New insights into transduction pathways that regulate boar sperm function

Theriogenology xxx (2015) 1–9

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Review

New insights into transduction pathways that regulate boar sperm function A. Hurtado de Llera, D. Martin-Hidalgo, M.C. Gil, L.J. Garcia-Marin, M.J. Bragado* Research Group of Intracellular Signaling and Technology of Reproduction (SINTREP), School of Veterinary Medicine, University of Extremadura, Caceres, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2015 Received in revised form 4 May 2015 Accepted 4 May 2015

Detailed molecular mechanisms mediating signal transduction cascades that regulate boar sperm function involving Ser/Thr and tyrosine phosphorylation of proteins have been reviewed previously. Therefore, this review will focus in those kinase pathways identified recently (<10 years) in boar spermatozoa that regulate different functional spermatozoa processes. AMP-activated protein kinase (AMPK) is a cell energy sensor kinase that was first identified in mammalian spermatozoa in 2012, and since then it has emerged as an essential regulator of boar sperm function. Signaling pathways leading to AMPK activation in boar sperm are highlighted in this review (PKA, CaMKKa/b, and PKC as well as Ca2þ and cAMP messengers as upstream regulators). Interestingly, stimuli considered as cell stress (hyperosmotic stress, inhibition of mitochondrial activity, absence of intracellular Ca2þ) markedly activate AMPK in boar spermatozoa. Moreover, AMPK plays a remarkable and necessary regulatory role in mammalian sperm function, controlling essential boar sperm functional processes such as motility, viability, mitochondrial membrane potential, organization and fluidity of plasma membrane, and outer acrosome membrane integrity. These mentioned processes are all required under fluctuating environment of spermatozoa when transiting through the female reproductive tract to achieve fertilization. An applied role of AMPK in artificial insemination techniques is also suggested as during boar seminal doses preservation at 17  C, physiological levels of AMPK activity markedly increase (maximum on Day 7) and result essential to maintain the aforementioned fundamental sperm processes. Moreover, regulation of sperm function exerted by the glycogen synthase kinase 3 and Src family kinase pathways is summarized. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: AMP-activated protein kinase Glycogen synthase kinase 3 Src family kinase Signaling pathway Sperm function

1. Introduction Most of the epididymal boar sperm acquire progressive motility in the middle (corpus) and terminal (cauda) regions. In this later terminal region of epididymis, boar sperm are stored in a quiet state to minimize possible premature membrane instability that could lead to nonphysiological acrosome reaction [1]. After ejaculation, mammalian sperm

* Corresponding author. Tel.: þ34927257160; fax: þ34927257110. E-mail address: [email protected] (M.J. Bragado). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.05.008

initiate flagellar beating and become actively motile although they are still unable to penetrate the egg layers. Motility activation is partially regulated by changes in the ionic media surrounding the spermatozoa. This motility pattern is later on modified to achieve hyperactivation, which exact role is not totally defined yet, although it is suggested to be related to spermatozoa release from the oviduct reservoirs and to aid sperm to penetrate the extracellular matrix of the oocyte. The physiological modifications that take place in sperm after epididymal maturation during their transit through the female reproductive tract that confers on sperm the ability to fertilize the oocyte have

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been collectively named as capacitation, which was first independently reported by Austin [2] and Chang [3]. Molecular events accompanying capacitation are still poorly understood; however, some processes are mostly pointing to a key spermatozoa compartment: the plasma membrane. Thus, capacitation correlates with increased plasma membrane fluidity and hyperpolarization, cholesterol efflux from the sperm plasma membrane as well as changes in intracellular ion concentrations and increased protein tyrosine phosphorylation [4]. These mentioned sperm physiological processes are necessary for the subsequent stimulation of hyperactivation and acrosome reaction [1]. The sperm progressive motility achieved after ejaculation occurs in response to exposure to the extracellular medium stimuli, especially bicarbonate which enters the spermatozoa through the plasma membrane via the actions of carbonic anhydrase, sodium bicarbonate cotransporter, and bicarbonate–chloride exchanger [5]. In the cytosol, bicarbonate binds and stimulates soluble adenylyl cyclase 10, also called sAC or SACY, which catalyzes the production of cAMP using ATP as substrate. The produced cAMP represents an intracellular messenger for the protein kinase A (PKA) that becomes allosterically activated and subsequently mediates downstream signaling pathways by stimulation of Ser/Thr phosphorylation of different substrate proteins [5,6]. More detailed molecular events in those transduction cascades mediated by cAMP involving PKA, PKC, PDK1, PLCgamma1IP(3), and also tyrosine phosphorylation of proteins in boar sperm have been reviewed before [5]. Recently, it has been reported the presence of integral membrane water channels, aquaporins 7 and 11 in boar sperm. The amount of aquaporin 11 shows a correlation with sperm motility and membrane integrity [7]. Besides motility, the transit through the female reproductive tract exposes spermatozoa to different stimuli able to trigger those physiological and biochemical modifications that accompany the sperm capacitation process and ultimately confer on it the ability to fertilize the oocyte. Very recently, a proteomic approach in identifying numerous capacitation-related proteins in boar sperm has been performed [8]. Regarding kinases, it has been identified the extracellular signal–regulated kinases pathway in boar sperm where it regulates tyrosine phosphorylation during capacitation [9] and also the activation of AKT induced by dopamine along this process [10]. General signaling mechanisms underlying sperm modifications during capacitation have been the main subject of previous reviews [5,11,12]. Special relevance deserves recent studies aimed to elucidate mechanisms involved in boar sperm hyperactivation [5,13,14], changes in the plasma membrane organization [15], which may be induced by endocannabinoid-binding type-1 cannabinoid receptor CB1 and transient receptor potential vanilloid 1 (TRPV1) [16], Ser/Thr phosphorylation [17], tyrosine phosphorylation [13,18], and finally those leading to the acrosome reaction [5,12,19,20], which may be partially mediated by reactive oxygen species (ROS) and phospholipase A [21]. Besides phosphorylation, another posttranslational modification of proteins, ubiquitination, modulates boar sperm capacitation [22]. This review will focus in recent (<10 years) research signaling pathways that regulate different aspects of boar

