Identification and function of exchange proteins activated directly by cyclic AMP (Epac) in mammalian spermatozoa.

The role of cAMP in spermatic functions was classically thought to be mediated exclusively through the activation of Protein Kinase A (PKA). However, it has recently been shown that cAMP also exerts its effects through a PKA-independent pathway activating a family of proteins known as Epac proteins. Therefore, many of the spermatic functions thought to be regulated by cAMP through the activation of PKA are again under study. We aimed to identify and to investigate the role of Epac proteins in spermatozoa using a specific permeable analog (8-Br-2'-O-Me-cAMP). Also, we aimed to study its relationship with E-cadherin, an adhesion protein involved in fertility. Our results demonstrate the presence and sub-cellular distribution of Epac 1 and Epac 2 in mammalian spermatozoa. Capacitation and the acrosome reaction induced a change in the localization of Epac proteins in sperm. Moreover, incubation with 8-Br-2'-O-Me-cAMP prompted an increase in Rap1 activation, in the scrambling of plasma membrane phospholipids (necessary for the capacitation process), the acrosome reaction, motility, and calcium mobilization, when spermatozoa were incubated in acrosome reaction conditions. Finally, the activation of Epac proteins induced a change in the distribution of E-cadherin. Therefore, the increase in the acrosome reaction, together with the increase in calcium (which is known to be essential for fertilization) and the Epac nteraction with E-cadherin, might indicate that Epac proteins have an important role in gamete recognition and fertilization

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

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

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

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

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

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

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