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

AMP-Activated Kinase AMPK Is Expressed in Boar Spermatozoa and Regulates Motility

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca2+ and bicarbonate TCM, time from 1–24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca2+ and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization

Expresión y función de la proteína quinasa activada por AMP, AMPK, en la célula germinal masculina de cerdo

Tesis doctoral con la Mención de "Doctor Internacional"El espermatozoide es una célula germinal altamente especializada que debe experimentar unos procesos funcionales característicos para lograr su función primordial: la fecundación del ovocito. Estos procesos funcionales son por un lado dependientes del estado energético, y por otro están regulados por mecanismos bioquímicos como la fosforilación de proteínas. La proteína quinasa activada por AMP, AMPK, actúa como una molécula sensora de la carga energética celular, y su activación provoca una regulación del metabolismo. Los resultados de la presente Tesis nos permiten elaborar las siguientes conclusiones: 1- AMPK, se expresa en el espermatozoide de cerdo y se encuentra enzimáticamente activa en condiciones fisiológicas. 2- AMPK se localiza principalmente en el acrosoma y en la pieza intermedia del flagelo. 3- La ruta de señalización que provoca la activación de AMPK está regulada por calcio, cAMP, por la proteína quinasa A (PKA), proteína quinasa C (PKC) y las proteínas quinasas α y/o β de la CAMKK. 4- AMPK está involucrada en la regulación de la movilidad del espermatozoide. 5- AMPK desempeña una importante función en las membranas del espermatozoide. 6- AMPK está implicada en la regulación del potencial de membrana mitocondrial. 7- AMPK se activa por fosforilación en repuesta a determinados tipos de estrés celular. En resumen, consideramos que los resultados de esta Tesis Doctoral permiten proponer a la AMPK como una quinasa esencial que regula la capacidad de la célula germinal masculina para adaptarse a las fluctuantes condiciones externas, como ocurre durante el tránsito a través del tracto reproductor femenino.Spermatozoon is a very specialized germ cell that undergoes characteristic functional processes prior accomplishing its essential function: fertilization of the oocyte. These processes depend on the energy charge of this germ cell and are also controlled by biochemical mechanisms, such as protein phosphorylation. AMP-activated kinase AMPK is a protein kinase that detects cell energy state and upon activation regulates metabolism. Results of the Thesis allow us to establish the following conclusions: 1- AMP-activated protein kinase AMPK is expressed in boar spermatozoa and is active under physiological conditions. 2- AMPK protein is mainly localized in the acrosome and in the middle piece of boar spermatozoa flagellum. 3- Intracellular mechanisms leading to AMPK activation by phosphorylation in boar spermatozoa involved calcium and cAMP, as well as signalling pathways of the protein kinase A (PKA), protein kinase C (PKC), protein kinase α and/or β of CaMKK. 4- AMPK protein Kinase is involved in the regulation of motility in boar spermatozoa. 5-AMPK protein plays a relevant function in spermatozoa membranes. 6- AMPK protein kinase is involved in the control of mitochondrial membrane potential in boar spermatozoa.7- AMPK protein kinase becomes activated by phosphorylation in boar spermatozoa in response to several germ cell stresses. In summary, we consider that results elaborated in this Thesis allows our proposal that AMPK acts as an essential kinase in the regulation of male germ cells ability for adapting to the changing conditions of the extracellular medium, as it occurs during their transit though the female reproductive tract for fertilization.Esta Tesis Doctoral ha sido financiada con cargo a los siguientes proyectos: - Ministerio de Ciencia e Innovación (AGL 2010-15188): “Función de la proteína quinasa activada por AMP, AMPK, en la célula germinal masculina: posible aplicación biotecnológica a la conservación de semen porcino”. - Gobierno de Extremadura (PRI 09A077): “Función de la proteína quinasa AMPK en la célula germinal masculina: aplicación a la optimización del protocolo de criopreservación de semen de cerdo ibérico”. - Gobierno de Extremadura (PDT 08A057): “Aplicación de nuevas tecnologías de contrastación seminal a la valoración de la calidad de dosis seminales comerciales de cerdo ibérico”. - Gobierno de Extremadura, Ayudas a Grupos (GR10156). - Universidad de Extremadura (Convenio 102/09): “Estudios de nuevos indicadores de calidad del semen del cerdo ibérico”. - Universidad de Extremadura, Ayudas a Grupos (GRU-09019/GRU-06116)

