AMP-activated kinase in human spermatozoa: Identification, intracellular localization, and key function in the regulation of sperm motility

AMP‑activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work’s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho‑Thr172‑AMPK were analyzed by Western blotting and indirect immunofluorescence. High‑ and low‑quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho‑Thr172‑AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho‑Thr172‑AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.Trabajo patrocinado por: Mutua Madrileña. Beca Junta de Extremadura y Fondos FEDER. Ayudas JUEX‑IBI13121, PCJ1008, GR10125, y GR10156 Fundación Tatiana Pérez Guzmán el Bueno. Beca para Violeta Calle Guisado Ministerio de Educación y Ciencia. Beca Predoctoral FPU para Violeta Calle GuisadopeerReviewe

N-ACETYLCYSTEINE DOES NOT IMPROVE SPERM MOTILITY OF LIDIA BULL AFTER PROLONGED EPIDIDYMAL STORAGE / LA N-ACETILCISTEINA NO MEJORA LA MOTILIDAD ESPERMÁTICA EN TOROS DE LIDIA LUEGO DEL ALMACENAMIENTO EPIDIDIMARIO PROLONGADO

The Lidia bovine breed is considered a hallmark of Spanish cattle industry. Assisted reproductive techniques like cryopreservation of epididymal spermatozoa could be considered as an important tool to obtain more offspring and store its genetics. As these bulls are not selected by their reproductive performance or sperm freezability, the quality of their ejaculates is poor and addition of antioxidants prior cryopreservation could exert beneficial effects on the post-thaw sperm quality. The aim of this study was to evaluate the effect of the supplementing a tris-fructose-egg yolk based freezing extender with1 mMand2.5 mMof N-acetylcysteine to sperm recovered from epididymis stored at4ºCfor 24, 48, 72 or 96 hours prior cryopreservation. Motility values and sperm kinematic parameters were compared against control (epididymis stored for 24 hours and no antioxidant addition). Our results showed that N-acetylcysteine addition did not improve sperm motility parameters at any of the time points or dosages tested. In addition, storage of bullfight epididymis up to 96 hours did not significantly affect sperm kinematic parameters or total and progressive motility.RESUMENLa raza de Lidia es considerada una insignia de la industria ganadera española. Las tecnologías de reproducción asistida como la criopreservación de espermatozoides epididimarios podría ser considerara como una herramienta importante para obtener más crías y conservar su genética. Ya que estos toros no son seleccionados por su desempeño reproductivo o congelabilidad espermática, la calidad de sus eyaculados es pobre y la adición de antioxidantes antes de la criopreservación podría tener efectos beneficiosos sobre la calidad del semen descongelado. El propósito de este estudio fue evaluar el efecto de la suplementación del medio a base de tris-fructosa y yema de huevo con 1 mM y 2,5 mM de N-acetilcisteína a espermatozoides recuperados de epidídimos almacenados a 4°C por 24, 48, 72 o 96 horas antes de la criopreservación. Los valores de movilidad y los parámetros de cinética espermática fueron comparados con el control (epidídimos almacenados por 24 horas y sin la adición de antioxidantes). Nuestros resultados muestran que la adición de N-acetilcisteína no mejora los parámetros de motilidad espermática en ninguno de los momentos o dosis evaluadas. Además, el almacenamiento de epidídimos refrigerados hasta 96 horas no afecta significativamente los parámetros de cinética espermática o la movilidad total o progresiva.

Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine

P. 1-10The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.S

Outlining adequate protocols for Lidia bull epididymal storage and sperm cry-opreservation: use of glycerol, dimethylformamide and N-acetylcysteine