sperm function, including motility, viability, mitochondrial membrane potential, plasma membrane fluidity, and acrosome membrane integrity. 2. AMP-activated protein kinase 2.1. Structure and upstream signaling AMP-activated protein kinase (AMPK) is a cellular fuel gauge that acts regulating energy balance at the cellular and whole body levels [23,24]. AMP-activated protein kinase is present in all eukaryotes as heterotrimeric complexes comprising a catalytic a subunit and regulatory b and g subunits, each of which occurs in mammals as alternate isoforms encoded by distinct genes [25]. In mammalian cells, there are two isoforms of the a subunit, two isoforms of the b subunit, and three isoforms of the g subunit [23]. The a subunit contains a typical serine/threonine protein kinase domain at the N-terminus and a Cterminal regulatory domain. Within the b subunit, there is a domain that has been termed the glycogen-binding domain or carbohydrate-binding module. The C-terminal region of the b subunit interacts with the a and g subunits, acting as a scaffold for the interaction of the heterotrimeric complex [23]. Finally, the g subunit contains four copies of a cystathionine-b-synthase domain [26] which forms four potential adenine nucleotide–binding sites [27]. Like most kinase cascades, AMPK is activated by phosphorylation of a residue (Thr172) within the activation loop of the kinase domain [28]. Several kinases have been described to phosphorylate AMPK at Thr172: LKB1 [29], a and b isoforms of the calcium-calmodulin kinase kinase (CaMKK) [30], TGF-beta-activating kinase 1, [31], and kinase suppressor of Ras 2 [32]. Moreover, AMPK becomes also activated by AMP via a tripartite mechanism: (1) promotion of Thr172 phosphorylation; (2) inhibition of Thr172 dephosphorylation and (3) allosteric activation. Only the second effect is mimicked by ADP, although all three AMP effects are antagonized by ATP [24]. 2.2. AMPK functions A vast majority of works investigating cellular functions of AMPK have been conducted in somatic cells [23–25]. Given its key role as a sensor of the cell energy status (through its activation by high AMP levels), one of the main known functions of AMPK is the control of metabolic pathways under different energy or stressful conditions. This metabolic regulator role of AMPK has been reported in different tissues including cardiac and skeletal muscle, adipose tissue, pancreas, liver, and brain [33,34]. In the reproduction realm, AMPK role has been studied in hypothalamic–hypophysis axis [35,36], epididymis and vas deferens function [37], and mouse oocyte meiotic maturation [38]. 2.3. AMPK in boar spermatozoa Until 2012, no works aimed to study the function of AMPK in spermatozoa had been conducted, although some AMPK-related kinases had been reported in these germ

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cells. Thus, in 2008, it was described that a shorter isoform of LKB1, called LKB1s, is expressed predominantly in haploid sperm cells from testes of mammals [39]. LKB1s knockout mice have a dramatic reduction in the number of mature spermatozoa in the epididymis, and the few spermatozoa produced are not motile, have abnormal head morphology, and result sterile [39]. These data suggested that this variant of the LKB1 has a crucial role in spermiogenesis and fertility (motility) in mice. In addition, members of the Ser/Thr kinase testis-specific TSSK family, which belongs to the AMPK branch in the human kinome tree, have been identified in human spermatozoa: TSSK2, TSKS, and SSTK [40]. Deletion of TSSK1 and 2 causes male infertility in chimera mice due to haploinsufficiency [41]. These studies suggested that some AMPK-related kinases might play a crucial role in the spermatozoa 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]. To date, AMPK has also been studied later in spermatozoa from other species such as mice [45], stallions [46], and chickens [47]. To study the functional role of AMPK in boar spermatozoa, two pharmacologic compounds have been used: (1) the kinase activator, A769662, which has been found that rapidly increases Thr172 phosphorylation of AMPK in boar spermatozoa without causing any sperm cytotoxic effects [48]; (2) the kinase inhibitor compound C (CC), which effectively blocks Thr172 phosphorylation of AMPK in boar spermatozoa [42]. 2.4. Role of AMPK pathway in boar spermatozoa motility According to the key role of AMPK in the control of cell energy homeostasis, AMPK has recently emerged as a new kinase that regulates those sperm functions critically dependent on energy levels, such as motility [42,44,48], one of the most important functional processes for spermatozoa ultimate function: fertilization. Different conclusive experiments show that inhibition of AMPK in boar spermatozoa using CC pretreatment causes a marked reduction of any spermatozoa motility parameter. Thus, AMPK inhibition significantly decreases the percentage of motile spermatozoa, significantly reduces spermatozoa curvilinear velocity, the average path velocity, and subsequently decreases the percentage of rapid spermatozoa (average path velocity, >80 mm/s) and also affects other motility parameters and coefficients analyzed by computer-assisted sperm analysis system [42]. On the basis of these clear effects on motility, it can be conceivable that AMPK might act by phosphorylating downstream protein substrates involved in the axoneme central apparatus or in

