Phosphorylation of PI3K in FSHRwt, FSHRmt, and FSHRwt/mt cells following treatment with FSH.

<p>Phosphorylation of PI3K p85 in 293T cells transfected with empty vector, FSHRwt, FSHRmt, or FSHRwt/mt followed by treatment without (FSH 0) and with 100 mIU/mL FSH (FSH 100) were analyzed by immunoblotting using antibodies against phospho-PI3K p85 and total PI3K p85. Each band was quantified with ImageJ® software. Phosphorylation level of PI3K p85 was normalized to the intensity of the total PI3K p85 expression band. Data express the mean ± SEM, obtained from three independent experiments. Single and double asterisks show the significant difference compared to FSHRwt cells treated without and with FSH, respectively (<i>P</i> <0.05).</p

Isolation and cell surface marker characterization of ESP and EMP.

<p>(<b>A</b>) Summary of procedures for the preparation of epithelium-enriched and stroma-enriched fractions from cycling human endometrium and isolation of ESP and EMP cells from the mixture of both fractions (SDECs). The two right-hand panels illustrate the representative images of flow cytometric distribution of ESP and EMP in SDECs stained with Hoechst 33342 in the absence (upper) and presence of 50 µM reserpine (lower). (<b>B</b>) Flow cytometric analysis of the expression patterns of surface markers for epithelial cells (CD326), stromal cells (CD10), endothelial cells (CD31 and CD34), and endometrial mesenchymal stem-like cells (CD140b, CD146 and W5C5) in ESP (red), EMP (green) and unstained controls (blue). Histograms are representatives of six independent recipients. Values are expressed as means ± SEM. * <i>P</i><0.005. ** <i>P</i><0.01. (<b>C</b>) The percentage of CD140B⁺CD146⁺ cells in ESP (left) and EMP cells (right). Dotplots are representatives of six independent recipients. Values are expressed as means ± SEM. * <i>P</i><0.05.</p