Expression of cannabinoid receptor type-1 at implantation and pseudopregnancy.

<p>Cannabinoid receptor type-1 (CB1) messenger (A and C) and protein (B and D) were detected during peri-implantation (A and B) and on day 5 of pseudopregnancy (C and D). Results are shown as means ± S.E.M. N = 4–6 for each point. a: p<0.001 vs the rest. d4: day 4, d5: day 5, d6: day 6, IM: implantation sites, II: inter-implantation sites, psp: pseudopregnancy.</p

NOS activity in the rat uterus during peri-implantation.

<p>NOS activity was determined (A) on days 4 (d4), 5 (d5) and 6 of gestation (implantation (d6IM) and inter-implantation sites (d6II) were assessed separately on day 6 of pregnancy), (B) in the delayed implantation model and (C) during pseudopregnancy (psp). NOS activity is expressed as pmoles citrulline mg prot⁻¹ h⁻¹. a: p<0,001 vs the rest. N = 4–6 for each point. d4: day 4, d5: day 5, d6: day 6, IM: implantation sites, II: inter-implantation sites.</p

Summary of cannabinoid receptors mediated effects of AEA on NOS activity.

<p>For details see description in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018368#s4" target="_blank">Discussion</a> section.</p

Localization of cannabinoid receptor type 2 during implantation.

<p>Immunolocalization of cannabinoid receptor and type-2 (CB2) in uteri from day 4 (A: luminal and B: glandular), day 5 (C), day 6 implantation sites (D) and day 6 inter-implantation sites (E: luminal, F: glandular). Tissue sections were processed by the immunoperoxidase technique using a polyclonal antibody directed against CB2. No staining was observed in the luminal and glandular epithelium when the first antibody was omitted (G). Black arrows denote specific staining. The scale bar indicates 20 µm.</p

Expression of cannabinoid receptor type-2 at implantation and pseudopregnancy.

<p>Cannabinoid receptor type-2 (CB2) messenger (A and C) and protein (B and D) were detected during peri-implantation (A and B) and on day 5 of pseudopregnancy (C and D). Results are shown as means ± S.E.M. N = 4–6 for each point. a: p<0.01 vs day 5, b: p<0.05 vs day 4. d4: day 4, d5: day 5, d6: day 6, IM: implantation sites, II: inter-implantation sites, psp: pseudopregnancy.</p

Effect of cannabinoid receptors selective antagonists on NOS activity.

<p>Cannabinoid receptors selective antagonists SR141716A (type 1, CB1) or SR144528 (type 2, CB2) were incubated alone for 30 min with day 5 pseudopregnant rat uterus and NOS activity was determined. Results are expressed as pmoles citrulline mg prot⁻¹ h⁻¹. N = 4–6 for each point.</p

Effect of AEA on NOS activity on day 5 of pseudopregnancy.

<p>(A) Concentration-response curve of anandamide (AEA). (B) Effect of selective cannabinoid receptors antagonists on AEA inhibitory action. (C) Effect of URB-597 (a selective inhibitor of FAAH, the enzyme that degrades AEA) and selective cannabinoid receptors antagonists. NOS activity is expressed as pmoles citrulline mg prot⁻¹ h⁻¹. a: p<0,001 vs the rest, b: p<0.001 vs the rest, c: p<0.05 vs the rest. N = 4–6 for each point. antCB1: CB1 selective antagonist (SR141716A), antCB2: CB2 selective antagonist (SR144528).</p

Effect of AEA on NOS activity in the uterus from pregnant rats.

<p>(A) Uterine tissue from day 5 pregnant rats was incubated for 30 min with AEA 10⁻⁹ M. (B) Implantation sites from day 6 of pregnancy were incubated for 15 min with AEA 10⁻⁹ M alone or pre-incubated with selective cannabinoid receptors antagonists. (C) Inter-implantation sites from day 6 of pregnancy were incubated with selective cannabinoid receptors antagonists. NOS activity is expressed as pmoles citrulline mg prot⁻¹ h⁻¹. a: p<0,001 vs the rest, b: p<0,001 vs control, antCB2 and antCB1+CB2, c: p<0.05 vs control and antCB1+antCB2. N = 4–6 for each point. antCB1: CB1 selective antagonist (SR141716A), antCB2: CB2 selective antagonist (SR144528).</p

Primer sequences and PCR conditions.

<p>Cb1: cannabinoid receptor type-1; Cb2: cannabinoid receptor type-2.</p

Effect of LPA on decidualization and vascularization markers in the rat uterus during the window of implantation.

<p>Uterine strips from rats pregnant on day 5 of gestation (implantation window) were incubated with LPA 50 µM for 6 and 12 h and the expression of IGFBP-1 (A) and IL-10 (B and C) was studied. In A: *** p<0,001 vs the rest; In C: *** p<0,001 vs the rest, ** p<0.01 vs C. Results are shown as means ± sem. N = 4–6 for each point. C: control.</p