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.

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.

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.

Protein kinase C activity in boar sperm.

Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

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.

A new Bayesian network-based approach to the analysis of sperm motility: application in the study of tench (Tinca tinca) semen.

In this study a Bayesian network (BN) has been built for the study of the objective motility of Tinca tinca spermatozoa (spz). Semen from eight 2-year-old sexually mature male tenchs was obtained and motility analyses were performed at 6-17, 23-34 and 40-51 s after activation, using computer-assisted sperm analysis (CASA) software. Motility parameters rendered by CASA were treated with a two-step cluster analysis. Three well-defined sperm subpopulations were identified, varying the proportion of spermatozoa contained in each cluster with time and male. Cluster, cinematic and time variables were used to build the BN to study the probabilistic relationships among variables and how each variable influenced the final sperm classification into one of three predefined clusters. Both network structure and conditional probabilities were calculated based on the collected data set. Results shown that almost all the variables were directly or indirectly related to each other. By doing probabilistic inference we observed that the cluster distribution corresponded to the definition provided by the cluster analysis. Also, velocity and time variables determined the cluster to which each spermatozoon belonged with a high degree of accuracy. Thus, BNs can be applied in the study of sperm motility. The construction of a BN that include fertility data opens a new way to try to clarify the roles of motility and other sperm quality indicators in fertilization.

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.

Inter- and intra-breed comparative study of sperm motility and viability in Iberian and Duroc boar semen during long-term storage in MR-A and XCell extenders.

During boar semen liquid preservation, extender is one of the factors that influence storage tolerance of spermatozoa. However, there are few studies about intra-breed variation in the preservation of semen quality during storage in different extenders. Similarly, boar breed is generally not considered a possible factor influencing variation in the semen storage tolerance in a particular extender. The aim of this study was to compare boar semen storage potential, in terms of the ability to maintain sperm viability and motility, of two currently used long-term extenders, MR-A and XCell. Extended semen from two breeds, Iberian and Duroc that had been stored at 17°C for up to 7 days was used. Intra- and inter-breed effect was studied. On Days 1, 4 and 7 (Day 0=day of semen collection), motility parameters and the percentage of total motile sperm and progressively motile sperm using a CASA system was evaluated. Viability (SYBR-14/PI) was evaluated by flow cytometry. Within each breed and for each storage day, there were differences between extenders, although semen tolerance to preservation was more influenced by the extender in the Iberian than in the Duroc breed. Neither breed nor extender influenced the percentage of viable spermatozoa during the storage time. Moreover, differences in motility parameters were observed between breeds, although the differences were greater when the XCell extender was used. In conclusion, both extender and breed influence motility characteristics of liquid-stored boar semen, so both aspects have to be considered in the design of comparative studies about stored boar semen quality from different breeds or with different extenders. Further studies are needed to corroborate these findings.

Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction.

Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 µm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.

The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C.

Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 µM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 µM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen.

Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3alpha.

Sperm functions are critically controlled through the phosphorylation state of specific proteins. Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (alpha and beta), the enzyme activity of which is inhibited by serine phosphorylation. Recent studies suggest that GSK3 is involved in the control of bovine sperm motility. Our aim was to investigate whether GSK3 is present in porcine spermatozoa and its role in the function of these cells. This work shows that both isoforms of GSK3 are present in whole cell lysates of porcine sperm and are phosphorylated on serine in spermatozoa stimulated with the cAMP analog, 8Br-cAMP. A parallel increase in serine phosphorylation of the isoform GSK3alpha, but not in the isoform GSK3beta, is observed after treatments that also induce a significant increase in porcine sperm velocity parameters. Therefore, a significant positive correlation among straight-line velocity, circular velocity, average velocity, rapid-speed spermatozoa, and GSK3alpha serine phosphorylation levels exists. Inhibition of GSK3 activity by alsterpaullone leads to a significant increase in the percentage of rapid- and medium-speed spermatozoa as well as in all sperm velocity parameters and coefficients. Moreover, pretreatment of porcine spermatozoa with alsterpaullone significantly increased the percentage of capacitated porcine spermatozoa and presents no effect in the number of acrosome-reacted porcine spermatozoa. Our work suggests that the isoform GSK3alpha plays a negative role in the regulation of porcine sperm motility and points out the possibility that sperm motile quality might be modulated according the activity state of GSK3alpha.

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

Hepatocyte growth factor activates several transduction pathways in rat pancreatic acini.

