Figure 1.
Detection of the OT system in the E15 mouse embryo by immunocytochemistry.
Somite staining with OT-GKR antibody (A) and selective antibodies for OT (B) and OTR (C). TUNEL reaction displaying apoptosis (D). Control staining of somites with OT-GKR-specific antibody reabsorbed with OT-GKR (E) Control staining of whole embryo with OT-GKR-specific antibody reabsorbed with OT-GKR (F). Immunodetection of OT-GKR in the fetal mouse heart at day E15 (arrow) (G). Polyclonal rabbit antibody specifically recognizing OT-GKR peptide was applied to detect OT-GKR. Staining was revealed by the biotin-streptavidin method. Higher magnification of OT-GKR staining in the fetal heart (H). Immunofluorescence of OT-GKR (Texas Red) in cryostat sections section of cardiac tissue (stained green by troponin Alexa Fluor 488 antibody) by (I). Immunodetection in the heart of OTR (J), OT (K), AVP (L), and V1R (M). Control staining with OT-GKR-specific antibody reabsorbed with OT-GKR (N).
Figure 2.
Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.
(A) The front upright view position (side view) of the OTR-OT- GKR complex structure. (A1) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (B) side view of the V1aR-OT- GKR complex, (B1) V1aR top view. (C1) detail of docking view of OTR-OT-GKR complex, and (C2) detail of V1aR-OT-GKR complex. D displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (E) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in Table 1.
Table 1.
List of the OTR, V1aR residues, involved in the List of the OTR interactions with OT-GKR, and OT; * - AVP agonist binding site; ** - OTA antagonist binding site.
Figure 3.
Generation of beating cell colonies of EC P19 cells after induction with different OT forms.
Micrographs (100× magnification) show cultures at day 10 with dotted lines encircling beating colonies and induced by: (A) Non-inducer, (B) OT, (C) OT-G, (D) OT-GK, (E) OT-GKR. (F) Time course of appearance of beating cell colonies. At day 5, cultures were transferred into a 24-well tissue culture dish and scored every 2 days for the presence of beating cell colonies. The generation of beating cell colonies was induced by treatment of EBs (from day 1 to 4) with OT, OT-G, OT-GK or OT-GKR in the presence of antagonists for OTR and V1aR (see Materials and Methods). The results are expressed as means ± SEM of 3 independent experiments. All treatments produced significantly different numbers of beating cell colonies than non-induced controls (NI). G demonstrates OTR immunodetection in cells at day 2 in control EC P19 cultures and (H) in cells subjected to OTR−/− siRNA treatment, (I) detection of V1aR in control, and (J) in these cells subjected to V1aR−/− siRNA treatment. (K) OTR and V1aR inhibition by siRNA in EC P19 cells decreases their ability to generate beating cell colonies after induction with OT-GKR (10−6 M). *p<0.05, NI – not induced control.
Figure 4.
Schematic structure of the OT-GKR-IRES-EGFP DNA construct.
Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; OT, oxytocin. (A) pcDNA3.1/Amp-OT-GKR-IRES-EGFP transfection to EC P19 cells stimulates the expression of green fluorescence (B) and produces OT-GKR protein marked with VA-18 antibody in red (Texas Red) (C). Blue DAPI staining of cell nuclei (D) and merged photo (E). Time course of appearance of beating cell colonies in non-induced EC P19 cells transfected with OT-GKR-IRES-EGFP and in normal EC P19 cells stimulated with OT-GKR (10−6 M), and non-induced controls (NI) (F).
Figure 5.
In response to treatment with OT or OT-X forms, GFP-P19Cl6 cells express green fluorescence driven by the transcriptional promoter of myosin light chain-2 ventricular (MLC-2v).
Fluoromicrograph (100× magnification) of day 8 cell cultures in non-induced conditions (A) and upon induction with OT (B), OT-G (C), OT-GK (D) or OT-GKR (E). Representative flow cytometry charts of fluorescence emitted by GFP-P19Cl6 cells at day 6 in non-induced conditions (A1) and in cultures induced by OT (B1), OT-G (C1), OT-GK (D1), and OT-GKR (E1). Bars present quantitative analysis of fluorescence emitted by differently-stimulated GFP-P19Cl6 cells at day 6 in 3 independent experiments (F).
Figure 6.
RT-PCR analysis of GATA-4 (A), Mef2c (B), myogenin (C) and MyoD (D) transcripts in EC P19 cells induced by OT or OT-GKR in the presence or absence of OT antagonist (OTA).
NI indicates non-induced controls.
Figure 7.
Immunofluorescence of cell differentiation markers in EC P19 cells of non-induced controls (A1-D1) and in cells stimulated to differentiate with OT (A2-D2) and OT-GKR (A3-D3).
(A4) Staining of OCT-4, a marker of the undifferentiated state. (B4) Expression of the cardiac muscle marker dihydropyridine receptor-α1 (DHPRα1). (C4) Ventricular myosin light chain-2 ventricular (MLC-2v) protein. (D4) Immunocytochemistry shows staining of α-actinin protein in cardiac and skeletal muscles. A4-D4 represent quantitative analysis of corresponding markers expressed as fluorescent areas. Fluoromicrographs of cells on differentiation day 6 presented at ×100 magnification.