A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa

Significance

Spermatozoon swimming performance is critical for fertilization success in fishes, yet the cellular mechanisms that regulate this vital trait are poorly understood. Here, we discovered that a water channel protein, acting as an H₂O₂ channel (peroxiporin) to detoxify the mitochondria, directly regulates the velocity and progressive motility of both freshwater and marine spermatozoa. The mitochondrial insertion of the peroxiporin is controlled by an increasingly sophisticated hierarchy of intracellular signaling cascades, which evolved into a multiplier stress-activated pathway in modern marine species. These adaptive solutions maximize the postactivated swimming performance of sperm under conditions of high osmotic and oxidative stress. Our findings thus reveal that the pathways regulating the peroxiporin transport in fish spermatozoa provide an advantage for competitive fertilization success.

Abstract

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H₂O₂ efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca²⁺-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca²⁺- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

For many dioecious animals, spermatozoon velocity, progressivity, and duration of motility are vital determinants of reproductive success and are thus major selection criteria for sperm evolution (1–6). Maximizing such kinematic properties contributes to spermatozoon vigor (7); however, due to the limitations in sperm ATP stores, which provide the chemical energy for flagellar contractions, a trade-off between swimming fast and for extended periods typically exists (8). Optimal combinations of traits that improve spermatozoon vigor are nevertheless important in polyandrous vertebrates facing sperm competition, which represents a powerful form of postcopulatory sexual selection (9–15). Since the phenomenon of sperm competition was first recognized (16), investigators have sought to understand the underlying mechanisms that could explain advantageous trait selection (17). To date, however, most research has focused on the physical and morphological properties involved in sperm competition, and very little is known concerning the molecular and genetic mechanisms underpinning spermatozoon performance (15, 18, 19).

One positively selected morphological change in respect of spermatozoon velocity and longevity in vertebrates as diverse as fishes, birds, and mammals, has been the increase in the spermatozoon midpiece size and the number or scale of mitochondria therein (20–22). Such changes have logically been associated with increased mitochondrial production of ATP for improved flagellar motility. However, the biochemical reactions that lead to increased ATP synthesis also generate elevated levels of hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that inhibits mitochondrial function and suppresses flagellar motility (23–27). With osmotic stress of the exposed ejaculate generating additional ROS (26, 28), it has been unclear how sperm evolved molecular mechanisms that surmount such signaling conflicts.

A solution to this apparent paradox was recently discovered in the spermatozoa of a marine teleost, in which a water channel protein, now termed Aqp8bb (a peroxiporin ortholog of mammalian aquaporin-8) (29), is rapidly (<1 s) trafficked to the inner mitochondrial membrane upon activation in seawater (SW) to facilitate H₂O₂ efflux and the maintenance of ATP production and flagellar motility (26). The importance of Aqp8bb, which mainly functions as a peroxiporin in these germ cells, was demonstrated through immunological inhibitory experiments, which highlighted the channel-trafficking mechanism as a critical regulator of the spermatozoon velocity and motility (26). To date, however, the signal transduction pathways that regulate peroxiporin trafficking in vertebrate spermatozoa remain completely unknown.

Among externally fertilizing vertebrates, the highest known intensity of sperm competition occurs in true bony fishes (teleosts) (6), and we therefore focused our investigations on these model organims. In contrast to amniotic vertebrates, in which ejaculates become gelatinous when emitted (30), the ejaculates of freshwater (FW) and marine teleosts are not only rapidly diluted, but respectively face tremendous and opposing osmotic stresses, which in most species activate sperm motility (31–33). To understand the significance of such harsh environments for peroxiporin signal transduction pathway evolution, we selected model species from ancient and modern lineages of teleosts, including the FW ostariphysan zebrafish (Danio rerio), the FW protacanthopterygian Atlantic salmon (Salmo salar), and the modern marine acanthomorph gilthead seabream (Sparus aurata). Using a combination of pharmacological, molecular, and physiological approaches, we uncover the evolution of increasingly sophisticated peroxiporin signal transduction pathways powering their spermatozoa. The findings provide insight into the underlying hierarchy of systemic molecular traits that regulate the velocity, progressivity and duration of spermatozoon motility.

Results

Activated Piscine Spermatozoa Traffic the Aqp8bb Peroxiporin to Mitochondria.

To investigate whether Aqp8bb is trafficked to the mitochondria of activated spermatozoa of FW teleosts as observed in the marine gilthead seabream (26), we first confirmed that the Aqp8bb orthologs of Atlantic salmon and zebrafish are expressed in intratesticular and ejaculated spermatozoa (SI Appendix, Figs. S1 and S2). Immunofluorescence microscopy of sperm maintained in the corresponding nonactivating medium (NAM), previously loaded with the mitochondrion-specific vital dye MitoTracker Red CMXRos (MTR), and using species-specific affinity-purified Aqp8bb antibodies, showed that the seabream Aqp8bb peroxiporin was distributed in the midpiece region and anterior part of the flagellum in immotile sperm, whereas in salmon and zebrafish Aqp8bb was mostly located in more discrete areas surrounding the spermatozoon head (Fig. 1 A–C). Upon SW- (seabream) or FW- (salmon and zebrafish) activation of sperm motility, Aqp8bb was rapidly accumulated in the mitochondria of each species (Fig. 1 A–C). Quantification of immotile and motile spermatozoa showing colocalization of Aqp8bb and MTR signals indicated that >75% of activated spermatozoa from each species showed Aqp8bb mitochondrial accumulation (Fig. 1 D–F), revealing that the peroxiporin trafficking mechanism during sperm motility is a conserved trait in teleosts.

Fig. 1.

The piscine Aqp8bb peroxiporin is transported to the mitochondria during spermatozoon motility activation. (A–C) Representative bright field images (Left) and immunodetection (Right) of Aqp8bb (green) in immotile (diluted in NAM) and activated spermatozoa of SW and FW spawning teleosts, using species-specific Aqp8bb antibodies. The mitochondria are labeled with MTR (red), whereas the spermatozoa nucleus are counterstained with DAPI (blue). Colocalized signals in mitochondria are yellow. (Scale bars, 5 µm; Insets, 1 µm.) (D and F) Percentage of spermatozoa showing Aqp8bb mitochondrial localization after incubation in NAM or upon activation in SW or FW in each species. Data are the mean ± SEM (n = 4 to 5 fish in D and E, and n = 3 pools of 5 different males each in F). Significance was measured by an unpaired Student’s t test with respect to immotile sperm. ***P < 0.001.

Signaling Pathways Involved in Aqp8bb Intracellular Trafficking in SW Spermatozoa.

To uncover the signaling pathways involved in the Aqp8bb trafficking mechanism of seabream spermatozoa, we used a battery of protein kinase inhibitors and activators (Fig. 2 and SI Appendix, Table S1). Immunofluorescence microscopy and immunoblotting data showed that the transit of Aqp8bb to the SW-activated sperm mitochondrion was strongly inhibited in a dose-dependent manner by the JNK inhibitor SP600125 with respect to spermatozoa treated with dimethyl sulfoxide (DMSO) vehicle (control), whereas CHIR99021 and BIM-II, typical blockers of glycogen synthase kinase 3 (GSK3) and protein kinase C (PKC), respectively, also reduced Aqp8bb transport but were less effective (Fig. 2 A and B). In contrast, inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase A (PKA), such as SB202190 and BIRB796, and H-89, respectively, showed no effect (Fig. 2 A and B). We further tested the effect of inhibitors of several JNK upstream regulators, including the MAPKKKs MAP3K7/TAK1, germinal center kinases (GCKs), apoptosis signal-regulating kinase 1 (ASK1), and mitogen-activated protein kinase kinases (MKKs) (34), on Aqp8bb trafficking, using the compounds NG25, GNF-7, NQDI-1, and PD98059. The data indicated that only ASK1 and MKKs inhibitors can reduce Aqp8bb mitochondrial accumulation in a dose-dependent manner (Fig. 2 A and B), suggesting that a canonical ASK1-MKK-JNK cascade, as well as PKC and GSK3 signaling pathways, regulate Aqp8bb trafficking in activated seabream spermatozoa.