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other related structures that are essential for spermatozoa flagellar motility, as it has been previously reported for its related kinase TSSK2, expressed in spermatozoa [40]. Thus, TSKK2 phosphorylates in vitro the axoneme central apparatus protein called SPAG16L, which is essential for flagellar motility in mouse spermatozoa [49]. Surprisingly, we have found that a sustained increase in AMPK activity over physiological levels, using the AMPK activator A769662, also negatively affects boar sperm motility [48]. Therefore, we have proposed that either up or down fluctuations of mammalian sperm AMPK activity away from energy charge–regulated physiological levels lead to a negative role in sperm motility [48]. Thus, physiological AMPK activity is necessary to maintain optimal boar sperm motility, likely adequate to the changing extracellular conditions 2þ (presence or absence of HCO 3 , Ca , and BSA). As the concentration of these stimuli fluctuates within the female reproductive tract, we suggest that this relevant function of AMPK in boar spermatozoa motility might likely occur in vivo as well, where three following levels of AMPK activity could exist: (1) Physiological levels of AMPK activity, which are the optimal to maintain the spermatozoa motility that is appropriate to fluctuating composition of extracellular medium and therefore to assure that spermatozoa are able to respond to the different environmentdepending demands of energy required for sperm function during transit through the female reproductive tract [42]; (2) Second level that would occur in vivo whenever sperm AMPK activity falls below the physiological range (experimentally approached using CC): Under these conditions, AMPK is mainly inactive [42], i.e., unable to respond by producing metabolic adjustments necessary to control and maintain ATP cell levels required for spermatozoa under each condition. This sustained lack of control in sperm energy maintenance causes that motility, one of the main ATP-dependent sperm functions, results negatively affected. The idea that a certain (physiological) level of AMPK activity is necessary for proper sperm motility is supported by a study performed in transgenic mice lacking the catalytic subunit a1 gene (a1AMPK knockout) which present a great reduction in sperm motility [45]; (3) a third level that would occur in vivo whenever sperm AMPK activity rises over physiological levels (experimentally approached using activator A769662). Under these conditions, a sustained increase in sperm AMPK activity would lead to a deregulation of sperm metabolism caused by a prolonged stimulation of ATP-generating catabolic pathways and by a sustained inhibition of ATP-consuming anabolic pathways. This A769662-induced deregulation of metabolic pathways is not adequate for the maintenance of proper spermatozoa motility under any extracellular conditions, as reported by Hurtado de Llera et al. [48]. The important role of AMPK in boar sperm motility is also supported by the localization of important levels of AMPK active (Thr172 phosphorylated) at the midpiece of the flagellum [43], where one of the main ATP-generating organelles, mitochondria, is exclusively located in spermatozoa. Besides boar sperm [5,42,44,48], the relevant role of AMPK in spermatozoa motility has been confirmed in other mammalian species, such as mice [45] and also in avian sperm [47].

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2.5. Role of AMPK pathway in the sperm acrosome and plasma membranes As mentioned, mammalian spermatozoa require a fine regulation of energy levels to maintain cellular structure, stability, and function of their membranes and intracellular ions composition during changing extracellular conditions, such as those leading to oocyte fertilization within the female reproductive tract [1,5]. In line with this, the energy charge–sensor kinase AMPK is involved in the maintenance of boar spermatozoa plasma membrane fluidity and organization at physiological temperature [43]. A well-known parameter that contributes to the correct function of spermatozoa is the degree of lipid organization of their plasma membrane. Experimental conditions that include either an inhibition (using CC) or a sustained AMPK activation over physiological levels (using A769662) lead to a marked lipid disorganization in the plasma membrane, which suggests that AMPK signaling pathway is likely involved in the maintenance of the proper lipid organization and fluidity in boar sperm plasma membrane [43,44]. Moreover, it is well known that physiological levels of HCO 3 produce a rapid collapse of the asymmetry of the boar sperm plasma membrane attributable to the activation of scramblases, enzymes that translocate membrane phospholipids, such as phosphatidylethanolamine and phosphatidylserine (PS) outward of the plasma membrane [50]. The phosphatidylserine externalization indicates plasma membrane scrambling which physiologically occurs in relevant spermatozoa functions. Interestingly, the inhibition of AMPK in boar spermatozoa incubated in the presence of HCO 3 causes a significant inhibition of the outward exposure of PS (measured by annexin-V binding using flow cytometry) in the spermatozoa plasma membrane, suggesting that inhibition of AMPK, at least at short time, might be leading to a downstream inhibition of scramblase(s) activity [43]. Furthermore, an increased AMPK activity (for 24 hours) induces a significant PS translocation to the outer leaf of the plasma membrane [48]. Functional effects of AMPK in boar sperm plasma membrane including lipid disorganization and the outward exposure of PS are likely occurring in the plasma membrane surrounding the most apical part of the acrosome, where a majority of active phospho-Thr172 AMPK is localized [43]. The role of AMPK in boar sperm membranes is supported by the fact that AMPK lies downstream of cAMP/PKA [51], a key signaling pathway in the regulation of boar sperm plasma membrane lipid architecture [52,53]. In parallel to a functional role at the spermatozoa plasma membrane, AMPK is involved in the regulation of acrosome membrane integrity, where, as mentioned, important levels of phospho-Thr172-AMPK active are located at physiological conditions [43]. Different experimental approaches show that any fluctuation either up (using A769662) or down (using CC) of AMPK activity away from physiological level causes loss of the outer acrosome membrane integrity evaluated by flow cytometry using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) as probe [43,48]. In conclusion, physiological levels of AMPK activity are essential to maintain the correct physiological plasma membrane lipid organization including a relevant

