Main signaling pathways involved in the control of fowl sperm motility

Main signaling pathways involved in the control of fowl sperm motility Thi Mong Diep Nguyen∗,†,1 ∗

reservation. In this article, we will briefly describe the roles of several signaling pathways involved in the motility of fowl sperm as compared to mammalian sperm. This review is intended to facilitate the design of future studies in fowl male fertility and improve the quality of frozen-thawed sperm.

ABSTRACT Motility is an essential function of the mature male gamete and is widely used as an indicator of semen quality. Knowledge of signal transduction pathways regulating sperm motility is therefore important to gain insight into new methods to increase the fertilizing ability of semen samples, especially those subjected to biotechnological processes such as cryop-

Key words: fowl sperm, mammalian sperm, sperm motility, signaling pathways 2018 Poultry Science 0:1–11 http://dx.doi.org/10.3382/ps/pey465

INTRODUCTION

means that avian spermatozoa can effectively gain full motility twice, first during copulation and then when released from sperm storage tubules (SST) when the female produces eggs ready for fertilization (Bakst, 1987). Sperm flagellar movement is produced by the sliding of tubulin and ATP-driven dynein motor protein in the axoneme (Summers and Gibbons, 1971). It is known that there is low oxygen and high lactic acid concentrations in quail SST and that flagellar quiescence can be induced by lactic acid through flagellar dynein ATPase inactivation following medium acidification (
Motility is one of the main parameters of spermatozoa, thus contributing highly to a successful fertilization. Sperm mobility, the net movement of a sperm cell population against fluid resistance at body temperature, is a quantitative trait of domestic fowl fertility (Froman and McLean, 1996; Froman and Feltmann, 1998). When spermatozoa are ejaculated, many factors contribute to their mobility including changes in osmolality (Morisawa and Suzuki, 1980), extracellular potassium concentration (Morisawa et al., 1983), and dilution of a putative sperm inactivation substance from seminal plasma (Pellicer and Combarnous, 1998). Indeed, in avian species, it is heritable (Froman and Feltmann, 2000; Bowling et al., 2003), and is the only trait recognized as positively correlated with fertility (Froman and Feltmann, 1998; Froman et al., 1999; King et al., 2000). Unlike mammal sperm, chicken sperm acquire their motility (Ashizawa and Sano, 1990) and part of their fertilizing ability as soon as they leave the testis (Howarth, 1983). Their fertilizing ability further increases in the epididymis (Munro, 1937). Moreover, fowl sperm may remain for a very long time in the female genital tract (up to 3 wk in the hen) before reaching the site of fertilization and undergoing the acrosome reaction to penetrate the inner perivitelline layer that surrounds the oocyte (Okamura and Nishiyama, 1978). However, in contrast to mammals, there is neither capacitation nor hyperactivation in chicken sperm (Horrocks et al., 2000; Lemoine et al., 2008). Remarkably, it

Extracellular Ca2+

 C 2018 Poultry Science Association Inc. Received June 21, 2018. Accepted November 9, 2018. 1 Corresponding author: [email protected]

The extracellular Ca2+ has been known since the late 1930s, to be required for fowl sperm motility (Ashizawa et al., 2000). However, unlike mammalian 1

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Faculty of Biology-Agricultural Engineering, Quy Nhon University, Binh Dinh 55000, Vietnam; and † Physiologie de la Reproduction et des Comportements, INRA, Nouzilly, France

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Intracellular Ca2+ / CaM Sperm motility can be modulated by a variety of signaling pathways. In mobile sperm, it is enabled by calcium cycling, which drives phospholipase A2 activity and thereby controls the release of endogenous fatty acids for beta-oxidation within mitochondria (Froman, 2007). The involvement of intracellular free Ca2+ and/or CaM-bound Ca2+ in sperm motility has been previously described (Ashizawa et al., 1994a). Intracellular free Ca2+ is essential for the maintenance of chicken sperm motility and CaM appears to modulate many, although not all, the effects of Ca2+ on fowl sperm motility. Demembranated sperm motility was inhibited in Ca2+ -free medium and was restored by the subsequent addition of Ca2+ alone at 30◦ C (Ashizawa et al., 1992b). CaM is a highly conserved, ubiquitous protein involved in mediating the influence of Ca2+ on cellular processes such as signal transduction, cell motility, and membrane fusion through its ability to activate key intracellular enzymes (Means et al., 1982). In fowl sperm, CaM can be located in the cytoplasmic matrix, the mitochondria, or the membrane, but it is not retained in the axoneme, since inhibition of motility of intact sperm by the addition of W-7 and trifluoperazine was not observed in demembranated sperm. Explaining the inhibition of fowl sperm motility by the addition of W-7 and trifluoperazine, the authors reported that this might be due to the inhibition of Ca2+ /CaM-regulated enzyme activities. However, it has been suggested that CaM may be involved in the control of axonemal func-

