Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

RESEARCH ARTICLE Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions Thi Mong Diep Nguyen1,2,3,4, Yves Combarnous1,2,3,4, Christophe Praud5, Anne Duittoz1,2,3,4, Elisabeth Blesbois1,2,3,4* 1 INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France, 2 CNRS, UMR7247, F-37380 Nouzilly, France, 3 Université François Rabelais de Tours, F-37000 Tours, France, 4 IFCE, F-37380 Nouzilly, France, 5 INRA, Unité de Recherches Avicoles, F-37380 Nouzilly, France * OPEN ACCESS Citation: Nguyen TMD, Combarnous Y, Praud C, Duittoz A, Blesbois E (2016) Ca2+/CalmodulinDependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions. PLoS ONE 11(1): e0147559. doi:10.1371/journal.pone.0147559 Editor: Alexander J. Travis, Cornell University College of Veterinary Medicine, UNITED STATES Received: July 4, 2015 Accepted: January 5, 2016 Published: January 25, 2016 Copyright: © 2016 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was performed with the financial support of the French Agence Nationale de la Recherche (http://www.agence-nationale-recherche. fr/) INRA (http://www.inra.fr), and the French National Science Infrastructure CRB-Anim. Thi Mong Diep Nguyen is a PhD student supported by a grant from the Vietnam Education Ministry (http://www.moet.gov. vn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/ CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells. PLOS ONE | DOI:10.1371/journal.pone.0147559 January 25, 2016 1 / 22 AMPK Activation by CaMKKs in Chicken Sperm Competing Interests: The authors have declared that no competing interests exist. Introduction Biological sperm functions such as motility and ability to undergo acrosome reaction (AR) are central to male fertility. These functions are highly dependent on energetic metabolism which is itself largely controlled by 5’-AMP activated protein kinase (AMPK) signaling. The activity of this kinase is regulated by calcium through signaling pathways [1–2] that are not yet determined in chicken sperm. Bird fertilization exhibits numerous specificities comprising oviparity, complex internal fertilization and long term sperm storage in specific oviductal storage tubules, making it a unique model in fertilization studies [3–4] and important for the sake of comparison with other vertebrate species. Chicken sperm also shows very rapid signaling reactions that make them unique for metabolic signal transduction studies and was chosen as model for the present studies [5–6]. The PKA, PKB and PKC signaling pathways have previously been shown to be involved in chicken motility regulation [7–8] and PKA, PI3K and ERK2 have proved to be key proteins for chicken sperm AR [5]. We have recently demonstrated the involvement of AMPK in chicken sperm regulation of motility and acrosome reaction [6]. Nevertheless, relationships between sperm functions regulation by AMPK and calcium signaling remained to be explored. The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) were initially identified as novel members of the protein serine/threonine kinases CaMK family, with 2 forms, CaMKKα and CaMKKβ (also named CaMKK1 or CaMKK2, respectively), both expressed in the nervous system, in endothelial cells of many areas of the brain, in hematopoietic cells, and at lower levels in testis, spleen, lung, liver and skeletal muscle [9–14]. In other tissues, such as kidney, intestine, and heart, the evidence for expression remains less clear [11–12, 14]. Upon interaction with calcium-bound calmodulin (Ca2+-CaM), CaMKKs activate two calmodulindependent protein kinases: CaMKI through phosphorylation at Thr177 and CaMKIV through phosphorylation at Thr196 [15–17]. CaMKKs can also phosphorylate and activate PKB/Akt [18] and AMPK [1–2]. CaMKKβ was identified as being an AMPK kinase which phosphorylates AMPK at Thr172 in response to an increase in intracellular Ca2+ [1–2]. CaMKK (α and β) inhibition causes a drop in AMPK phosphorylation in boar sperm [19]. However, the full characterization of the mechanisms involved in the regulation of AMPK phosphorylation and activity in sperm, including those involving CaMKKs, remains to be explored. Calcium signaling pathways are essential in regulating cellular processes such as muscle contraction, neurotransmitter release, cellular metabolism, gene expression, and cell proliferation [20–21]. In sperm, Ca2+ plays a prominent role during fertilization in all animal species. In mammals, extracellular Ca2+ is required for epididymal acquisition of sperm motility in mice, ra (...truncated)