3',5'-Cyclic adenosine monophosphate in the physiology of sea urchin spermatozoa

This paper was supported by PAPIIT-UNAM (IN206016) and CONACyT Fronteras de la ciencia 71 (2016-2018). Special thanks to Dr. Alberto Darszon for his critical reading of this review; and to José Luis de la Vega Beltrán, Francisco Fabio Herrera Rodríguez, Paulina Torres Rodríguez, J. Antonio Blancas Naranjo, and Leonel Linares Labastida for their technical support; and Xochitl Alvarado Affantranger and Andrés Saraleguí Amaro for their help in confocal microscopy.

1. Introduction

The concentration of cAMP in cells depends on the adenylyl cyclase activity (ACs) (Cooper, 2003; Cooper and Crossthwaite, 2006; Hanoune and Defer, 2001) that synthesizes it, and on the phosphodiesterase activity (PDE), which hydrolyzes it (reviewed in Ahmad et al., 2015; Francis et al., 2011; Maurice et al., 2014). This second messenger may act directly, joining ion channels (Kaupp and Seifert, 2001; Matsumoto et al., 2003; Santoro et al., 1998; Zufall et al., 1997), activating the guanine nucleotide exchange protein (EPAC) (De Rooij et al., 1998), or indirectly, through the cAMP dependent protein kinase (PKA), which phosphorylates proteins, regulating their activity.

2. Adenylyl cyclases (ACs)

The ACs are enzymes with ubiquitous distribution. In mammalian somatic cells, there are two kinds of ACs –the membrane kind (ACm) and the soluble kind (sAC). There are nine different genes that codify for the ACm (ACm1-9), whose expression and regulation pattern is different in said somatic cells (Gilman and Taussig, 1995). The ACm have the MgATP as substrate and are regulated in a differential way by G proteins, by forskolin (a non-physiological compound extracted from the Coleus forskohlii plant [Hanoune and Defer, 2001]), by kinases (PKA and/or protein kinase stimulated by diacylglycerol [PKC]), or by other signaling molecules, such as Ca2+ and calmodulin (CaM). The sAC (Buck et al., 1999), originally described in rat testicle extracts (Braun and Dods, 1975), is stimulated by bicarbonate and by Ca2+, and, unlike the ACm, its substrate is the MnATP (Chen, 2000; Jaiswal and Conti, 2003; Litvin et al., 2003; Zippin et al., 2003).

3. AC activity in the sea urchins spermatozoa (SUS)

The AC activity was initially studied in the sea urchins spermatozoa (SUS) (Garbers and Kopf, 1980; Garbers, 1989; Kopf and Garbers, 1980), when only the ACm were thought to exist (Cooper, 2003; Cooper and Crossthwaite, 2006; Hanoune and Defer, 2001). At that time it was observed that the SUS had an unusually high AC activity, compared to that of the somatic cells (reviewed in Mourelle et al., 1984). Additionally it was found that, both in this system (Garbers, 1981) as in another species’ spermatozoa, the MnATP dependent activity was much higher than the MgATP dependent. It is known that the AC activity in SUS is regulated by G proteins (Capasso et al., 1990), by pH and by Ca2+ (Bookbinder et al., 1990; Cook and Babcock, 1993; Kopf and Garbers, 1980; Mourelle et al., 1984; Watkins et al., 1978), as well as by the plasma membrane hyperpolarization (Beltrán et al., 1996). Although the rise in cAMP levels induced by the spermatozoa hyperpolarization is Ca2+ independent, it is clear that said levels do rise in presence of Ca2+ and do precede the RA (Beltrán et al., 1996), as was observed on isolated sperm heads (Garbers, 1981). Since two components of the ovum surrounding hyperpolarize the spermatozoa –a sulfated fucose polymer (FSP) and the speract decapeptide (GFDLNGGGVG; regulator of sperm motility)–, it was thought possible that the AC activation took part in the RA regulation, the motility and the chemotaxis of the SUS as in mammal (Esposito et al., 2004; Spehr et al., 2003; Visconti et al., 1995).

4. The soluble adenylyl cyclases (ACs) in the SUS

The most important cAMP source (~94%) in the SUS is the soluble AC (SUsAC) (reviewed in Vacquier et al., 2014). This partially purified enzyme (PM ~190 kDa) from Strongylocentrotus purpuratus SUS (Bookbinder et al., 1990) was cloned and sequenced out of a testicle library from the same species (SUsAC) (Nomura et al., 2005). In S. purpuratus spermatozoa, the enzyme is distributed along the spermatozoon including the acrosome and the mitochondrion areas (Beltrán et al., 2007b; Bookbinder et al., 1990). The SUsAC, such as the mammalian one (Chen, 2000; Jaiswal and Conti, 2003; Litvin et al., 2003), is stimulated by bicarbonate, but unlike it, the one from the sea urchin is also stimulated by alkaline pH and not by Ca2+ (Nomura et al., 2005). In spite of the SUsAC having 5 possible sites of phosphorylation by PKA, and regardless of the fact that in in vitro conditions it is phosphorylated by PKA and it joins CaM-agarose (Bookbinder et al., 1990; Bookbinder et al., 1991), its activity is not stimulated by PKA nor by CaM (Nomura et al., 2005).

5. The membrane ACs (ACm) in the SUS

The S. purpuratus urchin genome  contains five predicted protein isoforms from the ACm (ACm1, XP_787811; ACm2, XP_780688; ACm3, XP_011667569.1; ACm5, XP_787809; and ACm9, XP_798394), four of which (ACm1 [Fig. 1], ACm2, ACm5 and ACm9) were located with immunofluorescence (IF) experiments, distributed in a differential way in SUS (Vacquier et al., 2014), and with western blot (WB) in head membranes or in isolated flagellum membranes from same species spermatozoa (Beltrán et al., 2007b). In that same paper we, the authors, proved in a functional way that the AC activity from the particulate fraction (precipitate of the 200,000 xg) of full S. purpuratus spermatozoa is stimulated with NaF (which stimulates G proteins, which modulate ACm), and with forskolin, which activates the ACm-18 isoforms. We observed that the forskolin stimulation is prevented by 2’,5’-dideoxyadenosine (DDA; permeable inhibitor of ACm). We also observed stimulation of the AC activity by forskolin in the purified membranes of spermatozoa flagella, corroborating the presence of ACm in them (Beltrán et al., 2007b). There is evidence of the presence of ACm both in SUS Paracentrotus lividus (Capasso et al., 1990) as in mammals (Baxendale and Fraser, 2003; Leclerc et al., 1996; Liguori et al., 2004). Since the SUsAC in the SUS is not stimulated by Ca2+, and since the only ACm isoform which can be specifically found in the acrosome area of the SUS is the ACm1 (Fig. 1), whose mammal ortholog belongs to the group I (ACm1, ACm3 and ACm8) of those susceptible to be stimulated by Ca2+ and by CaM (reviewed in Vacquier et al., 2014), it is possible that the ACm1 be, at least partially, the enzyme responsible for the rise in cAMP levels when the SUS are hyperpolarized in presence of Ca2+ (Beltrán et al., 1996). This supports the ACm participation in the RA of the SUS.

Figure 1. ACm1 located in the SUS acrosome area lessens with the acrosome reaction. A) Immunofluorescence (confocal microscopy) of spermatozoa marked with anti-ACm1 (red) and with anti-phalloidin (green, which binds to actin [Su et al., 2005]) from both control spermatozoa and reacted spermatozoa (B). In both cases (A and B), the inserts show the superposition of the phase contrast with the fluorescence (modified from Romero, 2013).

6. cAMP and the acrosome reaction (RA)

The goal of the spermatozoon is to fecundate the ovum. In order to do so, the cell must undergo the acrosome reaction (RA). The RA is a complex process that must befall on the spermatozoa of all species with an acrosome (vesicle with acidic pH), so as to be able to fecundate the counterpart ova. This process is maintained from marine invertebrates to mammals, and is made up by a series of morphological and physiological changes. The first series of changes consists of the acrosome vesicle exocytosis, and of the intracellular pH dependent actin polymerization (pHi) (Tilney et al., 1978), which leads to the formation of the acrosome tubule, exposing a new membrane covered by the binding protein that will interact with the ovum (Zigler et al., 2005). Physiologically, the RA is induced in a specific-species kind of way by the sulfated fucose polymers (PFS) on the egg jelly cover (EJ) (Alves et al., 1997). In sea urchins the PFS is the natural RA inducer that, as it interacts with the egg jelly receptor (suREJ1) in the sperm plasma membrane (Moy et al., 1996; SeGall and Lennarz, 1979; Vacquier and Moy, 1997), produces both changes in the membrane potential (Em) which depend on K+, and rises in the pHi, the [Ca2+]i, and the sodium ([Na+]i) levels, as well as in those of cAMP, inositol 1,4,5-triphosphate (IP3), and nicotinic acid adenine dinucleotide phosphate (NAADP) (reviewed in Darszon et al., 2011). The union between the PFS and its receptor also stimulates the activities of the nitric oxide synthase, the phospholipase D, the AC, and the PKA, which at the same time phosphorylates proteins (reviewed in Neill and Vacquier, 2004).

In the lab, the RA can be induced in the SUS in a natural way through the counterpart ovum surrounding (egg jelly, or EJ), or through the PFS contained in it (Hirohashi et al., 2008). In this model (SUS) the RA can also be induced artificially, either by augmenting the extracellular pH to 9.0, or through the Ca2+ ionophores, A23187 or the K+/H+ antiporter, or nigericin (Hinkley et al., 1986; Kopf and Garbers, 1980) –they all raise the cAMP levels depending on external Ca2+ (Garbers, 1989; reviewed in Beltrán et al., 2007b; Neill et al., 2004). The isolated SUS heads contain AC, PDE of CAMP, PKA and CaM. Both in these as in the intact cells (Beltrán et al., 1996) the rises in CAMP precede the morphological changes associated with the RA, indicating the CAMP participation in the RA. However, this conclusion will have to be sustained with better temporal resolution experiments.

Using S. purpuratus spermatozoa (Beltrán et al., 2007b), we showed that the exposition of spermatozoa to DDA partially inhibits the rises both in cAMP levels (37%), and in EJ induced RA (49%), which implies the ACm participation in the RA (Beltrán et al., 2007b), as it occurs in mammalian spermatozoa (Leclerc et al., 1996; Liguori et al., 2004; Livera et al., 2005; Wertheimer et al., 2013).

It is worth noting that mice spermatozoa have AC activity significantly stimulated through forskolin, as compared to that of mice with null ACm3, and that the results of immunofluorescence experiments reveal that said enzyme is located on the acrosome area of the spermatozoon (Livera et al., 2005), similarly to the ACm1 (Fig. 1) in the SUS (Beltrán et al., 2007b). Added to this, the fact that mice lacking the gene that codifies for the ACm3 are infertile, due to a decrease in motility and a rise in the spontaneous RA (Livera et al., 2005), suggests that this enzyme takes part in both functions. Interestingly enough, when the Lytechinus pictus SUS are hyperpolarized with egg jelly (EJ; from -43.6±1.7 to -52.0±0.0 mV), or artificially, diluting them in seawater with no K+ but containing valinomycin (ionophore of K+; -43.6±1.7 to -130.4±6.7 mV), the levels of cAMP are stimulated 2.9 and 2.2 times, respectively. However, the rise in cAMP levels lessens from 2.2 to just 1.9 when the Ca2+ is eliminated from the seawater without K+ (Beltrán et al., 1996). Besides, the results of double immunofluorescence staining experiments (Fig. 1), with anti-ACm1 and anti-phalloidin coupled to Alexa 495 that marks actin (an indicator of RA in SUS) (Su et al., 2005), show that when the RA is induced, the signal of correspondent fluorescence to the ACm1 diminishes, corroborating the presence of ACm1 in the SUS acrosome area (Romero, 2013). Added to this, the fact that the ACm1 belongs, as it was mentioned, to the group of Ca2+ and CaM stimulable ACm (reviewed in Vacquier et al., 2014) strongly suggests that the ACm1 could participate in the RA of the SUS as it was suggested in mice spermatozoa. It has been reported that the Gs reactivity is lost in the reacted mice spermatozoa (Wertheimer et al., 2013), which is consistent with the location of both proteins, ACm1 and Gs, in the acrosome area.

7. cAMP and the spermatozoa motility

Since the eighties, the CAMP has been considered to regulate the motility in a SUS model permeated with the Tritón X-100 detergent (Murofushi et al., 1986; Tash, 1989; Tash et al., 1986; Tash and Means, 1983). The SUS are immobile in the gonads due to a low pHi because of the high CO2 concentration. When the spermatozoa are freed into the seawater which pH is 8.0, there is an H+ efflux increasing the pH from 7.2 to 7.6 (Lee et al., 1983). This pHi increase activates both the SUsAC (Nomura et al., 2005) and the dyneins, ATPasas activated with alkaline pH (7.5-8.0) (Christen et al., 1983) and the main consumers of the ATP that is synthesized in the only sperm mitochondria (Fig. 2). From this it can be found that both motility and breathing are linked by the pHi regulation (Christen et al., 1982; Shapiro et al., 1985). Since the spermatozoon specific Na+/H+ exchanger (sNHE) can be activated through the spermatozoon hyperpolarization (Lee, 1984, 1985), it is possible that the stimulation of the AC activity by hyperpolarization (Beltrán et al., 1996) is mediated by the rise in pHi caused by the activation of the sNHE that has a voltage sensor domain (Wang et al., 2003).

The speract is the first spermatozoa activator peptide (SAP) (reviewed in Beltrán et al., 2007; Darszon et al., 2008; Darszon et al., 2011; Nishigaki et al., 2014; Suzuki, 1995) that was purified and characterized from the surroundings of S. purpuratus, L. pictus and Hemicentrotus pulcherrimus ova (Hansbrough and Garbers, 1981; Suzuki et al., 1981). Picomolar concentrations of this SAP stimulate the motility, the breathing and the metabolism of the spermatozoa phospholipids (Hansbrough et al., 1980; Harumi et al., 1992), and they also induce rises in the GMPc and cAMP levels (Kopf et al., 1979). The levels of both nucleotides are determined by the activities of guanylyl cyclase (GC) and adenylyl cyclase (AC) that synthetize them respectively, and by the phosphodiesterase (PDE), which hydrolyzes them. Although it was initially reported that the PDE activity is restricted to the flagella (Sano, 1976; Toowicharanont and Shapiro, 1988), this is unlikely to be so, since the head as well has AC activity (Garbers, 1981). It is possible that the activity referred to in previous papers corresponds to the PDE5, which is specific for GMPc (produced by the GC), and is in fact located only in the spermatozoon flagellum. We know that the flagellum of S. purpuratus spermatozoa contains the SUsAC and at least 2 ACm: ACm2 and ACm9 (Beltrán et al., 2007b; Vacquier et al., 2014), and we know that the activity of sAC participates mainly in the motility of the spermatozoa of this species (Vacquier et al., 2014).

As mentioned, from the 9 ACm isoforms described in somatic cells (Hanoune and Defer, 2001), there are at least 4 in the S. purpuratus SUS, out of which both ACm2 and ACm9 have a similar distribution to the SUsAC including the flagellum (Beltrán et al., 2007b; Vacquier et al., 2014). Besides, the DDA (permeable inhibitor with IC50 of 45 μM that in a non-competitive way joins the P site of the ACm [Schuh et al., 2006]) modifies the L. pictus spermatozoa swimming pattern (Loza-Huerta, 2007). The results of those experiments in which S. purpuratus SUS were preincubated for 30 minutes with 75-300 μM of DDA concentration showed that said inhibitor does not affect the spermatozoa circular swimming speed (Loza-Huerta, 2013). On the contrary, same species spermatozoa incubation during 10 minutes with SQ22536 (another ACm inhibitor with IC50 of 200 μM and same action mechanism as DDA) (Schuh et al., 2006) inhibits scantily but significantly (1 mM ~25% and 2 mM ~30%) the circular swimming speed of said cells.

This and the fact that ACm2 is regulated by PKC (Cooper, 2003; Cooper and Crossthwaite, 2006; Hanoune and Defer, 2001; reviewed in Vacquier et al., 2014), that the spermatozoon motility is also susceptible to PKC inhibitors (White et al., 2007), and that the SUS contains phosphorylated substrates of PKC (White et al., 2007), all suggest the participation of ACm in the motility. It is important to consider that the effects of ACm inhibitors and PKC were only evaluated in the circular swimming speed (Loza- Huerta, 2013), and that spermatozoa have at least three motility sorts (circular, vibratory and rectilinear [Loza-Huerta, 2007]). As mentioned in the previous section, the mice with null ACm3 are subfertile and their spermatozoa have diminished motility (Livera et al., 2005), which supports the participation of ACm in the SUS motility.

7.1 cAMP and the spermatozoa chemotaxis

The sea urchin is the first animal model in which the chemotaxis phenomenon was documented (Lillie, 1912) –the capacity of the spermatozoa to swim towards the ova in response to chemical signals (Darszon et al., 2008; Hussain et al., 2016; Kaupp and Álvarez, 2016; Miller, 1985; Yoshida and Yoshida, 2011). In this model, spermatozoa are attracted to the ova by spermatozoa activating peptides (SAPs) which spread the egg jelly surrounding (reviewed in Darszon et al., 2008; Nishigaki et al., 2014; Suzuki, 1995). In Arbacia punctulata (Kopf et al., 1979; Suzuki et al., 1984; Ward et al., 1985) and L. pictus (Guerrero et al., 2010; Hansbrough and Garbers, 1981; Kopf et al., 1979), resact and separact are, respectively, the two best characterized SAPs with chemoattractant properties, which, just like the RA, depends on Ca2+. The speract induces Ca2+ fluctuations in the S. purpuratus (Kaupp et al., 2003) and L. pictus (Granados-González et al., 2005) spermatozoa flagella, which have also been observed in A. punctulata and the Asterias amurensis starfish in response to their respective chemoattractants, resact and asterosap (Bohmer et al., 2005). In A. punctulata spermatozoa populations, resact induces a rise of biphasic Ca2+, where the first is mediated by GMPc and the second by cAMP (Kaupp et al., 2003). In the same paper, but using individual cells, the authors showed that the Ca2+ spikes in the flagella make the spermatozoa turn (Kaupp et al., 2003), and suggested that both the first rise in cAMP and the second rise in Ca2+ might be involved in the adaptation of the spermatozoa to the chemoattractant (Kaupp et al., 2003).

It has been proposed that CatSper (the sperm-specific ion channel) both mediates the Ca2+ affluence evoked by the chemoattractant, and controls the chemotactic direction; and that the concomitant alkalization produced by speract works as a cooperative mechanism that allows CatSper to transform the periodical voltage changes into Ca2+ bursts (Espinal-Enríquez, et al., 2017; Seifert et al., 2015). Besides, as mentioned, the rise in speract triggered pH also stimulates the SUsAC, raising the cAMP levels which at the same time regulate CatSper. Due to this, it’d be expected that sAC and/or ACm inhibitors at least partially inhibit the chemotaxis. In Ciona intestinalis the results of KH7 experiments suggest that the sAC participates in the frequency of the flagellar shake and the ovum dependent chemotaxis, SAAF or sperm activating and attracting factor (Shiba and Inaba, 2014).

