In nematodes, spermiogenesis is a process of sperm activation in which nonmotile spermatids are transformed into crawling spermatozoa. Sperm motility acquisition during this process is essential for successful fertilization, but the underlying mechanisms remain to be clarified. Herein, we have found that extracellular adenosine-5′-triphosphate (ATP) level regulation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein (MSP) filament dynamics and sperm motility in the nematode Ascaris suum. During sperm activation, a large amount of ATP was produced in mitochondria and was stored in refringent granules (RGs). Some of the produced ATP was released to the extracellular space through innexin channels. MIG-23 was localized in the sperm plasma membrane and contributed to the ecto-ATPase activity of spermatozoa. Blocking MIG-23 activity resulted in a decrease in the ATP hydrolysis activity of spermatozoa and an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected sperm migration. Overall, our data suggest that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

Unlike mammalian sperm, which are flagellated via the tubulin cytoskeleton, nematode sperm crawl via the major sperm protein (MSP)-based cytoskeleton. Nematode sperm migration requires both protrusion of the leading edge and retraction of the cell body, coupled with adhesion to the substrate (Prass et al., 2006). Previous studies have demonstrated that the assembly and disassembly of MSP filaments regulate sperm motility directly. The dynamics of MSP filaments are modulated by factors such as membrane tension, MSP filament-associated proteins and phosphorylation level. Sperm membrane tension and sperm-specific Na+/K+ -ATPase regulate cell migration by controlling MSP-based cytoskeletal dynamics in Caenorhabditis elegans (Batchelder et al., 2011; Wang et al., 2021). MSP-associated protein phosphorylation and dephosphorylation regulate nematode sperm motility (Yi et al., 2009). Sperm-specific PP1 phosphatases (GSP-3 and GSP-4), which are spatial regulators of MSP disassembly, modulate sperm motility in Caenorhabditis elegans (Wu et al., 2012). In addition, intracellular Ca2+, cholesterol and the biosynthesis of glycosphingolipids have been found to be required for nematode sperm migration (Dou et al., 2012a; Shang et al., 2013). Here, we find that regulation of the extracellular ATP level by MIG-23, which is a homolog of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), is required for sperm migration in Ascaris suum.

ATP, which is an energy molecule or signaling molecule, is required for various physiological functions, including cellular metabolism, cell adhesion, activation, proliferation, differentiation and migration (Fujii et al., 2012). ATP produced by mitochondria is released from cells into the extracellular space via stimulated exocytosis and ATP channels (Lazarowski, 2012; Praetorius and Leipziger, 2009). The released ATP plays a role in cell functions. Human keratinocytes release ATP, and the released ATP acts as feedback to activate its receptors to further elevate the intracellular calcium concentration (Ho et al., 2013). Released ATP also regulates various key physiological processes. For example, it modulates central synaptic transmission in neurons and microglial migration (Dou et al., 2012b; Zhang et al., 2003). In ATP release-defective transgenic mice, synaptic crosstalk is affected and results in heterosynaptic depression (Pascual et al., 2005). Both ATP release and ATP hydrolysis are involved in neutrophil chemotaxis (Chen et al., 2006; Corriden et al., 2008). Extracellular ATP in the female reproductive tract facilitates the bovine spermatozoa acrosome reaction, and this process favors sperm fusion with oocytes (Luria et al., 2002). Extracellular ATP also is involved in spermatozoa chemotaxis in Ascaris suum. (Wang et al., 2022). Intracellular ATP is released into the extracellular space via ATP channels and exocytosis (Dou et al., 2012b; Zhang et al., 2007). ATP that is stored in lysosomes is released through exocytosis in astrocytes (Zhang et al., 2007). Connexin and pannexin are hemichannels that mediate ATP release (Romanov et al., 2007). On the astrocyte cell surface, ATP release channels are colocalized with ecto-ATPase (Joseph et al., 2003). Furthermore, the E-NTPDase family is responsible for hydrolyzing extracellular ATP to ADP and AMP. E-NTPDases control the concentrations of extracellular nucleotides. Eight members of the E-NTPDase family have been identified in mammals. NTPDase1, NTPDase2, NTPDase3 and NTPDase8 are localized on the plasma membrane. These membrane-bound NTPDases are glycoproteins and are present in various tissues, such as the brain, lung, heart and kidney. NTPDase1 is detected in the testis, and NTPDase6 is putatively localized in acrosome vesicles of round spermatids in mice (Martín-Satué et al., 2009). NTPDase1, NTPDase2 and NTPDase8 are localized in the rat uterus (Milošević et al., 2012). NTPDases are involved in multiple and diverse physiological processes, such as pathogen-host interactions, lipid glycosylation, eye development and taste-bud function (Massé et al., 2007; Schachter et al., 2015; Vandenbeuch et al., 2013). Rat NTPDase1 inhibits platelet aggregation and favors blood flow (Zebisch et al., 2012). Deficiency of E-NTPDase1 in mice affects male fertility by reducing the concentration of spermatozoa in the semen (Kauffenstein et al., 2014). Ecto-ATPases, which determine extracellular ATP homeostasis, are present in cells of various types and modulate important physiological functions. MIG-23 in Caenorhabditis elegans, which is an NDPase that interacts with MIG-17 [an ADAM (a disintegrin and metalloprotease)], regulates distal tip cell (DTC) migration (Nishiwaki et al., 2004). Here, we find that in the nematode Ascaris suum, MIG-23, which is a homolog of human NTPDase, is involved in sperm migration by modulating extracellular ATP levels.