Fig. 2.

Pharmacological identification of the major signal transduction pathways involved in Aqp8bb mitochondrial trafficking in seabream spermatozoa. (A) Dose–response inhibition of the percentage of activated spermatozoa showing Aqp8bb and MTR mitochondrial colocalization after treatment with DMSO alone (vehicle) or containing different protein kinase inhibitors determined by immunofluorescence microscopy. (B) Representative Aqp8bb immunoblots in mitochondrial extracts (Upper), and corresponding quantitation normalized to Phb (Lower), from sperm in A treated with 10 µM of the different drugs. (C) Total and phosphorylated JNK and GSK3 representative immunoblots (Upper) in sperm from B, and densitometric analysis of kinase phosphorylation normalized to the corresponding total kinase blot (Lower). (D and E) Activation of mitochondrial Aqp8bb trafficking in immotile spermatozoa maintained in NAM and exposed to activators of JNK (ANS), PKC (PMA), or GSK3 (MK2206) (black color) determined by immunofluorescence microscopy (D) and immunoblotting (E). Inhibition of ANS (10 µM)-induced Aqp8bb transport by inhibitors of GSK3 (CHIR99021) and PKC (BIM-II) is shown in red color. (F) MKK4, JNK, and GSK3 activation in sperm treated as in D. (G) Proposed model of the JNK and GSK3 signaling pathways controlling Aqp8bb mitochondrial transport in activated seabream spermatozoa. The kinase inhibitors (red color) and activators (green color) used are indicated. In A–F, data (mean ± SEM; n = 3 to 7 fish) were statistically analyzed by one-way ANOVA (P values for each compound are indicated in A and D). ***P < 0.001; **P < 0.01; *P < 0.05, with respect to control spermatozoa (treated with DMSO vehicle) or as indicated in brackets, or with respect to sperm treated with ANS alone (E and F in parenthesis).

Immunoblotting experiments confirmed the expression of ASK1, MKK4, JNK, and GSK3α/β kinases in immotile seabream spermatozoa (SI Appendix, Fig. S3A). Upon the hyperosmotic shock in SW, the phosphorylation of MKK4^T261, JNK^T183/Y185, and GSK3α/β^Y279/216 increased, whereas the inhibitory phosphorylation of ASK1^S966 and GSK3α^S21 decreased, suggesting that the catalytic activities of all of these kinases are activated (35–37). The inhibitory phosphorylation of GSK3β^S9 seems not to be involved during seabream sperm activation, since phosphorylated GSK3β^S9 was detected in testis extracts but not in spermatozoa (SI Appendix, Fig. S3B). Phosphorylation of JNK^T183/Y185 in SW-activated sperm was not affected by inhibitors of JNK, p38 MAPK, PKA, or GSK3 kinetic activity (Fig. 2C). However, JNK^T183/Y185 phosphorylation was reduced by PKC inhibition, although not completely, whereas that of Gsk3α/β^Y279/216 was partially decreased by both PKC and JNK blockage (Fig. 2C). Upon SW activation, the respective inhibition of the JNK upstream kinases ASK1 and MKK4 decreased MKK4^T261, JNK^T183/Y185, and GSK3α/β^Y279/216 phosphorylation (Fig. 2C), which is in accordance with the known role of ASK1 phosphorylating MKK4 (34) and the ability of PD98059 to prevent MKKs activation (38). Together, these data suggest that the ASK1-MKK4-JNK cascade can partially activate GSK3, and that PKC activity is required for full JNK and GSK3 activation. This combination can explain the somewhat lower potency of PKC inhibition to prevent Aqp8bb mitochondrial transport. However, the strong reduction of Aqp8bb trafficking by JNK inhibition suggests an additional role of JNK on channel trafficking that is not mediated by GSK3.

In order to assess the potential interaction of the JNK, PKC, and GSK3 pathways in the regulation of Aqp8bb mitochondrial transport, we investigated whether this mechanism could be stimulated in immotile spermatozoa. For this, NAM-maintained sperm were treated with the JNK/p38 MAPK phosphorylation-inducer anisomycin (ANS) and the PKC analog activator phorbol 12-myristate 13-acetate (PMA), as well as with MK2206 and SH-5, which are AKT/PKB inhibitors that can indirectly activate GSK3 (39, 40). The data show that all of these drugs stimulated Aqp8bb mitochondrial trafficking, while treatment with the PKC inactive enantiomer 4α-PMA had no effect (Fig. 2 D and E and SI Appendix, Fig. S4A). However, the percentage of spermatozoa showing Aqp8bb localization in the mitochondrion after exposure to ANS was similar to that observed in SW-activated sperm, whereas the other compounds only promoted channel mitochondrial accumulation in approximately half of the cells (Fig. 2 D and E and SI Appendix, Fig. S4A). The stimulatory effect of ANS on Aqp8bb trafficking, which occurs through JNK and not p38 MAPK (SI Appendix, Fig. S4 B and C), was completely blocked by GSK3 inhibition, but only partially reduced by the PKC inhibitors BIM-II and calphostin C (Fig. 2 D and E and SI Appendix, Fig. S4A). This suggests that PKC activation by JNK is necessary to drive Aqp8bb mitochondrial transport, but that this mechanism can still be triggered to some extent by JNK independently of PKC. The positive effects of PMA and MK2206 on Aqp8bb transport were not additive and could be reduced by JNK and GSK3 inhibition (SI Appendix, Fig. S4 D and E), suggesting that PKC and GSK3 activate the same mechanism for Aqp8bb trafficking, with both kinases dependent on active JNK.

Activation of JNK and PKC by ANS and PMA treatments, respectively, in immotile spermatozoa induced the phosphorylation of JNK^T183/Y185 and GSK3α/β^Y279/216, thus supporting that JNK and GSK3 can be activated via PKC, and that activation of GSK3 by JNK occurs during Aqp8bb trafficking (Fig. 2F). In contrast, MK2206 activated GSK3 but not JNK, indicating that GSK3 is downstream of JNK (Fig. 2F). However, as noted earlier, active JNK was still necessary for MK2206-induced Aqp8bb transport, thus supporting a role of JNK regulating mitochondrial Aqp8bb transport independently of GSK3. The ANS-mediated JNK^T183/Y185 phoshorylation was not affected by PKC or GSK3 inhibition, while that of GSK3α/β^Y279/216 was moderately reduced by PKC inhibition but it was not affected by the GSK3 blocker (Fig. 2F). Therefore, this also confirms that JNK can partially activate GSK3 in the absence of PKC activation.

Taken together, our findings suggest that a cross-talk between the PKC and JNK signaling pathways triggers the GSK3-mediated trafficking of the Aqp8bb peroxiporin to the inner mitochondrial membrane of the seabream spermatozoon (Fig. 2G). Moreover, a direct additive action of JNK, which bypasses the GSK3 pathway, acts as a multiplier for the rapid trafficking mechanism (Fig. 2G).

GSK3 and JNK Control Aqp8bb-Mediated Mitochondrial Detoxification in Activated Seabream Spermatozoa.

