Identificación, regulación y estudio funcional de la proteína quinasa N-terminal de c-Jun (JNK) en espermatozoides de mamíferos

En este trabajo de investigación demostramos por primera vez la presencia y la localización subcelular de la quinasa N-terminal de c-Jun (JNK), en lisados de espermatozoides de mamíferos, incluida la especie humana. Mediante la utilización de anticuerpos específicos, hemos estudiado la regulación de la actividad de esta quinasa en los espermatozoides mantenidos tanto en situaciones de estrés celular, como frente a estímulos fisiológicos que están relacionados con los cambios de tipo capacitante y la reacción acrosómica. Además, hemos valorado la participación de esta quinasa en diversas funciones de los espermatozoides maduros eyaculados de caballo. También se han estudiado los cambios producidos en la actividad de JNK cuando los espermatozoides son sometidos a procesos biotecnológicos, como el almacenamiento en refrigeración (5º, hasta 5 días) y la criopreservación, relacionando la JNK con los cambios deletéreos que se producen en esos procesos. Hemos estudiado también la implicación de JNK en el mantenimiento de la supervivencia o la muerte celular, así como su participación en los mecanismos moleculares que regulan esos procesos en los espermatozoides. Por último, hemos evaluado los cambios en la fosforilación de JNK en espermatozoides humanos procedentes de donantes fumadores y no fumadores, correlacionando la actividad de JNK con la viabilidad espermática.In this research work we have demonstrated for the first time the presence and subcellular localization of the N-terminal kinase of c-Jun (JNK) in lysates of mammalian spermatozoa, including humans. By using specific antibodies, we have studied the regulation of JNK activity in stallion sperm when are subjected to external stresses as well as under physiological conditions which are related to capacitation and acrosome reaction. In addition, we have evaluated the involvement of this kinase in various physiological functions of the ejaculated stallion spermatozoa. We have also studied the changes in the activity of JNK when sperm are submitted to biotechnology processes, such as refrigeration (5 ºC, up to 5 days) and cryopreservation, linking JNK with the deleterious changes that occur in the spermatozoa during these processes. We have also studied the involvement of JNK in cell survival or cell death in sperm and the involvement of this kinase in the molecular mechanisms that regulate these processes in sperm. Finally, we have evaluated the changes in the phosphorylation of JNK in human sperm from smokers and nonsmokers donors, correlating the JNK activity with sperm viability.Ministerio de Ciencia y Tecnología – FEDER. Proyecto de investigación (BFU2007-62423 (BFI)) y Beca de Formación de Personal Investigador (FPI) BES-2008-002106. Ministerio de Economía y Competitividad BFU2011-30261. Junta de Extremadura (Proyecto de investigación PCE1002). Junta de Extremadura - FEDER (Proyecto de investigación GR10010)

Identificación, regulación y estudio funcional de la proteína quinasa N-terminal de c-Jun (JNK) en espermatozoides de mamíferos

En este trabajo de investigación demostramos por primera vez la presencia y la localización subcelular de la quinasa N-terminal de c-Jun (JNK), en lisados de espermatozoides de mamíferos, incluida la especie humana. Mediante la utilización de anticuerpos específicos, hemos estudiado la regulación de la actividad de esta quinasa en los espermatozoides mantenidos tanto en situaciones de estrés celular, como frente a estímulos fisiológicos que están relacionados con los cambios de tipo capacitante y la reacción acrosómica. Además, hemos valorado la participación de esta quinasa en diversas funciones de los espermatozoides maduros eyaculados de caballo. También se han estudiado los cambios producidos en la actividad de JNK cuando los espermatozoides son sometidos a procesos biotecnológicos, como el almacenamiento en refrigeración (5º, hasta 5 días) y la criopreservación, relacionando la JNK con los cambios deletéreos que se producen en esos procesos. Hemos estudiado también la implicación de JNK en el mantenimiento de la supervivencia o la muerte celular, así como su participación en los mecanismos moleculares que regulan esos procesos en los espermatozoides. Por último, hemos evaluado los cambios en la fosforilación de JNK en espermatozoides humanos procedentes de donantes fumadores y no fumadores, correlacionando la actividad de JNK con la viabilidad espermática.In this research work we have demonstrated for the first time the presence and subcellular localization of the N-terminal kinase of c-Jun (JNK) in lysates of mammalian spermatozoa, including humans. By using specific antibodies, we have studied the regulation of JNK activity in stallion sperm when are subjected to external stresses as well as under physiological conditions which are related to capacitation and acrosome reaction. In addition, we have evaluated the involvement of this kinase in various physiological functions of the ejaculated stallion spermatozoa. We have also studied the changes in the activity of JNK when sperm are submitted to biotechnology processes, such as refrigeration (5 ºC, up to 5 days) and cryopreservation, linking JNK with the deleterious changes that occur in the spermatozoa during these processes. We have also studied the involvement of JNK in cell survival or cell death in sperm and the involvement of this kinase in the molecular mechanisms that regulate these processes in sperm. Finally, we have evaluated the changes in the phosphorylation of JNK in human sperm from smokers and nonsmokers donors, correlating the JNK activity with sperm viability.Ministerio de Ciencia y Tecnología – FEDER. Proyecto de investigación (BFU2007-62423 (BFI)) y Beca de Formación de Personal Investigador (FPI) BES-2008-002106. Ministerio de Economía y Competitividad BFU2011-30261. Junta de Extremadura (Proyecto de investigación PCE1002). Junta de Extremadura - FEDER (Proyecto de investigación GR10010)

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

Sperm membrane integrity (SYBR-14/PI) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>LIVE % (SYBR-14+ sperm), DEAD% (PI+ sperm), DAMAGED, (SYBR-14+/PI+sperm). Within a row values with different superscript differ statistically a-b p<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Mitochondrial membrane potential (Δφm) and lipid peroxidation (LPO) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>(Means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion). High spermatozoa depicting high Δφm, High and Low spermatozoa depicting simultaneously mitochondria with low and high Δφm, Low spermatozoa with low Δφm, LPO spermatozoa showing peroxidation of the lipids of their membranes.</p

Membrane intactness and subtle changes in sperm membrane integrity of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>Intact spermartozoa are those not stained and thus represent spermatozoa with completely intact membranes. YoPro+ are early apoptotic sperm depicting an increase in sperm membrane permeability, YoPro+/Eth+ are late apoptotic and Eth+ are necrotic spermatozoa. Within a row values with different superscript differ statistically a-c p<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Sperm motility and kinematics after computer assisted sperm analysis (CASA) of stallion spermatozoa stored during five days (day 1 D1 to day 5 D5) at 5°C FE fresh extended sperm, CC sperm processed through colloidal centrifugation.

<p>TM% total motile sperm, LM% linear motile sperm, RS% rapid sperm, VCL circular velocity, VSL straight line velocity, VAP average velocity, ALH lateral head displacement, BCF beat cross frequency. Within a row values with different superscripts differ statistically a-e, P<0.01. (means ± SD) Results are derived from 28 identical experiments (7 stallions, 4 ejaculates per stallion).</p

Changes in LC3B processing in stallion spermatozoa stored under refrigeration (5°C) for five days, after single layer centrifugation (Filtrated) or unprocessed (native sperm).

<p>In native sperm storage induced a significant increase in LC₃B processing at day 5 indicating that autophagy was activated during the period of storage. On the other hand, filtration of sperm selected a subpopulation of spermatozoa in which autophagy was already activated at the beginning of the period of storage and did not change over the time. Results are representative of 28 identical experiments (seven stallions, four ejaculates per stallion) * p<0.01.</p

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective