Participation of CB1 and TRPV1 in NO production during bull sperm capacitation by AEA.

<p>Sperm samples were incubated for 60 min at 38.5°C in 0.3% BSA sp-TALP containing 0.1 mM L-Arginine and 5 µM of DAF-FM diacetate and untreated (control) or treated with MetAEA (1.4 nM) and/or SR141716A (SR1: CB1 antagonist (0.1 nM)) or Capsazepine (CZP: TRPV1 antagonist (10 nM)). Spermatozoa were fixed and the fluorescent complex was measured by flow cytometry. Fluorescence data are expressed as mean fluorescence (percentage of control at 45 min incubation, control adjusted to 100%). Data are expressed as mean ± SEM (n = 5). a≠b, p<0.05.</p

Effect of L-NAME or Hemoglobin on bull sperm capacitation induced by AEA.

<p>Spermatozoa were incubated for 45 min at 38.5°C in 0.3% BSA sp-TALP medium with AEA (1 nM) and L-NAME (1 µM) or Hb (30 µg/ml). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). <b>A:</b> Assessment of sperm capacitation by CTC; T0 and T45: 0.3% BSA sp-TALP at 0 and 45 min (control) incubation respectively (n = 6). <b>B:</b> Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. T45: 0.3% BSA sp-TALP (control). After capacitation, spermatozoa were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 6). Data are expressed as mean ± SEM. a≠b, p<0.05.</p

Determination of NO levels in bull spermatozoa.

<p>Sperm samples were incubated for 60 min at 38.5°C in 0.3% BSA sp-TALP containing 0.1 mM L-Arginine and 5 µM of DAF-FM diacetate and untreated (control) or treated with MetAEA (1.4 nM), MetAEA+L-NAME (1 µM). Spermatozoa were fixed and the fluorescent complex was measured by flow cytometry. A) A representative photograph showing fluorescence in spermatozoa, indicating the content of intracellular NO (Magnification, ×600); B) Fluorescence data are expressed as mean fluorescence (percentage of control at 45 min incubation, control adjusted to 100%). Data are expressed as mean ± SEM (n = 8). a≠b, p<0.05.</p

Effect of L-NAME or Hemoglobin on bull sperm capacitation induced by MetAEA.

<p>Spermatozoa were incubated for 45 min at 38.5°C in 0.3% BSA sp-TALP medium with MetAEA (1.4 nM) and L-NAME (1 µM) or Hb (30 µg/ml). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). <b>A:</b> Assessment of sperm capacitation by CTC; T0 and T45: 0.3% BSA sp-TALP at 0 and 45 min (control) incubation respectively (n = 6). <b>B:</b> Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. T45: 0.3% BSA sp-TALP (control). After capacitation, spermatozoa were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 6). Data are expressed as mean ± SEM. a≠b, p<0.05.</p

Measurement of NO levels in bovine oviductal cells.

<p><b>(a) Assessment of NO₂⁻/NO₃⁻ production.</b> Oviductal cultures were incubated for 60 min at 38.5°C and 5% CO₂ in the presence of M199 medium (control) or MetAEA (1.4 nM). Then, once removed the culture medium, cells were incubated in M199 for 24 h at 38.5°C with 5% CO₂ to allow the accumulation of NO₂⁻ and NO₃⁻ in culture supernatants. NO production (NO₃⁻ plus NO₂⁻) was measured using the Griess assay; (n = 26; p>0.05).</p><p><b>(b) Assessment of NO synthase activity.</b> Confluent cell monolayers were incubated for 30 min at 38.5°C and 5% CO₂ in the presence of: 1) M199 medium (control) or 2) MetAEA (1.4 nM). Control and treated cells were removed by trypsinization, centrifuged and incubated for 15 min at 37°C in a HEPES buffer containing L-[¹⁴C]-Arginine and NADPH (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030671#s4" target="_blank">material and methods</a>); (n = 7; p>0.05).</p><p>Data are expressed as mean ± SEM.</p

Assessment of participation of the nitrergic system on sperm release induced by anandamide.

<p>Sperm cells and BOEC were co-cultured and then incubated for 15 min with BSA-free sp-TALP alone (control), AEA (1 nM) or MetAEA (1.4 nM) and L-NAME (1 µM; NO synthase inhibitor) or Hemoglobin (Hb) (30 µg/ml; NO scavenger). Bars indicate the number of spermatozoa that remained attached to the monolayers and represent the mean ± S.E.M. of bound spermatozoa/0.11 mm² monolayer (n = 6), a≠b p<0.05.</p

Effect of LPA and COX inhibitors on the production of PGE2.

a<p>:p<0.001 vs the rest.</p><p>Uterine strips from rats pregnant on day 5 of gestation were incubated for 6 h with LPA 50 µM alone or in the presence of indomethacin 1 µM (a non selective COX-1 and COX-2 inhibitor) or NS-398 1 µM (a selective COX-2 inhibitor). In another set of experiments, the tissue was incubated for 6 h with indomethacin or NS-398 alone. The production of PGE2 was determined by radioimmunoassay.</p

Expression of LPA3 receptor and Lyso-PLD enzyme in the rat uterus during the window of implantation.

<p>LPA3 messenger (A) and protein (B) and Lyso-PLD messenger (D) and protein (E) were detected by RT-PCR and Western Blot. Also, LPA3 localization (C) was determined by immunhistochemistry in day 5. Black arrows indicate positive staining (200x). In A *** p<0.001 day 4 vs day 5, ** p<0,01 day 6IM vs day 6II. Results are shown as means ± sem. N = 4–6 for each point. d5: uterus from rats pregnant on day 5 of gestation; IM: implantation sites; II: inter-implantation sites.</p

Effect of DGPP 10 µM on the production of PGE2 and on the expression of COX-2.

<p>Uterine strips from rats pregnant on day 5 of gestation were incubated for 6 h with DGPP 10 µM. The production of PGE2 was determined by radioimmunoassay. COX-2 mRNA and protein expression was analyzed by RT-PCR and Western Blot respectively.</p

First and the second antibody dilutions employed in Western Blot analyses.

<p>First and the second antibody dilutions employed in Western Blot analyses.</p