SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets.

Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr-induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca2+ homeostasis described in neonatal platelets. Both Ca2+ mobilization and Ca2+ influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca2+ mobilization in response to the PAR-1 agonist (SFLLRN) or by blocking SERCA3 function with tert-butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half-life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca2+ entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca2+ accumulation in the intracellular stores and Thr-evoked Ca2+ entry through either capacitative channels or non-selective channels are altered in neonatal platelets, contributing to deregulated Ca2+ homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects.

The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa.

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.

Phosphoproteomics for the identification of new mechanisms of cryodamage: the role of SPATA18 in the control of stallion sperm function†.

Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protein 4 (AKAP4), A-kinase anchoring protein 3 (AKAP3) and the Family with sequence similarity 71 member B (FAM71B). These proteins belong to the gene ontology (GO) terms sperm fibrous sheath (GO: 0035686), and sperm principal piece (GO: 0097228). The regulatory interactions between kinases and phosphorylation sites on the proteins that were affected most were also investigated, and the potential kinases (based on human orthologs) involved in the regulation of these phosphoproteins identified were: PKCß for SPATA18 and GSK3ß for CABYR. Kinase inhibition assays were also conducted showing that kinases phosphorylating the above-mentioned proteins play an important role in their activity and thus, phosphorylation controls the activity of these proteins and their role in the regulation of the functionality and viability of stallion spermatozoa. In conclusion, the data reported here contribute to the understanding of the fact that the dephosphorylation of certain proteins is a molecular lesion induced by cryopreservation in the stallion spermatozoa.

Antibiotic resistance among clinical Ureaplasma isolates from Cuban individuals between 2013 and 2018.

Introduction. Acquired resistance against the antibiotics that are active against Ureaplasma species has been described.Hypothesis/Gap Statement. Diagnostics combined with antimicrobial sensitivity testing are required for therapeutic guidance.Aim. To report the prevalence of antimicrobial resistance among Cuban Ureaplasma isolates and the related molecular mechanisms of resistance.Methodology. Traditional broth microdilution assays were used for antimicrobial sensitivity testing in 262 clinical Ureaplasma species isolates from Cuban patients between 2013 and 2018, and a subset of samples were investigated in parallel with the commercial MYCO WELL D-ONE rapid culture diagnostic assay. The underlying molecular mechanisms for resistance were determined by PCR and sequencing for all resistant isolates.Results. Among the tested isolates, the tetracycline and erythromycin resistance rates were 1.9 and 1.5%, respectively, while fluoroquinolone resistance was not found. The tet(M) gene was found in all tetracycline-resistant isolates, but also in two tetracycline-susceptible Ureaplasma clinical isolates. We were unable to determine the underlying mechanism of erythromycin resistance. The MYCO WELL D-ONE kit overestimated tetracycline and erythromycin resistance in Ureaplasma spp. isolates.Conclusions. Although low levels of antibiotic resistance were detected in Cuban patients over a 5-year period, continued surveillance of the antibiotic susceptibility of Ureaplasma is necessary to monitor possible changes in resistance patterns.

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility.

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SIGNIFICANCE: Proteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions.

First Report of Macrophomina phaseolina Causing Charcoal Rot of Peanut (Arachis hypogaea L.) in Mexico.

