Distinct Roles of Soluble and Transmembrane Adenylyl Cyclases in the Regulation of Flagellar Motility in Ciona Sperm

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.


Introduction
In most animal species, sperm are immotile in testis or sperm duct. Flagellar motility is activated and regulated by external cues such as specific ions, osmotic change or factors from the egg or female genital tract at fertilization [1]. In the ascidian Ciona intestinalis and C. savignyi, a sulfated steroid called sperm activating and attracting factor (SAAF) induces both sperm motility activation and chemotaxis [2]. Cyclic AMP (cAMP) is one of the most important intracellular factors in the signaling pathway for SAAF-induced sperm activation. A 21 kDa light chain of outer arm dynein (LC2) and a 26 kDa axonemal protein are phosphorylated, and dynein intermediate chains IC2 and IC116 are dephosphorylated in a cAMP-dependent manner, resulting in activation of flagellar motility [3,4].
Adenylyl cyclases (ACs) are the key enzyme that synthesizes cAMP at the onset of the signaling pathway to activate axonemal dyneins through protein phosphorylation. In sea urchin, frog and mammalian sperm, both soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclases (tmACs) are present and play important roles in sperm function [5][6][7][8][9][10]. However, the roles of two types of ACs are controversial. tmACs are suggested to be involved in the regulation of sperm motility, in particular during chemotactic behavior [11,12]. However, tmACs are shown localized at the acrosome [9,13]. sAC-deficient mice are infertile, lacking sperm motility [8]. In fact, sAC is localized in sperm flagella in mouse [8] and sea urchin [14]. In this study we first characterized sperm ACs in the ascidian C. intestinalis. Using inhibitors specific to each type of AC, we demonstrate that tmAC or sAC critically plays a distinct role in the basic activation of flagellar motility or increase in flagellar beat frequency and Ca 2+ -dependent regulation of flagellar waveform during chemotactic movement, respectively.

Expression of Adenylyl Cyclase (AC) Genes in Ciona Testis
Ten isoforms of AC have been cloned and characterized in mammals; nine are tmACs and the other is sAC [5,6]. Their distributions and regulations differ among isoforms. To clarify the functions of AC in Ciona sperm, we first examined the expression of AC genes in Ciona tesits. A search for AC genes in the genome of Ciona intestinalis revealed the presence of three tmAC genes and one sAC gene. Phylogenetic analysis suggested that three tmAC genes are grouped with mammalian AC5/6, AC8 and AC9 ( Figure 1). Next, we examined the expression of AC genes by reverse-transcriptase PCR (RT-PCR) using PCR primers for these four AC genes. Recently, AC5/6 was reported to be expressed in the intestine of Ciona juvenile [15]. From the analysis in the present study, it turned out that all AC isoforms were expressed in testis but the expression of AC8 is only testis-specific. sAC was expressed in testis at a high level but was also significantly expressed in ovary ( Figure 2).

Sperm Activating and Attracting Factor (SAAF)-Induced Motility Activation
Ciona sperm show less motility in seawater but become activated by an egg-derived factor, sperm activating and attracting factor (SAAF) [16]. SAAF induces Ca 2+ influx, membrane hyperpolarization and activation of adenylyl cyclase to produce cAMP [16]. Transient increase of intracellular cAMP and subsequent cAMP-dependent phosphorylation of dynein subunits are observed in response to the addition of SAAF [3,17]. Thus, cAMP is one of important factors for SAAF-induced sperm motility activation. To specify an AC isoform that is involved in SAAF-induced motility activation, we examined the effect of AC inhibitors specific to tmAC or sAC on motility activation and cAMP synthesis ( Figure 3). The tmAC inhibitor, MDL12330A, completely blocked SAAF-induced motility activation and transient increase of cAMP. MDL12339A did not affect any changes on the motility Triton X-100-demembranated sperm (data not shown). In contrast, the sAC inhibitor, KH-7, did not inhibit SAAF-induced motility activation or transient increase of cAMP synthesis. However it reduced swimming velocity to half of the control and brought basal cAMP level to almost zero in 5 min. Swimming velocity is closely related to the flagellar beat frequency [18]. These results indicate that tmAC has a major role in the signaling pathway in SAAF-induced motility activation and that sAC is involved in the increase in beat frequency and in the maintenance of basal level of cAMP. This agrees with the observation in mouse sperm that a sAC activator HCO 3 − induces the increase of flagellar beat frequency through cAMP-dependent protein kinase (protein kinase A; PKA) [19]. curvilinear velocity (B) and intracellular cAMP level (C) were measured before, 1 min and 5 min after the addition of 100 nM SAAF. Sperm were treated with 0.5% dimethyl sulfoxide (DMSO, control), 100 μM MDL12330A or 10 μM KH-7 respectively for 3 min before SAAF addition. Values are means ± standard error (SE) of the results from three experiments.