La raza bovina Lidia es un sello distintivo importante de la industria ganadera española. Los toros se seleccionan en función de la agresividad y la crioconservación de espermatozoides epididimales es la forma de obtener y almacenar su genética. Todavía no hay protocolos diseñados específicamente para realizar la crioconservación de espermatozoides Lidia bull. El presente estudio tuvo como objetivo determinar si un medio extensor de tris-fructosa-citrato-huevo (20% v / v; TFY) suplementado con 7% de glicerol (TFY1) o 3,5% de glicerol más 3,5% de dimetilformamida (DMF; TFY2) son medios adecuados Para la crioconservación de epididimales Lidia esperma de toro. Además, se evaluó el efecto de la N-acetilcisteína (NAC), un potente antioxidante. Los epidídimos se almacenaron a 4 ° C durante 24, 48, 72 o 96 h, y ambos medios de congelación se probaron como tales o se complementaron con 1 o 2,5 mM de NAC. Nuestros datos demostraron que la viabilidad posterior a la descongelación se mantuvo bien (TFY1: 50.8% ± 1.9 a las 24 h y 52.4% ± 0.8 a las 96 hy TFY2: 52.6% ± 1.6 a las 24 h y 56.1% ± 1.8 a las 96 h; media% ± SEM; p> 0.05) al igual que la motilidad espermática total y progresiva, el potencial de membrana mitocondrial alto, la producción de ROS, el estado del ADN y la intactidad acrosomal del esperma de toro Lidia hasta 96 h de almacenamiento de epidídimo, todas las variaciones del extensor son similares (p> 0.05 ). En conclusión, el uso de medio TFY suplementado con un 7% de glicerol solo o la combinación de un 3,5% de glicerol y un 3,5% de DMF fue una elección igualmente segura para la crioconservación de espermatozoides Lidia epididimales, y la adición de NAC no mejoró significativamente la calidad del esperma después del deshielo.The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.• Ministerio de Economía, Industria y Competitividad. Proyecto ITC-2015-1342 • Ministerio de Economía y Competitividad. Beca Juan de la Cierva IJCI- 2014-19428, para Beatriz Macías García • Centro de Cirugía de Mínima Invasión Jesús Usón. Beca para Elvira Matilla Pinto • Ministerio de Economía, Industria y Competitividad y Fondos FEDER. Proyecto AGL2015-73249-JIN (I+D+i) • Fundação para a Ciência e a Tecnologia (Portugal)/ESF/Ministerio de Ciencia, Tecnología y Educación Superior. Beca SFRH/BPD/85532/2012, para Lauro González Fernández • Ministerio de Educación, Cultura y Deportes. Beca FPU-014/03449, para Violeta Calle GuisadopeerReviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

La proteína quinasa activada por AMP, AMPK, en el espermatozoide humano

El análisis de la motilidad del espermatozoide mediante el programa informático ISAS permite evaluar de una manera objetiva la velocidad y calidad del movimiento de los espermatozoides. Mediante el estudio digitalizado y el análisis computarizado del movimiento del espermatozoide se obtiene una aproximación objetiva a las características de la movilidad de estas células germinales. El uso de la citometría de flujo está introduciéndose con fuerza en los últimos años en los laboratorios de andrología debido al elevado número de aplicaciones derivadas de esta técnica, su facilidad de utilización con estas células germinales y el alto número de células analizadas en muy poco tiempo, lo que hace aumentar significativamente la fiabilidad de los análisis.The analysis of sperm motility using the ISAS software allows an objective evaluation of the speed and quality of spermatozoa movement. The digitised study and computerised analysis of spermatozoa movement provides an objective approximation of the motility characteristics of these germ cells. The use of flow cytometry has been strongly introduced in recent years in andrology laboratories due to the high number of applications derived from this technique, its ease of use with these germ cells and the high number of cells analysed in a very short time, which significantly increases the reliability of the analyses

