Metformin inhibits human spermatozoa motility and signalling pathways mediated by protein kinase A and tyrosine phosphorylation without affecting mitochondrial function.

Metformin is a leading antidiabetic drug that is used worldwide in the treatment of diabetes mellitus. This biguanide exerts metabolic and pleiotropic effects in somatic cells, although its invitro actions on human spermatozoa remain unknown. The present study investigated the effects of metformin on human sperm function. Human spermatozoa were incubated in the presence or absence of 10mM metformin for 8 or 20h, and motility was measured by computer-aided sperm analysis (CASA); other parameters were evaluated by flow cytometry. Metformin significantly reduced the percentage of motile, progressive and rapid spermatozoa and significantly decreased sperm velocity. Metformin did not affect viability, mitochondrial membrane potential (MMP) or mitochondrial superoxide anion generation of human spermatozoa at any time studied. However, metformin clearly inhibited the protein kinase (PK) A pathway and protein tyrosine phosphorylation at 8 and 20h, key regulatory pathways for correct sperm function. In summary, metformin treatment of human spermatozoa had a detrimental effect on motility and inhibited essential sperm signalling pathways, namely PKA and protein tyrosine phosphorylation, without affecting physiological parameters (viability, MMP, mitochondrial superoxide anion generation). Given the growing clinical use of metformin in different pathologies in addition to diabetes, this study highlights an adverse effect of metformin on spermatozoa and its relevance in terms of human fertility in patients who potentially could be treated with metformin in the future.

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.

HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress.

Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode's basal medium or TBM) for 3, 8 and 24h or in capacitating conditions (Tyrode's complete medium or TCM) for 4h at 38.5°C or 40°C (Heat stress) in the presence or absence of 5 or 20µM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7±4.1 vs. 1.9±1.1; % mean±SEM), PM (3.1±0.9 vs. 0) and high mitochondrial membrane potential (19.5±2.2 vs. 11.8±0.8) decreased significantly in sperm incubated at 40°C for 24h in the presence of 20µM 17-AAG (control vs. 20µM 17-AAG, respectively; p<0.05). In sperm incubated at 38.5°C only a mild decrease in TM was observed (48.7±3.1 vs. 32.1±4.8; control vs. 20µM 17-AAG, respectively; p<0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.