Since JNK and GSK3 appear to be key regulators of Aqp8bb trafficking, we investigated whether the pharmacological blockage of their activities can impair Aqp8bb insertion into the spermatozoon inner mitochondrial membrane and hamper H₂O₂ efflux. An Aqp8bb immunoblot of inner membrane mitochondrial extracts from SW-activated spermatozoa confirmed that the amount of mitochondrial Aqp8bb was lower in sperm treated with the JNK and GSK3 inhibitors compared to controls (Fig. 3A). The low mitochondrial abundance of Aqp8bb in these groups was associated with a reduced ability of mitochondria to transport H₂O₂ as measured with the ROS-sensitive, cell permeable fluorescent dye 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Fig. 3B). As a consequence, a higher accumulation of H₂O₂ in the spermatozoa exposed to the JNK and GSK3 inhibitors with respect to controls was observed during activation (Fig. 3C). The increased intracellular ROS was harmful to sperm function since time-course monitoring of sperm motion kinetics using computer-assisted sperm analysis (CASA) for up to 180 s revealed that both inhibitors reduced the percentage of motility and progressivity of the spermatozoa, as well as their curvilinear velocity (VCL), with respect to sperm treated with DMSO alone (Fig. 3D). However, the three sperm motion parameters measured within the first 30 s postactivation were more diminished when the JNK pathway is blocked (17 ± 1% and 35 ± 5% inhibition with the GSK3 and JNK blockers, respectively, with respect to the controls; n = 9, P = 0.003, Student’s t test). The addition of the mitochondria-targeted antioxidant Mito-TEMPO recovered sperm motion kinetics within 60 to 70 s, except there was reduction in the percentage of motile spermatozoa induced by GSK inhibition (Fig. 3E). This suggests that GSK3 likely plays other roles to maintain sperm motility in addition to mitochondrial Aqp8bb trafficking. These observations are thus consistent with a role of the JNK and GSK3 signaling pathways regulating Aqp8bb mitochondrial detoxification and the kinematic properties of the spermatozoa.

Fig. 3.

JNK and GSK3 inhibition prevents Aqp8bb insertion into the seabream spermatozoa inner mitochondrial membrane upon SW activation. (A) Aqp8bb immunoblot in the inner mitochondrial membrane from SW-activated spermatozoa (n = 3 fish) exposed or not to 10 µM of the JNK (SP600125) and GSK3 (CHIR99021) inhibitors. Phb was used as a marker for even loading. (B) H₂O₂ uptake (mean ± SEM, n = 6 fish) by mitochondria isolated from NAM and SW spermatozoa treated with or without the inhibitors. (C) ROS levels (mean ± SEM, n = 8 fish) in spermatozoa treated as in B. Statistical differences were measured by one-way ANOVA. ***P < 0.001, with respect to NAM or SW-activated sperm not treated with the inhibitors, or as indicated in brackets. (D) Percentage of motility and progressivity, and VCL, of spermatozoa (mean ± SEM, n = 4 fish) treated with DMSO in the presence or absence of SP600125 and CHIR99021. Statistical differences with respect to control spermatozoa (only during the first 60 s for motility and progressivity) were determined by the Mann–Whitney U test (P values indicated in each panel). (E) Effect of the mitochondria-targeted antioxidant Mito-TEMPO (MTPO; 50 µM) on the SP600125 (SP) or CHIR99021 (CHIR) inhibition of sperm kinetics (mean ± SEM, n = 7 fish) at 60 to 70 s postactivation (marked with a gray bar in D). Data are statistically analyzed by one-way ANOVA (minus MTPO), and by the unpaired Student’s t test between sperm untreated and treated with MTPO. *P < 0.05; **P < 0.01; ***P < 0.001, with respect to nontreated sperm, or as indicated in brackets.

Intracellular Ca²⁺ and ROS Trigger Seabream Aqp8bb Mitochondrial Transport.

We previously demonstrated that prevention of the intracellular Ca²⁺ surge that normally occurs in seabream sperm upon SW activation partially inhibits mitochondrial Aqp8bb trafficking (41), and that the SW-induced hyperosmotic shock increases ROS levels in the spermatozoa (26). Since it is known that the ASK1-MAPK signaling pathway can be activated by cellular oxidative stress (42), we hypothesize that elevated intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) and ROS linked to the hyperosmotic shock in SW are the upstream signals in seabream spermatozoa triggering JNK and GSK3 activation and Aqp8bb intracellular trafficking.

To investigate this hypothesis, we first determined the contribution of Ca²⁺-activated signaling pathways in the regulation of Aqp8bb mitochondrial transport by treating immotile sperm with 3 mM Ca²⁺ in the presence of 10 µM of the Ca²⁺ ionophore A23187, which generates similar levels of [Ca²⁺]_i to those determined in SW-activated sperm (Fig. 4A). Under these conditions, Aqp8bb transport to the mitochondria was enhanced, but interestingly, the percentage of spermatozoa showing mitochondrial localization of the channel was approximately half of that observed in SW-activated sperm (Fig. 4B). Ca²⁺-triggered Aqp8bb trafficking was not affected by inhibitors of ASK1 and MKK, whereas it was reduced to control levels by blockers of PKC, JNK, and GSK3 (Fig. 4C and SI Appendix, Fig. S5). After Ca²⁺ treatment, the inhibitory phosphorylation of ASK1^S966 did not change, and MKK4 was not activated, while phosphorylation of JNK^T183/Y185 and GSK3α/β^Y279/216 was enhanced (Fig. 4D). The activation of JNK and GSK3 was reduced by PKC inhibition, while the blockage of JNK only partially diminished GSK3 activation (Fig. 4D). These data suggest that a [Ca²⁺]_i surge alone can partially trigger JNK- and GSK3-mediated Aqp8bb mitochondrial transport through PKC activation of JNK and GSK3, and thus independently of the ASK1-MKK4 signaling pathway.

Fig. 4.

An intracellular Ca²⁺ surge partially drives Aqp8bb transport to the mitochondrion in nonactivated seabream spermatozoa. (A) Intracellular Ca²⁺ levels in sperm maintained in NAM and treated with 3 mM Ca²⁺ and/or 10 µM of the Ca²⁺ ionophore A23187, and in SW-activated sperm. (B) Percentage of spermatozoa showing Aqp8bb mitochondrial localization after exposure to Ca²⁺ and A23187 in NAM, or upon activation in SW. (C) Representative immunoblot of mitochondrial Aqp8bb (Left), and corresponding quantitation normalized to Phb (Right), from sperm incubated with or without Ca²⁺ and A23187 in NAM, and treated with 10 µM of different kinase inhibitors. (D) Total and phosphorylated ASK1, MKK4, JNK, and GSK3α/β representative immunoblots (Upper) in spermatozoa treated as in C. (Lower) Depiction of the densitometric analysis of kinase phosphorylation normalized to the corresponding band from the total kinase blot. Data are the mean ± SEM (n = 4 to 6 fish) and are statistically analyzed by one-way ANOVA. ***P < 0.001; **P < 0.01; *P < 0.05, with respect to nontreated sperm, or treated with Ca²⁺+A23187 without inhibitors (in parenthesis), or as indicated in brackets.

