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The 5'-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

Nguyen TM, Seigneurin F, Froment P, Combarnous Y, Blesbois E - PLoS ONE (2015)

Bottom Line: MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased.CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters.Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.

View Article: PubMed Central - PubMed

Affiliation: INRA-CNRS, Unité Mixte de Recherche de Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France; Université François Rabelais, Tours, France.

ABSTRACT
Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5'-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.

No MeSH data available.


Related in: MedlinePlus

Modulators effects on AMPK phosphorylation in frozen-thawed chicken sperm.Sperm lysates were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr172-AMPKα and anti-AMPKα antibody. Bands for phospho-Thr172-AMPKα were detected at 62kDa (top lanes). Total AMPKα was used as loading control (62kDa) (bottom lanes) and the phosphorylated protein AMPKα (Thr172)/ total AMPKα ratio is shown at the bottom. Cryopreserved sperm were treated in the presence of an AMPK activator (2mM AICAR (in light gray) or 1mM MET (in diagonals)) or an AMPK inhibitor (5μM CC (in dark gray)) or Control (Ctrl, in white); Fresh semen (checkered pattern). Values represent means ± SEM from 6 different experiments (4 samples/experiment). Different superscripts indicate significant differences between Ctrl and treatments (AICAR, MET or CC) in frozen-thawed semen (a,b,c, P<0.05). Asterisks indicate statistically significant differences between fresh and frozen-thawed semen (*, P<0.05).
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pone.0134420.g001: Modulators effects on AMPK phosphorylation in frozen-thawed chicken sperm.Sperm lysates were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr172-AMPKα and anti-AMPKα antibody. Bands for phospho-Thr172-AMPKα were detected at 62kDa (top lanes). Total AMPKα was used as loading control (62kDa) (bottom lanes) and the phosphorylated protein AMPKα (Thr172)/ total AMPKα ratio is shown at the bottom. Cryopreserved sperm were treated in the presence of an AMPK activator (2mM AICAR (in light gray) or 1mM MET (in diagonals)) or an AMPK inhibitor (5μM CC (in dark gray)) or Control (Ctrl, in white); Fresh semen (checkered pattern). Values represent means ± SEM from 6 different experiments (4 samples/experiment). Different superscripts indicate significant differences between Ctrl and treatments (AICAR, MET or CC) in frozen-thawed semen (a,b,c, P<0.05). Asterisks indicate statistically significant differences between fresh and frozen-thawed semen (*, P<0.05).

Mentions: Western blot analyses using antibodies against phospho-Thr172-AMPKα and total AMPKα (as control) were performed on chicken sperm incubated in the absence or presence of 2mM AICAR, 1mM MET, or 5μM CC during freezing and thawing (Fig 1).


The 5'-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

Nguyen TM, Seigneurin F, Froment P, Combarnous Y, Blesbois E - PLoS ONE (2015)

Modulators effects on AMPK phosphorylation in frozen-thawed chicken sperm.Sperm lysates were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr172-AMPKα and anti-AMPKα antibody. Bands for phospho-Thr172-AMPKα were detected at 62kDa (top lanes). Total AMPKα was used as loading control (62kDa) (bottom lanes) and the phosphorylated protein AMPKα (Thr172)/ total AMPKα ratio is shown at the bottom. Cryopreserved sperm were treated in the presence of an AMPK activator (2mM AICAR (in light gray) or 1mM MET (in diagonals)) or an AMPK inhibitor (5μM CC (in dark gray)) or Control (Ctrl, in white); Fresh semen (checkered pattern). Values represent means ± SEM from 6 different experiments (4 samples/experiment). Different superscripts indicate significant differences between Ctrl and treatments (AICAR, MET or CC) in frozen-thawed semen (a,b,c, P<0.05). Asterisks indicate statistically significant differences between fresh and frozen-thawed semen (*, P<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519194&req=5

pone.0134420.g001: Modulators effects on AMPK phosphorylation in frozen-thawed chicken sperm.Sperm lysates were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr172-AMPKα and anti-AMPKα antibody. Bands for phospho-Thr172-AMPKα were detected at 62kDa (top lanes). Total AMPKα was used as loading control (62kDa) (bottom lanes) and the phosphorylated protein AMPKα (Thr172)/ total AMPKα ratio is shown at the bottom. Cryopreserved sperm were treated in the presence of an AMPK activator (2mM AICAR (in light gray) or 1mM MET (in diagonals)) or an AMPK inhibitor (5μM CC (in dark gray)) or Control (Ctrl, in white); Fresh semen (checkered pattern). Values represent means ± SEM from 6 different experiments (4 samples/experiment). Different superscripts indicate significant differences between Ctrl and treatments (AICAR, MET or CC) in frozen-thawed semen (a,b,c, P<0.05). Asterisks indicate statistically significant differences between fresh and frozen-thawed semen (*, P<0.05).
Mentions: Western blot analyses using antibodies against phospho-Thr172-AMPKα and total AMPKα (as control) were performed on chicken sperm incubated in the absence or presence of 2mM AICAR, 1mM MET, or 5μM CC during freezing and thawing (Fig 1).

Bottom Line: MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased.CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters.Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.

View Article: PubMed Central - PubMed

Affiliation: INRA-CNRS, Unité Mixte de Recherche de Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France; Université François Rabelais, Tours, France.

ABSTRACT
Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5'-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.

No MeSH data available.


Related in: MedlinePlus