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.

5’-AMP-Activated Protein Kinase Regulates Goat Sperm Functions via Energy Metabolism In Vitro

“…1C). These observations were different with Hurtado de Llera et al [30], who reported that phospho-Thr172-AMPK was specially present in the most apical region of the acrosome and in the equatorial subsegment of the head, and also in the midpiece of boar sperm after being incubated with TBM or TCM. Moreover, phospho-Thr172-AMPK was present in the principal piece of tail and subequatorial region in the head of stallion sperm [14,31], mainly in the flagellum and acrosome and with a lower intensity in the midpiece of chicken sperm [15].…”

“…AMPK was expressed in the acrosome, midpiece and the entire flagellum of goat sperm, which was in consistence with that in boar [30], human [16,17] and chicken [15] sperm. However, this observation was different to the report by Swegen et al [31] who demonstrated that AMPK was predominantly present in the midpiece and the entire flagellum of stallion sperm.…”

“…AMPK have been regarded as one of the regulators of ΔΨm in chicken [15], mouse [41], and boar [30] sperm. In present study, AMPK up-activation yielded higher ΔΨm, while AMPK inactivation reduced ΔΨm.…”

New insights into transduction pathways that regulate boar sperm function

“…However, the role of AMPK protein in spermatozoa had not been investigated until 2012, where we identified for the first time the presence of AMPK protein in mammalian spermatozoa using an AMPK antibody against the a catalytic subunit [42]. Immunolocalization technique reveals that AMPK is localized at the entire acrosome and in the midpiece of the flagellum in boar spermatozoa [43,44]. Interestingly, under physiological conditions of boar spermatozoa, part of AMPK becomes phosphorylated (active) at Thr172 [42] and then is specifically restricted to the most apical part of the acrosome and to the subequatorial segment, remaining in the midpiece of the flagellum [43].…”

“…Immunolocalization technique reveals that AMPK is localized at the entire acrosome and in the midpiece of the flagellum in boar spermatozoa [43,44]. Interestingly, under physiological conditions of boar spermatozoa, part of AMPK becomes phosphorylated (active) at Thr172 [42] and then is specifically restricted to the most apical part of the acrosome and to the subequatorial segment, remaining in the midpiece of the flagellum [43]. To date, AMPK has also been studied later in spermatozoa from other species such as mice [45], stallions [46], and chickens [47].…”