To examine the effect of oxidative stress on Aqp8bb intracellular trafficking in spermatozoa, independently of the Ca²⁺ signal, we followed two different approaches: The generation of ROS in immotile sperm by exposure to the xanthine-xanthine oxidase (X-XO) system (43), and the treatment of SW-activated sperm with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) in the presence or absence of diphenyleneiodonium (DPI), a compound that limits ROS production by inhibiting membrane-bound NADPH oxidases (NOXes) and other flavoproteins (44). External treatment of nonactivated sperm with X-XO produced an increment of ROS in a dose–response manner, the highest X-XO dose generating similar ROS levels to those measured in SW-activated sperm (Fig. 5 A and D). Upon SW activation, treatment with BAPTA abolished the [Ca²⁺]_i surge in a dose-dependent manner, which completely inhibited motility (SI Appendix, Fig. S6A), while ROS accumulation in the spermatozoa was not affected (Fig. 5 B and D). Conversely, exposure of activated sperm to DPI decreased ROS levels but did not affect the increase of [Ca²⁺]_i (Fig. 5 C and D). Interestingly, as previously observed in Ca²⁺-treated immotile sperm, treatment of immotile spermatozoa with the highest dose of X-XO stimulated Aqp8bb mitochondrial trafficking in approximately half of the spermatozoa with respect to SW-activated sperm exposed to DMSO alone (SI Appendix, Fig. S6B). The same result was observed in sperm activated in SW containing BAPTA (SI Appendix, Fig. S6C).

Fig. 5.

Oxidative stress-induced Aqp8bb mitochondrial transport in seabream spermatozoa. (A) ROS levels in immotile sperm treated with increasing doses of X-XO (X-XO1, X-XO2, and X-XO3), and in SW-activated spermatozoa. (B and C) Intracellular Ca²⁺ (red line) and ROS levels (bars) in nonactivated and activated sperm in the presence of BAPTA (B) or DPI (C). (D) Epifluorescence photomicrographs of nonactivated and activated spermatozoa treated with X-XO3, 500 µM BAPTA, 50 µM DPI, or BAPTA+DPI, and labeled with the CM-H₂DCFDA dye (green). Nuclei are counterstained with DAPI (blue). (Scale bars, 5 µm.) (E) Immunoblot of mitochondrial Aqp8bb in sperm maintained in NAM and exposed to X-XO3 (Upper), in the presence or absence of 10 µM of kinase inhibitors, and corresponding quantitation normalized to Phb (Lower). (F) Total and phosphorylated ASK1, MKK4, JNK, and GSK3α/β immunoblots (Left), and densitometric analysis of phosphorylated forms normalized to the corresponding nonphosphorylated bands (Right), in spermatozoa treated as in E. (G) Percentage of spermatozoa showing Aqp8bb in mitochondria after treatment with X-XO3 in NAM, or upon activation in SW with or without BAPTA and/or DPI. (H) Immunoblot of mitochondrial Aqp8bb in immotile sperm or activated in SW with or without BAPTA and/or DPI (Left) and quantitation normalized to Phb (Right). (I) Immunoblots of kinase activation in sperm treated as in H (Left) and quantitation of phosphorylated forms. In all panels, data are displayed as mean ± SEM (n = 4 to 6 fish) and are statistically analyzed by one-way ANOVA. ***P < 0.001; **P < 0.01; *P < 0.05, with respect to nontreated spermatozoa in NAM, spermatozoa exposed to NAM plus X-XO3 or SW without inhibitors (in parenthesis), or as indicated in brackets (G and E).

In both ROS- and SW+BAPTA-treated spermatozoa, Aqp8bb mitochondrial accumulation was completely inhibited by blockers of ASK1, MKK, JNK, and GSK3, whereas inhibition of PKC only partially decreased Aqp8bb transport (Fig. 5E and SI Appendix, Fig. S6 B–D). The ROS generated in immotile and SW spermatozoa activated ASK1 by deposhosphorylation of the inhibitory ASK1^S966 site, which was not affected by any of the downstream kinase inhibitors, while it enhanced the phosphorylation of MKK4^T261, JNK^T183/Y185, and GSK3α/β^Y279/216 (Fig. 5F and SI Appendix, Fig. S6E). Activation of MKK4 was reduced to control levels by inhibitors of ASK1 and MKK, but not by inhibition of downstream kinases, whereas ROS-induced JNK and GSK3 activation was completely blocked by ASK1 and MKK inhibitors, and only partially prevented by PKC inhibition (Fig. 5F and SI Appendix, Fig. S6E). However, JNK inhibition strongly reduced ROS-mediated GSK3 activation (Fig. 5F and SI Appendix, Fig. S6E), which indicates that the ROS-triggered ASK1-MKK4-JNK pathway can activate GSK3 independently of PKC. The data, however, also suggest that ROS-induced PKC activity may be a positive regulator of JNK, as previously observed after Ca²⁺ treatment alone, but that in this case this mechanism requires previous activation of JNK by the canonical upstream kinase MKK4.

Consistent with this model, when either the [Ca²⁺]_i or ROS increments in SW-activated sperm were completely abolished by BAPTA and DPI, respectively, the percentage of spermatozoa showing mitochondrial localization was similar to that elicited by ROS treatment of immotile spermatozoa, and was approximately half of that observed in SW-activated sperm (Fig. 5 G and H). However, when both inhibitors were present upon SW activation, Aqp8bb mitochondrial accumulation was strongly reduced to the level observed in immotile spermatozoa (Fig. 5 G and H). Kinase phosphorylation analysis showed that while ASK1 activation in SW-activated spermatozoa was not affected by BAPTA treatment, DPI reduced both ASK1 and MKK4 activation (Fig. 5I). In contrast, each of the BAPTA and DPI treatments only partially prevented JNK and GSK3 activation (Fig. 5I). In the presence of BAPTA plus DPI, the activation state of ASK1 and MKK4 did not change with respect to that observed in the presence of DPI alone, whereas the activation of JNK and GSK3 was strongly diminished (Fig. 5I).

Taken together, these findings suggest that the intracellular Ca²⁺ and ROS signals occurring in SW-activated seabream spermatozoa respectively trigger alternative and additive PKC and ASK1-MAPK pathways, which cross-talk to activate JNK and GSK3 and direct Aqp8bb mitochondrial insertion.

JNK and GSK3 Can Phosphorylate Seabream Aqp8bb In Vitro.

In silico analysis of the seabream Aqp8bb amino acid sequence revealed the presence of three Ser residues (Ser¹⁶, Ser¹⁹, and Ser²⁰) and one Thr (Thr²⁴) residue in the N terminus of the channel as potential phosphorylation recognition sites for GSK3 and proline-directed kinase (such as p38 MAPK and JNK), respectively (SI Appendix, Fig. S7A). An additional Ser residue (Ser⁹¹) showing a low score for GSK3 and proline-directed kinase phosphorylation was also identified in the first cytoplasmic loop (SI Appendix, Fig. S7A). To experimentally assess whether GSK3 and JNK can phosphorylate seabream Aqp8bb, we employed in vitro phosphorylation assays using mouse recombinant JNK1 (rJNK1) and human recombinant GSK3β (rGSK3). In these experiments, Flag-tagged Aqp8bb (Aqp8bb-Flag) transiently expressed in embryonic kidney cells 293T (HEK293T) was immunoprecipitated using the seabream Aqp8bb specific antibody and incubated with rJNK1 or rGSK3β in the presence or absence of ATP. Immunobloting of immunoprecipitated Aqp8bb-Flag using phosphorylated Ser- and Thr-specific antibodies confirmed the ATP-dependent specific phosphorylation of Aqp8bb Ser and Thr residues by rJNK1, and only of Ser residues by rGSK3β (SI Appendix, Fig. S7B). To determine which specific channel residues are phosphorylated by the kinases, the same assays were carried out using wild-type Aqp8bb-Flag (Aqp8bb-Flag-WT) or Aqp8bb-Flag constructs in which all Ser and Thr residues in the N terminus, as well as Ser⁹¹, were independently mutated into Ala. The results showed that Aqp8bb-Flag-T24A and -S91A prevented rJNK1 Thr and Ser phosphorylation, respectively, whereas Aqp8bb-Flag-S16A abolished Ser phosphorylation by rGSK3β (SI Appendix, Fig. S7C). These data therefore indicate that Thr²⁴ and Ser⁹¹ are the residues phosphorylated by rJNK1, while only Ser¹⁶ is the target site for rGSK3β.