7.2 The speract signaling path

Based on the results of experiments made throughout 35 years by different groups (reviewed in Espinal-Enríquez et al., 2017; García-Rincón et al., 2016), the proposed model for the speract triggered signaling path is as follows: the union between the speract and its receptor (Cardullo et al., 1994), coupled to the guanylyl cyclase (GC) in the spermatozoon flagellum membrane, raises the GMPc levels, which opens the K+ tetra-KCNG ion channel, producing a K+ exit and a transient hyperpolarization (the Em diminishes, that is, it turns more negative) of the spermatozoon (Galindo et al., 2000; Lee and Garbers, 1986). Said hyperpolarization activates the Na+/H+ exchanger, raising the pHi (Lee, 1985; reviewed in Nishigaki et al., 2014). The rise in pHi stimulates the soluble AC (SUsAC), raising the cAMP levels (reviewed in Vacquier et al., 2014), the ATPase dynein (Christen et al., 1983), the carnitina palmitoil transferasa I (CPT-I; associated with the mitochondria external membrane) (Bezaire et al., 2004), the sperm-specific Ca2+ channel, CatSper (the most complex known ion channel, whose absence causes infertility among mice) (Chung et al., 2017; Espinal-Enríquez et al., 2017; Ren et al., 2001; Seifert et al., 2015) to feed the -oxidation (Eaton et al., 1996) and raise the free fatty acids (FFA) (Jezek et al., 1998) that enter the mitochondria, respectively. This raises the levels of NADH transferring its electrons to the electron transport chain (CTE), stimulating the oxygen consumption and generating the change in the mitochondrial membrane potential (Emit; Fig. 2) used by the F0F1- ATPsintasa for the ATP synthesis. The initial hyperpolarization, triggered by the speract and the rise in cAMP levels synthesized by the SUsAC, opens the Na+ channel, SpHCN, producing a Na+ entrance and a depolarization of the spermatozoon. This depolarization, along with the rises in cAMP, activates the CatSper channel that induces Ca2+ oscillations in the cell flagellum. Finally, the dynein hydrolyzes the ATP in concert with the [Ca2+] oscillations in order to regulate the spermatozoon swim (the motility and the chemotaxis) (modified from (García-Rincón et al., 2016).

Figure 2. Rhodamine-123 marks the mitochondria of the S. purpuratus spermatozoa. Spermatozoa marked with 10 μM of rhodamine-123. A) Distribution of the fluorescence observed in a confocal microscope. B) Superimposition of the fluorescence with the phase.

The use of the sAC specific inhibitor, KH7, allowed us to demonstrate that said enzyme participates chiefly in the SUS motility (Vacquier et al., 2014). These results match the fact that the spermatozoa from mice with null sAC do not fecundate the in vitro ova, because the cells don’t move (Esposito et al., 2004). Besides, the immobile spermatozoon gets its motility back when exposed to permeable analogue cAMP (Wang et al., 2003), confirming that the sAC is involved in generating the cAMP needed for the mice spermatozoa motility. In the C. intestinalis there is also functional evidence of the ACm participation in the activation of basal flagellar motility mediated by PKA (Shiba and Inaba, 2014).

8. cAMP and the PKA dependent protein phosphorylation in the motility and in the RA of the SUS

The reversible phosphorylation of proteins is a post-translational modification identified as the main mechanism for intracellular events control in eukaryotic cells, and for the spermatozoon it has been proposed as a regulatory mechanism of great importance for mitochondrial bioenergetics (Mizrahi and Breitbart, 2014). As mentioned before, one of the cAMP main targets is the cAMP dependent protein kinase, or PKA, that phosphorylates proteins and regulates their activity. In eukaryotic cells, PKA is one of the first discovered and best characterized kinases (Walsh et al., 1968), and is also a heterotetramer integrated by two regulatory (R) and two catalytic (C) subunits, with four different types of R subunits (RI, RI, RII and RII) and four different types of C subunits (C, C, Cand PrKX) (Taylor et al., 2004; Zimmermann et al., 1999).

PKA was the first protein kinase to be identified in SUS (Garbers and Kopf, 1980; Garbers et al., 1980; Lee and Iverson, 1976), and from that moment on, it is a known fact that when the cAMP concentration rises, the PKA activates, certain flagellum axoneme proteins are phosphorylated, and the flagellar motility starts, which lasts until the spermatozoon fuses with the ovum (Garbers and Kopf, 1980; Garbers, 1989).

It has been proposed that changes in the state of phosphorylation of the ~250 proteins that make up the axoneme (Inaba, 2003) regulate the spermatozoa motility, because of the rises in cAMP and calcium levels (Tash, 1989; reviewed in Darszon et al., 2008), although just a few have been identified at the molecular level.

In SUS the PKA (Fig. 3; González-Mora, 2016), such as the ACs (Beltrán et al., 2007b; Vacquier et al., 2014), are distributed along the spermatozoon. In this model the authors initially found that the axoquinin (flagellum axoneme protein) is phosphorylated depending on the levels of cAMP when the spermatozoa motility is activated with ATP (Tash et al., 1986). They also identified a RII of the PKA (Paupard et al., 1988) and CaM (Tash and Means, 1982) as phosphorylated proteins associated with the spermatozoa motility. Subsequently, the exposure of intact spermatozoa to different ionic conditions to keep them immobile or mobile revealed that the activation of motility increased the phosphorylation of only 4 proteins of the flagellum (32, 45, 130 and 500 kDa), which could be subunits of the ATPase dynein, considering its solubility properties (Bracho et al., 1998).

The increase in pH triggered by the union between the speract and its receptor inactivates the GC, dephosphorylating its catalytic site (Ward et al., 1985), and raises the PDE5 activity (Su and Vacquier, 2006), which is activated through GMPc (Rybalkin et al., 2003) and through PKA mediated phosphorylation when SUS are exposed to EJ, natural inducer of RA (Su and Vacquier, 2006).

There is evidence that voltage-regulated Ca2+ channels (Cav) are also regulated by phosphorylation mediated by different kinases (reviewed in Darszon et al., 2011), among which is the PKA (Catterall, 2016). The use of commercial antibodies against rat Cavs (anti-CavPan, anti-Cav1.2 and anti- Cav2.3) and immunofluorescence experiments allowed us to prove that the Cav1.2 and Cav2.3 are differentially distributed in the flagella and in the mitochondria and acrosome areas of S. purpuratus SUS (Granados-González et al., 2005). With the same antibodies and WB experiments, we corroborated that said channels are in the flagella membranes of spermatozoa. In addition, we observed that the antagonists of Cav channels, nifedipine and nimodipine, which inhibit RA, reduce the intracellular increase of Ca2+ induced by a depolarization caused by K+ in L. pictus spermatozoa treated with the ionophore of K+, valinomycin. This suggests that the Cav1.2 and Cav2.3 channels could participate in the RA and/or in the motility of the SUS (Granados-González et al., 2005). Reports show that the induction of the RA from SUS with EJ increases the activity of AC, cAMP and PKA mediated phosphorylation (Su et al., 2005). In the cited paper, the use of the PKA inhibitor, H89, plus a permeable analogue of cAMP and the PDEs inhibitor, IBMX, allowed to demonstrate that PKA is necessary for the RA to be carried out. These results match the PKA distribution in the SUS that, unlike that reported for mice spermatozoa (Wertheimer et al., 2013), can also be found in the head (Fig. 3). Furthermore, by means of an antibody that detects PKA phosphorylated substrates (anti-PKAs) and mass spectroscopy (MS/ MS), six proteins phosphorylated by this enzyme were identified when the spermatozoa was exposed to EJ, among which is the PDE5 (GMPc-specific) and/or PDE11, that degrades both cAMP and GMPc. We know that cAMP is involved in all SUS important functions, such as motility, chemotaxis and RA, and that GMPc participates in the motility and chemotaxis of this cell. They also identified an adenylate kinase (AK) that can be either AK1 or AK5. Actin was another of the identified proteins, which we know is polymerized during RA. Another identified protein was creatine kinase (CK), structural protein linked to both the flagellum membrane and the axoneme, and among the SUS there is the hypothesis that it participates in a shuttle of phosphocreatine, re- phosphorylating the dynein produced ADP in order to reestablish the ATP. In fact, in SUS, this is the proposed mechanism for transporting the ATP, 40 μm from the mitochondria (Fig. 2) where it is produced until the end of the flagellum (Wothe et al., 1990). Finally, they also identified EPS8, a substrate of the epidermal growth factor (EGF) receptor pathway (Su et al., 2005).