tion by regulating a number of key enzymes, including dynein ATPase, myosin light chain kinase, cyclic nucleotide phosphodiesterases (PDE), and adenylate cyclase (AC; Tash and Means, 1983). Another possible explanation for the influence of CaM on sperm mobility is that, in spermatozoa, it might stimulate the activity of AC responsible for generating cAMP, which is known to be essential for the stimulation of mammalian sperm mobility (Gross et al., 1987). However, unlike mammalian sperm, demembranated fowl sperm mobility could not be restored by the addition of cAMP at 40◦ C (Ashizawa et al., 1989, 1992a). In mammalian sperm, CaM is present in the principal piece of the flagellum and possibly in the fibrous sheath, an insoluble accessory structure that is found exclusively in the principal piece of the mammalian flagellum. Inhibition of sperm mobility by the addition of the CaM inhibitor W-7 provides a more direct evidence for a role of CaM in the regulation of sperm mobility (Schlingmann et al., 2007).

Adenylyl Cyclase/Cyclic AMP Cyclic AMP is a primary second messenger in all somatic and germ cells. Its intracellular concentration is dependent on the balance between its synthesis by AC and its degradation by nucleotide PDE. Among the 10 AC types, nine (AC1-9) are transmembrane proteins exhibiting 12 transmembrane passages and only one (AC10) is cytosolic and also named “soluble AC” (Steegborn, 2014). Both soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclases (tmAC) are present and play important roles in sperm function (Shiba and Inaba, 2014). Soluble AC has been identified in the genomes of many eukaryotic organisms. The sAC gene has been found in many animals such as chicken, ciona intestinalis, corals, mosquito, honey bee, dogfish, and all analyzed mammals (human, chimpanzee, dog, cow, rabbit, mouse, and rat; Kamenetsky et al., 2006; Tresguerres et al., 2010; Barott et al., 2013). Soluble AC appears to be the predominant AC type in sperm, and its direct activation by bicarbonate provides a mechanism for generating the cAMP required to complete the bicarbonate-induced processes necessary for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction. The evidence suggests that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. Spermatozoa from sAC KO mutant mice are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro (Esposito et al., 2004; Xie et al., 2006). Transmembrane ACs are also detected in sperm but mainly localized at the acrosome (Buffone et al., 2014) where protein Gα subunit is also found (Wertheimer et al., 2013). These AC types are therefore more suspected to be involved in capacitation (Fernandez and Cordoba, 2017) or acrosome reaction (Shiba and Inaba,

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sperm, chicken sperm display the unique phenomenon of reversible temperature-dependent immobilization: in most synthetic diluents, they become immobile at the avian body temperature of 40◦ C to 41◦ C, but regain motility when the temperature is lowered below 35◦ C (Ashizawa and Nishiyama, 1978; Ashizawa and Wishart, 1987; Wishart and Ashizawa, 1987; Ashizawa et al., 1989). On the one hand, it has been suggested that this phenomenon involves a loss of intracellular Ca2+ towards the suspending medium at 40◦ C, whereas retention of calcium by sperm, with consequent enhancement of motility, increased as the temperature was lowered from 40◦ C to 30◦ C. This phenomenon appears due to the inactivation of a calcium ATPase promoting calcium-efflux. On the other hand, sperm flagellar motility appears to be controlled by various protein phosphorylation-dephosphorylation systems (Tash and Means, 1983; Brokaw, 1987; Lindemann and Kanous, 1989; Tash, 1989; Majumder et al., 1990). Studies during the 1990s showed a regulatory role for Ca2+ in axonemal function (Ashizawa et al., 2000). Sperm contain the major components known to be involved in the action of cyclic adenosine 3’,5’ monophosphate (cAMP) and Ca2+ in the control of cell motility, namely cAMP-dependent PKA, calmodulin (CaM), and myosin light chain kinase (Tash and Means, 1983).