The majority of GSK3 substrates require prephosphorylation at a residue 4 or 5 amino acids C-terminal to the GSK3 target site for full catalytic activity, a phenomenon referred to as “priming” (45). The seabream Aqp8bb shows two such potential priming sites, Ser¹⁹ and Ser²⁰, located three and four residues C-terminal to the Ser¹⁶ GSK3 phosphorylation site (SI Appendix, Fig. S7A). In addition, Thr²⁴ could also play a role since some substrates of GSK3 show a priming site much further from the target site (45). To investigate whether a priming mechanism regulates rGSK3β-mediated Aqp8bb phosphorylation at Ser¹⁶, we first established by immunoblotting the lowest amount of rGSK3β to yield detectable levels of Ser phosphorylation in Aqp8bb (SI Appendix, Fig. S7D), and subsequently test if Aqp8bb-Flag-S19D, -S20D, and -T24D mutants, which mimic a phosphorylated state, increase rGSK3β-mediated channel phosphorylation. The results show that Ser¹⁶ phosphorylation of Aqp8bb-Flag by rGSK3β was increased by ∼27 times in Aqp8bb-Flag-S19D and -S20D mutants with respect to the wild-type, whereas the Aqp8bb-Flag-T24D was phosphorylated at Ser¹⁶ ∼12 times more efficiently (SI Appendix, Fig. S7E). These findings therefore suggest that Aqp8bb phosphorylation of Ser¹⁹, Ser²⁰, and to a lesser extent Thr²⁴, can prime the channel N terminus for Ser¹⁶ phosphorylation by GSK3 (SI Appendix, Fig. S7F).

GSK3 and JNK Phosphorylation of Ser¹⁶ and Thr²⁴, Respectively, in Seabream Aqp8bb Regulate Channel Trafficking.

Transcriptional and translational activity in ejaculated sperm is very low or completely absent (46). Therefore, in order to investigate the molecular basis of Aqp8bb mitochondrial trafficking, we examined whether the signaling pathways controlling this mechanism in spermatozoa can be reproduced in cultured mammalian cells. For these tests, we selected the human hepatocarcinoma-derived cell line HepG2 in which the AQP8 ortholog is expressed in the inner mitochondrial membrane (47). The results of these experiments showed that the same Ca²⁺-activated PKC-GSK3 and H₂O₂-activated JNK-GSK3 signaling cascades controlling Aqp8bb mitochondrial transport in the seabream spermatozoon can be replicated in HepG2 cells, except that in this case the cross-talk between PKC and JNK does not occur (SI Appendix, Supplementary Text S1 and Fig. S8).

Using the HepG2 cells as a surrogate system for functional analyses, we investigated the role of Aqp8bb phosphorylation on mitochondrial channel trafficking. To this end, we transfected cells with Aqp8bb-Flag-WT or single phosphomimetic mutants at each Ser and Thr residue in the N terminus of the channel (Aqp8bb-Flag-T2D, -T15D, -S16D, -S19D, -S20D, -T24D), and at Ser⁹¹ (Aqp8bb-Flag-S91D), or with a S16D/T24D double mutant, and subsequently determined mitochondrial targeting of the encoded proteins by immunofluorescence microscopy. The data showed that Aqp8bb-Flag-WT and the Aqp8bb-Flag-S91D mutant mainly remained in the cell plasma membrane, whereas the Aqp8bb-Flag-T2D and -T15D channels were retained in the cytoplasm (Fig. 6A and SI Appendix, Fig. S9A). In contrast, the Aqp8bb-Flag-S19D and -S20D mutants were partially targeted to the mitochondria, which appears to be enhanced for the Aqp8bb-Flag-S16D, -T24D and S16D/T24D channel constructs (Fig. 6A). Immunoblot analysis showed that the wild-type and mutant channels were expressed at similar levels, and confirmed that each of the Aqp8bb-Flag-S16D and -T24D mutants were targeted to the mitochondria, unlike the Aqp8bb-Flag-WT (Fig. 6B). The Aqp8bb-Flag-S19D and -S20D constructs also accumulated in the mitochondria but to a lesser amount than the Aqp8bb-Flag-S16D and -T24D mutants (Fig. 6B). However, the data also revealed that the Aqp8bb-Flag-S16D/T24D double mutant was more concentrated in the mitochondria than each of the corresponding single mutants (Fig. 6B), suggesting that phosphorylation of Ser¹⁶ and Thr²⁴ residues in the channel are additive for activating mitochondrial transport.

Fig. 6.

Dual N terminus phosphorylation of seabream Aqp8bb by GSK3 and JNK control mitochondrial channel transport in HepG2 cells. (A) Double staining of Aqp8bb-Flag (green) and mitochondria (MTR, red) in cells transiently expressing wild-type Aqp8bb-Flag (WT) or phospho-mimetic mutants as indicated. Arrows point to the plasma membrane, whereas arrowheads indicate colocalized signals in the mitochondria. (Scale bars, 10 µm; Insets, 5 µm.) (B) Representative immunoblot of total and mitochondrial Aqp8bb-Flag-WT and mutants, using tubulin (TUBA) and PHB as loading controls, respectively (Left). Mitochondrial Aqp8bb-Flag quantitation is normalized to PHB (Right). (C) Immunoblot of mitochondrial Aqp8bb-Flag-WT and -S16C, -T24A, and -S16C/T24A mutant channels, and Ser and Thr phosphorylation of the corresponding immunoprecipitated proteins, in cells treated with Ca²⁺ and H₂O₂. (Right) The amount of each construct in the the mitochondria, normalized to PHB, and their Ser and Thr phosphorylation state normalized to the total immunoprecipitated protein. (D) Effect of coexpression of Aqp8bb-Flag-WT and dominant-negative forms of JNK1 and GSK3β (dnJNK1 and dnGSK3β, respectively) on the mitochondrial transport and Ser/Thr phosphorylation of the channel in cells treated as in C. (E) Immunoblot of nonactivated and activated GSK3 in cells coexpressing Aqp8bb-Flag-WT and dnJNK1 and treated with Ca²⁺ and H₂O₂ (Left), and densitometric analysis of phosphorylated GSK3 forms (Right). In B and C, quantifications are displayed as mean ± SEM (n = 3 separate experiments), and data are statistically analyzed by one-way ANOVA (B–D) or by an unpaired Student’s t test (E). ***P < 0.001; **P < 0.01; *P < 0.05, with respect Aqp8bb-Flag-WT or as indicated in brackets.

To further investigate the role of Ser¹⁶ and Thr²⁴ phosphorylation driving Aqp8bb mitochondrial trafficking, cells were transfected with Aqp8bb-Flag-WT or mutant channels in which one or both Ser¹⁶ and Thr²⁴ were mutated into Cys or Ala, respectively, and treated with 10 mM Ca²⁺ and 100 µM H₂O₂. Immunoblot analyses of total protein extracts showed that the three Aqp8bb-Flag-S16C, -T24A, and -S16C/T24A mutants were equally expressed than the Aqp8bb-Flag-WT (SI Appendix, Fig. S9B). However, mitochondrial trafficking in response to Ca²⁺ and H₂O₂ treatment was similarly reduced in both Aqp8bb-Flag-S16C and -T24A constructs with respect to that shown by the Aqp8bb-Flag-WT, whereas channel transport of the Aqp8bb-Flag-S16C/T24A double mutant was almost completely abolished (Fig. 6C). Immunoprecipitation followed by immunoblot analysis of phosphorylated Ser and Thr showed that Aqp8bb-Flag-WT was phosphorylated at Ser and Thr residues in response to Ca²⁺ and H₂O₂ uptake (Fig. 6C). However, only Ser or Thr phosphorylation was reduced in the Aqp8bb-Flag-S16C and -T24A constructs, respectively, with respect to that seen in the Aqp8bb-Flag-WT, while phosphorylation of both Ser and Thr residues was decreased in the Aqp8bb-Flag-S16C/T24A double mutant (Fig. 6C). These data therefore support that Ser¹⁶ and Thr²⁴ phosphorylation in the Aqp8bb N terminus are both necessary for maximum mitochondrial channel transport.