Substantial ATP is produced and stored in refringent granules during nematode sperm maturation

Sperm maturation in Ascaris suum is associated with the formation of a motile pseudopodium, fusion of membranous organelles (MOs) with the plasma membrane and coalescence of refringent granules (RGs) (Abbas and Cain, 1981). In addition to these changes in morphology and physiology, we found that the mitochondrial membrane potential (MMP) increased significantly during sperm maturation (Fig. 1A). The MMP reflects the mitochondrial activity (Lachance et al., 2013). It was monitored with a fluorescent indicator, JC-1 dye, which is a monomer molecule permeable to the mitochondrial membrane that emits green fluorescence. JC-1 aggregates emit red fluorescence when MMP levels increase (Chazotte, 2011). JC-1 can be used to measure the MMP of spermatids (nonmature) and spermatozoa (mature) in flow cytometric analysis. As shown in Fig. S1A, the displacement of the spermatozoa population from green fluorescence (FL1 channel) to red fluorescence (FL2 channel) is greater than its displacement the spermatid population. Compared with spermatids, spermatozoa showed a higher intensity of red fluorescent JC-1 aggregates, which suggests that the MMP level in spermatozoa was much higher. The MMP of spermatozoa increased nearly threefold compared with that of spermatids (Fig. 1A). This indicates that spermatozoa generate a much greater supply of ATP to meet needs such as protein phosphorylation, plasma membrane extension, vesicle fusion and cytoskeleton assembly. To measure cytosolic ATP levels during sperm maturation, ATP concentrations were measured using a luciferin-luciferase assay. The results showed that cytosolic ATP levels rose at the beginning of sperm maturation (∼5 min) and subsequently declined (∼15-30 min) (Fig. 1B). However, at the same time point, spermatozoa maintained a higher MMP (∼15-30 min) (Fig. S1B). This raises the interesting issue of why mitochondrial activity is higher but the cytosolic ATP level is lower. Three possible pathways may result in a lower ATP level in the cytosol. First, physiological activities, such as cytoskeleton assembly, protein phosphorylation and sperm motility, consume ATP during sperm maturation (Miao et al., 2003). Second, some ATP may be stored in vesicles. Third, some ATP may be released into the extracellular space. We tested whether nematode sperm are able to store ATP. Quinacrine is an ATP storage marker and indicates ATP localization in cells (Haanes et al., 2014). We found that quinacrine accumulated in dispersed vesicles in spermatids (Fig. 1C). In spermatozoa, quinacrine accumulated in a large organelle (Fig. 1C). The quinacrine intensity of spermatozoa was stronger than that of spermatids. Flow cytometry analyses demonstrated that the fluorescence intensity of quinacrine in spermatozoa was more than twofold higher than that in spermatids (Fig. 1D). The dynamic process of ATP storage was consistent with RG fusion. The quinacrine intensity of sperm increased significantly during sperm maturation. The process was observed and images were obtained with a confocal microscope (Fig. 1E, Movie 1). Next, we tested the possibility of ATP release. The released ATP (in medium) of spermatids and spermatozoa that were activated at the 5th minute was measured (Fig. 2D). Compared with the control, the ATP concentration in the medium increased significantly in spermatids and spermatozoa. This indicates that both spermatids and spermatozoa are able to release ATP from the cytosol into the extracellular space.