SIGNALING PATHWAYS IN FOWL SPERM MOTILITY

PKA and Protein Phosphatases Phosphorylation and dephosphorylation of structural and regulatory proteins are involved in the regulation of most cellular processes in eukaryotes, particularly in signal transduction, and have an essential role in the coordination and regulation of intricate intracellular pathways (Sun and Tonks, 1994; Hunter, 2007). The level of protein phosphorylation is tightly regulated by the coordinated and opposed activity of PKA and protein phosphatases (Figure 1). PKA transfer a phosphate group from ATP to their specific target proteins, typically at serine, threonine, or tyrosine residues, while protein phosphatases catalyze removal

of phosphate groups from these residues and revert proteins to their nonphosphorylated conformation (Sun and Tonks, 1994). Similar to somatic cells, PKA and phosphatases have essential roles in signal transduction and cell recognition in sperm. Previous studies have demonstrated the presence in sperm of a number of serine/threonine kinases involved in the regulation of motility processes, including protein kinase A (PKA; de Lamirande and Gagnon, 1993; Holt and Harrison, 2002; Lemoine et al., 2009; Shahar et al., 2011), Protein kinase C (PKC; Rotem et al., 1990a, b; Ashizawa et al., 1994b), phosphatidylinositol 3-kinase/Protein kinase B (PI3K/PKB; Tash and Bracho, 1994; Vijayaraghavan et al., 2000; NagDas et al., 2002; Luconi et al., 2004; Lemoine et al., 2009), Mitogen-activated protein kinases (MAPK; Ashizawa et al., 1997a; Almog et al., 2008; Lemoine et al., 2009), AMP-activated protein kinase (AMPK; Hurtado de Llera et al., 2012; 2013; Cordova et al., 2014; Nguyen et al., 2014; Calle-Guisado et al., 2017) and other less well characterized serine/threonine kinases in sperm. This number is rapidly increasing with the findings of recent studies (Bailey et al., 2005; Cao et al., 2006; Lalancette et al., 2006; Martinez-Heredia et al., 2006, 2008; Baker et al., 2008; ; Peddinti et al., 2008; Labas et al., 2015) based on proteomic techniques, which have described additional serine/threonine kinases in sperm involved in functions such as metabolism regulation, proteolysis, motility, and cell cycle regulation. Concurrently, serine/threonine phosphatases have emerged as critical components of signaling pathways involving serine/threonine protein phosphorylation in spermatozoa, demonstrating an essential role for Ser/Thr phosphorylation level in the regulation of sperm biological functions including motility (Ashizawa et al., 1994c, 1997b, 2004; Han et al., 2007). Protein Kinase A. In mammalian cells, the main actions of cAMP are mediated through the activation of the cAMP-dependent PKA. PKA is a tetrameric enzyme consisting of two catalytic subunits (C), which are maintained in an inactive state by binding to a regulatory (R) subunit homodimer. Thus, two PKA subtypes exist depending on the RI or RII regulatory subunits forming the regulatory homodimeric component. Upon binding of cAMP to the regulatory subunits of type I- or type II-holoenzyme, the catalytic subunits are released as active serine/threonine kinases and can phosphorylate their specific substrates, initiating a cascade of signaling events inside the cell (Corbin et al., 1973; Niu et al., 2001; Kim et al., 2005). In mammalian sperm, motility is critically controlled through modulation of PKA activity. For example, PKA mediates light-induced hyperactivated motility in human sperm (Shahar et al., 2011). This type of motility arises during the capacitation process when sperm motility changes from progressive to hyperactivated (de Lamirande and Gagnon, 1993). This change permits more efficient penetration into the zona pellucida than with non-hyperactivated sperm (Stauss et al., 1995). If hyperactivated motility

