title: AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa.
abstract: Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein kinase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (∆Ψm), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30μM), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPKα protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr(172)-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa. Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high ∆Ψm. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa. This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function.
authors.affiliation: Research Group of Intracellular Signaling and Technology of Reproduction (SINTREP), School of Veterinary Medicine, University of Extremadura, Caceres, Spain. anahl@unex.es
authors.forename: Ana
authors.initials: A
authors.lastname: Hurtado de Llera
authors.forename: David
authors.initials: D
authors.lastname: Martin-Hidalgo
authors.forename: Joan E
authors.initials: JE
authors.lastname: Rodriguez-Gil
authors.forename: M Cruz
authors.initials: MC
authors.lastname: Gil
authors.forename: Luis J
authors.initials: LJ
authors.lastname: Garcia-Marin
authors.forename: M Julia
authors.initials: MJ
authors.lastname: Bragado
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D055372:AMP-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D000177:Acrosome / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D020101:Acrosome Reaction / Q000187:drug effects; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013552:Swine
chemical_list: D007527:Isoenzymes; D011720:Pyrazoles; D011743:Pyrimidines; C516138:dorsomorphin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000001:Calcimycin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases
keywords: AMPK; Acrosome membrane; Lipid disorganization; Phosphatidylserine externalization; Spermatozoa plasmalemma