role regulating the suitable outward translocation of PS. Physiological AMPK activity is also necessary to maintain the integrity of acrosome membrane at a level adequate to the changing extracellular conditions at which boar spermatozoa are physiologically exposed. 2.6. Role of AMPK pathway in the mitochondrial membrane potential of boar spermatozoa Spermatozoa energy in the form of ATP is obtained mainly from two metabolic pathways: glycolysis and mitochondrial oxidative phosphorylation. These metabolic pathways are localized in the fibrous sheath and midpiece of spermatozoa’s flagellum, respectively [54]. AMPactivated protein kinase has emerged as one of the regulators of the mitochondrial membrane potential, DJm, in spermatozoa from boars [43] and mice [45]. Thus, inactivation of AMPK by two different experimental approaches causes a decrease in DJm, which in boar spermatozoa is 2þ modulated by stimuli such as HCO [43] and in 3 and Ca a1AMPK knockout mice, it is concomitant to a reduced basal oxygen consumption [45]. Moreover, an AMPK up-activation prevents at the short time the fall in the percentage of boar sperm showing high DJm under HCO 3and Ca2þ-stimulated conditions [48], which additionally supports a regulatory role of AMPK activity in the maintenance of sperm DJm. The consequences of an AMPK upactivation in boar sperm DJm depend on extracellular conditions, supporting the idea that AMPK serves as a metabolic checkpoint by integrating extracellular stimuli– triggered signaling with spermatozoa metabolism. Taking into consideration the aforementioned results and the fact that part of AMPK activated form (phospho-Thr172) is found in the midpiece of boar spermatozoa tail where mitochondria are also located [43], it is concluded that a specific level of AMPK activity is essential to maintain a proper mitochondrial membrane potential in boar spermatozoa according to the fluctuating extracellular stimuli. 2.7. Role of AMPK pathway in the viability of boar spermatozoa Some extracellular conditions lead to a loss in boar sperm viability, such as high concentration of calcium [50,55] or those that represent germ cell stresses. Under specific extracellular conditions, the activity of AMPK might be involved in the modulation of spermatozoa viability [42,44,48]. Thus, the inhibition of AMPK in a medium without extracellular Ca2þ exerts a protective effect in boar spermatozoa viability [42]. However, the inhibition of AMPK on boar spermatozoa incubated in Ca2þ- and HCO 3stimulated conditions has no effect on spermatozoa viability [42]. In contrast, sustained AMPK up-activation partially prevents at the short time the loss in boar sperm 2þ viability induced by HCO [48]. In conclusion, 3 and Ca under those extracellular conditions leading to a decrease in boar sperm viability, such as high concentration of Ca2þ or those that represent sperm stresses [44], the activity of AMPK, at least at the short term, would be crucial to the maintenance of sperm viability.

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2.8. Role of the AMPK pathway during boar semen preservation. Possible use of AMPK to implement assisted reproduction techniques Nowadays, in the most industrialized countries, the percentage of sows inseminated by artificial insemination varies between 80% and 98%. Moreover, this rate is increasing year after year in the rest of nations [56]. For this reason, AI is fundamental in pig breeding, where 99% of inseminations worldwide are performed using chilled boar seminal doses [57]. As mentioned, since 2012, AMPK is been the aim of basic research in boar sperm physiology and currently is also being the goal of applied research in some assisted reproduction techniques such as artificial insemination [44] or cryobiology [46,58]. Thus, during boar seminal doses preservation at 17  C, phospho-Thr172 AMPK (active) levels clearly increase from Day 0, where is barely detected, to Day 10 reaching maximum levels on Day 7 of storage [44]. Moreover, the inhibition of AMPK during boar seminal doses preservation at 17  C exerts storage time– depending effects in spermatozoa. Thus, at short term, AMPK inhibition by CC causes slight modifications in sperm motility parameters, whereas at long-term storage, AMPK inhibition leads to a clear reduction in sperm motility parameters (mainly velocities and percentage of motile sperm) and causes deleterious effects on plasma membrane and acrosome membrane integrity [44]. Therefore, there is a clear relation between AMPK activity and the aging process in extended boar semen during long-term preservation, in which spermatozoa are subjected to stressful conditions such as storage below the physiological temperature, dilution of protective agents of seminal fluid, reduction of intracellular pH, or diminished oxygen availability [57]. In brief, correct physiological levels of AMPK activity are essential to maintain fundamental sperm functional processes, such as motility, mitochondrial activity, plasma membrane organization, and acrosome membrane integrity, during boar seminal doses preservation at 17  C, which definitely represents stressful conditions for spermatozoa [44]. As AMPK has emerged as a new relevant signaling cascade in spermatozoa physiology (Fig. 1), further studies focused on sperm AMPK pathway (i.e., using AMPK activators) might definitely help to improve assisted reproduction techniques not only in pigs but also in other species. 2.9. Signaling pathways underlying AMPK activity in boar spermatozoa The role of AMPK as an essential regulator of boar spermatozoa function has been highlighted previously. Therefore, an important issue in spermatozoa physiology is to elucidate the intracellular signaling pathways leading to AMPK activity. It has been clearly reported using different experimental approaches that SACY, cAMP [42], and PKAmediated pathway are upstream regulators of AMPK activity in boar spermatozoa [43,51]. The sperm AMPK activation by cAMP/PKA might occur through its upstream kinase LKB1, which in somatic cells can be directly phosphorylated at Ser431 by PKA in response to activation of adenylate cyclase by forskolin [59,60] or IBMX [59]. To date, it is

Fig. 1. AMP-activated protein kinase (AMPK) regulates main boar spermatozoa functional processes. The regulation of sperm motility, viability, mitochondrial membrane potential, plasma membrane organization and fluidity, acrosome membrane integrity mediated by AMPK is indicated. A possible role of AMPK pathway in applied research to improve artificial insemination technology is also highlighted.