Valinomycin-and Theophylline-Induced Motility Activation
Valinomycin is a potassium ion-selective ionophore and induces motility activation of Ciona sperm by causing membrane hyperpolarization [17]. In contrast, an increase in intracellular cAMP levels was induced by a phosphodiesterase inhibitor theophylline, resulting in the motility activation [16]. To elucidate if sAC participates in SAAF-induced signaling pathway in the motility activation, we examined the effect of AC inhibitors on valinomycin-and theophylline-induced motility activation in the presence or absence of Ca 2+ (Figure 4). Both valinomycin and theophylline activated sperm motility in ASW but valinomycin-treated sperm swam with lower velocity ( Figure 4A,B). The tmAC inhibitor MDL12330A completely suppressed both motility and swimming velocity in artificial seawater (ASW) ( Figure 4A,B). These results suggest the involvement of tmAC in the motility of valinomycin-treated sperm. Valinomycin-treated sperm were immotile in Ca 2+ -free seawater (CaFSW), as also previously reported [20], suggesting that Ca 2+ -influx is necessary for tmAC and motility activation ( Figure 4C).
Theophylline-treated sperm showed activated in both ASW and CaFSW. The sAC inhibitor KH-7 showed ~40% suppression of the motility at 1 min but the suppression became not significant at 5 min ( Figure 4B). The velocity of theophylline-treated sperm decreased to half of the control by KH-7 in ASW ( Figure 4B), supporting the idea that sAC is involved in the increase of flagellar beat frequency. In contrast, theophylline-treated sperm showed motility activation even in CaFSW. The motility was suppressed by KH-7 ( Figure 4D), suggesting that sAC is involved in the sperm motility in Ca 2+ -free condition. The sAC in human sperm shows synthesis of cAMP in the absence of Ca 2+ but is activated by Ca 2+ in a concentration-dependent manner [21]. Therefore, it is likely that the activation of sperm motility in CaFSW by theophylline is caused by basal activity of sAC in the absence of Ca 2+ . Ca 2+ -activated sAC could be involved in other process, such as regulation of intracellular pH (see 2.

Sperm Motility Is Activated by HCO 3 − but not by Forskolin
To further test the roles of two types of ACs in the regulation of sperm motility, we examined the effects of two activators for ACs, forskolin and HCO 3 − . Forskolin is a diterpene that effectively activates tmAC1 to tmAC8 [22,23] and is shown to increase cAMP and activate motility in mammalian sperm [13,24]. In Ciona sperm, forskolin showed no effect on the activation of sperm motility even at 100 μM ( Figure 5A), suggesting that either Ciona tmAC5/6 or tmAC8 does not participate in SAAF-dependent activation of sperm motility. sAC is directly activated by HCO 3 − and Ca 2+ [25,26], both of which are two important factors for capacitation of mammalian sperm [27]. Although it is known that CO 2 inhibits motility of sperm from marine animals showing external fertilization, such as sea urchin [28,29] and flatfish [30], activation of sperm motility by HCO 3 − has not been reported. Here, we observed that extracellular HCO 3 − activated the motility in Ciona sperm ( Figure 5B). Because insoluble CaCO 3 formed by the addition of high concentration of HCO 3 − into sea water disturbed the observation of sperm motility, we used the artificial sea water containing 1 mM CaCl 2 (low calcium sea water; LCSW). The activation was observed in LCSW but not in CaFSW ( Figure 5B), indicating that extracellular Ca 2+ is required for HCO 3 − mediated motility activation. To see if the action of HCO 3 − is due to the change in intracellular pH, we used an antiporter of H + and K + , nigericin, to bring intracellular pH to the same pH of extracellular solution. Nigericin itself activated sperm motility in ASW at pH 7.5, but the level of activation was lower than of sperm activated by HCO 3 − ( Figure 5C). The motility of HCO 3 − -activated sperm was reduced by nigericin, suggesting that intracellular pH of HCO 3 − -activated sperm was higher than 7.5. KH-7 inhibited both nigericin-activated sperm and HCO 3 − -activated sperm. These results suggest that the action of HCO 3 − is mediated by sAC in a mechanism closely coupled with the increase in intracellular pH. It is likely that the activity of Ciona sAC is highly dependent on pH like the case in sea urchin sperm [14] and that cAMP produced by sAC activates a Na + /H + exchanger (sNHE) through cyclic nucleotide binding site to raise intracellular pH [31,32] (see [33] for review).