Fig. 1.

Substantial ATP is produced during sperm maturation. (A) Mitochondrial membrane potentials (MMPs) in spermatids and spermatozoa were summarized and then normalized. To avoid interference from inter-worm variation, when measuring sperm from every individual worm, the MMP of spermatozoa was normalized by dividing the JC-1 red/green fluorescence ratio of spermatozoa by the value from spermatids from the same worm. The MMP of spermatids was defined as 1. (B) The cytosolic ATP levels were measured with the luciferin-luciferase method at a few time points during sperm activation and were normalized to cytosolic ATP concentration at the beginning of the measurements. (C) Spermatids and spermatozoa were loaded with quinacrine. ATP was stored in refringent granules (RGs) in spermatids and spermatozoa. (D) Fluorescence intensity of quinacrine. Spermatids and spermatozoa were stained with quinacrine and analyzed using a flow cytometer. (E) The dynamics of ATP storage and RG fusion during sperm maturation. Data are mean±s.e.m. **P<0.01 (Student's t-test). Scale bars: 5 µm.

Fig. 1.

Substantial ATP is produced during sperm maturation. (A) Mitochondrial membrane potentials (MMPs) in spermatids and spermatozoa were summarized and then normalized. To avoid interference from inter-worm variation, when measuring sperm from every individual worm, the MMP of spermatozoa was normalized by dividing the JC-1 red/green fluorescence ratio of spermatozoa by the value from spermatids from the same worm. The MMP of spermatids was defined as 1. (B) The cytosolic ATP levels were measured with the luciferin-luciferase method at a few time points during sperm activation and were normalized to cytosolic ATP concentration at the beginning of the measurements. (C) Spermatids and spermatozoa were loaded with quinacrine. ATP was stored in refringent granules (RGs) in spermatids and spermatozoa. (D) Fluorescence intensity of quinacrine. Spermatids and spermatozoa were stained with quinacrine and analyzed using a flow cytometer. (E) The dynamics of ATP storage and RG fusion during sperm maturation. Data are mean±s.e.m. **P<0.01 (Student's t-test). Scale bars: 5 µm.

Fig. 2.

Refringent granules are lysosome-related and ATP-containing organelles. (A) Both quinacrine and LysoTracker were enriched in no-fused refringent granules (RGs) in spermatids and in fused RG in spermatozoa. Signals of quinacrine and LysoTracker were attenuated by GPN. Scale bar: 5 μm. (B) Fluorescence intensities of quinacrine and LysoTracker in spermatids (T) and spermatozoa (Z) were analyzed using flow cytometry and then quantified. Compared with control, GPN treatment significantly decreased fluorescence intensities of quinacrine and LysoTracker in spermatids and spermatozoa. (C) Transmission electron microscope images show the ultrastructure of the spermatid, the spermatozoon in vitro and the spermatozoa in the female reproductive tract. RGs (indicated by yellow arrows) show different morphology in these three cell types. Mitochondria are indicated by red arrows. A mitochondrion in the spermatid is indicated by a green arrow. A fused mitochondrion in the spermatozoon is indicated by a green arrowhead. (D) Both spermatids (T) and spermatozoa (Z) released ATP into extracellular space. Data are mean±s.e.m. **P<0.01 (Student's t-test).