Peanut (Arachis hypogaea L.) is the third most important oilseed crop in the world. The cultivated area in Mexico is currently 52,046 ha with a production of 91,109 ton in 2018 (FAO, 2020). Puebla state ranks third in the national production with 9,313 ton (SIAP, 2020). In September 2019, typical symptoms of charcoal rot (Macrophomina phaseolina (Tassi) Goid.) were observed in about 50% of cultivar Virginia Champs peanuts, and it affecting 1.5 ha located in Chietla (18° 27' 39" N; 98° 37' 11" W), Puebla, Mexico. Diseased plants showed brown discoloration in stem and root rot, with chlorotic foliage, dark microsclerotia were observed on the stem and premature dying. To isolate the causal agent of these symptoms, 20 infected plants were recovered and processed in the laboratory. Ten pieces of stem and root tissue were selected from each plant, cut into small pieces 5-mm in length, superficially disinfested with 1% sodium hypochlorite for 3 min, followed by three rinses with sterile distilled water. Later, dried on sterile paper and placed on Petri plates containing potato dextrose agar (PDA) medium, which were kept at 28°C for 7 days (12 h light and 12 h dark). Four colonies were purified via hyphal tip culture, fungus was consistently isolated from the analyzed tissues; additional microcultures were prepared to observe phenotypic characteristics. Colonies showed dense growth, with a gray initial mycelium, becoming black after 7 days. Microesclerotia with spherical to oblong in shape were observed after 5 days on PDA, with a black coloration, measuring an average of 74 µm width × 110 µm length (n=40). Phylogenetic analysis was conducted by amplification and sequencing of the internal transcribed spacer (ITS) region with the ITS5 and ITS4 primers (White et al. 1990). The obtained sequences were deposited in GenBank database under accession numbers: MW585378, MW585379, MW585380, and MW585381 containing approximately 601 bp of the ITS1-5.8S-ITS2 region (complete sequence); they were 99% identical with the reference sequence of Macrophomina phaseolina (GenBank accession KF951698) isolated in Phaseolus vulgaris from Mexico. Based on the symptoms in the field, colony morphology, color, and shape of the microsclerotia, and molecular identification, the fungus was identified as M. phaseolina (Tassi) Goid. The pathogenicity test was performed on peanut plants cultivar Virginia Champs grown on plastic pots with an autoclaved peat/soil mixture under greenhouse conditions (70% relative humidity and 28°C). Fifty two-month-old peanut plants were inoculated using the toothpick method. The toothpicks were previously sterilized and then placed in Petri plates with each of the four colonies of M. phaseolina until colonization. Small wounds were made with those toothpicks in the roots, and a sterile toothpick was used in the control plants, the assays were performed twice. After three weeks, the inoculated plants exhibited symptoms of wilting chlorosis on the leaves and brown to dark brown discoloration of the vascular ring, while control plants remained healthy. M. phaseolina was re-isolated from symptomatic root tissues and identified by phylogenetic approach, fulfilling Koch's postulates. To date, this fungus affects at least 372 hosts globally causing yield losses. Although in Mexico this fungus has been documented in Glycine max, Ipomoea batatas, Phaseolus vulgaris, Physalis ixocarpa, Saccharum officinarum, Sesamum indicum, Solanum melongena, S. tuberosum, and Sorghum bicolor (Farr and Rossman 2021). However, there are no reports of M. phaseolina as a potential pathogen on peanut; therefore, according to our knowledge, this is the first report of this fungus affecting A. hypogaea in Mexico.

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression.

In this study, the effects of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.

Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation.

Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/MS/MS was used to study the sperm proteome under these two distinct conditions and bioinformatic enrichment analysis conducted. Gene Ontology (GO) and pathway enrichment analysis were performed revealing dramatic changes as consequence of cryopreservation. The terms oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport and electron transport chain were significantly enriched in fresh samples (P = 5.50 × 10-12, 4.26 × 10-8 and 7.26 × 10-8, respectively), while were not significantly enriched in frozen thawed samples (P = 1). The GO terms oxidation reduction process and oxidoreductase activity were enriched in fresh samples and the enrichment was reduced in frozen thawed samples (1.40 × 10-8, 1.69 × 10-6 versus 1.13 × 10-2 and 2-86 × 10-2 respectively). Reactome pathways (using human orthologs) significantly enriched in fresh sperm were TCA cycle and respiratory electron transport (P = 1.867 × 10-8), Respiratory electron transport ATP synthesis by chemiosmosis coupling (P = 2.124 × 10-5), Citric acid cycle (TCA cycle)(P = 8.395 × 10-4) Pyruvate metabolism and TCA cycle (P = 3.380 × 10-3), Respiratory electron transport (P = 2.764 × 10-2) and Beta oxidation of laurolyl-CoA to decanoyl CoA-CoA (P = 1.854 × 10-2) none of these pathways were enriched in thawed samples (P = 1). We have provided the first detailed study on how the cryopreservation process impacts the stallion sperm proteome. Our findings identify the metabolic proteome and redoxome as the two key groups of proteins affected by the procedure. SIGNIFICANCE: In the present manuscript we investigated how the cryopreservation of stallion spermatozoa impacts the proteome of these cells. This procedure is routinely used in horse breeding and has a major impact in the industry, facilitating the trade of genetic material. This is still a suboptimal biotechnology, with numerous unresolved problems. The limited knowledge of the molecular insults occurring during cryopreservation is behind these problems. The application and development of proteomics to the spermatozoa, allow to obtain valuable information of the specific mechanisms affected by the procedure. In this paper, we report that cryopreservation impacts numerous proteins involved in metabolism regulation (mainly mitochondrial proteins involved in the TCA cycle, and oxidative phosphorylation) and also affects proteins with oxidoreductase activity. Moreover, specific proteins involved in the sperm-oocyte interaction are also affected by the procedure. The information gathered in this study, opens interesting questions and offer new lines of research for the improvement of the technology focusing the targets here identified, and the specific steps in the procedure (cooling, toxicity of antioxidants etc.) to be modified to reduce the damage.

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3.

BACKGROUND: The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. OBJECTIVES: Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. MATERIALS AND METHODS: Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 µM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor). RESULTS: Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01). CONCLUSION: We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology.

The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa†.

Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation-reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37°C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility.

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.

Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 µM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.

Identification and function of exchange proteins activated directly by cyclic AMP (Epac) in mammalian spermatozoa.

The role of cAMP in spermatic functions was classically thought to be mediated exclusively through the activation of Protein Kinase A (PKA). However, it has recently been shown that cAMP also exerts its effects through a PKA-independent pathway activating a family of proteins known as Epac proteins. Therefore, many of the spermatic functions thought to be regulated by cAMP through the activation of PKA are again under study. We aimed to identify and to investigate the role of Epac proteins in spermatozoa using a specific permeable analog (8-Br-2'-O-Me-cAMP). Also, we aimed to study its relationship with E-cadherin, an adhesion protein involved in fertility. Our results demonstrate the presence and sub-cellular distribution of Epac 1 and Epac 2 in mammalian spermatozoa. Capacitation and the acrosome reaction induced a change in the localization of Epac proteins in sperm. Moreover, incubation with 8-Br-2'-O-Me-cAMP prompted an increase in Rap1 activation, in the scrambling of plasma membrane phospholipids (necessary for the capacitation process), the acrosome reaction, motility, and calcium mobilization, when spermatozoa were incubated in acrosome reaction conditions. Finally, the activation of Epac proteins induced a change in the distribution of E-cadherin. Therefore, the increase in the acrosome reaction, together with the increase in calcium (which is known to be essential for fertilization) and the Epac nteraction with E-cadherin, might indicate that Epac proteins have an important role in gamete recognition and fertilization.

Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration.

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.

