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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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Related in: MedlinePlus

Short time effect (0–4 h) of the AMPK inhibition in the percentage of rapid spermatozoa.Spermatozoa were incubated in TBM (A, circles) or TCM medium (B, squares) at 38.5°C in the absence (white) or presence (black) of the AMPK inhibitor, compound C, (CC 30 µM) during 4 h. Samples at 17°C were considered as time 0. The percentage of those motile spermatozoa with VAP>80 µm/s, defined as rapid spermatozoa, was measured. This experiment was performed at least 6 times and the results express the percentage of rapid spermatozoa from the total spermatozoa motile analyzed (4.000–5.000). Statistical differences were considered when p<0.05 and showed with an asterisk.
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pone-0038840-g004: Short time effect (0–4 h) of the AMPK inhibition in the percentage of rapid spermatozoa.Spermatozoa were incubated in TBM (A, circles) or TCM medium (B, squares) at 38.5°C in the absence (white) or presence (black) of the AMPK inhibitor, compound C, (CC 30 µM) during 4 h. Samples at 17°C were considered as time 0. The percentage of those motile spermatozoa with VAP>80 µm/s, defined as rapid spermatozoa, was measured. This experiment was performed at least 6 times and the results express the percentage of rapid spermatozoa from the total spermatozoa motile analyzed (4.000–5.000). Statistical differences were considered when p<0.05 and showed with an asterisk.

Mentions: Rapid spermatozoa are defined as the percentage of those motile spermatozoa with velocity VAP higher than 80 µm/s. As seen in Figure 4 and according to previous literature [21], the time-course of the percentage of rapid spermatozoa incubated in TBM (Fig. 4A) clearly differs from the time-course in TCM (Fig. 4B). The inhibition of AMPK by CC in either TBM or TCM leads to a significant reduction in the percentage of those motile spermatozoa that move in a rapid manner in a time-dependent manner. However, whereas in TBM the reduction is detected at 2 h and maximum at 4 h of AMPK inhibition (reduction of number of rapid spermatozoa by 44%), in TCM the CC inhibitory effect is detected as rapid as 1 h and remains constant at any time studied (about 30% of reduction of the percentage of rapid spermatozoa).


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)

Short time effect (0–4 h) of the AMPK inhibition in the percentage of rapid spermatozoa.Spermatozoa were incubated in TBM (A, circles) or TCM medium (B, squares) at 38.5°C in the absence (white) or presence (black) of the AMPK inhibitor, compound C, (CC 30 µM) during 4 h. Samples at 17°C were considered as time 0. The percentage of those motile spermatozoa with VAP>80 µm/s, defined as rapid spermatozoa, was measured. This experiment was performed at least 6 times and the results express the percentage of rapid spermatozoa from the total spermatozoa motile analyzed (4.000–5.000). Statistical differences were considered when p<0.05 and showed with an asterisk.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375287&req=5

pone-0038840-g004: Short time effect (0–4 h) of the AMPK inhibition in the percentage of rapid spermatozoa.Spermatozoa were incubated in TBM (A, circles) or TCM medium (B, squares) at 38.5°C in the absence (white) or presence (black) of the AMPK inhibitor, compound C, (CC 30 µM) during 4 h. Samples at 17°C were considered as time 0. The percentage of those motile spermatozoa with VAP>80 µm/s, defined as rapid spermatozoa, was measured. This experiment was performed at least 6 times and the results express the percentage of rapid spermatozoa from the total spermatozoa motile analyzed (4.000–5.000). Statistical differences were considered when p<0.05 and showed with an asterisk.
Mentions: Rapid spermatozoa are defined as the percentage of those motile spermatozoa with velocity VAP higher than 80 µm/s. As seen in Figure 4 and according to previous literature [21], the time-course of the percentage of rapid spermatozoa incubated in TBM (Fig. 4A) clearly differs from the time-course in TCM (Fig. 4B). The inhibition of AMPK by CC in either TBM or TCM leads to a significant reduction in the percentage of those motile spermatozoa that move in a rapid manner in a time-dependent manner. However, whereas in TBM the reduction is detected at 2 h and maximum at 4 h of AMPK inhibition (reduction of number of rapid spermatozoa by 44%), in TCM the CC inhibitory effect is detected as rapid as 1 h and remains constant at any time studied (about 30% of reduction of the percentage of rapid spermatozoa).

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