Expression and function of the AMP-activated kinase, AMPK, in human spermatozoa

Tesis por compendio de publicacionesEn la presente Tesis Doctoral nos planteamos la hipótesis de que la proteína AMPK puede representar una nueva vía de señalización intracelular que podría regular las funciones del espermatozoide humano. Los objetivos que nos planteamos fueron los siguientes: 1) Identificar y estudiar la localización intracelular de la proteína AMPK y de su forma activa (fosforilada) en el espermatozoide humano. 2) Investigar la relación entre la actividad de la AMPK y la calidad del espermatozoide humano. 3) Estudiar la implicación de la vía de la AMPK en los principales procesos funcionales del espermatozoide humano. 4) Investigar el efecto del activador indirecto de la AMPK, metformina, en la fisiología del espermatozoide humano, debido a su amplio uso clínico. Los resultados obtenidos nos han permitido concluir que: 1) La proteína AMPK y su forma activa han sido identificadas en el espermatozoide humano y se localiza en el acrosoma, la pieza media y el flagelo mientras que la forma activa se restringe al flagelo y al acrosoma. 2) La AMPK está más activa en la población de espermatozoides humanos de mayor calidad. 3) La proteína AMPK regula la motilidad del espermatozoide humano, aunque no está implicada en el mantenimiento de la viabilidad, del potencial de membrana mitocondrial, de la integridad de la membrana acrosomal y tampoco en la producción mitocondrial de especies reactivas de oxígeno ni en la traslocación de fosfatidilserina hacia la cara externa de la membrana plasmática. 4) El fármaco metformina provoca una disminución de la motilidad en el espermatozoide humano.The present Doctoral Thesis hypothesized that the AMPK may represent an intracellular signalling pathway that regulates human sperm function. The objectives were: 1) To identify and determine the intracellular location of AMPK and its active form (phosphorylated) in human spermatozoa. 2) To investigate the possible relationship between AMPK activity and human sperm quality. 3) To study the role of the AMPK pathway in the main physiological processes of human spermatozoa. 4) To investigate the effect of the indirect activator of AMPK, metformin, in the physiology of human spermatozoa, due to its current clinical use. The scientific data included in this Doctoral Thesis allowed us to reach the following conclusions: 1) AMPK, as well as its active form, were identified in human spermatozoa and is located in the acrosome, midpiece and flagellum and its phosphorylated whereas her active form is restricted to the flagellum and the acrosome. 2) AMPK is more active in human spermatozoa exhibiting higher quality. 3) AMPK regulates motility in human spermatozoa. Nevertheless, AMPK is not involved in other functional processes of human spermatozoa, such as spermatozoa viability, mitochondrial membrane potential maintenance, acrosomal membrane integrity, mitochondrial reactive oxygen species production or translocation of phosphatidylserine to the outer leaf of the membrane. 4) Metformin causes a decrease in sperm motility.• Fundación Tatiana Pérez de Guzmán el Bueno: PRE/FT2014-28, en 2014 • Ministerio de Educación, Cultura y Deporte (MECD): Contrato predoctoral , dentro del Programa de Formación del Profesorado Universitario (FPU14/03449) en 2015 • European Molecular Biology Organization (EMBO): estancia predoctoral en el “Laboratorio di Biologia dello spermatozoo di DMSC-Sezione di medicina critica e meidicine specialistiche” en la Universitá degli Studi de Firenze • Fondo Social Europeo (FSE) y Fondo Europeo de Desarrollo Regional (FEDER

Estudiando el espermatozoide, de la ciencia básica a las técnicas de reproducción asistida

Se analizan las dianas moleculares que detectan el estado de energía y regulan el metabolismo del espermatozoide de cerdo, lo que permite generar conocimiento acerca de la fisiología, bioquímica y biología molecular del mismo. Los estudios se centran en la proteína AMPK, quinasa activada por AMP. Esta proteína se identificó en los años 70 y es una molécula muy conservada desde el punto de vista evolutivo, que actúa como un sensor del estado energético celular y, de manera consecuente, regula el metabolismo. Detecta el estado energético celular al activarse alostéricamente por AMP, aunque también se activa por fosforilación en la Thr172 de su subunidad catalítica inducida por diversos estímulos dependiendo del tipo celular estudiado, como el calcio intracelular o varios tipos de estrés. La consecuencia metabólica de la actividad de la AMPK es la inducción de rutas catabólicas que producen ATP y la inhibición de rutas anabólicas que consumen ATP.Molecular targets that detect the energy status and regulate the metabolism of pig spermatozoa are analysed, generating knowledge about the physiology, biochemistry and molecular biology of the spermatozoa. The studies focus on the AMPK protein, AMP-activated kinase. This protein was identified in the 1970s and is an evolutionarily highly conserved molecule that acts as a sensor of cellular energy status and consequently regulates metabolism. It senses cellular energy status by being allosterically activated by AMP, although it is also activated by phosphorylation at Thr172 of its catalytic subunit induced by various stimuli depending on the cell type studied, such as intracellular calcium or various types of stress. The metabolic consequence of AMPK activity is the induction of ATP-producing catabolic pathways and the inhibition of ATP-consuming anabolic pathways