Fig. 2.

Refringent granules are lysosome-related and ATP-containing organelles. (A) Both quinacrine and LysoTracker were enriched in no-fused refringent granules (RGs) in spermatids and in fused RG in spermatozoa. Signals of quinacrine and LysoTracker were attenuated by GPN. Scale bar: 5 μm. (B) Fluorescence intensities of quinacrine and LysoTracker in spermatids (T) and spermatozoa (Z) were analyzed using flow cytometry and then quantified. Compared with control, GPN treatment significantly decreased fluorescence intensities of quinacrine and LysoTracker in spermatids and spermatozoa. (C) Transmission electron microscope images show the ultrastructure of the spermatid, the spermatozoon in vitro and the spermatozoa in the female reproductive tract. RGs (indicated by yellow arrows) show different morphology in these three cell types. Mitochondria are indicated by red arrows. A mitochondrion in the spermatid is indicated by a green arrow. A fused mitochondrion in the spermatozoon is indicated by a green arrowhead. (D) Both spermatids (T) and spermatozoa (Z) released ATP into extracellular space. Data are mean±s.e.m. **P<0.01 (Student's t-test).

In addition, we tested whether ATP that is produced by mitochondria is required for sperm maturation. Inhibitors of mitochondrial function, such as CCCP (an uncoupler of oxidative phosphorylation), antimycin (an inhibitor of cellular respiration, specifically oxidative phosphorylation) and oligomycin (an inhibitor of ATP synthase) were used to treat sperm. All these reagents inhibited ATP production (Fig. S1E) and blocked the sperm pseudopodia extension and MO fusion that are induced by SASs (sperm-activated substrates), which were extracted from vas deference (Abbas and Cain, 1979) (Fig. S1C,D). These results suggest that ATP is essential for sperm maturation.

RGs are specialized organelles that are enriched with amino acids, polypeptides and sugars in Ascaris suum sperm (Abbas and Cain, 1981, 1984). During sperm maturation, the small RGs fused with each other and formed a large organelle. Therefore, the sizes and shapes of the RGs differed between spermatids and spermatozoa (Movies 2 and 3). Furthermore, the sizes of the RGs in spermatozoa in vitro and in vivo were different. The RGs of spermatozoa in the female reproductive tract disappeared (Fig. 2C). This indicates that RGs may play a role in sperm fertilization with oocytes. However, the characteristics and functions of RGs remain unknown. To further characterize ATP storage vesicles in Ascaris suum sperm, spermatids and spermatozoa were stained with quinacrine and LysoTracker, a fluorescent dye for labeling acidic organelles. ATP storage vesicles that were visualized with quinacrine could also be labeled with LysoTracker (Fig. 2A). This indicates that the RGs are acidic organelles. To further study the characteristics of the RGs, the lysosomotropic compound glycyl-L-phenylalanine 2-naphthylamide (GPN), which is a lysosome-disrupting cathepsin C substrate that causes reversible permeabilization of lysosomes by osmotic swelling, was used to treat sperm. After spermatids and spermatozoa were treated with GPN, the fluorescence intensities of quinacrine and LysoTracker were attenuated significantly (Fig. 2A). To study the effect of GPN on the storage of ATP and protons, flow cytometry analysis was performed to quantify the fluorescence intensities of quinacrine and LysoTracker in sperm treated with or without GPN. The storage of ATP and protons decreased by almost 40% in spermatids and spermatozoa upon GPN treatment (Fig. 2B). Our data suggest that RG is not only an ATP-containing organelle but also a lysosome-related organelle. Therefore, ATP is stored in acidic organelles in Ascaris sperm. This indicates that an appreciable amount of ATP is produced by mitochondria and is stored in RGs.

