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 Ca2+ 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 Ca2+ 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.
Highlights
The spermatozoon is a germ cell that is highly specialized for cellular processes, motility, capacitation, hyperactivation and acrosome reaction that promote its essential function of oocyte fertilization
Results show that no band is detected with the secondary antibody and confirm that bands visualized are due to the AMPKa antibody used
It is interesting to mention that the expression level of AMPK protein detected in male germ cells (7 mg proteins were loaded into the SDS-PAGE) is likely higher than in somatic cells derived from other porcine tissues (20 mg proteins)
Summary
The spermatozoon is a germ cell that is highly specialized for cellular processes, motility, capacitation, hyperactivation and acrosome reaction that promote its essential function of oocyte fertilization. All these cellular processes are dependent on the energetic cellular state, determined by the ratio between cellular AMP and ATP, [1], [2] and regulated by biochemical mechanisms such as phosphorylation of proteins. The AMP activated protein kinase AMPK is an evolutionarily conserved serine/threonine kinase that acts as a sensor that detects the cell energy state and subsequently regulates metabolism [3]. One of the essential features of the AMPK kinase as a sensor and metabolic regulator is its extreme sensitivity to AMP, as any increase in the ratio AMP/ATP that means a decrease in cellular energy state, activates AMPK [3], [4]
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