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2014). In spite of this localization, tmAC have been claimed to be regulated by a protein exhibiting motility stimulation in buck sperm (Dey et al., 2014). But this protein was later shown to stimulate ACs in sperm so the involvement of tmAC in motility is not firmly established. Cyclic AMP is indispensable for the initiation of epididymal sperm motility at ejaculation in mammals (Ishida et al., 1987). White and Voglmayr, in 1986, routinely added cAMP to the reactivation medium, although the reactivation of ejaculated ram sperm was usually as good with ATP alone (White and Voglmayr, 1986). However, it was logical to presume that the effects of cAMP and Ca2+ on sperm motility was in some way regulated by protein phosphorylation (Tash and Means, 1983), since these components regulate motility by producing changes in protein phosphorylation (Dedman et al., 1979). The earliest observations suggesting a role of cAMPdependent phosphorylation in the activation of sperm motility were made in mammalian sperm. The phosphorylation of a 55 kDa protein is apparently related to the motility state of bovine sperm (Brandt and Hoskins, 1980), and axokinin, a soluble 56 kDa phosphoprotein, seems to play a key role in mediating the cAMP response in dog sperm (Tash et al., 1986). Demembranated ejaculated bull sperm was most motile in the presence of both ATP and cAMP (Lindemann, 1978). Mohri and Yanagimachi, in 1980, also reported that cAMP stimulated the ATP-induced activity of demembranated hamster sperm (Mohri and Yanagimachi, 1980). However, cAMP-independent phosphoproteins have also been identified in human (Huacuja et al., 1977), rat (Chulavatnatol et al., 1982), and dog (Tash and Means, 1982) sperm. The rat sperm 42.7 kDa phosphoprotein undergoes phosphorylation in the absence of cAMP (Chulavatnatol et al., 1982). Three major cAMP-independent phosphoproteins of 98 kDa, 43 kDa, and 26 kDa have been identified in dog sperm [Tash and Means, 1982; 1983). In fowl sperm, phosphorylation of the 43 kDa protein was not affected by the addition of cAMP but nevertheless appears to be associated with the maintenance of motility (Ashizawa et al., 1992a).

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is prevented, fertilization cannot occur (Amieux and McKnight, 2002). In boar sperm, PKA activation is achieved by alterations in the intracellular concentration of the messenger molecule, cAMP, brought about by changes in adenylyl cyclase activity (Holt and Harrison, 2002). In fowl sperm, the presence of PKA pathway and its role in the control of sperm motility is evidenced by the observation that the specific PKA inhibitor H89 negatively affects most of motility parameters (Lemoine et al., 2009). Protein Kinase C. PKC, a serine/threonine kinase family of related subspecies, is a key regulatory enzyme in signal transduction mechanisms that governs various cell responses (Nishizuka, 1992). PKC has been implicated in synaptic transmission, memory, learning, growth, differentiation, metabolism, contraction, exocytosis, gene expression, regulation of ion channels, and transformation (Nishizuka, 1992). PKC is localized in the equatorial segment in a distinct band and in the principal piece of the tail in human sperm (Rotem et al., 1990a,b), and in the postacrosomal region of the sperm head and a minor staining of the midpiece in bovine sperm (Chaudhry and Casillas, 1992). In contrast, potentially active PKC is not present in ram sperm (Roldan and Harrison, 1988). Functional studies also suggest the involvement of PKC in the control of mammalian flagellar motility (Naor and Breitbart, 1997). The presence and role of PKC in fowl

sperm have been determined (Ashizawa et al., 1994b) and found to be located in the cytoplasmic matrix or the membrane, but not retained in the axoneme. Indeed, inhibition of motility found in intact sperm by the addition of PKC activators was not observed in demembranated sperm. The observations of authors have yielded conflicting results with mammalian sperm: the concentration increases of 5-Chloro-N-(6-phenylhexyl)1-naphthalenesulfonamide (SC-9) or 1-Oleoyl-2-acetylsn-glycerol (OAG; PKC activators) reduced the motility of intact fowl sperm incubated at 30◦ C, and was restored by reducing the concentrations of these activators (Ashizawa et al., 1994b). In contrast, Rotem et al. found that the addition of PKC activators, such as the potent tumour promoter phorbol ester 12-Otetradecanoyl phorbol-13-acetate and the membranepermeable diacylglycerol analogue OAG, stimulated human sperm motility (Rotem et al., 1990a,b). To explain these discreapancies, the authors gave several lines of evidence pointing towards differential regulation of sperm motility in fowl and mammals. First, demembranated fowl sperm can be motile even in the presence of millimolar concentrations of Ca2+ (Ashizawa et al., 1989), whereas at such high concentrations of Ca2+ , the motility of demembranated mammalian sperm is inhibited (White and Voglmayr, 1986; Feng et al., 1988). Second, cAMP is indispensable for the initiation and stimulation of flagellar motility of mammalian sperm

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Figure 1. Schematic summary of main signaling pathways involved in fowl sperm motility. The signaling pathways could be activated mainly by HCO3 − /Ca2+ and pharmacological conditions. The increase in bicarbonate and Ca2+ activates in turn adenyl cyclase (AC) with increased generation of cAMP and subsequent activation of protein kinase A (PKA). Moreover, the increase of Ca2+ causes the activation of CaM, leading itself to the activation of CaMKs and AMPK. However, protein kinases can also be directly activated by the pharmacological agent and phosphatases can cause protein dephosphorylation. So the activation of intracellular pathways MAPK, PI3K, PKA, PKC, or AMPK are all involved in sperm mobility. Solid arrows and blocked arrows illustrate more established relationships between stimuli, signals, improved metabolic status, and control of fowl sperm functions; dashed gray arrows are used for hypotheses. Solid arrows and blocked arrows illustrate more established relationships between stimuli, signals, improved metabolic status, and control of fowl sperm functions. Dashed gray arrows are used for hypotheses.