Nematode sperm release ATP into the extracellular space via innexin channels

Neural or immune cells are able to release ATP via channels or exocytosis (Guthrie et al., 1999; Junger, 2011; Loiola and Ventura, 2011). Innexins in invertebrates and pannexins in vertebrates mediate ATP release (Dahl et al., 2013; Pinheiro et al., 2013; Samuels et al., 2010; Sandilos et al., 2012). We tested whether nematode sperm release ATP into the extracellular space through innexin channels. First, we measured ATP concentration of sperm medium (Fig. 3D). Spermatids were washed with buffer, and the ATP concentration in the medium was measured at two time points (0 and the 10th minutes). The ATP concentration in the medium of spermatids (2×107 cells in 500 μl of solution) was 1.93 μM initially and increased to 2.66 μM by the 10th minute (Fig. 3D); this indicates that nematode spermatids are able to release ATP through autocrine signaling. Second, we tested whether innexins mediated ATP release. Spermatids were treated with carbenoxolone (CBX, 100 μM), which is an innexin channel blocker. CBX attenuated ATP release from 2.66 μM to 1.51 μM at the 10th minute (Fig. 3D), suggesting that ATP release in spermatids was probably mediated by innexins. There are 25 members of the innexin family in Caenorhabditis elegans, three of which are expressed in sperm (Altun et al., 2009). In Ascaris suum, transcripts of innexin-3 and innexin-11 were detected in the testis (Fig. 3A). We raised Innexin-3 antibody to detect its localization on sperm. Innexin-3 (GenBank: ERG82140.1) is composed of 429 amino acids, and its molecular weight is ∼50 kDa. In an immunoblotting assay, a band of ∼50 kDa was detected in the plasma membrane but not in the cytosol of Ascaris sperm (Fig. 3B and Fig. S2A). Moreover, these signals in the plasma membrane decreased dramatically when the antibody was preincubated with peptide antigen. No bands were detected when samples were probed with preimmune serum (Fig. 3B). In the immunostaining assay, as shown in Fig. 3C, Innexin-3 was localized on the plasma membrane of spermatids and spermatozoa. The cytosolic signals were probably nonspecific because they were not blocked by peptide antigen. We cannot rule out the possibility that the Innexin-3 antibody we raised also binds other proteins, considering that the antigen peptide was not able to completely block the non-specific signals in immunoblotting (Fig. 3B) and immunofluorescence (Fig. 3C) assays. However, the signals in the plasma membrane could be blocked specifically by the Innexin-3 antigen peptide, which provides compelling evidence for the localization of Innexin-3 in the plasma membrane. To further elucidate the role of ATP release in sperm maturation, CBX (100 μM), which was demonstrated to inhibit ATP release, was used, and we found that CBX blocked more than 50% of sperm activation (Fig. S2B,C). This indicates that innexin-mediated ATP release plays a role in modulating sperm activation.

Fig. 3.

ATP is released through innexin channels. (A) RT-PCR results demonstrated that transcripts of innexin-3 and innexin-11 were detected in testis. (B) In western blotting analysis, Innexin-3 was detected in the plasma membrane fractions of spermatids and spermatozoa using an Innexin-3 antibody. Peptide antigen and preimmune serum were used to distinguish signals of Innexin-3 from nonspecific signals. PM, plasma membrane; cyto, cytosol. (C) Immunofluorescence analysis of Innexin-3 expression in spermatids and spermatozoa. Innexin-3 was expressed in sperm plasma membrane. Peptide antigen was used to distinguish signals of Innexin-3 from nonspecific signals. Scale bars: 5 µm. (D) ATP levels in spermatid medium were measured and found to increase after 10 min. CBX, an ATP channel blocker, blocked this process. Data are mean±s.e.m. **P<0.01 (Student's t-test).