Figure 3. PKA is distributed throughout the spermatozoon including the mitochondria and acrosome areas. (A) Distribution of the PKA fluorescence (anti-PKA; 1:100) in the SUS. (A’). Superimposition of the fluorescence with the spermatozoa white light observed in an Olympus FV1000 2P inverted confocal microscope with a 60x objective. (B y B’) The SUS incubated with only the secondary antibody (A’1) is the fluorescence of the spermatozoon indicated in (A’) amplified and (A’2) is the superimposition of the fluorescence with white light of the same spermatozoon. In every panel the scale represents 10 μm (modified from González-Mora, 2016).

More recently, we showed that there is a differential phosphorylation of some PKA and PKC substrates associated with the isolated BL (see the proteins identified in section IX.2. The lipid rafts [BL] in SUS), starting from S. purpuratus spermatozoa in different motility conditions: immobile, mobile and speract stimulated (Loza-Huerta et al., 2013). This suggests that some mitochondrial proteins regulated by PKA and by PKC can influence the SUS motility (Loza-Huerta et al., 2013). These results match the fact that the union of the speract to its receptor depolarizes the mitochondria of the spermatozoon by increasing the pH triggered by the peptide, which, along with the increase in Ca2+, modulates the mitochondrial metabolism to regulate motility (García-Rincón et al., 2016).

8.1 PKA and AKAPs

Different scaffold proteins for A kinases (AKAPs) perform a dual function; as a PKA anchor to different subcellular locations near the substrates of PKA, to selectively phosphorylate them; and as a scaffold to the signalosome with different proportions of PKA, ACs, phosphatase, other sorts of kinase, Epac, PDEs and CaM, among other effector proteins (Aggarwal-Howarth and Scott, 2017; Maurice et al., 2014; Scott and McCartney, 1994). Signalosomes are multimolecular signage/regulation complexes located in specific intracellular sites that group signaling, regulatory and effector molecules, where they enable the compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions (Ahmad et al., 2015; Monterisi and Zaccolo, 2017). In spermatozoa, the location of the AKAPs is critical, since it is a highly compartmentalized cell where they participate in motility, training and RA (Carr and Newell, 2007; Vizel et al., 2015). In this system, different AKAPs have been detected, among which are: AKAP1, initially called AKAP8, which anchors the PKAIIto the mitochondria in the spermatozoon flagellum; AKAP3 (Amaral et al., 2014; Vizel et al., 2015), also called AKAP110 (Carr et al., 2001); AKAP4, or AKAP82 (Ben-Navi et al., 2016); AKAP8, that anchors PKA to the spermatozoon nucleus; AKAP11, AKAP220, MAP2 –another AKAP that participates in the regulation of capacitation and/or the acrosomal reaction (reviewed in Carr and Newell, 2007); and RSP3, a name derived from “radial spoke protein” (Gaillard et al., 2001; Smith and Yang, 2004).

Through the proteomic analysis (MS/MS) of solubilized S. purpuratus spermatozoa passed by a Co2+ column, or by agarose attached lectin affinity columns (concanavalin A or wheat germ agglutinin [WGA]), we identified 6, 11 and 12 unique peptides, respectively, from the protein SpRSP (gi|2905895; 63kDa) of the axoneme (Beltrán, unpublished article). In addition, in a protein band of a flagella sample separated in a denaturing mini polyacrylamide-sodium dodecyl sulfate gel (PA-SDS) in which PKA was detected by WB, 13 SpRSPl (gi|780158049; 31 kDa) and 11 SpRSP9 (gi|115733045) unique peptides were identified through MS/MS (González- Mora, 2016). In the same band detected with anti-PKA but in a different experiment, we also identified 5 unique peptides of the X1 isoform of SpRSP3 (gi|72009354; 45 kDa), which has high identity (67%) and homology (84%) with the AKAP of human sperm, corresponding to AKAP4.

It has been proposed that in mammalian spermatozoa, AKAP10 could act as a scaffold for phosphatase PP1 of serine/threonine and for rho, a small G protein, as well as its RHOK effector (serine/threonine kinase) (Carr et al., 2001). Also through the proteomic analysis of S. purpuratus samples, we identified the PP1 (Beltrán, unpublished article), and it has been reported that both rho (which regulates actin-based cellular processes) and its RHOK effector participate in the RA of the SUS (Castellano et al., 1997; Urióstegui et al., 2007). This also suggests the participation of AKAPs in the SUS. There is a model that proposes that the central pair of axoneme microtubules distribute the chemical signals to RSP, which modify the phosphorylation status of the axoneme proteins to activate/inactivate particular dynein arms that promote a coordinated sliding of the microtubules in the axoneme (reviewed in Yoshimura et al., 2007).

9. ACs in signaling platforms/lipid rafts

9.1 Lipid rafts (BL)

The microdomains in native membranes, also known as BL or “lipid rafts”, LD-DIM (low density detergent insoluble membranes), DRM (detergent resistant membranes), or DIGs (insoluble domains in detergents enriched in glycolipids), are defined as signaling platforms enriched in sphingolipids, cholesterol, and signaling proteins resistant to extraction with cold nonionic detergents (Pike, 2004). Lipid rafts (BL) are linear microdomains that include caveolae, flask-shaped plasma membrane invaginations (50-100nm) (Gheber and Edidin, 1999) enriched with the caveolin protein, a scaffold protein (Lisanti et al., 2004; Martens et al., 2000; Zheng et al., 2008).

