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AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility.

Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ - PLoS ONE (2012)

Bottom Line: We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa.Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients.CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation.

View Article: PubMed Central - PubMed

Affiliation: Research Group of Intracellular Signalling and Technology of Reproduction, Veterinary School, University of Extremadura, Caceres, Spain.

ABSTRACT
The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca(2+) and bicarbonate TCM, time from 1-24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca(2+) and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.

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Phosphorylation of AMPK at Thr172 is regulated at physiological temperature in boar spermatozoa and is effectively inhibited by the compound C.2A: Spermatozoa were incubated in TBM or TCM medium at 38.5°C for indicated times and then lysed. Samples at 17°C were considered as time 0. Proteins (20 µg) from lysates were analyzed by western blotting using anti-phospho-Thr172-AMPKα as primary antibody. Arrow indicates the cross-reactive band of phospho-Thr172AMPK, recognized by the anti-AMPKα. This experiment was performed 6 times and a representative film is shown. 2B: AMPK phosphorylation was evaluated in spermatozoa incubated in TBM in the presence (+) or absence (−) of AMPK inhibitor, compound C (CC 30 µM) at 38.5°C during 24 h. This experiment was performed 3 times and a representative film is shown. Loading controls were performed for each experiment in the same membranes (with different time of exposure) using anti-GSK3β antibody and are showed at lower panels in 2A and 2B.
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pone-0038840-g002: Phosphorylation of AMPK at Thr172 is regulated at physiological temperature in boar spermatozoa and is effectively inhibited by the compound C.2A: Spermatozoa were incubated in TBM or TCM medium at 38.5°C for indicated times and then lysed. Samples at 17°C were considered as time 0. Proteins (20 µg) from lysates were analyzed by western blotting using anti-phospho-Thr172-AMPKα as primary antibody. Arrow indicates the cross-reactive band of phospho-Thr172AMPK, recognized by the anti-AMPKα. This experiment was performed 6 times and a representative film is shown. 2B: AMPK phosphorylation was evaluated in spermatozoa incubated in TBM in the presence (+) or absence (−) of AMPK inhibitor, compound C (CC 30 µM) at 38.5°C during 24 h. This experiment was performed 3 times and a representative film is shown. Loading controls were performed for each experiment in the same membranes (with different time of exposure) using anti-GSK3β antibody and are showed at lower panels in 2A and 2B.

Mentions: The level of phosphorylation of AMPK in Thr172 was analyzed at physiological temperature of boar spermatozoa (38.5°C) as an assessment of its enzymatic activity. As seen in Figure 2A, two cross-reactive bands are detected with anti-phospho-Thr172-AMPK antibody, being the upper band the AMPK phosphorylated at Thr172, as i) the molecular weight is the proper to the α subunit of AMPK and ii) this upper band is also recognized with the anti AMPKα antibody used in Figure 1. Negative control was also performed omitting the primary antibody and blot was probed with secondary antibody (anti-rabbit-HRP) only. No bands are detected with the secondary antibody and confirm that bands visualized are due to the anti-phospho-Thr172-AMPK antibody used (data not shown). As shown in Figure 2A, a clear band of phospho-Thr172-AMPK is detected when spermatozoa are incubated at 38.5°C in TBM or in a medium with calcium and bicarbonate (TCM). The intensity of the AMPK phosphorylated band results dependent on the incubation time at 38.5°C, reaching highest levels of phosphorylation between 30–60 minutes. When the same pools of spermatozoa are incubated either in a TBM or TCM at lower temperature, 17°C (considered as minute 0 in the Figure), which is the routine value for porcine semen preservation with low energy consumption, AMPK phosphorylation is very low or even not detectable. This effect is independent of the incubation time of spermatozoa at 17°C (data not shown). A loading control of protein is showed in lower panel of Figure 2A using an anti-GSK3β antibody, as we have previously shown that level of this protein does not change under these experimental conditions [30].


AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility.

Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ - PLoS ONE (2012)

Phosphorylation of AMPK at Thr172 is regulated at physiological temperature in boar spermatozoa and is effectively inhibited by the compound C.2A: Spermatozoa were incubated in TBM or TCM medium at 38.5°C for indicated times and then lysed. Samples at 17°C were considered as time 0. Proteins (20 µg) from lysates were analyzed by western blotting using anti-phospho-Thr172-AMPKα as primary antibody. Arrow indicates the cross-reactive band of phospho-Thr172AMPK, recognized by the anti-AMPKα. This experiment was performed 6 times and a representative film is shown. 2B: AMPK phosphorylation was evaluated in spermatozoa incubated in TBM in the presence (+) or absence (−) of AMPK inhibitor, compound C (CC 30 µM) at 38.5°C during 24 h. This experiment was performed 3 times and a representative film is shown. Loading controls were performed for each experiment in the same membranes (with different time of exposure) using anti-GSK3β antibody and are showed at lower panels in 2A and 2B.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375287&req=5

pone-0038840-g002: Phosphorylation of AMPK at Thr172 is regulated at physiological temperature in boar spermatozoa and is effectively inhibited by the compound C.2A: Spermatozoa were incubated in TBM or TCM medium at 38.5°C for indicated times and then lysed. Samples at 17°C were considered as time 0. Proteins (20 µg) from lysates were analyzed by western blotting using anti-phospho-Thr172-AMPKα as primary antibody. Arrow indicates the cross-reactive band of phospho-Thr172AMPK, recognized by the anti-AMPKα. This experiment was performed 6 times and a representative film is shown. 2B: AMPK phosphorylation was evaluated in spermatozoa incubated in TBM in the presence (+) or absence (−) of AMPK inhibitor, compound C (CC 30 µM) at 38.5°C during 24 h. This experiment was performed 3 times and a representative film is shown. Loading controls were performed for each experiment in the same membranes (with different time of exposure) using anti-GSK3β antibody and are showed at lower panels in 2A and 2B.
Mentions: The level of phosphorylation of AMPK in Thr172 was analyzed at physiological temperature of boar spermatozoa (38.5°C) as an assessment of its enzymatic activity. As seen in Figure 2A, two cross-reactive bands are detected with anti-phospho-Thr172-AMPK antibody, being the upper band the AMPK phosphorylated at Thr172, as i) the molecular weight is the proper to the α subunit of AMPK and ii) this upper band is also recognized with the anti AMPKα antibody used in Figure 1. Negative control was also performed omitting the primary antibody and blot was probed with secondary antibody (anti-rabbit-HRP) only. No bands are detected with the secondary antibody and confirm that bands visualized are due to the anti-phospho-Thr172-AMPK antibody used (data not shown). As shown in Figure 2A, a clear band of phospho-Thr172-AMPK is detected when spermatozoa are incubated at 38.5°C in TBM or in a medium with calcium and bicarbonate (TCM). The intensity of the AMPK phosphorylated band results dependent on the incubation time at 38.5°C, reaching highest levels of phosphorylation between 30–60 minutes. When the same pools of spermatozoa are incubated either in a TBM or TCM at lower temperature, 17°C (considered as minute 0 in the Figure), which is the routine value for porcine semen preservation with low energy consumption, AMPK phosphorylation is very low or even not detectable. This effect is independent of the incubation time of spermatozoa at 17°C (data not shown). A loading control of protein is showed in lower panel of Figure 2A using an anti-GSK3β antibody, as we have previously shown that level of this protein does not change under these experimental conditions [30].

Bottom Line: We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa.Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients.CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation.

View Article: PubMed Central - PubMed

Affiliation: Research Group of Intracellular Signalling and Technology of Reproduction, Veterinary School, University of Extremadura, Caceres, Spain.

ABSTRACT
The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca(2+) and bicarbonate TCM, time from 1-24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca(2+) and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.

Show MeSH
Related in: MedlinePlus