unknown whether mature spermatozoa express LKB1 although its short splice variant LKB1s is highly expressed in haploid spermatids in mice testes [39] where it has a relevant role in spermiogenesis and fertility. Another possible mechanism that might explain AMPK activation by an increase in cAMP levels is via regulation of cAMP degradation to AMP, as occurs in somatic cells [61]. Therefore, any stimulus leading to an increase in intracellular cAMP in spermatozoa could result in AMPK activation either by direct activation of PKA or by a phosphodiesterases-induced increase in AMP levels, which activate allosterically AMPK, or both mechanisms (Fig. 2). An essential regulator of any spermatozoa functional process is the intracellular messenger Ca2þ, which through the subsequent activation of the specific SACY and downstream through PKA potently activates AMPK in boar spermatozoa [43,51]. Additionally, Ca2þ might also lead to the phosphorylation of boar sperm AMPK through the activation of Ca2þ/calmodulin–dependent kinase kinases II, CaMKKa/b [51], which lie upstream of AMPK in somatic cells [29,30]. Moreover, the facts that AMPK phosphorylation is stimulated by direct activation of PKC with phorbol 12myristate 13-acetate and that the PKC inhibitor Ro-322þ 0432 inhibits HCO 3 - and Ca -induced AMPK activation [51] indicate that at least one isoform of PKC is upstream of AMPK in boar spermatozoa. Among PKC isoforms that have been identified in mammalian spermatozoa are PKCa and PKCbI in bovine [62], PKC-zeta in hamster [63] and mouse sperm [64]. It is therefore plausible that PKC-zeta might play in boar spermatozoa a similar role than in somatic cells as showed by Xie et al. [65], leading to AMPK activation through the phosphorylation of LKB1. An alternative explanation describing the pathway by which PKC is upstream of sperm AMPK activity is based on the fact that PKC activity lies downstream of PKA in the control pathway of boar sperm motility [66]. Previously, Harayama and Miyake [67] reported that cAMP/PKA signaling can induce the activation of calcium-sensitive PKCs, which are responsible

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Fig. 2. Signaling transduction pathways leading to regulation of AMP-activated protein kinase (AMPK) activity in boar spermatozoa. Intracellular mechanisms detailed in the text that are involved in AMPK phosphorylation at Thr172 are triggered by Ca2þ, HCO 3 , 8Br-cAMP, PMA, A769662, and different types of cell stresses (absence of Ca2þ, hyperosmotic stress, inhibition of mitochondrial activity). Inhibitors of different AMPK upstream kinases (H89, IBMX, STO-609 and RO-32-0432) are also indicated.

for boar sperm hyperactivation. Therefore, we have proposed that another PKC isoform(s) besides PKC-zeta, which is not calcium sensitive, could be likely mediating AMPK activation, at least in response to an elevation of cAMP levels, in boar spermatozoa [51]. In addition to the mentioned physiological mimicking conditions, AMPK becomes markedly activated in boar spermatozoa under different stimuli considered as cell stress (Fig. 2), such as inhibition of spermatozoa mitochondrial activity by blocking electron transport chain and sorbitol-induced hyperosmotic stress [51]. In somatic cells, cell stress–induced AMPK activation can be mediated by (1) an increase in AMP levels, and (2) ROS generation that act as signaling molecules to activate AMPK [68] through LKB1 and CaMKK pathways. Surprisingly, the absence of intracellular Ca2þ in boar spermatozoa by incubation with 1,2-bis(2aminophenoxy)ethane-N,N,N0 ,N0 - tetraacetic acid/acetoxy methyl (BAPTA-AM) in a Ca2þ-free media leads to a strong increase in AMPK activity [51], which may be mediated through an increase in nitric oxide NO$ production. In this sense, de Lamirande et al. [69] reported in human sperm that BAPTA-AM promotes the production of an ROS, the nitric oxide NO$. Accordingly, AMPK activation is also directly influenced by cellular redox status in somatic cells, as H2O2 activates AMPK through oxidative modification of cysteine residues in the AMPKa subunit [70]. An alternative or simultaneous explanation is that NO$ produced by BAPTA-AM in boar spermatozoa might interacts with the cAMP pathway as it occurs in humans [69] leading to AMPK activity. In our opinion, the exact molecular mechanisms

involved in the signaling pathway(s) leading to AMPK activity and the identity of AMPK downstream targets that ultimately control boar sperm function undoubtedly deserve future investigations. 3. Glycogen synthase kinase 3 Special relevance in past 10 years of research in the signaling transduction pathways controlling boar sperm physiology deserves glycogen synthase kinase 3 (GSK3). This kinase was discovered as one of the Ser/Thr kinases that phosphorylates and inactivates glycogen synthase, although currently, GSK3 is recognized as a key component of a large number of cellular processes implicated in cell adhesion, division, survival, and apoptosis [71]. Glycogen synthase kinase 3 exists as two isoforms, GSK3a and GSK3b, which share 97% sequence similarity and are ubiquitously expressed in mammalian tissues [71]. Unlike other kinases, GSK3 activity is significantly reduced by phosphorylation of an N-terminal serine, Ser9, in GSK3b and Ser21 in GSK3a [71]. The presence of GSK3 in spermatozoa was initially reported in humans [72] where only GSK3b was detected and simultaneously in bovine sperm by Somanath et al. [73] where both isoforms were identified. In boar spermatozoa, both isoforms of GSK3 were identified by our group in 2007 [74]. In response to stimuli that induce the activation of the cAMP/PKA pathway in boar sperm, GSK3a and GSK3b isoforms are clearly phosphorylated in Ser21 and Ser9, respectively [74], indicating that PKA is an upstream regulator of GSK3 in boar spermatozoa.

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Furthermore, we have shown that GSK3 phosphorylation is totally dependent on PKA activity [66] and that is also downstream of the PKC and PI3K pathways [66]. Our proposed model of GSK3 signaling in boar spermatozoa implies the initial activation of SACY by sperm stimuli that in turn increases cAMP levels and subsequently activates PKA, which might lead to (1) direct phosphorylation of GSK3a in Ser21, as it occurs in somatic cells [75], or (2) activation of PKC, as suggested by Harayama and Miyake [67]. PKC activation could lead to GSK3 phosphorylation either directly or indirectly by an intermediary unknown kinase. A different signaling pathway involved in the control of GSK3 phosphorylation is PI3K, which through the regulation of cAMP levels lies upstream of PKA in boar sperm [66]. Studies aiming to elucidate the functional role of GSK3 phosphorylation in boar sperm motility reported a significant positive correlation between GSK3a inactivation and increases in the curvilinear velocity, straight linear velocity, and subsequently in the percentage of sperm showing rapid velocity [74]. Additionally, a pharmacologic approach confirmed a negative role of GSK3 on the regulation of boar spermatozoa motility as GSK3 enzymatic blockade caused a clear increase in the previously mentioned motility parameters [74]. Besides motility, GSK3 is likely involved in the regulation of other functional processes of boar sperm such as capacitation [74]. In summary, a relevant function of GSK3 in the regulation of boar sperm physiology, especially in motility, is established, although the exact molecular mechanisms involved in the signaling pathway leading to GSK3 signaling and the identity of GSK3 downstream targets that ultimately control boar sperm motility still need future research.