For many years the existence of BL has been a point of controversy in terms of their size, stability, and physiological importance (Edidin, 2003; Munro, 2003). However, the use of different strategies such as biochemical techniques, fluorescent probes, microscopy visualization, functional approaches (for instance, the investigation of the effects of the destruction of the BL in the ion channels functioning [reviewed in Dart, 2010]), the apparent ability of said microdomains to selectively add signaling molecules that interact with each other, and the implication that they could be involved in the spatial-temporal organization of cellular signaling pathways (Patel et al., 2008; Simons and Toomre, 2000), all are evidence that said microdomains exist and that they are essential in the cells physiology.

9.2 Lipid rafts (BL) in the SUS

BL from a gamete (Ohta et al., 1999) were isolated and characterized for the first time in the SUS of three species (H. pulcherrimus, S. purpuratus, and Anthocidaris crassispina). This was done by solubilizing the spermatozoa with 1% of Triton-X100 at 4° and and separating the BL in a sucrose gradient (Ohta et al., 1999). In a later paper, the same authors (Ohta et al., 2000), through western blot (WB) experiments, identified in the BL of S. purpuratus SUS the speract receptor, the egg jelly receptor (SuREJ1), a 63 kDa protein (anchored to glycosylphosphatidylinositol, a BL marker), the  subunit of the stimulatory heterotrimeric G protein (Gs), adenylyl cyclase (that, as was later learned, corresponds to the sAC [reviewed in Vacquier et al., 2014]), guanylyl cyclase (GC) and PKA. In addition, since they noticed that the speract receptor, the 63 kDa protein anchored to GPI, and the Gs, all co-immunoprecipitate, the authors proposed that said BL could be the interaction site of the speract and its receptor, as well as of the subsequent signaling pathway involved in the induction of respiration, motility, and possibly the acrosomal reaction of the SUS (Ohta et al., 2000).

Ionic fluxes are essential in spermatozoa physiology and there is evidence that BL regulates the function of ion channels in different ways, either by direct protein-lipid interactions or by altering the physical properties of the lipid bilayer (Dart, 2010). Since there is controversy regarding the existence of BL, and because it has been suggested that said rafts could be artifacts of solubilization with detergents (Chamberlain, 2004), Esperanza Mata-Martínez (2010) in her undergraduate thesis took on the task of obtaining BL through three different strategies (non- ionic detergent, sonication and sonication/alkaline pH), and through WB experiments, confirming the presence of Gs, PKA and SUsAC. She showed as well that these BL also contain two isoforms of transmembrane adenylyl cyclase (ACm2 and ACm9), the TetraKCNG, Cav1.2 and SpHCN ion channels, and flotillin 2, a 45 kDa protein and BL marker that may or may not be in caveolae (Banning et al., 2011). In the BL of the SUS that were obtained solubilizing with 1% of Triton X100, no caveolin was found, although we know that the spermatozoa of this species contains it (Mata- Martínez, 2010). There is evidence (biochemical, microscopic, and functional) that virtually all types of ion channels are associated with BL (Martens et al., 2000; reviewed in Dart, 2010), which supports our findings. Later, Miriam Cerrillos, in her undergraduate thesis (Romero, 2013), proved that the BL of isolated flagella also contain ACm2 and ACm9, confirming the presence of said enzymes in the flagellum as shown by immunofluorescence experiments (Beltrán et al., 2007b). In the same paper it was proved that the destabilization of lipid rafts of S. purpuratus SUS with the BL disassembler, methyl--cyclodextrin (MC), inhibits ~90% of the acrosome reaction (RA) and stimulates ~20% of the spermatozoa motility (Romero, 2013). The fact that the C. intestinalis BL participate in the signaling of Ca2+, responsible for the activation of spermatozoa motility and chemotaxis (Zhu e Inaba, 2011) backs up the results in SUS.

As mentioned in section 8, looking for proteins whose phosphorylation pattern changes with the motility of the spermatozoon, BL of mobile immobile SUS were isolated and stimulated with the motility regulator, speract (Loza-Huerta et al., 2013). The proteins were separated in two- dimensional (2-D) mini gels, and, through WB, the proteins were analyzed with specific antibodies against substrates phosphorylated by PKA (anti- sPKA) or by PKC (anti-sPKC), so as to be identified at the molecular level by mass spectroscopy (MS/MS) (Loza-Huerta et al., 2013). This was done both in BL derived from whole cells and in isolated flagella. Interestingly, in the first case (BL of SUS), only one spot detected with anti-sPKA and 3 with anti-sPKC changed the level of phosphorylation with motility, and that in the spot detected with anti-sPKA (Table I), mainly mitochondrial proteins were identified: ATP synthase, kinase creatine, NADH dehydrogenase (ubiquinone), flavoprotein 2, succinate- CoA ligase, and the voltage-dependent anion channel 2 (VDAC-2), plus the PKA RII proteins, the chain of the tubulin and the actin Cy I (Table 1) (Loza-Huerta et al., 2013). On the other hand, in the BL derived from the isolated flagella, 16 spots were detected with anti-sPKA (Table II), out of which 22 substrates were identified (Table II) and only one spot with anti-sPKC (Loza-Huerta, 2013). Among the identified PKA substrates are: the speract receptor, PDE5, phosphatase 1A (PP1A), PKA RII, flotillin (BL marker), and axoneme proteins, among others (Table II). This paper suggests that SUsAC, PKA, and PKC participate in the SUS motility by changing the phosphorylation levels of some proteins, and that PKA and PKC show a cross communication during this event (Loza-Huerta, 2013). Interestingly again, through proteomic analysis (MS/MS) of a protein band of flagella sample detected with anti-SUsAC (Mr ~190 kDa), we identified 57 unique peptides from the tetraKCNG ion channel (gi|126506318), 3 from the PDE of GMPc (gi|780164106), 7 from CatSper (gi|780129264), and 2 from the SpHCN channel (gi|74136757), all of which suggests that SUsAC interacts directly or indirectly with some of these in the membrane of the SUS, confirming the results obtained in BL of said spermatozoa (Beltrán, unpublished article). Furthermore, in another protein band from the same sample of flagella, but detected with anti-PKA, we identified 10, 9, and 6 peptides unique to the ser/treo PP1 phosphatase (gi|780178928), in three different preparations respectively, and in one of said preparations 6 unique peptides of the PPA2 phosphatase (gi|115675671) (Beltrán, unpublished article). These results contribute in an important way to understand how the SUS motility is regulated. Moreover, the fact of having identified mitochondrial proteins as substrates of PKA and PKC confirms the participation of this organelle in fertilization (Ardón et al., 2009; García-Rincón et al., 2016). BL have also been described in mammalian sperm (Treviño et al., 2001) and they have been implicated in the capacitation (Cross, 2004; Sleight et al., 2005; Travis et al., 2001) and interaction between the spermatozoa and the ovum zona pellucida (Miranda et al., 2009; Zitranski et al., 2010).