SIGNALING PATHWAYS IN FOWL SPERM MOTILITY

ways and are widely used throughout evolution in many physiological processes (Widmann et al., 1999). MAPK comprise the extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino (N)-terminal kinases 1/2/3 (JNK1/2/3), p38 isoforms (α, β , γ , and δ ), and ERK5 (Chen et al., 2001). In avian species, possible stimulating effect of MAPK pathways on motility of demembranated fowl sperm have been presented (Ashizawa et al., 1997a). This study shows that the activities of MAPK/p34cdc2 kinase (that compete with endogenous physiological substrates and therefore inhibit endogenous MAPK activity) inhibited the flagellar motility of demembranated fowl sperm, since the addition of MAPK/p34cdc2 kinase substrate peptides completely inhibited the percentage of motile demembranated sperm at 30◦ C, thus suggesting a role for MAPK in motility (Ashizawa et al., 1997a). It was suggested that MAPK may phosphorylate axonemal and/or cytoskeletal proteins, which are required for motility induction (Ashizawa et al., 1997a). On the contrary, p34cdc2 kinase seems to act differently in human sperm; the addition of antibodies to cyclins and p34cdc2 kinase did not have any significant effect on the percentage of motile cells (Naz et al., 1993). More evidence regarding the role of ERK in sperm motility was demonstrated in the maturation and acquisition of motility of mice sperm in the epididymis (Lu et al., 1999). ERK was shown to be gradually activated throughout the transition of sperm from the caput (immotile) to the vas deferens (fully motile) (Lu et al., 1999). Furthermore, a positive role for MAPK1 was suggested (Lemoine et al., 2009), MAPK1 pathways were affected in the motility mechanism of chicken sperm, since addition of U0126 significantly decreased the percentage of motile, rapid, and progressive sperm, as well as parameters of sperm velocity VCL, VSL, and VAP, but none of the motility parameters was significantly changed by the addition of SB 202190 (MAPK14 inhibitor; Lemoine et al., 2009). However, what triggers the activation of MAPK during induction of motility in mammalian sperm is still unclear. An opposite report concluded that ERK inhibits motility (Weidinger et al., 2005). The author shows that tryptase, a product of mast cells, acts via the ERK1/2 pathway to inhibit motility of human sperm, whereas others show evidence that ERK1/2 stimulates both flagellar sperm motility and hyperactivated motility, and that p38 MAPK serves as its counterpart, namely inhibits both types of motility (Almog et al., 2008). AMPK Pathway. Recently, it has been indicated that the AMPK, an evolutionarily conserved serine/threonine kinase that acts as a cell energy sensor and subsequently regulates metabolism (Hardie et al., 2006), is implicated in male reproductive functions. In fowl sperm, AMPK activity likely plays an important role in the regulation of optimal sperm motility (Nguyen et al., 2014). The authors suggested the presence of AMPK pathway in fowl sperm and its role in the control of sperm motility, since Compound C, an