4. Src family of tyrosine kinases The key role of PKA in the signaling pathways controlling boar sperm physiology is emphasized by the fact that in addition to induce downstream phosphorylation in Ser/ Thr residues, PKA is also an essential regulator of the protein tyrosine phosphorylation that physiologically occurs mainly during the sperm capacitation process [6,13,18]. Among the possible kinases responsible for this tyrosine phosphorylation, the Src family of protein tyrosine kinases (SFKs) has been recently included in boar [76], mouse [77], and also in human spermatozoa [78–80]. In 2012, we have identified the presence of different members of the SFK in boar spermatozoa: c-Lyn and c-Yes, named SFK1 and SFK2, respectively, which become activated (phosphorylated in Tyr416) during sperm capacitation [76]. The SFK pathway is not likely involved in the biochemical control of neither motility nor viability in boar spermatozoa. However, SFK inhibition causes increases in both nonstimulated and calcium-induced acrosome reaction and a decrease in the F-actin content in boar capacitated spermatozoa [76]. We have proposed a model in which members of SFK, which become activated (Tyr416 phosphorylated) during boar sperm capacitation, play an inhibitory role in the regulation of the physiological process that occurs immediately after the capacitation: the acrosome reaction [76]. This might be explained at least partially by the involvement of SFK in

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actin polymerization observed during boar sperm capacitation [76]. While writing this review, a novel transduction pathway has been identified in boar spermatozoa, the Wnt1 ligand– modulated Wnt/b-catenin signaling pathway, which plays a regulatory role in both in vitro capacitation and in subsequent progesterone-induced acrosome reaction [81]. 4.1. Conclusions In summary, AMPK, which is upstream regulated by PKA, CaMKKa/b, and PKC kinases and by Ca2þ and cAMP messengers (Fig. 2), plays a remarkable and necessary regulatory role in mammalian spermatozoa function (Fig. 1). This kinase controls main boar sperm functional processes (motility, viability, mitochondrial membrane potential, organization and fluidity of plasma membrane, outer acrosome membrane integrity), which are all required under the fluctuating environment of spermatozoa when transiting through the female reproductive tract to achieve fertilization. Further research is necessary to clearly elucidate the key role of AMPK as well as GSK3 and SFK in boar spermatozoa function. Acknowledgments This work was supported by a national grant, AGL201015188 (from the Spanish Ministry of Education and Science) and by regional grants: PRI09A077, IB13121, and GR10156 (from Junta de Extremadura, Spain). Martin-Hidalgo D. received a PhD fellowship award from the Junta de Extremadura, Spain. The authors Hurtado de Llera A. and Martin-Hidalgo D contributed equally to this review. References [1] Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil JD, editors. The physiology of reproduction. New York: Raven press; 1994. p. 189–317. [2] Austin CR. The capacitation of the mammalian sperm. Nature 1952; 170:326. [3] Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 1951;168:697–8. [4] Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci U S A 2009;106:667–8. [5] Harayama H. Roles of intracellular cyclic AMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa. J Reprod Dev 2013;59:421–30. [6] Buffone MG, Wertheimer EV, Visconti PE, Krapf D. Central role of soluble adenylyl cyclase and cAMP in sperm physiology. Biochim Biophys Acta 2014;1842:2610–20. [7] Prieto-Martinez N, Vilagran I, Morato R, Rodriguez-Gil JE, Yeste M, Bonet S. Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality. Reprod Fertil Dev 2014. http://dx.doi.org/10.1071/RD14237. [Epub ahead of print]. [8] Kwon WS, Rahman MS, Lee JS, Kim J, Yoon SJ, Park YJ, et al. A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa. BMC Genomics 2014;15:897. [9] Awda BJ, Buhr MM. Extracellular signal-regulated kinases (ERKs) pathway and reactive oxygen species regulate tyrosine phosphorylation in capacitating boar spermatozoa. Biol Reprod 2010;83:750–8. [10] Ramirez AR, Castro MA, Angulo C, Ramio L, Rivera MM, Torres M, et al. The presence and function of dopamine type 2 receptors in boar sperm: a possible role for dopamine in viability, capacitation, and modulation of sperm motility. Biol Reprod 2009;80:753–61. [11] Signorelli J, Diaz ES, Morales P. Kinases, phosphatases and proteases during sperm capacitation. Cell Tissue Res 2012;20.

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A. Hurtado de Llera et al. / Theriogenology xxx (2015) 1–9