Expression and function of the AMP-activated kinase, AMPK, in human spermatozoa

Tesis por compendio de publicacionesEn la presente Tesis Doctoral nos planteamos la hipótesis de que la proteína AMPK puede representar una nueva vía de señalización intracelular que podría regular las funciones del espermatozoide humano. Los objetivos que nos planteamos fueron los siguientes: 1) Identificar y estudiar la localización intracelular de la proteína AMPK y de su forma activa (fosforilada) en el espermatozoide humano. 2) Investigar la relación entre la actividad de la AMPK y la calidad del espermatozoide humano. 3) Estudiar la implicación de la vía de la AMPK en los principales procesos funcionales del espermatozoide humano. 4) Investigar el efecto del activador indirecto de la AMPK, metformina, en la fisiología del espermatozoide humano, debido a su amplio uso clínico. Los resultados obtenidos nos han permitido concluir que: 1) La proteína AMPK y su forma activa han sido identificadas en el espermatozoide humano y se localiza en el acrosoma, la pieza media y el flagelo mientras que la forma activa se restringe al flagelo y al acrosoma. 2) La AMPK está más activa en la población de espermatozoides humanos de mayor calidad. 3) La proteína AMPK regula la motilidad del espermatozoide humano, aunque no está implicada en el mantenimiento de la viabilidad, del potencial de membrana mitocondrial, de la integridad de la membrana acrosomal y tampoco en la producción mitocondrial de especies reactivas de oxígeno ni en la traslocación de fosfatidilserina hacia la cara externa de la membrana plasmática. 4) El fármaco metformina provoca una disminución de la motilidad en el espermatozoide humano.The present Doctoral Thesis hypothesized that the AMPK may represent an intracellular signalling pathway that regulates human sperm function. The objectives were: 1) To identify and determine the intracellular location of AMPK and its active form (phosphorylated) in human spermatozoa. 2) To investigate the possible relationship between AMPK activity and human sperm quality. 3) To study the role of the AMPK pathway in the main physiological processes of human spermatozoa. 4) To investigate the effect of the indirect activator of AMPK, metformin, in the physiology of human spermatozoa, due to its current clinical use. The scientific data included in this Doctoral Thesis allowed us to reach the following conclusions: 1) AMPK, as well as its active form, were identified in human spermatozoa and is located in the acrosome, midpiece and flagellum and its phosphorylated whereas her active form is restricted to the flagellum and the acrosome. 2) AMPK is more active in human spermatozoa exhibiting higher quality. 3) AMPK regulates motility in human spermatozoa. Nevertheless, AMPK is not involved in other functional processes of human spermatozoa, such as spermatozoa viability, mitochondrial membrane potential maintenance, acrosomal membrane integrity, mitochondrial reactive oxygen species production or translocation of phosphatidylserine to the outer leaf of the membrane. 4) Metformin causes a decrease in sperm motility.• Fundación Tatiana Pérez de Guzmán el Bueno: PRE/FT2014-28, en 2014 • Ministerio de Educación, Cultura y Deporte (MECD): Contrato predoctoral , dentro del Programa de Formación del Profesorado Universitario (FPU14/03449) en 2015 • European Molecular Biology Organization (EMBO): estancia predoctoral en el “Laboratorio di Biologia dello spermatozoo di DMSC-Sezione di medicina critica e meidicine specialistiche” en la Universitá degli Studi de Firenze • Fondo Social Europeo (FSE) y Fondo Europeo de Desarrollo Regional (FEDER

The Role of Exercise to Reduce the Impact of Diabetes in the Seminal Quality: A Systematic Review

Background and Objectives: One of the most relevant consequences of diabetes mellitus is the temporal or complete infertility which can happen in young individuals. Therefore, the current systematic review aimed to investigate the effects of exercise to reduce the impact of Type 2 Diabetes Mellitus (T2DM) in seminal quality and related parameters. Materials and Methods: A systematic search was conducted in Pubmed and Web of Science databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines (PRISMA). The inclusion criteria were: (1) the study included at least one experimental and one comparison group, (2) the sample of the study was comprised of humans or animals with diabetes mellitus, (3) an intervention based on physical exercise was conducted, and (4) the study reported variables related to the seminal quality. Results: A total of 115 articles were identified. However, only six accomplished the inclusion and exclusion criteria. This systematic review includes a sample size of 260 participants (180 rats and 80 humans). Intervention ranged from 6 to 14 weeks, with 3–6 days per week. All interventions performed endurance training (50–70% VO2max or maximum heart rate). Physical exercise increased sperm count, motility, and morphology, as well as improved testosterone, Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH) levels. Moreover, physical exercise intervention reduced the percentages of sperms with negative Tubular Differentiation Index (TDI) and Spermiogenesis Index (SPI), DNA fragmentation, and also ameliorated the diabetes-induced apoptosis and improved sperm apoptosis index. Conclusions: Physical exercise could ameliorate diabetic pathological effects on sperm quality and related parameters that cause infertility or subfertility conditions. However, further homogeneous studies are needed to confirm these findings