Dose dependence of the inhibition by 17βE of the ATP induced plasma membrane depolarization in human sperm

<p><b>Copyright information:</b></p><p>Taken from "Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action"</p><p></p><p>Purinergic Signalling 2005;1(4):369-375.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096547.</p><p></p> Isolated sperm were suspended in the presence of 200 nM bis-oxonol as described in the Materials and methods section. Sperm suspensions were stimulated with 17βE at different doses (0.01, 0.1, 1.0 and 10.0 µM) for 2 min before addition of ATP (2.5 mM). Plasma membrane depolarization is expressed as percentage of the plasma membrane depolarization induced by ATP determined in the absence of 17βE (100%). Results are mean T S.D. of three separate experiments

Mineralizing ability of human osteoblasts stimulated with INSL3.

<p>Mineralized nodule formation was detected by Alizarin red-S staining and the effect of INSL3 (100 nM) was compared to that of 1,25-vitamin D (100 nM) used as positive control.</p

INSL3 effects on protein phosphorylation in human osteoblasts.

<p>Western blot analysis for the phosphorylation of proteins involved in MAPK pathway (cRAF, MEK, ERK), integrin pathway (PYK, Src), β-catenin pathway, calcium pathway (PLCβ3), Akt pathway (Akt, GS3β) and NF-κB pathway (NF-κB, IKKα, IKKβ, IκBα). Cells were stimulated with different INSL3 concentrations (1 nM, 10 nM and 100 nM) for a short time period (5, 10, 15, 30 and 45 minutes). β-actin was used as endogenous control to normalize protein quantity. Control is obtained without stimulation. Figures are representative of three independent experiments. Ctrl: Control.</p

Quantitative RT-PCR on osteoblasts stimulated with INSL3.

<p><b>A</b>. <i>COL1A1</i>, <b>B</b>. <i>COL6A1</i>, <b>C</b>. <i>Osteonectin</i>, <b>D</b>. <i>Osteopontin</i>, <b>E</b>. <i>TGF-β</i>, <b>F</b>. <i>M-CSF</i>, <b>G</b>. <i>OPG</i>, <b>H</b>. <i>AR</i>, <b>I</b>. <i>VDR</i>, <b>J</b>. <i>PTHR</i>. Quantification was normalized to the expression of the housekeeping gene β₂-microglobulin on control (no stimulation). Data are shown as the mean ± standard deviation (SD) of the mean of three different experiments performed in triplicate. * P<0.05 vs control. nRQ: normalized Relative Quantity. Ctrl: Control.</p

Quantitative phosphorylation analysis of Akt, MEK, and ERK in human osteoblasts induced by INSL3.

<p>Quantitative analysis on Akt phosphorylation: <b>A</b>. ELISA at S473, <b>B</b>. densitometric analysis at S473 and <b>C</b>. densitometric analysis at T308. Quantitative analysis on MEK phosphorylation: <b>D</b>. ELISA and <b>E</b>. densitometric analysis. Quantitative analysis on ERK phosphorylation: <b>F</b>. ELISA and <b>G</b>. densitometric analysis. ELISA data are shown as the mean ± standard deviation (SD) of the mean of three different experiments performed in triplicate. Densitometric data are shown as the mean ± standard deviation (SD) of the mean of three different experiments. * P<0.05 vs control.</p

WGA analysis and aCGH of single sperm.

<p><b>a</b>. Gel electrophoresis of WGA products from single human sperm. Ct: cycle threshold as determined by Real-Time PCR with SYBR Green. <b>b</b>. Real-Time PCR of the same WGA products. The difference in fluorescence (fluorescence at the end of each cycle minus baseline fluorescence level) is plotted against the cycle number. Hybridization failed for samples with a cycle threshold >10. <b>c</b>. Molecular karyotype of a normal 23,X sperm co-hybridized with female (upper panel) and male reference (lower panel). <b>d</b>. Molecular karyotype of a normal 23,Y sperm co-hybridized with a female (upper panel) and a male reference (lower panel).</p

Subject characteristics and summary of aCGH results on single sperm.

<p>Subject characteristics and summary of aCGH results on single sperm.</p

Flow diagram of the protocol developed for aCGH on single human sperm.

<p>Flow diagram of the protocol developed for aCGH on single human sperm.</p

Detection and localization of HPV in human sperm.

<p><b>a</b>. Fluorescence in situ hybridization (fluorescence microscope) for HPV DNA on sperm from a patient with HPV16 in semen. Infected and noninfected sperm are shown. Red: HPV DNA (Texas red); blue: nuclear staining (DAPI). <b>b</b>. Immunofluorescence (confocal fluorescence microscope) for HPV16 capsid protein L1 on sperm from a control (left) and a patient with HPV16 in semen (right). Upper panel, L1 antibody; central panel, L1 antibody and Pisum Sativum (acrosome); lower panel, L1 antibody and Pisum Sativum after induction of the acrosome reaction. Red: HPV16 L1; green: Pisum Sativum; blue: nuclear staining (DAPI). <b>c</b>. PCR for HPV E7 gene from sperm DNA. Lane M: DNA marker (100 bp); 1: negative control (no template); 2: positive control (sperm transfected with recombinant plasmid pIRES2-AcGFP1-E6E7); 3: sperm from a patient with HPV16 in semen; 4: sperm from a control subject.</p