Table 1. PKA substrates in BL of S. purpuratus spermatozoa whose phosphorylation pattern changes with mobility.

Proteins identified via MS/MS in a spot detected in 2-D gels through WB experiments with the anti-PKAs antibody.  TP: Number of total peptides.  NUP: Number of unique peptides.

Table 2. PKA substrates in BL of isolated flagella from S. purpuratus spermatozoa whose phosphorylation pattern changes with motility.

9.3 Speract modifies the phosphorylation pattern of PKA substrates and/or PKC substrates in the BL of SUS

As mentioned, the speract receptor is coupled to a membrane guanylyl cyclase (GCm) in the sperm flagellum, whose activity is regulated both by changes in its phosphorylation status and by pH induced changes through the binding of the speract to its receptor (Bentley et al., 1986; Ramarao and Garbers, 1985; Suzuki and Garbers, 1984; Ward et al., 1985). In order to investigate whether speract induces changes in the phosphorylation of other proteins besides GCm under conditions similar to the physiological ones, the SUS were exposed for 10 seconds to different speract concentrations (0.1, 1 and 10 nM) in seawater (Loza-Huerta, 2013). The analysis of spermatozoa proteins exposed to these conditions by WB experiments and specific anti-PKA antibodies or anti-PKCs, that detect substrates phosphorylated by PKA or PKC respectively, showed that in basal conditions of motility PKA phosphorylates six substrates with PM of ~180, 120, 80, 70, 50 and 45 kDa, and that speract stimulation differentially modifies the phosphorylation patterns of these proteins. Overall, a decrease in the phosphorylation of PKA substrates was observed, except in the bands of 120 and 80 kDa whose phosphorylation increased with 10 nM of speract and in the band of 45 kDa, which increased with 1 nM of speract. Similarly, the speract also modified the degree of phosphorylation of different substrates of PKC (Loza-Huerta, 2013). The above results suggest that when the spermatozoa are swimming towards the ovum, the speract gradient modifies the phosphorylation of the substrates of PKA and PKC in a dose-dependent manner. An in silico analysis, by means of the NetPhosK program, indicated that most proteins involved in the speract signaling cascade have possible phosphorylation sites both by PKA (GCm, PDE specific for GMPc, tetraKCNG, Cav [Catterall, 2016]), and by PKC (GCm, tetraKCNG, NHE, Cav [Catterall, 2016], CatSper, ACm2 and sAC) (Loza-Huerta, 2013).

In S. purpuratus two ion channels activated by hyperpolarization and cyclic nucleotides (HCN), SpHCN1 (Gauss et al., 1998) and SpHCN2 (Galindo et al. 2005), were cloned, and both were detected in the spermatozoa flagella (Galindo et al., 2005; Gauss et al., 1998). The analysis of the SpHCN2 sequence shows that it has several possible phosphorylation sites: two by PKA (S54 and S84), five by PKC (T60, S104, S204, S385 and T409), and one by tyrosine kinase, and that the nucleotide binding domain has 135 amino acids, 44% of the identity of SpHCN1 (Galindo et al., 2005). A similar channel to SpHCN1, that activates directly by cAMP, and is three times more selective for K+ than for Na+, was studied in SUS swollen through “patch-clamp experiments” (Sánchez et al., 2001). Given its selectivity, under physiological conditions, when the channel opens, sodium enters by depolarizing the cell, which suggests its participation in the sperm response after the initial hyperpolarization was triggered by the speract. Since the HCN channels are involved in the periodicity in other cell types, it has been proposed that in SUS they could modulate the flagellar shake and participate in the chemotaxis (Kaupp and Seifert, 2001).

In the absence of protein synthesis, as in the spermatozoon, phosphorylation becomes an important mechanism for function regulation of many proteins. Therefore, the above mentioned results indicate that the proteins that participate in the speract stimulated motility modulate their function through changes in their phosphorylation state induced by PKA and/or by PKC. Additionally, the results of immunoprecipitation experiments and identification by mass spectroscopy showed that the SUsAC forms complexes with at least 10 proteins of the plasma membrane and the SUS axoneme (Nomura and Vacquier, 2006): the sperm-specific Na+/H+ exchanger, two heavy chains of dyneins 7 and 9, the ion channel SpHCN, the GCm, the PDE5, the CK, the speract receptor and -tubulins. These results led the group to propose that the SUsAC associated proteins could be important to unite the signaling of the plasma membrane to the use of energy in the regulation of spermatozoa motility (Nomura and Vacquier, 2006). Furthermore, in mice the sNHE has a putative binding site for cyclic nucleotides (Wang et al., 2003). This, along with the fact that SUsAC and sNHE are interacting in this cell, suggests that cAMP regulates sNHE. It is also important to remember that the independent elimination of the genes coding for the sAC (Esposito et al., 2004) or the sNHE cause infertility in mice, due to severe defects in the spermatozoa motility (Wang et al., 2003), which highlights the importance of both proteins in spermatozoa physiology.

10. Are sAC, PKA and Epac in the SUS mitochondria?

More than 60 mitochondrial phosphoproteins have been found in somatic cells (Pagliarini and Dixon, 2006), which suggests that the reversible phosphorylation of proteins is an important mechanism in the regulation of mitochondrial activity (Deng et al., 2011; Pagliarini and Dixon, 2006). It has been reported that synthesized cAMP within the mitochondria regulates oxidative phosphorylation (Acin-Pérez et al., 2010; Valsecchi et al., 2014), and although initially there was doubt about the presence of CA and PKA within the mitochondria, there is evidence that the complete signaling pathway CO2-HCO3--sAC-cAMP-PKA is inside mitochondrial somatic cells (Acin-Pérez et al., 2010; Valsecchi et al., 2014).

SUS have just one mitochondria responsible for generating most of the energy (in the form of ATP) that the cell needs to swim towards the ovum and fertilize it (reviewed in García-Rincón et al., 2016). We know that the stimulation of S. purpuratus SUS with agents that affect mitochondrial function by different mechanisms increase mitochondrial Ca2+ (Ardón et al. 2009), and that speract, through the rise in pHi, depolarizes the mitochondria (reduces its Emit) and increases the levels of NADH independently of external Ca2+ (García-Rincón et al., 2016). As well, as mentioned before, ATP is essential for the dyneins (flagellum motors) to function and the spermatozoa to swim (reviewed in Nishigaki et al., 2014). All of the above shows the importance of the only SUS mitochondria (Fig. 2) in spermatozoa motility and in fertilization.