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(Tash and Means, 1983; Lindemann and Kanous, 1989), but not for fowl sperm, especially at 40◦ C (Ashizawa et al., 1989). Besides, several lines of evidence suggest that PKC modulates ion conductance by phosphorylating membrane proteins such as channels, pumps, and ion-exchange proteins (Nishizuka, 1986). PI3K and MAPK Pathways. There is increasing evidence suggesting a role for other kinases and phosphatases regulating sperm motility (Tash and Bracho, 1994; Ashizawa et al., 1997a; Vijayaraghavan et al., 2000; NagDas et al., 2002; Luconi et al., 2004). PI3K has also been suggested to play an important role, since its pharmacological inhibition by LY294002 resulted in a significant increase in human (Luconi et al., 2004) and boar (Aparicio et al., 2005) sperm forward motility, whereas treatment with another inhibitor, wortmannin, inhibited capacitation and motility of hamster sperm (NagDas et al., 2002) but had no effect on human sperm capacitation and motility (du Nauc et al., 2004; du Plessis et al., 2004). In addition, inhibition of PI3K/AKT signal by wortmannin caused a reduced percentage of motile spermatozoa because of induction of apoptosis (Koppers et al., 2011). In contrast, PI3K is not involved in fowl sperm motility, since treatment of these sperm with LY294002 did not affect any of their motility parameters (Ashizawa et al., 2009; Lemoine et al., 2009). To explain the differences in the use of LY294002 and wortmannin inhibitors, Nauc V et al. observed that LY294002 and wortmannin had opposing effects on sperm capacitation and tyrosine phosphorylation, and on Thr-Glu motif phosphorylation of specific sperm proteins. LY294002 acted in a manner similar to fetal cord serum ultrafiltrate (FCSu) a capacitation inducer, whereas wortmannin decreased the phospho-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. In addition, while LY294002 increased calcium concentrations, wortmannin had no effect on calcium. Moreover, the inhibition of PI3K by LY294002 results in a significant increase in human sperm forward motility by interfering with the cAMP/PKA intracellular pathway. LY294002 induced by an increase in intracellular cAMP levels and tyrosine phosphorylation of the PKA anchoring protein AKAP3 (a PKA-anchoring protein that is mostly found in sperm tails) stimulated PKA recruitment to sperm tail through its regulatory RIIβ subunit, and increased sperm motility (Luconi et al., 2004). Recently, it was also found that the PI3K-AKT pathway is required for motility and hyper activation in human spermatozoa (Sagare-Patil et al., 2013) and that phosphoAKT preserved stallion spermatozoa motility (Gallardo Bolanos et al., 2014). MAPK, also seem to be involved in sperm mobility, but sometimes with contradictory effects. MAPK are protein Ser/Thr kinases that convert extracellular stimuli into a wide range of cellular responses. MAPK are among the most ancient signal transduction path-

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all sperm velocity parameters and also significantly increases the percentage of capacitated porcine spermatozoa (Aparicio et al., 2007). Male mice lacking GSK3α cannot accomplish the fertilization, probably because of impairments affecting sperm motility, velocity parameters, and flagellar beat (Bhattacharjee et al., 2015). Activated and hyperactivated sperm motility characterized by high-amplitude flagellar beat are essential for penetration and fertilization of the egg (Suarez and Ho, 2003). Sperm lacking GSK3α will also be unable to fertilize eggs in vitro, due to impaired capacitation and their inability to undergo hyperactivation (Bhattacharjee et al., 2015). GSK3 belongs to the Wnt/frizzled pathway where it is negatively controlled by Dishevelled (Dsh). It is also negatively controlled by the PI3K/AKT kinase pathway downstream of many growth factors. In both pathways the final targets are gene expression controls through β -catenin or MAPK respectively. Obviously, GSK3 cannot intervene in this way in spermatozoa although its activity has been shown to be correlated with sperm motility (Somanath et al., 2004). In fact, transcriptionally silent spermatozoa respond to Wnt signals released through exosomes from the epididymis during their transit through this organ (Koch et al., 2015). GS3K has yet to be explored in bird sperm, but the main GSK3 action concerning motility is to inhibit protein phosphatases and these proteins have been shown in bird sperm (see below). Type 1 and 2 Protein Phosphatases Since phosphorylation is required for the activation of the sperm motility, then proteins dephosphorylation by specific regulatory phosphatases also affects sperm motility. Such regulatory serine/threonine protein phosphatases are classified into four main enzymes: type 1 (PP1), type 2A (PP2A), type 2B (PP2B), also known as calcineurin (CaN), and type 2C (PP2C) (Cohen, 1989). With regard to fowl sperm motility, it has been proposed that the inhibition of sperm motility at body temperature (40◦ C) is due to the activation of PP1, one of the serine/threonine phosphatases present in fowl sperm axoneme and/or accessory cytoskeletal components, since the immobilization of demembranated sperm at 40◦ C can be reversed by the addition of inhibitors of PP1, such as okadaic acid, calyculin A (Ashizawa et al., 1997b). Furthermore, the addition of recombinant PP1 supplemented with Mn2+ /inhibited the motility of demembranated sperm at 30◦ C (Ashizawa et al., 1994c), whereas PP2B appears not to be involved in the regulation of the flagellar movement at body temperature of fowl sperm since the addition of specific inhibitors of PP2B did not significantly stimulate motility at 40◦ C (Ashizawa et al., 1997a). A similar result was found in mammalian spermatozoa, PP1 might take part in the inhibition of the sperm motility activation by interacting with AKAPs and CAMKII (Ashizawa et al., 2004). The sperm-specific PP2B/CaN isoform appears to be