[12] Ickowicz D, Finkelstein M, Breitbart H. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian J Androl 2012;14:816–21. [13] Harayama H, Noda T, Ishikawa S, Shidara O. Relationship between cyclic AMP-dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa. Mol Reprod Dev 2012;79:727–39. [14] Kojima A, Matsushita Y, Ogura Y, Ishikawa S, Noda T, Murase T, et al. Roles of extracellular Ca in the occurrence of full-type hyperactivation in boar ejaculated spermatozoa pre-incubated to induce the cAMP-triggered events. Andrology 2015;3:321–31. [15] Boerke A, Tsai PS, Garcia-Gil N, Brewis IA, Gadella BM. Capacitationdependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction. Theriogenology 2008;70: 1188–96. [16] Bernabo N, Palestini P, Chiarini M, Maccarrone M, Mattioli M, Barboni B. Endocannabinoid-binding CB1 and TRPV1 receptors as modulators of sperm capacitation. Commun Integr Biol 2012;5: 68–70. [17] Ramio-Lluch L, Fernandez-Novell JM, Pena A, Ramirez A, Concha II, Rodriguez-Gil JE. ‘In vitro’ capacitation and further ‘in vitro’ progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa. Reprod Domest Anim 2012;47:766–76. [18] Bravo MM, Aparicio IM, Garcia-Herreros M, Gil MC, Pena FJ, GarciaMarin LJ. Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa. Mol Reprod Dev 2005;71:88–96. [19] Adachi J, Tate S, Miyake M, Harayama H. Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/ threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog. J Reprod Dev 2008;54: 171–6. [20] Bragado MJ, Gil MC, Garcia-Marin LJ. Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction. Reprod Domest Anim 2011;46:943–9. [21] Awda BJ, Mackenzie-Bell M, Buhr MM. Reactive oxygen species and boar sperm function. Biol Reprod 2009;81:553–61. [22] Purdy PH. Ubiquitination and its influence in boar sperm physiology and cryopreservation. Theriogenology 2008;70:818–26. [23] Carling D, Thornton C, Woods A, Sanders MJ. AMP-activated protein kinase: new regulation, new roles? Biochem J 2012;445:11–27. [24] Hardie DG, Ashford ML. AMPK: regulating energy balance at the cellular and whole body levels. Physiology (Bethesda) 2014;29:99–107. [25] Gowans GJ, Hawley SA, Ross FA, Hardie DG. AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation. Cell Metab 2013;18: 556–66. [26] Bateman A. The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci 1997; 22:12–3. [27] Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin resistance, and the metabolic syndrome. J Clin Invest 2013;123: 2764–72. [28] Hardie DG, Alessi DR. LKB1 and AMPK and the cancer-metabolism linkdten years after. BMC Biol 2013;11:36. [29] Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 2003;13:2004–8. [30] Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, et al. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2005; 2:9–19. [31] Xie M, Zhang D, Dyck JR, Li Y, Zhang H, Morishima M, et al. A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMPactivated protein kinase energy-sensor pathway. Proc Natl Acad Sci U S A 2006;103:17378–83. [32] Costanzo-Garvey DL, Pfluger PT, Dougherty MK, Stock JL, Boehm M, Chaika O, et al. KSR2 is an essential regulator of AMP kinase, energy expenditure, and insulin sensitivity. Cell Metab 2009;10:366–78. [33] Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005;1:15–25. [34] Ramamurthy S, Ronnett GV. Developing a head for energy sensing: AMP-activated protein kinase as a multifunctional metabolic sensor in the brain. J Physiol 2006;574:85–93.

[35] Iwasaki Y, Nishiyama M, Taguchi T, Kambayashi M, Asai M, Yoshida M, et al. Activation of AMP-activated protein kinase stimulates proopiomelanocortin gene transcription in AtT20 corticotroph cells. Am J Physiol Endocrinol Metab 2007;292: E1899–905. [36] Mountjoy PD, Bailey SJ, Rutter GA. Inhibition by glucose or leptin of hypothalamic neurons expressing neuropeptide Y requires changes in AMP-activated protein kinase activity. Diabetologia 2007;50: 168–77. [37] Hallows KR, Alzamora R, Li H, Gong F, Smolak C, Neumann D, et al. AMP-activated protein kinase inhibits alkaline pH- and PKAinduced apical vacuolar Hþ-ATPase accumulation in epididymal clear cells. Am J Physiol Cell Physiol 2009;296:C672–81. [38] Downs SM, Ya R, Davis CC. Role of AMPK throughout meiotic maturation in the mouse oocyte: evidence for promotion of polar body formation and suppression of premature activation. Mol Reprod Dev 2010;77:888–99. [39] Towler MC, Fogarty S, Hawley SA, Pan DA, Martin DM, Morrice NA, et al. A novel short splice variant of the tumour suppressor LKB1 is required for spermiogenesis. Biochem J 2008;416:1–14. [40] Xu B, Hao Z, Jha KN, Digilio L, Urekar C, Kim YH, et al. Validation of a testis specific serine/threonine kinase [TSSK] family and the substrate of TSSK1 & 2, TSKS, as contraceptive targets. Soc Reprod Fertil Suppl 2007;63:87–101. [41] Xu B, Hao Z, Jha KN, Zhang Z, Urekar C, Digilio L, et al. Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency. Dev Biol 2008;319:211–22. [42] Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ. AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility. PLoS One 2012;7:e38840. [43] Hurtado de Llera A, Martin-Hidalgo D, Rodriguez-Gil JE, Gil MC, Garcia-Marin LJ, Bragado MJ. AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa. Biochim Biophys Acta 2013;1828:2143–51. [44] Martin-Hidalgo D, Hurtado de Llera A, Yeste M, Gil MC, Bragado MJ, Garcia-Marin LJ. Adenosine monophosphate-activated kinase, AMPK, is involved in the maintenance of the quality of extended boar semen during long-term storage. Theriogenology 2013;80: 285–94. [45] Tartarin P, Guibert E, Toure A, Ouiste C, Leclerc J, Sanz N, et al. Inactivation of AMPKalpha1 induces asthenozoospermia and alters spermatozoa morphology. Endocrinology 2012;153:3468–81. [46] Cordova A, Strobel P, Vallejo A, Valenzuela P, Ulloa O, Burgos RA, et al. Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5’ AMP-activated protein kinase (AMPK). Cryobiology 2014;69:473–81. [47] Nguyen TM, Alves S, Grasseau I, Metayer-Coustard S, Praud C, Froment P, et al. Central role of 5’-AMP-activated protein kinase in chicken sperm functions. Biol Reprod 2014;91:121. [48] Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ. AMPK up-activation reduces motility and regulates other functions of boar spermatozoa. Mol Hum Reprod 2014;21:31–45. [49] Zhang Z, Kostetskii I, Tang W, Haig-Ladewig L, Sapiro R, Wei Z, et al. Deficiency of SPAG16L causes male infertility associated with impaired sperm motility. Biol Reprod 2006;74:751–9. [50] Gadella BM, Harrison RA. The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane. Development 2000;127:2407–20. [51] Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ. The calcium/CaMKKalpha/beta and the cAMP/PKA pathways are essential upstream regulators of AMPK activity in boar spermatozoa. Biol Reprod 2014;90:29. [52] Gadella BM, van Gestel RA. Bicarbonate and its role in mammalian sperm function. Anim Reprod Sci 2004;82-83:307–19. [53] Harrison RA. Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate. Mol Reprod Dev 2004;67:337–52. [54] Ferramosca A, Zara V. Bioenergetics of mammalian sperm capacitation. Biomed Res Int 2014;2014:902953. [55] Martin G, Sabido O, Durand P, Levy R. Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction. Hum Reprod 2005;20:3459–68. [56] Riesenbeck A. Review on international trade with boar semen. Reprod Domest Anim 2011;46(Suppl 2):1–3. [57] Johnson LA, Weitze KF, Fiser P, Maxwell WM. Storage of boar semen. Anim Reprod Sci 2000;62:143–72.