To confirm that GSK3 and JNK are, respectively, involved in Ser¹⁶ and Thr²⁴ phosphorylation of Aqp8bb, HepG2 cells were cotransfected with Aqp8bb-Flag-WT and dominant-negative catalytically inactive forms of Xenopus laevis GSK3β (dnGSK3β) (48) or human JNK1 (dnJNK1) (49), and exposed to external Ca⁺² and H₂O₂. Both inhibitory constructs reduced the accumulation of Aqp8bb-Flag-WT in the mitochondria in response to external Ca²⁺ and ROS (Fig. 6D), thus further supporting the role of JNK and GSK3 in the Aqp8bb trafficking mechanism. However, while dnGSK3β expression decreased Ser phosphorylation of Aqp8bb-Flag-WT, but not Thr phosphorylation, the dnJNK1 construct completely abolished Ser and Thr phosphorylation of the channel (Fig. 6D). These findings suggest that JNK could activate GSK3 for Aqp8bb Ser phosphorylation, in addition to its potential role phosphorylating Thr²⁴ in the channel, as previously predicted to occur in sperm cells. To corroborate this hypothesis, activation of GSK3 was evaluated in HepG2 cells expressing Aqp8bb-Flag-WT and dnJNK1 and treated with Ca²⁺ and H₂O₂. The data confirmed that phosphorylation of GSK3α/β^Y279/216 was reduced in the presence of catalytically inactive JNK1, whereas that of GSK3α^S21, but not of GSK3β^S9, was increased (Fig. 6E). These results therefore suggest that mitochondrial trafficking of seabream Aqp8bb is regulated by Ser¹⁶ phosphorylation by JNK- and PKC-activated GSK3, as well as by JNK-mediated Thr²⁴ phosphorylation of the channel.

Aqp8bb Mitochondrial Transport in FW Spermatozoa Is Controlled by PKC or GSK3 Phosphorylation of an N-Terminal Ser Residue.

To examine whether the same signaling pathways regulating seabream Aqp8bb mitochondrial transport are present in FW spermatozoa, we initially assessed the effect of PKC, JNK, and GSK3 inhibitors (BIM-II, SP600125, and CHIR99021, respectively) on Aqp8bb trafficking in Atlantic salmon and zebrafish spermatozoa upon FW activation. In contrast to the seabream, the results showed that JNK inhibition did not affect mitochondrial channel transport in salmon and zebrafish sperm, indicating that the JNK signaling pathway is not involved in this mechanism (Fig. 7A). In contrast, both PKC and GSK3 inhibitors reduced the percentage of salmon spermatozoa showing Aqp8bb mitochondrial localization in a dose–response manner, whereas only the blockage of PKC elicited the same effect in zebrafish (Fig. 7A). These observations suggest a role of PKC and GSK3, and of PKC only, in the control of Aqp8bb transport in salmon and zebrafish spermatozoa, respectively.

Fig. 7.

Aqp8bb mitochondrial transport in FW spermatozoa is controlled by monophosphorylation of the N terminus of the channel by PKC or GSK3. (A) Percentage of salmon and zebrafish spermatozoa (mean ± SEM; n = 4 fish for salmon, and n = 3 pools of 5 fish for zebrafish) showing Aqp8bb mitochondrial localization after treatment with vehicle (DMSO), or JNK (SP600125), PKC (BIM-II), or GSK3 (CHIR99021) inhibitors. (B) ROS levels (mean ± SEM; n = 6 fish) in immotile and activated zebrafish spermatozoa treated with vehicle or BIM-II. (C) Motility, progressivity, and VCL of zebrafish activated spermatozoa (mean ± SEM, n = 7 fish) treated with vehicle or BIM-II. P values are calculated from the Mann–Whitney U test (during the first 60 s only for motility and progressivity). (D) Effect of MTPO (50 µM) on BIM-II inhibition of zebrafish sperm kinetics (mean ± SEM, n = 5 fish) at 30 to 40 s postactivation (gray bar in C). Data in B and D are statistically analyzed by one-way ANOVA (***P < 0.001; *P < 0.05, with respect to nontreated sperm). (E) Amino acid sequence alignment of seabream, salmon, and zebrafish Aqp8bb N terminus indicating the GSK3, PKC, and JNK phosphorylation sites. (F) Immunoblot of Ser phosphorylation in salmon and zebrafish Aqp8bb-Flag-WT and -S14A and -S16A mutants, respectively, after phosphorylation in vitro by rPKCα (0.08 µM) and rGSK3β (0.17 µM). (Lower) Quantitation of phosphorylated channel normalized to total immunoprecipitated protein. (G and I) Aqp8bb-Flag and MTR double staining of HepG2 cells expressing salmon (G) or zebrafish (I) Aqp8bb-Flag-WT and phosphomimetic mutants. Arrowheads indicate colocalized signals in the mitochondria. (Scale bars, 10 µm; Insets, 5 µm.) (H and J) Immunoblots of Aqp8bb-Flag-WT and mutants in mitochondrial extracts from cells shown in G and I. Data in F, H, and J are the mean ± SEM of three separate experiments, and are statistically analyzed by one-way ANOVA (F) or an unpaired Student’s t test (H and J). ***P < 0.001; **P < 0.01, as indicated in brackets.

To confirm that PKC is the signaling kinase controlling Aqp8bb-mediated mitochondrial detoxification in zebrafish spermatozoa, the levels of ROS in immotile and FW-activated sperm in the presence or absence of the PKC inhibitor BIM-II were determined. The results showed that the hypoosmotic shock in FW does not increase ROS levels in zebrafish sperm; however, when the transport of Aqp8bb to the mitochondria was impaired by PKC inhibition, ROS levels were increased (Fig. 7B). This reveals that an osmotic shock-induced ROS-MAPK-JNK signaling pathway controlling Aqp8bb trafficking is absent in zebrafish spermatozoa. As observed for seabream sperm, high levels of intracellular ROS induced by PKC inhibition also reduced the percentage of motility and progressivity and the VCL of zebrafish spermatozoa (Fig. 7C). However, in this case Mito-TEMPO treatment only partially recovered the kinematic properties (Fig. 7D), suggesting that PKC plays an additional role for sperm motility and velocity maintenance in zebrafish that is unrelated to the mitochondrial peroxiporin transport mechanism.

