Effect of the inhibition of Complex I of the electron transport chain on stallion sperm motility (CASA analysis).

<p>Stallion spermatozoa were extended in BWW media and incubated in presence of rotenone (0 nm (DMSO), 100 nm, 500 nm, 5 μM and 10 μM) and antimycin-A (0 vehicle DMSO, 100 nm, 500 nm, 5 μM and 10 μM) as indicated in the materials and methods section; motility was evaluated after 1 and three hours of incubation. TM%—percentage of total motile spermatozoa, PM %—percentage of progressive motile spermatozoa. Comparisons are made against controls. * P<0.05, ** P<0.01. The results are given as the means ± SD. The graphics represent two independent experiments (one for antimycin-A and one for rotenone) following a split sample design (the same ejaculate was split into the treatment and control groups).</p

Effect of the inhibition of glycolysis on stallion sperm motility and kinematics.

<p>Stallion sperm were incubated in presence of 0, 5, and 10 mM of 2-deoxy-D-glucose as described in the materials and methods. The percentages of total motile sperm and progressively motile sperm and the sperm velocities were analyzed using a CASA system. *p<0.05, ** p<0.001. The results are given as the means ± SD.</p

Effect of the inhibition of Complexes I and III of the electron transport chain on the percentage of stallion spermatozoa showing ROS production using CellRox deep red reagent.

<p>Stallion spermatozoa were extended in BWW media and incubated in the presence of rotenone (0 (DMSO), 100 nm, 500 nm, 5 μM and 10 μM) or antimycin (0 (DMSO), 100 nm, 500 nm, 5 μM and 10 μM) as indicated in the materials and methods. ROS were measured using flow cytometry after CellRox deep red staining as described in the materials and methods. * p<0.05, ** p<0.01. The results are given as the means ± SD.</p

Significant correlations between different indicators of the production of ROS and membrane intactness and mitochondrial functionality.

<p>** p<0.01, n.s non-significant</p><p>Mitosox+ spermatozoa show mitochondrial production of superoxide radical, H₂DCFDA—percentage of spermatozoa showing hydrogen peroxide production, CellRox (Live Cells)—percentage of live spermatozoa showing ROS production, HE—percentage of spermatozoa showing superoxide production, Intact sperm—percentage of spermatozoa with completely intact membranes, YoPro+—percentage of spermatozoa with intact membranes but with increased permeability, JC- high percentage of spermatozoa with high mitochondrial membrane potential.</p><p>Significant correlations between different indicators of the production of ROS and membrane intactness and mitochondrial functionality.</p

Significant correlations between Mitosox-positivity and sperm motility and velocity.

<p>Mitosox + spermatozoa showing mitochondrial production of superoxide radical, TM%—percentage of total motile sperm, VCL—curvilinear velocity, VAP—average path velocity, VSL—straight-line velocity.</p><p>*p<0.05</p><p>** p<0.01</p><p>Significant correlations between Mitosox-positivity and sperm motility and velocity.</p

Effect of the incubation of stallion spermatozoa in BWW complete media (BWW GLUC PYR) devoid of glucose (BWW PYR) or devoid of glucose and pyruvate (BWW) on on the ATP content of stallion spermatozoa.

<p>ATP was determined as described in the materials and methods. The results are given as the means ± SD.</p

Effect of the inhibition of Complex III of the electron transport chain on stallion sperm velocities (CASA analysis).

<p>Stallion spermatozoa were extended in BWW media and incubated in the presence of antimycin-A (0 vehicle DMSO, 100 nm, 500 nm, 5 μM and 10 μM) as indicated in the materials and methods; velocities were evaluated after 1 and three hours of incubation, VCL—curvilinear velocity (μM/s), VSL—straight-line velocity (μM/s), VAP—average path velocity (μM/s). * P<0.05, ** P<0.01. The results are given as the means ± SD.</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

Effect of the inhibition of Complexes I and III of the electron transport chain on the production of mitochondrial superoxide in stallion spermatozoa.

<p>Stallion spermatozoa were extended in BWW media and incubated in the presence of rotenone or antimycin as indicated in the materials and methods. Mitochondrial superoxide was measured using flow cytometry after Mitosox staining as described in the materials and methods. Data represent Mitosox positive sperm in the whole population. * p<0.05, ** p<0.01. The results are given as the means ± SD.</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