AMPK Function in Mammalian Spermatozoa

AMP-activated protein kinase AMPK regulates cellular energy by controlling metabolism through the inhibition of anabolic pathways and the simultaneous stimulation of catabolic pathways. Given its central regulator role in cell metabolism, AMPK activity and its regulation have been the focus of relevant investigations, although only a few studies have focused on the AMPK function in the control of spermatozoa’s ability to fertilize. This review summarizes the known cellular roles of AMPK that have been identified in mammalian spermatozoa. The involvement of AMPK activity is described in terms of the main physiological functions of mature spermatozoa, particularly in the regulation of suitable sperm motility adapted to the fluctuating extracellular medium, maintenance of the integrity of sperm membranes, and the mitochondrial membrane potential. In addition, the intracellular signaling pathways leading to AMPK activation in mammalian spermatozoa are reviewed. We also discuss the role of AMPK in assisted reproduction techniques, particularly during semen cryopreservation and preservation (at 17 °C). Finally, we reinforce the idea of AMPK as a key signaling kinase in spermatozoa that acts as an essential linker/bridge between metabolism energy and sperm’s ability to fertilize

Short time effect (0–4 h) of the AMPK inhibition in the percentage of rapid spermatozoa.

<p>Spermatozoa were incubated in TBM (A, circles) or TCM medium (B, squares) at 38.5°C in the absence (white) or presence (black) of the AMPK inhibitor, compound C, (CC 30 µM) during 4 h. Samples at 17°C were considered as time 0. The percentage of those motile spermatozoa with VAP>80 µm/s, defined as rapid spermatozoa, was measured. This experiment was performed at least 6 times and the results express the percentage of rapid spermatozoa from the total spermatozoa motile analyzed (4.000–5.000). Statistical differences were considered when p<0.05 and showed with an asterisk.</p

Short term effect (0–4 h) of the AMPK inhibition by compound C (CC) in boar spermatozoa motility parameters in TBM.

<p>Spermatozoa were incubated in TBM at 38.5°C in the presence or absence of the AMPK inhibitor, CC, (30 µM) during 4 hours. Samples measured at 17°C were considered as time 0. Spermatozoa motility parameters including straight-line velocity (VSL, expressed in µm/s) and coefficients LIN (Linearity coefficient in %), STR (Straightness coefficient in %), WOB (Wobble coefficient in %), ALH (Amplitude of lateral head displacement in µm) and BCF (Beat cross frequency in Hz) were measured by the ISAS system. This experiment was performed at least 6 times and results express the mean ± standard error of the mean. Statistical differences were considered when p<0.05.</p

Expression of AMP-activated kinase protein, AMPK, in boar spermatozoa

<p>. Protein from boar spermatozoa lysates (lane 1) were analyzed by western blotting using anti-AMPKα as primary antibody. Other porcine tissues were homogenized and used as positive controls (hearth, lane 2; brain, lane 3 and lung, lane 4). Molecular weight markers are indicated in kDa and the amount of protein (µg) loaded into each lane is shown at the bottom. This experiment was performed 7 times and a representative film is shown.</p

Short term effect (0–4 h) of the AMPK inhibition by compound C (CC) in boar spermatozoa motility parameters in TCM.

<p>Spermatozoa were incubated in TCM at 38.5°C in the presence or absence of the AMPK inhibitor, CC, (30 µM) during 4 hours. Samples measured at 17°C were considered as time 0. Spermatozoa motility parameters including straight-line velocity (VSL, expressed in µm/s) and coefficients LIN (Linearity coefficient in %), STR (Straightness coefficient in %), WOB (Wobble coefficient in %), ALH (Amplitude of lateral head displacement in µm) and BCF (Beat cross frequency in Hz) were measured by the ISAS system. This experiment was performed at least 6 times and results express the mean ± standard error of the mean. Statistical differences were considered when p<0.05.</p

Effect of the AMPK inhibition by compound C in spermatozoa viability.

<p>Spermatozoa were incubated in TBM or TCM medium at 17°C or 38.5°C in absence or presence of the AMPK inhibitor CC (30 µM) for different times (1–24 h). Spermatozoa viability was measured by flow cytometry using SYBR-14 and IP as probes. This experiment was performed at least 4 times and the results expressed as percentage of viable cells are shown as mean ± standard error of the mean. Statistical differences are shown as a,b when p<0.001 between treatments (columns).</p

Phosphorylation of AMPK at Thr¹⁷² is regulated at physiological temperature in boar spermatozoa and is effectively inhibited by the compound C.

<p>2A: Spermatozoa were incubated in TBM or TCM medium at 38.5°C for indicated times and then lysed. Samples at 17°C were considered as time 0. Proteins (20 µg) from lysates were analyzed by western blotting using anti-phospho-Thr¹⁷²-AMPKα as primary antibody. Arrow indicates the cross-reactive band of phospho-Thr¹⁷²AMPK, recognized by the anti-AMPKα. This experiment was performed 6 times and a representative film is shown. 2B: AMPK phosphorylation was evaluated in spermatozoa incubated in TBM in the presence (+) or absence (−) of AMPK inhibitor, compound C (CC 30 µM) at 38.5°C during 24 h. This experiment was performed 3 times and a representative film is shown. Loading controls were performed for each experiment in the same membranes (with different time of exposure) using anti-GSK3β antibody and are showed at lower panels in 2A and 2B.</p