Fig. 3.

ATP is released through innexin channels. (A) RT-PCR results demonstrated that transcripts of innexin-3 and innexin-11 were detected in testis. (B) In western blotting analysis, Innexin-3 was detected in the plasma membrane fractions of spermatids and spermatozoa using an Innexin-3 antibody. Peptide antigen and preimmune serum were used to distinguish signals of Innexin-3 from nonspecific signals. PM, plasma membrane; cyto, cytosol. (C) Immunofluorescence analysis of Innexin-3 expression in spermatids and spermatozoa. Innexin-3 was expressed in sperm plasma membrane. Peptide antigen was used to distinguish signals of Innexin-3 from nonspecific signals. Scale bars: 5 µm. (D) ATP levels in spermatid medium were measured and found to increase after 10 min. CBX, an ATP channel blocker, blocked this process. Data are mean±s.e.m. **P<0.01 (Student's t-test).

MIG-23, which is a homolog of E-NTPDase, contributes to the ecto-ATPase activity of spermatozoa

To further study the correlation between extracellular ATP and sperm function, we measured the extracellular ATP level at a few time points when spermatids were activated. As shown in Fig. 4A, the extracellular ATP level increased nearly twofold compared with the control 5 min after SASs were added. This was consistent with the intracellular ATP level (Fig. 1B). However, after 5 min, the extracellular ATP level decreased gradually (Fig. 4A). This raises the question of why extracellular ATP is attenuated significantly during sperm maturation but mitochondrial activity is higher in the same time? This suggests that released ATP is hydrolyzed by ecto-ATPase on spermatozoa. We hypothesize that ecto-ATPases on the sperm surface are activated during sperm maturation and degrade released ATP. To test this hypothesis, exogenous ATP (5 μM) was added to spermatozoa (107 cells in 500 μl solution). The spermatozoa hydrolyzed 80% of the exogenous ATP (5 μM) within 5 min (Fig. 4B). However, when exogenous ATP (5 μM) was added to spermatids (107 cells in 500 μl solution), they did not hydrolyze the exogenous ATP (Fig. 4B). This suggests that ecto-ATPase activity of spermatozoa is gained during sperm maturation. Ecto-nucleoside triphosphate diphosphohydrolase, ecto-phosphodiesterases/pyrophosphatases, ecto-5′-nucleotidase and alkaline phosphatases are able to hydrolyze extracellular ATP (Sansom et al., 2008). It has been reported that ZnCl2, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), reactive blue-2 (RB-2) and suramin decrease mammalian E-NTPDase activity (Baqi et al., 2009; Barros et al., 2000; Dowd et al., 1999; Iqbal et al., 2005; Leal et al., 2005). We tested whether these inhibitors also functioned in Ascaris suum sperm. Spermatozoa (2×107 cells in 500 μl solution) were treated with ZnCl2 (500 µM), DIDS (10 µM), RB-2 (50 µM) and suramin (10 µM) for 10 min, and exogenous ATP (5 µM) was added. As shown in Fig. S3, these inhibitors, especially RB-2, significantly impeded exogenous ATP degradation by spermatozoa. These data demonstrate that E-NTPDases contribute to the ecto-ATPase activity of spermatozoa in Ascaris suum.

Fig. 4.