A. Hurtado de Llera et al. / Theriogenology xxx (2015) 1–9 [58] Bertoldo MJ, Guibert E, Tartarin P, Guillory V, Froment P. Effect of metformin on the fertilizing ability of mouse spermatozoa. Cryobiology 2014;68:262–8. [59] Collins SP, Reoma JL, Gamm DM, Uhler MD. LKB1, a novel serine/ threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo. Biochem J 2000;345(Pt 3):673–80. [60] Sapkota GP, Kieloch A, Lizcano JM, Lain S, Arthur JS, Williams MR, et al. Phosphorylation of the protein kinase mutated in PeutzJeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell growth. J Biol Chem 2001;276:19469–82. [61] Omar B, Zmuda-Trzebiatowska E, Manganiello V, Goransson O, Degerman E. Regulation of AMP-activated protein kinase by cAMP in adipocytes: roles for phosphodiesterases, protein kinase B, protein kinase A, Epac and lipolysis. Cell Signal 2009;21:760–6. [62] Breitbart H, Naor Z. Protein kinases in mammalian sperm capacitation and the acrosome reaction. Rev Reprod 1999;4:151–9. [63] NagDas SK, Winfrey VP, Olson GE. Identification of Ras and its downstream signaling elements and their potential role in hamster sperm motility. Biol Reprod 2002;67:1058–66. [64] Jungnickel MK, Sutton KA, Wang Y, Florman HM. Phosphoinositidedependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction. Dev Biol 2007;304:116–26. [65] Xie Z, Dong Y, Zhang J, Scholz R, Neumann D, Zou MH. Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis. Mol Cell Biol 2009;29:3582–96. [66] Bragado MJ, Aparicio IM, Gil MC, Garcia-Marin LJ. Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase-3A serine 21 phosphorylation in boar spermatozoa. J Cell Biochem 2010;109:65–73. [67] Harayama H, Miyake M. A cyclic adenosine 3’,5’-monophosphatedependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa. Mol Reprod Dev 2006;73:1169–78. [68] Emerling BM, Weinberg F, Snyder C, Burgess Z, Mutlu GM, Viollet B, et al. Hypoxic activation of AMPK is dependent on mitochondrial ROS but independent of an increase in AMP/ATP ratio. Free Radic Biol Med 2009;46:1386–91. [69] de Lamirande E, Lamothe G, Villemure M. Control of superoxide and nitric oxide formation during human sperm capacitation. Free Radic Biol Med 2009;46:1420–7.

9

[70] Zmijewski JW, Banerjee S, Bae H, Friggeri A, Lazarowski ER, Abraham E. Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase. J Biol Chem 2010;285: 33154–64. [71] Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther 2015;148C:114–31. [72] Aquila S, Sisci D, Gentile M, Middea E, Catalano S, Carpino A, et al. Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J Clin Endocrinol Metab 2004;89:1443–51. [73] Somanath PR, Jack SL, Vijayaraghavan S. Changes in sperm glycogen synthase kinase-3 serine phosphorylation and activity accompany motility initiation and stimulation. J Androl 2004;25:605–17. [74] Aparicio IM, Bragado MJ, Gil MC, Garcia-Herreros M, GonzalezFernandez L, Tapia JA, et al. Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3alpha. Reproduction 2007;134:435–44. [75] Fang X, Yu SX, Lu Y, Bast Jr RC, Woodgett JR, Mills GB. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc Natl Acad Sci U S A 2000;97:11960–5. [76] Bragado MJ, Gil MC, Martin-Hidalgo D, Hurtado de LA, Bravo N, Moreno AD, et al. Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa. Reproduction 2012;144:67–75. [77] Baker MA, Hetherington L, Aitken RJ. Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitationassociated hyperactivation of murine spermatozoa. J Cell Sci 2006; 119:3182–92. [78] Lawson C, Goupil S, Leclerc P. Increased activity of the human sperm tyrosine kinase SRC by the cAMP-dependent pathway in the presence of calcium. Biol Reprod 2008;79:657–66. [79] Mitchell LA, Nixon B, Baker MA, Aitken RJ. Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa. Mol Hum Reprod 2008;14:235–43. [80] Varano G, Lombardi A, Cantini G, Forti G, Baldi E, Luconi M. Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa. Hum Reprod 2008;23:2652–62. [81] Covarrubias AA, Yeste M, Salazar E, Ramirez-Reveco A, Rodriguez Gil JE, Concha II. The Wnt1 ligand/Frizzled 3 receptor system plays a regulatory role in the achievement of the ‘in vitro’ capacitation and subsequent ‘in vitro’ acrosome exocytosis of porcine spermatozoa. Andrology 2015;3:357–67.