Mitochondria copy number.

<p>Total DNA was isolated from liver of control, hypothyroid and hypothyroid+T₂ treated rats. Quantitative Real time PCR was used to determine nuclear DNA and mtDNA contents by using specific primers, targeting the nuclear gene <i>Gadph</i> and the mt D-loop region. The mtDNA level was expressed as mtDNA copy number to the nDNA copy number ratio (mtDNA/nDNA). Data are means ± SD of four experiments (each sample was analyzed in triplet).</p

Effect of 3,5-diiodothyronine on rat-liver mitochondrial respiratory rates from lipid substrates.

<p>Palmitoyl-CoA+carnitine or L-palmitoylcarnitine were used as respiratory substrates. Mitochondrial respiratory activity in state 3, state 4 and with FCCP is expressed as natoms oxygen/min/mg mitochondrial proteins. Respiratory control index (RCI) represents the ratio between state 3 and state 4 respiration rate. Data are means ± SD of 5 experiments performed with duplicate samples.</p

Effect of T₂ on palmitate oxidation by isolated rat hepatocytes.

<p>Hepatocytes were obtained from euthyroid (□), hypothyroid ( ) and hypothyroid +T₂-treated rats (▪). Hepatocytes were incubated with [1-¹⁴C]palmitate. Total fatty acid oxidation was obtained as the sum of labeled CO₂ and total acid soluble products (ASP). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052328#s3" target="_blank">Results</a>, expressed as [1-¹⁴C]palmitate into products/h/10⁶ cells, are means ± SD of 5 experiments with duplicate samples. Values sharing a different symbol differ significantly (P<0.05 vs euthyroid).</p

OXPHOS complexes enzyme activities.

<p>(A) NADH: coenzyme Q oxidoreductase (Complex I); (B) Succinate: coenzyme Q oxidoreductase (Complex II); (C) Succinate: cytochrome c oxidoreductase (Complex II–III); (D) Cytochrome c oxidase (Complex IV). Euthyroid rats (□), hypothyroid rats ( ) and hypothyroid +T₂-treated rats (▪). Complex activities were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052328#s2" target="_blank">Materials and Methods</a> section. Complex I and Complex II activities are expressed as nmoles DCIP reduced/min/mg protein. Complex II–III and Complex IV activity: nmoles cytochrome c reduced/min/mg protein. Data are means ± SD of five separate experiments with duplicate samples.</p

Oxygen uptake in liver mitochondria isolated from euthyroid, hypothyroid and hypothyroid plus T₂-treated rats.

<p>Glutamate(5 mM)+malate (2.5 mM), succinate (5 mM) or a mixture of glutamate+malate+succinate (5, 2.5, 5 mM) were used as respiratory substrates. Oxygen consumption was measured by a Clark oxygen electrode. Respiratory activity of state 3, state 4 and FCCP-stimulated was expressed as natoms oxygen/min/mg of mitochondrial proteins. Respiratory control index (RCI) represents the ratio between state 3 and state 4 respiration rate. Data are means ± SD of 6 experiments performed with duplicate samples. Values sharing a different letter differ significantly. P<0.05.</p

Carnitine palmitoyltransferase I (CPT-I) activity from euthyroid, hypothyroid and T₂-treated hypothyroid rats (Hypothyroid+T₂).

<p>The CPT-I activity was assayed in permeabilized hepatocytes A), in whole liver homogenate, B) and in isolated liver mitochondria C) by measuring the incorporation of 0.5 mM L-[Methyl-¹⁴C]carnitine (2 Ci/mol) into [¹⁴C]palmitoylcarnitine using 50 µM palmitoyl-CoA as substrate. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052328#s3" target="_blank">Results</a> are means ± SD of 5 experiments with duplicate samples. Values sharing a different symbol differ significantly (P<0.01 vs Euthyroid).</p

Representative 2-D gels of proteins extracted from the plasma membrane head isolated from fresh (A), cryopreserved with DMSO (B), with DMSO plus AFPI (C) and with DMSO plus AFPIII (D) gilthead sea bream spermatozoa.

<p>Extracted flagellar proteins (60 µg) were loaded onto non-linear IPG strips pH 3–10, and following isoelectric focusing were separated by 12.5% SDS–PAGE. Gels were silver stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#s2" target="_blank">Materials and Methods</a>. Spots that changed their expression after cryopreservation procedure are highlighted with a circle.</p

Effects of different cryoprotectants on different motility and viability parameters in sea bream spermatozoa before freezing (fresh sperm) and after different thawing conditions.

<p>Values are expressed as means ± SD for % motility, motility duration and % vitality and as means ± SEM for VCL and VSL. Within each column, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p><p>VSL: Velocity Straight Line.</p><p>VCL: curvilinear velocity.</p

Differences in relative abundance of 10 sperm head membrane spots in fresh and cryopreserved sea bream spermatozoa.

<p>Spots are highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#pone-0099992-g002" target="_blank">Figure 2</a> and the <i>x</i> axis identified the experimental conditions (fresh semen or cryopreserved with DMSO, with DMSO plus AFPI and with DMSO plus AFPIII). The <i>y</i>-axis corresponds to the intensity of spots expressed as normalized spot volume. Spot intensity is expressed as mean (dark bars) ± SD (light grey bars) of normalized spot volume (N = 18, that correspond to 6 different fish and 3 replicates for each). Different letters in the same graph indicate that mean values are significantly different by Mann–Whitney test (p<0.05). The spots showing a decrease of their expression after cryopreservation procedure are reported in the group 1, and those showing an increase are reported in the group 2.</p

Effect of AFPI and AFPIII at three different concentrations (0.1, 1 and 10 µg/ml) on motility rate and viability before freezing (fresh sperm) and after thawing.

<p>Values are expressed as means ± SD.</p><p>Within each row, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p