Effects of sAC Inhibitors on Sperm Chemotaxis and Swimming Trajectory
Sperm chemotaxis in Ciona is controlled by transient increase in intracellular Ca 2+ concentration, followed by calaxin-mediated propagation of asymmetric flagella waveforms [34,35]. Next, we examined the effect of KH-7 on the chemotactic behavior ( Figure 6). The movement with quick "turn" toward the attractant was significantly suppressed by KH-7 ( Figure 6A,B). The calculated chemotactic index (LECI, [2]) demonstrates that sperm chemotaxis is significantly inhibited by KH-7 ( Figure 6C). We found that diameter of the circle of sperm trajectory became smaller in the presence of KH-7. An analysis of flagellar waveform asymmetry indicates that flagellar waveform become more asymmetric in the presence of KH-7 ( Figure 7). This suggests that intracellular Ca 2+ concentration increases by KH-7, most likely by inhibiting Ca 2+ -efflux system, such as Na + /Ca 2+ exchanger [36]. It was also reported in mammalian sperm that HCO 3 − -mediated sAC activity is involved in conversion into symmetrical flagellar waveform [27]. Alternatively, sAC could participate in the mechanism for the resumption from calaxin-mediated asymmetric waveform to symmetric waveform [35]. In either case, inhibition of sAC by KH-7 results in loss of efficient turn movement, which would cause suppression of chemotactic movement of sperm. It is still possible that the loss of efficient turn movement is secondarily resulted from KH-7-induced decrease in swimming velocity.

Effects of AC Inhibitors on cAMP-Dependent Protein Phosphorylation in Ciona Sperm
cAMP-dependent protein phosphorylation is prerequisite for activation of sperm flagellar motility [3,[37][38][39]. Next we examined the effects of AC inhibitors on the PKA-dependent protein phosphorylation in Ciona sperm (Figure 8). SAAF induced PKA-mediated phosphorylation of six proteins, including a high molecular mass (HMM) protein (possibly dynein heavy chain), 105 kDa protein, 80 kDa protein, 65 kDa protein, 48 kDa protein (possibly PKA regulatory subunit), and 26 kDa protein (Figure 8, asterisks). Phosphorylation of these proteins, except for the 48 kDa protein, was greatly diminished in sperm treated with MDL12330A in both the presence and absence of Ca 2+ . Phosphorylation of both the 105 and 80 kDa proteins were suppressed by KH-7 in ASW, suggesting that these are closely related to the function of sAC. In contrast, HMM, 65 and 26 kDa proteins were significantly phosphorylated in KH-7-treated sperm, suggesting that these proteins are involved more in basic activation of sperm motility than in the increase of beat frequency or in chemotactic behavior (see motility experiments in Figures 4-7). In CaFSW, only theophylline-treated sperm without AC inhibitors exhibit motility. The phosphorylation pattern well coincided with the motility data ( Figure 4); phosphorylation of all the six major proteins of theophylline-treated sperm in CaFSW was more prominent than the others (Figure 8).  In the present study, we showed distinct roles of tmAC and sAC in the regulation of sperm motility in Ciona. tmAC is thought to be involved in the basal pathway for activation of motility; it transiently synthesizes high level of cAMP in response to SAAF-induced Ca 2+ influx and membrane hyperpolarization, resulting in the activation of PKA that phosphorylates axonemal proteins for motility. In contrast, sAC is thought to mainly participate in the regulation of the asymmetry of flagellar waveforms and increase of beat frequency. The swimming velocity of SAAF-activated, KH-7-treated sperm showed half of the control sperm. This is also the case in sperm activated by valinomycin as well as theophylline-activated, KH-7-treated sperm. Thus, it is likely that flagellar motility is regulated by at least two pathways: SAAF-dependent tmAC pathway for basic activation for motility and SAAF-independent sAC pathway for full motility with high beat frequency and waveform conversion leading to sperm chemotaxis. Proteins that are differently phosphorylated with or without AC inhibitors may be the key to prove this model. Several studies demonstrate distinct localization of AC isoforms in mammalian [40][41][42] and sea urchin sperm [14]. Studies on immunolocalization of ACs in Ciona sperm would surely provide further evidence for the roles of ACs in the regulation of sperm motility.