As it was already established, the SUsAC is distributed throughout the spermatozoa and specific antibodies against the enzyme mark in a particular way the area of its mitochondria (Beltrán et al., 2007b; Vacquier et al., 2014). Due to this, Juan Pablo González-Mora (2016), in his undergraduate thesis, investigated whether the SUsAC can be found in the SUS mitochondria. Such research showed through specific antibodies and WB experiments, that the SUsAC and the PKA are in the fractions of mitochondria isolated in density gradients, beginning with S. purpuratus SUS, and the proteomic analysis of those fractions corroborated the presence of both enzymes in said organelles. In addition, immunofluorescence experiments allowed us to show that PKA (Fig. 3), like the SUsAC (Beltrán et al., 2007b), is distributed throughout the S. purpuratus spermatozoa, also marking the areas of the acrosome and of the mitochondria. The preliminary results of transmission electron microscopy corroborated the presence of SUsAC in the SUS mitochondria. This supports the fact that both SUsAC and PKA are found in the SUS mitochondria (González-Mora, 2016), as it was functionally shown (for the sAC) and by electron microscopy (for the PKA) in the bull spermatozoa (Mizrahi and Breitbart, 2014), from where they could modulate motility and acrosome reaction, essential spermatozoa functions.

Finally, as we already mentioned, another target for cAMP are the Epac, multidomain proteins whose C-terminal catalytic region possesses a guanine nucleotide exchange factor (GEF) specific for the small GTPasas, Rap1 and Rap2. Epac catalyzed the conversion of Rap1 and Rap2 from an inactive way (Rap-GDP) to an active one (Rap-GTP) (De Rooij et al., 1998; Kawasaki et al., 1998). Epac regulate countless cellular processes in different tissues (reviewed in Lewis et al., 2016), including adhesion, proliferation, differentiation, and cell survival, as well as regulation of exchangers such as isoform 3 of Na+/H+ (NHE) (Honegger et al., 2004), ion channels (Zhang et al., 2009), and Ca2+ mediated signaling (Kang et al., 2006; Morel et al., 2005; Schmidt et al., 2013).

There is evidence of Epac participation in different important events for fertilization in mammalian spermatozoa (Miro-Moran et al., 2012). In the head of mice spermatozoa, the cAMP-Epac signaling pathway was identified (Amano et al., 2007), and it was proven that Epac is involved in the flagellar shake of the hamster spermatozoa (Kinukawa et al., 2006). In addition, using a polyclonal anti-Epac antibody, generated in rabbit against the LREDNCHFLRVDK synthetic peptide (Branham et al., 2006), whose sequence is identical in the two human isoforms, corresponding to the residues of 285–297 of Epac1 (De Rooij, et al., 1998) and 438–450 of Epac2 (Ueno et al., 2001). Ruete et al. (2014) demonstrated that epac participates in the acrosome reaction of human sperm. The S. purpuratus genome contains the predicted sequences of two Epac2 isoforms; SpEpac2 X1 (XP_011668470.1; calculated molecular weight (PMc)=103119 Da), and SpEpac2 X2 (XP_784278.3; PMc=102075 Da), both of which have identities of 56 and 57%, and homologies of 73 and 74%, respectively,  with human Epac2 (Q8WZA2), also known as rap guanine nucleotide exchange factor 4. Given that the epitope sequence to generate the antibody against human Epac1/2 is highly conserved in the two isoforms of SpEpac2 (X1 and X2), we used said antibody (donated kindly by Dr. Claudia Tomes, from the National University of Cuyo, Mendoza, Argentina) and through WB experiments, detected a band of ~100 kDa both in flagella membranes (Beltrán, unpublished article) and in the mitochondrial fraction of S. purpuratus SUS (González-Mora, 2016), which could correspond to either of the two isoforms (X1 or X2) of SpEpac2. We found as well that a selective Epac inhibitor decreases little but significantly (~5-8%) the circular swimming speed of same species spermatozoa (Loza-Huerta, 2013). Although there are still some experiments to be done, the above results suggest the presence of Epac in the flagellum and in the mitochondria of S. purpuratus SUS, as has been proposed in other systems (Valsecchi et al., 2014), and that said cAMP target (Epac) also participates in the SUS physiology.

11. Final comment

It is clear that cAMP is vital for the essential functions of sea urchin spermatozoa (motility, chemotaxis and acrosome reaction), and that to fulfill its task, it needs to have production sources (ACs) strategically distributed throughout the cell. However, given that its action depends on a highly precise spatio-temporal location regulation, it is fundamental that both the ACs and the PDEs that hydrolyze the cAMP function at the precisely needed time and place. To achieve this, it is essential that the proteins that regulate (Ca2+ ion channels, CaM, PKA, PKC, heterotrimeric G proteins, NHE, phosphatases, G protein coupled receptors [GPCR] etc.) each of the ACs (sAC and ACm) and the PDE of CAMP, work coordinately. We know that both the ACs and the PDEs (Francis et al., 2011; Maurice et al., 2014) are associated with different sets of proteins in different membrane microdomains, however, they could also be associated as in mammals in different signalosomes (see section 8.1). In order to better understand the cAMP action mechanisms in spermatozoa physiology, it is crucial to identify the proteins with which the different ACs interact forming complexes, not only in the membrane, but inside the cell. In addition to showing us which proteins are inside the cells, subcellular proteomics helps us to understand where they reside. There is still a long way to go and experiments to be done to unravel the diversity and complexity of cAMP signaling in spermatozoa.

Abbreviations

AC: adenylyl cyclase
ACm: membrane adenylyl cyclase
AKAP: A kinase anchor protein
cAMP: 3'5'-cyclic adenosine monophosphate
ATP: adenosine triphosphate
BL: lipid raft
[Ca+2] : intracellular calcium concentration
CaM: calmodulin
DDA: 2’5’-dideoxyadenosine
EJ: egg jelly
Em: membrane potential
Epac: cAMP activated guanine nucleotide exchange protein
PDE, phosphodiesterase
Gs: stimulator G protein
GC: guanylyl cyclase
GCm: membrane guanylyl cyclase
GMPc: cyclic guanosine monophosphate
kDa: kilodaltons
KH7: propanoic acid 2-(1H-benzimidazole- 2-litio)-2-[(5-bromine-2- hydroxyphenyl) methylene] hydrazide
MS/MS: tandem mass spectrometry
mV: millivolts
NHE: Na+/H+ exchanger
sNHE: sperm-specific NHE
PFS: sulfated fucose polymer
pHi: intracellular hydrogen potential
PKA: cAMP-dependent protein kinase
PKC: protein kinase C stimulated by diacylglycerol
PM: molecular weight
b: acrosome reaction
RSP: AKAP whose name derives from “radial spoke protein”
Sx: serine where x= number of amino acids
sAC: soluble adenylyl cyclase
SAP: spermatozoa activating peptide
Sp: Strongylocentrotus purpuratus
SpHCN: ion channel regulated by hyperpolarization and Sp cyclic nucleotides
SUsAC: sea urchin sAC
Tx: threonine where x= number of amino acids
WB: western blot.