MIG-23 is required for sperm function. (A) Extracellular ATP levels at a few time points were measured after spermatids were activated. Extracellular ATP levels increased within 5 min and then decreased gradually. (B) Spermatozoa showed ecto-ATPase activity and degraded exogenous ATP. However, spermatids did not degrade exogenous ATP. (C) A transcript of mig-23 was detected in testis. −, no cDNA added; +, cDNA added. (D) Anti-MIG-23 antibody detected the signal in the plasma membrane (PM) but not in cytosol (Cyto) in the immunoblotting assay (left panels). PM and cytosol fractions were analyzed by SDS-PAGE (right panels). CBB, Coomassie Brilliant Blue (E) In the immunostaining assay, non-permeabilized spermatids and spermatozoa were labeled using anti-MIG-23 antibody. MIG-23 was localized on the plasma membrane. (F) MIG-23 antiserum partially attenuated the process in which exogenous ATP (5 μM) was degraded by spermatozoa. (G) Sperm treated with MIG-23 antiserum showed normal mitochondria fusion indicated by punctate signals of FM 1-43, a membrane probe, during sperm maturation. However, these sperm formed round pseudopodia in which major sperm protein (MSP) based filaments were not maintained. As a control, sperm treated with preimmune serum showed normal mitochondria fusion and pseudopodia. Data are mean±s.e.m. *P<0.05 (Student's t-test). Scale bars: 5 µm.

Fig. 4.

MIG-23 is required for sperm function. (A) Extracellular ATP levels at a few time points were measured after spermatids were activated. Extracellular ATP levels increased within 5 min and then decreased gradually. (B) Spermatozoa showed ecto-ATPase activity and degraded exogenous ATP. However, spermatids did not degrade exogenous ATP. (C) A transcript of mig-23 was detected in testis. −, no cDNA added; +, cDNA added. (D) Anti-MIG-23 antibody detected the signal in the plasma membrane (PM) but not in cytosol (Cyto) in the immunoblotting assay (left panels). PM and cytosol fractions were analyzed by SDS-PAGE (right panels). CBB, Coomassie Brilliant Blue (E) In the immunostaining assay, non-permeabilized spermatids and spermatozoa were labeled using anti-MIG-23 antibody. MIG-23 was localized on the plasma membrane. (F) MIG-23 antiserum partially attenuated the process in which exogenous ATP (5 μM) was degraded by spermatozoa. (G) Sperm treated with MIG-23 antiserum showed normal mitochondria fusion indicated by punctate signals of FM 1-43, a membrane probe, during sperm maturation. However, these sperm formed round pseudopodia in which major sperm protein (MSP) based filaments were not maintained. As a control, sperm treated with preimmune serum showed normal mitochondria fusion and pseudopodia. Data are mean±s.e.m. *P<0.05 (Student's t-test). Scale bars: 5 µm.

Mammalian E-NTPDases 1, 2, 3 and 8 localize on the plasma membrane. They regulate multiple and diverse physiological processes in cells, such as neutrophil chemotaxis, T-cell activation and set points of cardiac fibroblasts (Corriden et al., 2008; Knowles, 2011; Lu and Insel, 2013; Robson et al., 2006). We performed BLAST with the human E-NTPDase 2 sequence and obtained the homolog nucleoside-diphosphatase MIG-23 (GenBank: ERG84046.1) in Ascaris suum. MIG-23 has 551 amino acids, and its molecular weight is ∼60 kDa. It shares 41% similarity with mammalian E-NTPD1, 42% with E-NTPD2, 42% with E-NTPD3 and 41% with E-NTPD8. MIG-23 is conserved in various species. The alignments of MIG-23 with E-NTPD2 in C. elegans, Homo sapiens, Mus musculus and Xenopus tropicalis are shown in Fig. S4. The transcription of mig-23 was detected in the testis (Fig. 4C). MIG-23 peptide antibody was raised to investigate the precise localization of MIG-23 in sperm. In an immunoblotting assay, a band of ∼60 kDa was detected in the plasma membrane extracts but was absent in the cytosolic extracts (Fig. 4D). Minor bands were also detected by this antibody. This may be due to degradation of MIG-23 or binding of antibody to other proteins. However, the intensities of these minor bands were much lower than that of the major band, which suggests that the antibody binds to MIG-23 more efficiently. In immunofluorescence assays, when sperm were not permeabilized, MIG-23 signals were detected on the plasma membrane (Fig. 4E). MIG-23 is predicted to have two transmembrane domains and a large extracellular loop. The antigen peptide for antibody production was between the two transmembrane domains. Thus, our results of immunofluorescence (Fig. 4E) testify the predicted orientation of MIG-23 in the plasma membrane. Both N- and C-termini of MIG-23 are located intracellularly, and the loop region between the two transmembrane domains faces the extracellular space. To test whether the functional domain of MIG-23 is present on the surface of sperm, MIG-23 antiserum (1:100 dilution) was incubated with spermatozoa to block MIG-23 activity. The MIG-23 antiserum significantly attenuated the ability of spermatozoa to hydrolyze exogenous ATP (5 µM) at the 10th and the 15th minutes (Fig. 4F). This indicates that the functional domain of MIG-23 localizes in the extracellular region and that its activity can be blocked by MIG-23 antiserum.