Comparison of the deduced amino acid sequences of the Aqp8bb N terminus from seabream, salmon, and zebrafish revealed a Ser residue in position 14 or 16 in all three species, whereas the JNK Thr²⁴ phosphorylation site from seabream Aqp8bb was missing in the two FW spawning teleosts. In silico analysis revealed that Ser¹⁴ from salmon Aqp8bb is a putative phosphorylation site for GSK3, as is Ser¹⁶ from seabream Aqp8bb, including a potential priming site at Ser¹⁸, whereas Ser¹⁶ in zebrafish Aqp8bb is a presumed residue for PKC phosphorylation (Fig. 7E). To confirm that these target sites in salmon and zebrafish Aqp8bb are functional, in vitro phosphorylation assays were performed in HEK293 cells as described above. In this case, we tested rGSK3β and recombinant X. laevis PKCα (rPKCα) on Aqp8bb-Flag-WT and mutant channels, in which Aqp8bb Ser¹⁴ (salmon) or Ser¹⁶ (zebrafish) were replaced by Ala. Immunoblot results showed that salmon Aqp8bb-Flag-WT was equally phosphorylated by rPKCα and rGSK3β at Ser residues, whereas rGSK3β was no longer able to phosphorylate Ser residues in the Aqp8bb-Flag-S14A mutant, and only Ser phosphorylation by rPKCα was detected (Fig. 7F). Similarly, zebrafish Aqp8bb-Flag-WT was Ser-phosphorylated by rPKCα and rGSK3β, but the Aqp8b-Flag-S16A mutant could only be phosphorylated by rGSK3β (Fig. 7F). These data indicate that salmon Aqp8bb Ser¹⁴ and zebrafish Aqp8bb Ser¹⁶ are, respectively, the target residues of rGSK3β, and rPKCα. However, both salmon and zebrafish channels contain other residues that are phosphorylated by rPKCα and rGSK3β, respectively.

Finally, we transiently expressed salmon and zebrafish Aqp8bb-Flag-WT in HepG2 cells and determined the effect of external Ca²⁺ and/or H₂O₂ on mitochondrial channel localization. In contrast to seabream, Aqp8bb immunostaining of MTR-loaded cells, as well as immunoblotting of mitochondrial extracts, showed that only Ca²⁺ was able to traffic both salmon and zebrafish Aqp8bb-Flag-WT to the mitochondria (SI Appendix, Fig. S10 A and B). Since Ser¹⁴ and Ser¹⁶ from salmon and zebrafish Aqp8bb are, respectively, the target sites of Ca²⁺-activated GSK3 and PKC, we investigated whether phosphomimetic mutations at these sites could induce mitochondrial targeting of the channels. For salmon Aqp8bb, we also tested Ser²⁰ in case this residue could play a role as a priming site for GSK3 phosphorylation. All constructs were expressed at similar levels in HepG2 cells (SI Appendix, Fig. S10 C and D), but immunostaining and immunoblotting data indicated that only the salmon Aqp8bb-Flag-S14D and zebrafish Aqp8bb-Flag-S16D mutants were constitutively targeted to the mitochondria (Fig. 7 G–J and SI Appendix, Fig. S10C). Taken together, these data suggest that Ca²⁺-activated PKC or the PKC/GSK3 cascade are the effective pathways phosphorylating Ser¹⁶ or Ser¹⁴ in the N terminus of zebrafish and salmon Aqp8bb, respectively, which traffic the peroxiporin to the mitochondria.

Discussion

The present work reveals insight into the evolution of molecular mechanisms regulating spermatozoan kinematic properties in an infraclass of vertebrates that display intense levels of sperm competition. The data show that the rapid mitochondrial trafficking of an Aqp8bb peroxiporin, first discovered in a marine teleost (26), is consistently regulated through N-terminal phosphorylation in FW and SW teleosts. Such channel regulation thus represents a conserved mechanism of mitochondrial ROS detoxification for the maintenance of ATP production and improved spermatozoon performance.

The model arising from the data (Fig. 8) suggests that the signal transduction pathways regulating Aqp8bb trafficking in teleost spermatozoa evolved increasing levels of sophistication and cross-talk between the pathways through the inclusion of alternative kinase cascade modules. Thus, in older lineages of FW teleost in which spermatozoon motility is activated through hypoosmotic shock, a more direct Ca²⁺-PKC–mediated pathway phosphorylating the Aqp8bb N terminus suffices (Fig. 8). However, in the protacanthopterygian FW salmonids, in which the importance of sperm velocity under sperm competition is well known (50), a PKC-regulated GSK3 cascade is added and the channel is only phosphorylated by GSK3 to induce its trafficking (Fig. 8). The addition of the extra GSK3 regulatory layer may act as a multiplier if the expression levels of this kinase significantly exceed those of PKC, a facet that could explain the higher sperm velocities of sedentary parasitic males compared to anadromous “bourgeois” males (51). In contrast to the FW species, we find that marine spermatozoa have recruited an addtitional ROS-triggered JNK pathway to cope with the elevated hypertonic stress (Fig. 8). In this case, JNK directly activates GSK3, but also cross-talks with Ca²⁺- and ROS-activated PKC to trigger Aqp8bb N-terminal phosphorylation by GSK3, thus creating a multiplier pathway for the regulation of the peroxiporin trafficking. In addition, JNK bypasses the GSK3 pathway to directly phosphorylate the channel in a different N-terminal residue, further enhancing the peroxiporin trafficking.

Fig. 8.

A model for the evolution of the intracellular multiplier peroxiporin signal transduction pathway powering piscine spermatozoa. In spermatozoa from more primitive FW teleost lineages, such as in zebrafish and salmonids, a rise of intracellular Ca²⁺ ([Ca²⁺]_i) upon activation in FW triggers the PKC-GSK3 signaling pathway, leading to the direct PKC (zebrafish) or GSK3 (Atlantic salmon) phosphorylation of N-terminal Aqp8bb residues, promoting rapid channel trafficking to the mitochondria. In modern marine teleosts such as the seabream, which release the sperm into SW, a cross-talk between [Ca²⁺]_i-activated PKC and ROS-activated JNK and PKC signaling pathways converge to activate GSK3 with an additional JNK-mediated multiplier regulation of Aqp8bb mitochondrial trafficking to cope with a higher osmotic stress.

The conserved action of PKC in each of the pathways regulating Aqp8bb trafficking in FW and SW spermatozoa reveals how the multiplier mechanism evolved. In SW-activated spermatozoa, both Ca²⁺ and ROS induce PKC activity, with ROS also inducing JNK activity through the ASK1-MKK4 cascade to further enhance PKC catalysis which, together with the direct activation of GSK3 by JNK, multiplies the action of GSK3 to phosphorylate Aqp8bb (Fig. 8). The importance of this stress-induced multiplier pathway is clearly evident on the spermatozoon kinematic properties, where JNK inhibition more rapidly suppresses the degree of motility, progressivity and velocity of spermatozoa compared to GSK3 inhibition. Consequently, it seems likely that positive selection of the JNK pathway with efficient catalytic kinetics would be advantageous in the sperm competition of SW species, while those of the PKC and GSK3 pathways would be favorable for FW species.

Interestingly, with the exception of the cross-talk between JNK and PKC to activate GSK3, we were able to replicate the signaling pathways regulating mitochondrial Aqp8bb trafficking in piscine spermatozoa in human cells. This finding suggests that these signal-transduction pathways are ancient and were likely present in the last common ancestor of mammals and fishes (>435 million y ago) (52). However, although ROS transport is involved in normal human spermatozoa functioning, and AQP8 has been observed in the midpiece and likely in the mitochondria (53, 54), it is not yet clear whether AQP8 functions as a mitochondrial peroxiporin in mammalian sperm. If indeed AQP8 is an orthologous mitochondrial peroxiporin contributing to the kinematic properties of vertebrate spermatozoa, it will be revealing to identify whether the same JNK and GSK3 pathways are involved in AQP8 trafficking mechanisms.