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AMPK inhibitor, negatively affects most of the motility parameters in fowl sperm whereas 5-Aminoimidazole-4carboxamide ribonucleotide (AICAR) and metformin, two AMPK activators, significantly stimulated the motility parameters in fowl sperm (Nguyen et al., 2014). Furthermore, positive effects of AMPK’s stimulation are retained after cryopreservation, which supports the hypothesis of its ”restful” role after the gametes have been exposed to extreme thermal and osmotic stress conditions encountered during the freezing/thawing process (Nguyen et al., 2015). A similar result was found in human and boar sperm incubation with Compound C (Hurtado de Llera et al., 2012; 2013). However, the results obtained in stallion frozen-thawed sperm were disappointing (Cordova et al., 2014). On the basis of these clear effects on motility, the localization of important levels of AMPK at the flagellum (Hurtado de Llera et al., 2013; Nguyen et al., 2014; Calle-Guisado et al., 2017), it is conceivable that AMPK might act by phosphorylating downstream protein substrates involved in the axoneme central apparatus or in other related structures that are essential for spermatozoa flagellar motility, as it has been previously reported for its related kinase TSSK2, expressed in spermatozoa (Xu et al., 2007). Thus, TSKK2 phosphorylates in vitro the axoneme central apparatus protein called SPAG16L, which is essential for flagellar motility in mouse spermatozoa (Zhang et al., 2006). Moreover, 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) that present a great reduction in sperm motility (Tartarin et al., 2012). Moreover, AMPK activation in spermatozoa is potently stimulated by an elevation of cAMP levels and subsequent PKA activation (blocked by H89) or by Ca2+ that acts through two pathways, PKA and CaMKKalpha/beta (blocked by STO-609) or phosphoThr172-AMPK levels greatly increased upon PKC activation (Moreno et al., 2008). Other factors such as ATP deficiency (indicated by reduction in Na+/K+ ATPase activity), transforming growth factor (TGFβ )-activated kinase-1 activation, and AKT deactivation stimulated AMPK, which caused a decline in boar spermatozoa motility (Zhao et al., 2016). Glycogen Synthase Kinase-3 Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (α and β ) whose activity is regulated by tyrosine and serine/threonine phosphorylation. GSK3 is present in spermatozoa and is a key kinase for male gamete function and fertility (Vijayaraghavan et al., 2000; Bhattacharjee et al., 2015; Belenky and Breitbart, 2017; Dey et al., 2018). Stimulation of bovine sperm motility by isobutyl-methyl-xanthine, 2-chloro2 -deoxyadenosine, or calyculin A is accompanied by a dramatic increase in GSK3 serine phosphorylation (Somanath et al., 2004). Inhibition of GSK3 activity by alsterpaullone, a specific inhibitor of this kinase activity, leads to a significant increase in the percentage of rapid and medium-speed spermatozoa, as well as in

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SIGNALING PATHWAYS IN FOWL SPERM MOTILITY Table 1. Main signaling pathways involved in fowl sperm motility Effects on sperm motility

Localizations

Pharmacologies

Intracellular Ca2+/CaM

Cytoplasmic matrix, mitochondria, membrane

Inhibitor: W-7 or Trifluoperazine

AC/Cyclic AMP

Soluble AC is the key to synthesize cAMP in mammalian sperm cells and is essential for successful display of progressive motility but it has yet to be explored in bird sperm, although its gene has already been found in it.

Inhibition

References Tash and Means, 1983; Ashizawa et al., 1992b, 1994a Esposito et al., 2004; Kamenetsky et al., 2006; Tresguerres et al., 2010; Barott et al., 2013; Xie et al., 2006

cAMP/PKA

Head region

Inhibitor: H89

Inhibition

Lemoine et al., 2009

PKC

Cytoplasmic matrix or membrane

Activator: 5-Chloro-N(6-phenylhexyl) -1naphthalenesulfonamide (SC-9), 1-Oleoyl-2acetyl-sn-glycerol (OAG )

Inhibition

Ashizawa et al., 1994b

Phosphatidylinositol 3-kinase (PI3K)

Head region

Inhibitor LY294002

No effect

Lemoine et al., 2009.

