Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa.

Metformin is clinically used to treat diabetes. Given its role-impacting metabolism, metformin has been also added to semen cryopreservation media showing specie-dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin-including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ-population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.

Human sperm motility is downregulated by the AMPK activator A769662.

AMP-activated kinase (AMPK) plays a key function in maintaining cellular energy homeostasis. We recently identified and localized AMPK protein in human spermatozoa and showed that inhibition of AMPK activity significantly modified human sperm motility. Recently, AMPK has gained great relevance as prime target for pharmacological approaches in several energy-related pathologies and therefore pharmacological research is aimed to develop direct AMPK-activating compounds such as A769662. Our aim was to investigate the effect of A769662 in essential functional processes of human spermatozoa. Human spermatozoa were incubated in the presence or absence of the AMPK activator A769662 for different incubation times (0-20 h) and motility was evaluated by CASA system whereas other functional parameters were evaluated by flow cytometry. A769662 treatment significantly reduces the percentages of motile, progressive, and rapid spermatozoa starting at 2 h. Moreover, AMPK activator in human spermatozoa causes a significant reduction in any velocity measured, which is concomitant to a significant decrease in the percentage of rapid spermatozoa, both at short- (2-3 h) and long-time treatment (20 h). Treatment of human spermatozoa with A769662 does not significantly alter any of the following functional parameters: sperm viability, mitochondrial membrane potential, phosphatidylserine translocation to the outer leaf of plasma membrane, acrosome membrane integrity, or mitochondrial superoxide anion production. In summary, our results suggest that AMPK in human spermatozoa contributes to the regulation of sperm motility, without affecting basic physiological parameters of human spermatozoa (viability, mitochondrial membrane potential or reactive oxygen species production, acrosome membrane integrity, phosphatidylserine exposure at plasma membrane). As sperm motility is required in the female reproductive tract to achieve fertilization, we conclude that AMPK is an essential regulatory kinase of the human spermatozoa function. This conclusion needs to be taken into account when AMPK is elected as prime target in pharmacological approaches for several energy-related pathologies.

New insights into transduction pathways that regulate boar sperm function.

Detailed molecular mechanisms mediating signal transduction cascades that regulate boar sperm function involving Ser/Thr and tyrosine phosphorylation of proteins have been reviewed previously. Therefore, this review will focus in those kinase pathways identified recently (<10 years) in boar spermatozoa that regulate different functional spermatozoa processes. AMP-activated protein kinase (AMPK) is a cell energy sensor kinase that was first identified in mammalian spermatozoa in 2012, and since then it has emerged as an essential regulator of boar sperm function. Signaling pathways leading to AMPK activation in boar sperm are highlighted in this review (PKA, CaMKKα/ß, and PKC as well as Ca(2+) and cAMP messengers as upstream regulators). Interestingly, stimuli considered as cell stress (hyperosmotic stress, inhibition of mitochondrial activity, absence of intracellular Ca(2+)) markedly activate AMPK in boar spermatozoa. Moreover, AMPK plays a remarkable and necessary regulatory role in mammalian sperm function, controlling essential boar sperm functional processes such as motility, viability, mitochondrial membrane potential, organization and fluidity of plasma membrane, and outer acrosome membrane integrity. These mentioned processes are all required under fluctuating environment of spermatozoa when transiting through the female reproductive tract to achieve fertilization. An applied role of AMPK in artificial insemination techniques is also suggested as during boar seminal doses preservation at 17 °C, physiological levels of AMPK activity markedly increase (maximum on Day 7) and result essential to maintain the aforementioned fundamental sperm processes. Moreover, regulation of sperm function exerted by the glycogen synthase kinase 3 and Src family kinase pathways is summarized.

AMPK up-activation reduces motility and regulates other functions of boar spermatozoa.

We recently demonstrated that AMPK inhibition in spermatozoa regulates motility, plasma membrane organization, acrosome integrity and mitochondrial membrane potential. As AMPK activity varies in different energy conditions induced by sperm environment, this work investigates the functional effects of AMPK activation in boar spermatozoa. Spermatozoa were incubated under non-stimulating (TBM) or Ca(2+) and [Formula: see text]-stimulating (TCM) media in the presence/absence of AMPK activator, A769662, for different times. AMPK activity, evaluated as Thr(172) phosphorylation by western blot, is effectively increased by A769662 in spermatozoa. AMPK activation significantly reduces the percentage of motile spermatozoa under Ca(2+) and/or [Formula: see text]-stimulating conditions. Moreover, AMPK activation in spermatozoa incubated in TBM or TCM significantly reduces curvilinear VCL, straight-line VSL and average VAP velocities, which subsequently lead to a significant decrease in the percentage of rapid spermatozoa (VAP > 80 µm/s). The effect of AMPK activation on motility is intensified by the absence of BSA in the incubation medium. AMPK activation for a short time prevents the decline in cell viability and in the sperm population displaying high mitochondrial membrane potential which is induced by Ca(2+) and [Formula: see text]. Sustained (24 h) AMPK activation under TBM or TCM significantly increases both lipid disorganization and phosphatidylserine externalization in the sperm plasma membrane, and diminishes the acrosome membrane integrity. In summary, AMPK activation modifies essential sperm processes such as motility, viability, mitochondrial membrane potential, acrosome membrane integrity, and organization and fluidity of plasma membrane. As these spermatozoa processes are required under different environmental conditions when transiting through the female reproductive tract to achieve fertilization, we conclude that balanced levels of AMPK activity are essential for regulating sperm function.