The receptor of hepatocyte growth factor (HGF), c-met induces different physiological responses in several cell types. Little is known about the role of HGF in exocrine pancreas. However, abnormal HGF signaling has been strongly implicated in pancreatic tumorigenesis and association of HGF with pancreatitis has been demonstrated. We have studied the presence of c-met and activation of their intracellular pathways associated in rat pancreatic acini in comparison with cholecystokinin (CCK) and epidermal growth factor (EGF). C-met expression in rat exocrine pancreas was identified by immunohistochemistry and immunoprecipitation followed by Western analysis. Tyrosine phosphorylation of c-met is strongly stimulated as well as kinase pathways leading to ERK1/2 cascade. HGF, but not CCK or EGF, selectively caused a consistent increase in the amount of p85 regulatory subunit of PI3-K present in anti-phosphotyrosine immunoprecipitates. Downstream of PI3-K, HGF increased Ser473 phosphorylation of Akt selectively, as CCK or EGF did not affect it. HGF selectively stimulated tyrosine phosphorylation of phosphatase PTP1D. HGF failed to promote the well-known CCK effects in pancreatic acini such as amylase secretion and intracellular calcium mobilization. Although HGF shares activation of ERK1/2 with CCK, we demonstrate that it promotes the selective activation of intracellular pathways not regulated by CCK or EGF. Our results suggest that HGF is an in vivo stimulus of pancreatic acini and provide novel insight into the transduction pathways and effects of c-met/HGF in normal pancreatic acinar cells.

Protective effect of long term high fiber diet consumption on rat exocrine pancreatic function after chronic ethanol intake.

The effects of ethanol administration on exocrine pancreas have been widely studied, but little is known about the effect of dietary fiber in combination with chronic ethanol on exocrine pancreatic function. The aim of this work was to examine the chronic effects of a high fiber diet, ethanol ingestion, and a combination of both on the function of the rat exocrine pancreas. Four groups of rats were fed for six months the following diets: 1.- NW: standard laboratory diet; 2.- FW: high fiber diet (15% cellulose); 3.- NE: standard laboratory diet and 20% ethanol in the drinking water; and 4.- FE: high fiber diet and 20% ethanol. Cholecystokinin (CCK) and acetylcholine (Ach) effects on amylase release and intracellular calcium mobilization in pancreatic acini were studied. In rats fed a 20% ethanol (NE), both the basal amylase release and the basal [Ca(2+)](i) were significantly increased; nonetheless, CCK and Ach-induced amylase release were significantly reduced compared with control rats. Ach- but not CCK-stimulated [Ca(2+)](i) increase in NE rats was significantly decreased compared with NW. In rats fed a combination of ethanol and a high fiber diet (FE) all the parameters under study were not significantly affected compared to control rats (NW). In conclusion, high fiber consumption does not alter the function of the exocrine pancreas. However, it ameliorates the deleterious effect of chronic ethanol consumption on pancreatic amylase secretion and, at least partially, reverses the ethanol-induced alterations on [Ca(2+)](i) in the rat exocrine pancreas.

Regulation of the initiation of pancreatic digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo.

BACKGROUND & AIMS: Cholecystokinin (CCK) is known to stimulate the synthesis of digestive enzymes in the pancreas at the translational level. We investigated in vivo the biochemical regulation of initiation factors important for the stimulation of translation of digestive enzyme protein in rat pancreas by CCK. METHODS: Intraperitoneal injection of CCK or intragastric administration of a trypsin inhibitor to elicit endogenous CCK release was followed by removal and preparation of pancreas for protein evaluation. Isoelectric focusing was used to evaluate the phosphorylation of the initiation factor eIF4E, and Western blotting and immunoprecipitation followed by Western blotting were used to study the phosphorylation state and amount of other interacting factors. RESULTS: CCK treatment induced a time- and dose-dependent phosphorylation of pancreatic eIF4E and its binding protein (PHAS-I). Because the release of eIF4E from its binding protein as a result of phosphorylation is followed by formation of a messenger RNA cap-binding complex that includes the initiation factor eIF4G, we evaluated the association of eIF4G with released eIF4E and showed that it was increased by CCK. These events occurred over a range of CCK doses from 0.2 to 5 microg/kg. We also evaluated the effect of endogenous CCK by administering a synthetic trypsin inhibitor, camostat (100 mg/kg). Camostat treatment markedly increased the phosphorylation of both PHAS-I and eIF4E and the formation of eIF4E-eIF4G complex. Thus, both exogenous and endogenous CCK activate translational initiation factors in vivo. CONCLUSIONS: Activation of translational machinery necessary for initiation of protein synthesis likely contributes to the normal postprandial synthesis of pancreatic digestive enzymes.