In mammals, the Ser/Thr kinase GSK3 is encoded by two paralogous genes (GSK3α and GSK3β), the activities of which are regulated by posttranslational Ser²¹/Ser⁹ and Tyr²⁷⁹/Tyr²¹⁶ phosphorylation (37). It is now well recognized that the GSK3α enzyme plays an important role in the acquisition of sperm motility and acrosomal reaction in mammals (55–57). The inhibitory GSK3α^S21 phoshorylation is high during the epididymal sperm maturation stage, but subsequently decreases to activate the kinase activity during the hyperactivation phase in the female oviduct (58–61). This latter mammalian hyperactivation mechanism is reminiscent of the activation of motility in marine fish spermatozoa (31), which is also associated with the dephosphorylation of GSK3α^S21 as demonstrated here in the seabream. In teleosts, however, the role of GSK3 on spermatozoon function has remained largely unknown, although a recent study in zebrafish has shown that the knockout of miR-34a, which normally down-regulates GSK3, enhances sperm motility and the fertilization rates (62). Since we show that GSK3 is not involved in the peroxiporin trafficking mechanism in zebrafish spermatozoa, it seems likely that this kinase controls other elements of the motility. Indeed, the inability of the antioxidant mito-TEMPO to rescue seabream spermatozoa motility, when inhibited by the GSK3 blocker CHIR99021, supports the notion that GSK3 plays additional motility activation roles unrelated to Aqp8bb mitochondrial detoxification.

In contrast to GSK3, the role of the stress-activated JNK cascade for spermatozoon motility in vertebrates is less well known. This pathway is involved in both proapoptotic and antiapoptotic mechanisms in response to various stimuli, such as osmotic and oxidative stress (63). The data available for mammalian spermatozoa suggest a role for JNK in cell survival under osmotic stress (64), as well as during progesterone-induced hyperactivation (65). Such a role of JNK during osmotic stress is consistent with the results of the present study showing the involvement of the JNK signaling pathway controlling Aqp8bb-mediated mitochondrial detoxification in marine spermatozoa. Interestingly, in the nonflagelated spermatozoa of the nematode Caenorhabditis elegans, both Ca²⁺ and the MAPK cascade, which functions downstream of, or parallel with, the Ca²⁺ signaling, are necessary for sperm activation (66). This resembles the molecular scenario controlling Aqp8bb trafficking in seabream spermatozoa. In addition, pharmacological activation of JNK/p38 MAPK is sufficient to trigger sperm motility acquisition in C. elegans (66), while the same treatment of immotile seabream sperm bypasses the requirement of the Ca²⁺ signal for the induction of mitochondrial Aqp8bb transport. Therefore, although nematode and fish spermatozoa are morphologically distinct, and their motility is regulated by different molecular machineries, both types of spermatozoa appear to utilize conserved signaling pathways for different purposes, to modulate sperm maturation in the case of the nematode, or to insert a peroxiporin in the mitochondrion of the seabream spermatozoon as an antiapoptotic mechanism.

Our pharmacological studies in seabream sperm and HepG2 cells suggest a complex cascade of kinase phosphorylation events, where PKC- and JNK-mediate GSK3 activation by GSK3α/β^Y279/216 phosphorylation, and PKC activates JNK and vice versa. However, both PKC and JNK are Ser/Thr kinases, and in ejaculated mammalian spermatozoa PKC inhibits, rather than activates, GSK3 activity by GSK3α^S21 inhibitory phosphorylation (67–69). In contrast, in colon cancer cells the atypical PKCζ isoform can rapidly and transiently activate GSK3β activity through Ser¹⁴⁷ phosphorylation, which is also required to maintain the constitutive basal activity of GSK3β (69). Although a PKCζ ortholog is expressed in fish sperm (70), the role of this specific kinase regulating GSK3 and Aqp8bb mitochondrial transport requires further investigation. The PKC-mediated GSK3αβ^Y279/216 phosphorylation occurring in seabream sperm could also be an indirect mechanism, as it seems to occur during the activation of GSK3 by JNK, or the activation of JNK by PKC. The latter would resemble the Toll-like receptor 4 transient receptor potential channel 1-PKCα signaling pathway that is triggered as defense and proinflammatory response to bacterial infection (71), in which PKC may phosphorylate Ser¹²⁹ in JNK augmenting its phosphorylation by MKK4 (72). The JNK-mediated activation of GSK3 has been reported to take place in a hepatocyte cell line through the MAP3K mixed-lineage kinase 3 in a positive feedback mechanism (73). A similar indirect mechanism mediating the activation of GSK3 by JNK might occur through stimulation of the proline-rich tyrosine kinase 2, which is a Ca²⁺-activated tyrosine kinase in capacitated human sperm (74), or through the Src-related tyrosine kinase Fyn (75).

The present study provides evidence suggesting that piscine Aqp8bb is a physiological substrate of PKC, GSK3, and JNK, which regulate channel trafficking. This is supported by the demonstration that these kinases can phosphorylate the different teleost Aqp8bb orthologs in vitro, and that mutations of the respective target sites trigger or abolish mitochondrial channel transport. In the seabream model, it is also supported by the observation that inhibition of GSK3 and JNK activity through the expression of dominant-negative forms of the kinases in HepG2 cells specifically reduces the phosphorylation of the target sites in the channel and impairs mitochondrial trafficking. The phosphorylation of Aqp8bb by JNK at Thr²⁴ could play a role as a priming mechanism for subsquent GSK3 phosphorylation since our in vitro data show that mimicking a constitutive phosphorylated state of this site can enhance GSK3 phosphorylation of Ser¹⁶ in the channel. This would agree with the observation that under cell stress JNK can phosphorylate antiapoptotic proteins priming them for subsequent phosphorylation by GSK3 (45). However, we found that mutation of Thr²⁴ into Ala in seabream Aqp8bb to prevent its phosphorylation by JNK does not reduce GSK3-mediated Ser phosphorylation of the channel in HepG2 cells during Ca²⁺ and H₂O₂ stimulation. This therefore argues against a role of JNK as a priming kinase for GSK3. Rather, our data indicate that GSK3 and JNK phosphorylation of Aqp8bb are both required for maximum accumulation of the channel in the mitochondria.

In summary, this work presents a mechanism of spermatozoon motility regulation in animals. The data show that a conserved process of mitochondrial ROS detoxification evolved in piscine spermatozoa to enhance their kinematic properties. The detoxification process occurs through the rapid trafficking of an Aqp8bb peroxiporin to the inner mitochodrial membrane to facilitate H₂O₂ efflux and the continued production of ATP necessary for flagellar contractions. The signal transduction cascades controlling the trafficking mechanism are rapidly activated in ejaculated sperm through hydration in FW teleosts and dehydration in SW teleosts, leading to phosphorylation of Ser/Thr residues in the Aqp8bb N terminus. The data further reveal the evolution of an increasingly sophisticated hierarchy of kinases activating the trafficking mechanism with more direct Ca²⁺-PKC or Ca²⁺-PKC-GSK3 induction pathways in ancient lineages of FW teleosts to a cross-talk between the Ca²⁺-PKC-GSK3 signaling mechanism and a ROS-activated JNK multiplier pathway in modern lineages of marine teleosts. These findings uncover gene networks involved in postactivated spermatozoon swimming performance and thus provide insight into the molecular controls that may form selective traits for postcopulatory sexual selection.

Materials and Methods

Detailed information on the methods employed are provided in SI Appendix, Supplementary Materials and Methods.

Procedures relating to the care and use of animals and sample collection were approved by the Ethics Committee of the Institute of Agrifood Research and Technology, following the European Union Council Guidelines (86/609/EU), or in accordance with the regulations approved by the governmental Norwegian Animal Research Authority (http://www.fdu.no/fdu/).

Data Availability

All study data are included in the article and SI Appendix.