Sperm Capacitation associated increase in Tyrosine Phosphorylation: kinetic of the increase and novel members involved in the process.

Sperm capacitation is a post-ejaculatory maturational event required for successful fertilization. Specific molecular events are induced during capacitation: a cAMP-dependent activation of protein kinase A (PKA) leading downstream to the increase of tyrosine phosphorylation. Activation of PKA and tyrosine phosphorylation was shown to occur on different time scale. Here we showed that phosphorylation events during capacitation are tightly regulated over time, with fast activation of PKA and a late and continuous increase in the level of tyrosine phosphorylation to guarantee a pool of capacitated sperm at the right time/site of fertilization. Despite several studies on tyrosine phosphorylation, the identity of the tyrosine kinase(s) that mediate these increases has not been conclusively demonstrated. Recently a role for focal adhesion kinases (PYK2 and FAK) was proposed in stallion, based on the use of PF431396, a small molecule inhibitor directed against this kinase family, but critical loss of function experiment have not been reported. We used both pharmacological tools and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase of tyrosine phosphorylation in human and mouse sperm. PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation in both human and murine. On the other hand, Pyk2–/– mice showed a physiological capacitation-associated tyrosine phosphorylation and the specific inhibition of FAK by PF573228 showed no decrease in the levels of tyrosine phosphorylation, indicating that focal adhesion kinases are not responsible for this phosphorylation process. Here we show that PF431396 can also inhibit the tyrosine kinase FER and that sperm from mice targeted with a kinase inactivating mutation in FER failed to undergo capacitation-associated increases in tyrosine phosphorylation. While these mice are fertile, their sperm displayed normal levels hyperactivation but have reduced ability to fertilize metaphase-II arrested eggs in vitro

Effetti della neurotossina MPTP e protezione da parte della pargilina sui metaboliti energetici extracellulari e sui livelli di dopamina nello striato di ratti "freely moving"

The neurotoxin MPTP induces neurodegeneration of SNpc dopaminergic cells by mitocondrial impairment. Therefore, we deemed of interest the study of pargyline neuroprotective role on dopamine (DA) and energy metabolism. The study was carried out on Wistar rats using an asymmetric microdialysis allowing simultaneous monitoring of DA and energy substrates striatal levels. Samples were quantified via spectrophotometer and HPLC-EC tecniques. Moreover, amperometric microsensors and biosensors were used to real-time detect oxygen (O2), glucose (GLU) and lactate (LAT) variations. Both microdialisys probes and sensors were stereotaxically implanted in right striatum. Pargyline (15mg/Kg) pre-treatment was injected i.p. for 3 days 40 min before MPTP administration (25/15/10mg/Kg i.p.). First MPTP dose induced an increase of DA and O2 levels among all groups. A decrease of striatal DA levels following further MPTP administrations were observed. This trend was partially contrasted in pargyline pre-treated group. Furthermore, first MPTP dose led to a general increase of striatal energy substrates concentrations, while subsequent toxin administration at days 2 and 3 showed a progressive reduction of GLU and PYR and an increase of LAT striatal levels and L/P ratio. Pargyline pre-treatment partially preserved GLU and PYR decrease, as well as LAT and L/P ratio MPTP-related increase. These results ascertained pargyline neuroprotective effect on DA and energy metabolism by preventing MPTP insult

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.Fil: Alvau, Antonio. University of Massachussets; Estados UnidosFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gervasi, Maria Gracia. University of Massachussets; Estados UnidosFil: Navarrete, Felipe A.. University of Massachussets; Estados UnidosFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: De la Vega Beltran, José Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Greer, Peter. Queens University; CanadáFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados UnidosFil: Salicioni, Ana María. University of Massachussets; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados Unido

Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+, and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.Fil: Navarrete, Felipe A.. University of Massachusetts; Estados UnidosFil: García Vázquez, Francisco A.. University of Massachusetts; Estados Unidos. Universidad de Murcia; EspañaFil: Alvau, Antonio. University of Massachusetts; Estados UnidosFil: Escoffier, Jessica. University of Massachusetts; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Salicioni, Ana M.. University of Massachusetts; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University of Massachusetts; Estados Unido

Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca 2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca 2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca 2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.Fil: Navarrete, Felipe A.. University of Massachusetts; Estados UnidosFil: Alvau, Antonio. University of Massachusetts; Estados UnidosFil: Lee, Hoi Chang. University of Massachusetts; Estados UnidosFil: Levin, Lonny R.. Weill Cornell Medical College; Estados UnidosFil: Buck, Jochen. Weill Cornell Medical College; Estados UnidosFil: Leon, Patricia Martin De. University of Delaware; Estados UnidosFil: Santi, Celia M.. University of Washington; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mager, Jesse. University of Massachusetts; Estados UnidosFil: Fissore, Rafael A.. University of Massachusetts; Estados UnidosFil: Salicioni, Ana M.. University of Massachusetts; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Visconti, Pablo E.. University of Massachusetts; Estados Unido

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

Background: MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wildtype samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common. Methods: MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA. Results: The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP. Conclusions: The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable

Confluencias marginales — Márgenes que confluyen. Las intervenciones públicas de LUCE en Espadilla (Castellón)

La madrificación constante del tejido artístico contemporáneo invisibiliza con frecuencia narrativas marginales que se hilan en otras áreas de la Península Ibérica, impidiendo posibles discursos paralelos o contra-dominantes. En este sentido, no es inusual que la necesidad de repensar la marginalidad como nuevo (des)centro sea uno de los enfoques principales que impulsan muchas de las instituciones, artistas y proyectos españoles que se desarrollan fuera de la capital y que se han ido sumando en los últimos años con el fin de ocupar ese espacio vacante que les corresponde. El siguiente artículo se focaliza en definir la naturaleza de los llamados (des)centros, a través del prisma de las prácticas artísticas experimentales que se sitúan en espacios liminales como narrativa paralela a las lógicas aglomeradoras del centro. A este respecto, se incide en el caso de las intervenciones realizadas por el artista valenciano LUCE en el pueblo de Espadilla (Castellón), en el marco de Confluències, proyecto de intervenciones de tipo público-participativo del Instituto Valenciano de Arte Moderno desarrollado en los 24 pueblos de la Ruta 99 de la provincia de Castellón

Biosensing Technologies for Therapeutic Drug Monitoring

Background and Rationale: Therapeutic drug monitoring (TDM) is the clinical practice of measuring pharmaceutical drug concentrations in patients’ biofluids at designated intervals to allow a close and timely control of their dosage. This practice allows for rapid medical intervention in case of toxicity-related issues and/or adjustment of dosage to better fit the therapeutic demand. Currently, TDM is performed in centralized laboratories employing instruments, such as immunoassay analyzers and mass spectrometers that can be run only by trained personnel. However, the time required for the preparation, samples analysis, and data processing, together with the related financial cost, severely affects the application of TDM in medical practices. Therefore, a new generation of analytical tools is necessary to respond to the timely need of drug administration or reduction aiming at effectively treating oncologic patients. Aim of the Review: State-of-the-Art Technologies for TDM: Technological advances in the field of nanosciences and biosensors offer the unique opportunity to address such issues. The interest for the so-called nanobiosensors is considerably increasing, particularly in drug discovery and clinical chemistry, even though there are only few examples reporting their use for TDM. The techniques employing nanobiosensors are mainly based on electrochemical, optical, and mass detection systems. Conclusions: In this review, we described the most promising methodologies that, in our opinion, will bring TDM towards the next stage of clinical practice in the future

Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma

The implementation of therapeutic drug monitoring in the routine clinical practice in oncology is mainly limited by the lack of therapeutic indexes for the majority of the anticancer drugs, and by the absence of suitable analytical tools, which can accurately quantify in real time the concentration of the administered drugs and their relevant metabolites in biological fluids. In this work, a simple and efficient fluorimetric determination of SN-38, the active metabolite of the anticancer drug irinotecan, was developed and applied to human plasma samples. The intrinsic fluorescence of SN-38 allowed its quantification in the range 10–500 ng mL −1 with a LOQ of 5.0 ng mL −1 and a LOD of 1.5 ng mL −1 . Low interferences due to main metabolites of irinotecan and comedications, commonly associated with administration of irinotecan, were observed. A validation study, according to FDA and EMA guidelines for bioanalytical method validation, was carried out and, finally, blind samples were analyzed in parallel with a HPLC-MS method obtaining an excellent agreement between the two techniques