Resveratrol mobilizes Ca2+ from intracellular stores and induces c-Jun N-terminal kinase activation in tumoral AR42J cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin naturally found in grapes and red wine, is a redox-active compound endowed with significant positive activities. In this study, the effects of resveratrol on intracellular free Ca(2+) concentration ([Ca(2+)](c)) and on cell viability in tumoral AR42J pancreatic cells are examined. The results show that resveratrol (100 µM and 1 mM) induced changes in [Ca(2+)](c), that consisted of single or short lasting spikes followed by a slow reduction toward a value close to the resting level. Lower concentrations of resveratrol (1 and 10 µM) did not show detectable effects on [Ca(2+)](c). Depletion of intracellular Ca(2+) stores by stimulation of cells with 1 nM CCK-8, 20 pM CCK-8 or 1 µM thapsigargin, blocked Ca(2+) responses evoked by resveratrol. Conversely, prior stimulation of cells with resveratrol inhibited Ca(2+) mobilization in response to a secondary application of CCK-8 or thapsigargin. In addition, resveratrol inhibited oscillations in [Ca(2+)](c) evoked by a physiological concentration of CCK-8 (20 pM). On the other hand, incubation of cells in the presence of resveratrol induced a reduction of cell viability. Finally, incubation of AR42J cells in the presence of resveratrol led to activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase responsive to stress stimuli. Activation of JNK was reduced in the absence of extracellular Ca(2+). In summary, the results show that resveratrol releases Ca(2+) from intracellular stores, most probably from the endoplasmic reticulum, and reduces AR42J cells viability. Reorganization of cell's survival/death processes in the presence of resveratrol may involve Ca(2+)-mediated JNK activation.

The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway.

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 µm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the regulation of the viability of these cells.

Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa.

Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 µm). At the beginning and at the end of the incubation period, sperm motility (using computer-assisted sperm analysis), membrane integrity and permeability, fluidity of the sperm membrane, LPO, and mitochondrial membrane potential (Δψm) were flow cytometrically evaluated. Melatonin reduced changes in the spermatozoa related to apoptosis (increased sperm membrane permeability and lowered Δψm) (P < 0.05). Furthermore, LPO was dramatically reduced (P < 0.01) while no effect was observed on sperm motility or kinematics. Interestingly, melatonin helped maintain a more fluid sperm plasmalemma (P < 0.05). Our results clearly show the absence of MT1 and MT2 receptors in the stallion spermatozoa. It is concluded that melatonin is a useful tool to improve the quality of stored stallion sperm, increasing their life span and reducing premature aging, this likely relates to melatonin's antioxidant properties.

Melatonin reduces pancreatic tumor cell viability by altering mitochondrial physiology.

Melatonin reduces proliferation in many different cancer cell lines. Thus, melatonin is considered a promising antitumor agent, promoting apoptosis in tumor cells while preserving viability of normal cells. Herein, we examined the effects of melatonin on the pancreatic AR42J tumor cell line. We have analyzed cytosolic-free Ca(2+) concentration ([Ca(2+) ](c) ), mitochondrial-free Ca(2+) concentration ([Ca(2+) ](m) ), mitochondrial membrane potential (Ψm), mitochondrial flavin adenine dinucleotide (FAD) oxidative state, cellular viability and caspase-3 activity. Our results show that melatonin induced transient changes in [Ca(2+) ](c) and [Ca(2+) ](m) . Melatonin also induced depolarization of Ψm and led to a reduction in the level of oxidized FAD. In addition, melatonin reduced AR42J cell viability. Finally, we found a Ca(2+) -dependent caspase-3 activation in response to melatonin. Collectively, these data support the likelihood that melatonin reduces viability of tumor AR42J cells via its action on mitochondrial activity and caspase-3 activation.

Ethanol consumption as inductor of pancreatitis.

Alcohol abuse is a major cause of pancreatitis, a condition that can manifest as both acute necroinflammation and chronic damage (acinar atrophy and fibrosis). Pancreatic acinar cells can metabolize ethanol via the oxidative pathway, which generates acetaldehyde and involves the enzymes alcohol dehydrogenase and possibly cytochrome P4502E1. Additionally, ethanol can be metabolized via a nonoxidative pathway involving fatty acid ethyl ester synthases. Metabolism of ethanol by acinar and other pancreatic cells and the consequent generation of toxic metabolites, are postulated to play an important role in the development of alcohol-related acute and chronic pancreatic injury. This current work will review some recent advances in the knowledge about ethanol actions on the exocrine pancreas and its relationship to inflammatory disease and cancer.