Identification and subcellular distribution of Epac 1 and Epac 2 proteins in mammalian spermatozoa.

<p>Twenty µg of proteins from rat pancreas lysates and from human, stallion and boar spermatozoa lysates were resolved by SDS-PAGE, followed by Western blotting with anti-Epac 1 or anti-Epac 2 antibodies, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s4" target="_blank">Materials and Methods</a>. Immunolocalization was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s4" target="_blank">Materials and Methods</a> using specific antibodies against Epac 1 and Epac 2 proteins. A: Epac 1 immunolocalization with representative areas digitally augmented; B: Epac 2 immunolocalization.</p

Immunolocalization of Epac 1 and Epac 2 proteins in boar spermatozoa under non-capacitating and capacitating conditions.

<p>Immunolocalization was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s4" target="_blank">Materials and Methods</a> using specific antibodies against Epac 1 and Epac 2 proteins in spermatozoa previously incubated in TBM, TCM and TCM+A23187.. Representative images of spermatozoa showing Epac 1 localization in TBM (A), TCM (B, C) and TCM+A23187 (D) and Epac 2 localization in TBM (E), TCM (F) and TCM+A23187 (G). N = 3 replicates.</p

Effect of Epac activation on boar spermatozoa motility.

<p>The motility of spermatozoa, incubated in TBM, TCM and TCM+A23187 and in the presence or absence Me-cAMP was assessed by ISAS. Graphics show the percentage of motile spermatozoa. Columns with different superscripts are statistically different from each other, so that for (a–b–c) (P<0.05). N = 5 replicates.</p

Effect of Epac activation on the acrosomal status of boar spermatozoa.

<p>Acrosomal status was evaluated by flow cytometry using FITC-PNA (0.5 mg/ml) and IP (1.2 mM), which stains non-viable cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s2" target="_blank">Results</a> are represented as percentages of maximum of viable spermatozoa recorded as PNA positive cells ± SEM. Columns with different letters indicate significant differences (P<0.05). N = 5 replicates.</p

Colocalization of Epac 1 and E-cadherin.

<p>To study the co-localization of Epac 1 and E-cadherin, boar spermatozoa were incubated overnight with specific antibodies (anti Epac1 and E-cadherin) and, then, were washed and incubated with the appropriated secondary antibodies, which were labeled with Alexa 488 and Alexa 568. Representative images of spermatozoa showing Epac 1 (A), E-cadherin (B), and colocalization of both proteins (C) in TCM+A23187. Representative images of spermatozoa showing Epac 1 (D), E-cadherin (E), and colocalization both proteins (F) in TCM+A23187+Me-cAMP (50 µM). N = 3 replicates.</p

Rap1 identification and activation.

<p>Twenty µg of proteins from human and boar spermatozoa lysates were resolved by SDS-PAGE, followed by Western blotting with anti-Rap1 antibody (7A), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s4" target="_blank">Materials and Methods</a>. To study the immunolocalization of Rap1, boar spermatozoa were incubated overnight with specific anti-Rap1 antibody (7B). Rap1 activation was studied using a commercial kit according to the manufacturer's protocol. Western blotting was performed using an anti-Rap1 antibody, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s4" target="_blank">Materials and Methods</a> (7C). Graphic shows the fold-increase of Rap-GTP with respect to the control (TCM alone) ± SEM. N = 4 replicates.</p

Effect of Epac activation on the distribution of E-cadherin.

<p>To study the immunolocalization of E-cadherin, boar spermatozoa were fixed and incubated overnight with a specific antibody and then were washed and incubated with the appropriated secondary antibody, which was labeled with Alexa 488. Representative images of spermatozoa showing E-cadherin localization in: TCM+A23187 (A) and TCM+A23187+Me-cAMP (B).</p

Measurement of cytosolic free calcium concentration ([Ca²⁺]_i).

<p>Samples were incubated in TCM for 90 min in the presence or absence of Me-cAMP (50 µM). Then, cells were incubated in TCM with 2 µM for 30 min, and washed in Na-HEPES. Fluorescence of FURA-2-AM was recorded using a fluorescence spectrophotometer (Varian Ltd., Madrid, Spain), and changes in [Ca2+]i were monitored every second. A) [Ca2+]i values were calculated and expressed as nM. Traces are representative of 5 independent experiments. B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s2" target="_blank">Results</a> of the integral of the rise in [Ca2+]i of 5 independent experiments ± SEM. Column marked with * indicate significant differences compared to control (P<0.05). N = 7 replicates.</p

Effect of the Epac activation on the scrambling of plasma membrane phospholipids of boar spermatozoa.

<p>The scrambling of plasma membrane phospholipids was monitored by flow cytometry by using M540 (1.3 µM). Spermatozoa were also stained with YO-PRO-1 (25 nM) to distinguish viable and non-viable cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037713#s2" target="_blank">Results</a> are represented as the percentage of maximum of viable cells recorded with high M540 ± SEM. Columns with different letters indicate significant differences (P<0.05). N = 5 replicates.</p