Ciona Sperm
The ascidian Ciona intestinalis was supplied by the Education and Research Center of Marine Bio-Resources, Tohoku University, Onagawa, Japan, and the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Animals were kept in aquaria under constant light for accumulation of gametes without spontaneous spawning. Semen samples were collected by dissecting the sperm duct and kept on ice until use.

Assessment of Sperm Motility
Semen was suspended in 2000 volumes of ASW containing DMSO or inhibitors and incubated for 3 min. The sperm suspension was immediately placed on a glass slide coated with 1% BSA to avoid adhesion of sperm to the glass. The sperm movement was recorded at RT under a phase contrast microscope (BX51, Olympus, Tokyo, Japan) with a 10× objective (UPlan FLN, Olympus, Tokyo, Japan) and analyzed using Sperm Motility Analysis System (SMAS, DITECT Corporation, Tokyo, Japan). The percentage of motile sperm and curvilinear velocity were analyzed before, 1 and 5 min after the addition of SAAF, theophylline and valinomycin (final concentration; 100 nM, 1 mM and 10 nM respectively). Analysis of chemotactic behavior and flagellar waveform of sperm was performed by the method previously described [35]. Images were recorded with a high-speed CCD camera (HAS-D3, DITECT Corporation, Tokyo, Japan) and analyzed using Bohboh software (Bohboh Soft, Tokyo, Japan).

Measurement of cAMP
Levels of intracellular cAMP were assayed by using Amersham cAMP Biotrak Enzyme immunoassay (EIA) System (GE Healthcare, Buckinghamshire, UK). Semen was suspended in 2000 volumes of ASW containing DMSO (WAKO, Osaka, Japan) or inhibitors and incubated for 3 min, and SAAF (final concentration 100 nM) was added to the suspension. The sperm suspension before, 10 s, 1 min and 5 min after the addition of SAAF were transferred to a lysis buffer to stop the reaction. The lysate was centrifuged at 12,000× g for 10 min at RT and the supernatant was used for the measurement of cAMP.

Immunoblotting by Anti-Phospho-PKA Substrate Antibody
Semen was suspended in 100 volumes of ASW or CaFSW containing DMSO or inhibitors and incubated for 3 min, and SAAF, theophylline or valinomycin (final concentration; 100 nM, 1 mM or 10 nM respectively) were added to the suspension. After 1 min cells were solubilized in a sample buffer; 62.4 mM Tris-HCl, 2% SDS, 4% glycerol, 0.004% bromophenol-blue, pH 6.8 and boiled at 95 °C for 2 min. Proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride (PVDF) membrane and subjected to Western blotting with anti-phospho-cAMP-dependent protein kinase (PKA) substrate monoclonal antibody (#9624, Cell Signaling Technology, Beverly, MA, USA) as the primary antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) as the secondary antibody. The immunoreactive bands were detected using ECL-prime (GE Healthcare, Buckinghamshire, UK).

Conclusions
Three tmAC (AC5/6, AC8 and AC9) and one sAC are encoded in the genome of the ascidian Ciona intestinalis. All of these AC genes are expressed in the testis. From the analyses of sperm motility using inhibitors for tmAC and sAC, we show that tmAC and sAC play distinct roles of in the regulation of sperm motility. tmAC transiently synthesizes high level of cAMP in response to SAAF-induced Ca 2+ influx and membrane hyperpolarization, resulting in the activation of PKA that phosphorylates axonemal proteins for basal activation of sperm motility. In contrast, sAC mainly participates in the regulation of the asymmetry of flagellar waveforms and increase in beat frequency in a HCO 3 − /pH-dependent manner. Phosphorylation of 105 and 80 kDa proteins appears closely related to this process. Thus, flagellar motility is likely to be regulated by two pathways: (1) SAAF-dependent tmAC pathway for basic activation of motility and (2) SAAF-independent sAC pathway for full motility with high beat frequency and waveform conversion.