MAPK14 (p38 MAPK)

Head region

Inhibitor SB202190

No effect

Lemoine et al., 2009

MAPK1

Intermediate piece, head and flagellum

Inhibitor U0126

Inhibition

Lemoine et al., 2009

AMPK

Acrosomal region, midpiece, flagellum

Inhibitor: Compound C; Activator: AICAR or Metformin

AICAR and Metformin improve motility; Compound C decreases it

Nguyen et al., 2014, 2015

Glycogen synthase kinase-3 (GSK3) Type 1 and 2 protein Phosphatases

GSK3 has yet to be explored in bird sperm. Sperm axoneme Inhibitor: calyculin A and okadaic acid

Restores sperm motility in the absence of Ca(2+) at 40◦ C

Ashizawa et al., 1997b

required for mouse sperm motility (Miyata et al., 2015) as it confers midpiece flexibility during epididymal transit.

Others Although phosphorylation/dephosphorylation by PKA and phosphatases are the prominent switches in most signaling pathways, there are some other potentially important mechanisms. For example, in SST of female birds, sperm are typically immotile. Flagellar quiescence of sperm has been shown to be induced by SST-secreted lactic acid (Matsuzaki et al., 2015). Sperm immotility appears to be due to cytoplasmic acidification caused by luminal lactic acid accumulation and to the entry of extracellular protons by a still unclear mechanism. Since spermatozoa are transcriptionally inactive, progesterone cannot act at this level through its classical transcription factor re-

ceptor. It is more likely that plasma membrane (Lishko et al., 2010) or mitochondrial (Tantibhedhyangkul et al., 2014) progesterone receptors are involved. The plasma membrane progesterone receptor is the CatSper calcium channel induction of sperm motility in mammals that has been previously described (Lishko et al., 2011; Strunker et al., 2011) but it is not present in birds sperm (Cai and Clapham, 2008). Sperm motility is also directly dependent upon the availability of energy obtained through ATP hydrolysis (Ford, 2006; Ruiz-Pesini et al., 2007) and two metabolic pathways producing ATP, namely oxidative phosphorylation (OXPHOS; Bohnensack and Halangk, 1986) and anaerobic glycolysis. Numerous studies have provided evidence supporting the role of OXPHOS in sperm midpiece mitochondria as the main ATP provider for sperm motility (Ford, 2006; RuizPesini et al., 2007). Moreover, glycolysis takes place in the principal piece, which occupies the major part of the flagellum. Several glycolytic enzymes have been

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Signalling pathways

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T. M. D. NGUYEN

CONCLUDING REMARKS There are similarities but also clear-cut differences between the mechanisms of sperm motility in fowl and mammals. Cyclic AMP, CaM, and PKA and phosphatases, besides external effects such as calcium or temperature, play primary roles in sperm motility but often with contrasting effects. In mammalian sperm, motility-associated cAMP-dependent or independent phosphorylation is related to modulation of the calcium, whereas in fowl sperm, phosphorylation of protein was not affected by the addition of cAMP. Moreover, sperm motility is also affected by the indirect action of Ca2+ /CaM signaling. In particular, the activation of proteins kinases and phosphatases systems was strongly involved in sperm motility. The knowledge of signaling pathways involved in fowl sperm motility is still limited compared to mammalian sperm (Table 1). Many signaling pathways have been shown in mammals, such as GSK3 (Aparicio et al., 2007; De Robertis and Ploper, 2015; Koch et al., 2015) or tyrosine kinases (Ickowicz et al., 2012), but not (or not yet) in fowl sperm. Future analyses characterizing the functionality of new proteins and their importance in fowl sperm is thus mandatory to improve the quality of fertilization and reproduction of these species.

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identified, mainly in the fibrous sheath of the principal piece in mammalian species (Turner, 2006) and birds (Mezquita et al., 1998). Respiration has been historically regarded as the main source of ATP production for sperm motility, relegating glycolysis to a secondary role. However, recent studies revealed that glycolysis works as the major process for supplying ATP in the principal piece, since this is where glycolysis enzymes are localized, some of them being tightly associated with the cytoskeletal structure in it. We observed that extracellular glucose was highly utilized for the entire flagellar bending motion with a high beat frequency compared to pyruvate, a substrate for respiration (Nguyen et al., 2015). Moreover, the localizations of phosphodiesterase 4, primarily on the midpiece of human sperm (Fisch et al., 1998), and of PDE3A, on the post-acrosomal region of the sperm head (Lefievre et al., 2002), suggest that they may have roles in sperm motility and capacitation, respectively, since cAMP is not only a substrate but also a regulator of PDE in mammals (Conti et al., 2003). However, this is still a hypothesis to study in the future.

SIGNALING PATHWAYS IN FOWL SPERM MOTILITY

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