MIG-23 is required for MSP-based filament assembly and sperm migration

To further evaluate the role of MIG-23 in sperm function, MIG-23 activity was blocked by MIG-23 antiserum (1:100 dilution). Spermatids that were preincubated with MIG-23 antiserum were not activated by SASs normally, although MOs fused with the plasma membrane (Fig. 4G). These sperm showed round pseudopodia without leading edge protrusions during early sperm activation (Fig. 4G), thereby indicating abnormal MSP filament assembly and disassembly in these sperm. Moreover, spermatozoa that were pretreated with MIG-23 antiserum were not able to migrate in vitro (Fig. 5, Movies 4 and 5). Nematode spermatozoa migration is based on the MSP filament cytoskeleton. Cytoskeleton assembly and disassembly are tightly coupled with sperm migration (Bottino et al., 2002; Wolgemuth et al., 2005). To examine the effects of MIG-23 on MSP filament dynamics, we captured images of MSP filament dynamics in vitro (Movies 6 and 7). In a cell-free system, MSP fibers reconstituted using extracts of sperm pretreated with MIG-23 antiserum grew more slowly than fibers reconstituted using extracts of preimmune serum-treated sperm (Movies 6 and 7), which suggests that the balance between disassembly and assembly of MSP filaments was destroyed because of the lack of MIG-23 activity. Thus, the activity of MIG-23 is required for MSP cytoskeleton dynamics. Together, these data indicate that the membrane protein MIG-23 plays an essential role in regulating sperm migration. Cytoskeletal dynamics are modulated precisely by factors such as MSP-associated proteins, protein phosphorylation and dephosphorylation (Yi et al., 2009). MIG-23, which is a membrane protein with a functional domain localized on the outer membrane, regulates sperm MSP filament dynamics and migration by modulating extracellular ATP levels.

Fig. 5.

MIG-23 regulates motility of sperm. (A) Time-lapse images of sperm migration on the glass slide. Sperm treated with MIG-23 antiserum showed impaired motility. Representative cells are indicated by different colored asterisks to show the motility of individual cells. Scale bar: 5 µm. (B) Statistical analysis of velocities of sperm treated with preimmune serum or with MIG-23 antiserum. Data are mean±s.e.m. **P<0.01 (Student’s t-test).

Fig. 5.

MIG-23 regulates motility of sperm. (A) Time-lapse images of sperm migration on the glass slide. Sperm treated with MIG-23 antiserum showed impaired motility. Representative cells are indicated by different colored asterisks to show the motility of individual cells. Scale bar: 5 µm. (B) Statistical analysis of velocities of sperm treated with preimmune serum or with MIG-23 antiserum. Data are mean±s.e.m. **P<0.01 (Student’s t-test).

Previous work has demonstrated that, during sperm maturation in Ascaris suum, the MSP-based cytoskeleton is assembled, pseudopodia form, membranous organelles fuse with the plasma membrane and granular organelles fuse with each other. In addition, we found that the MMP increased significantly and that a large amount of ATP was produced to satisfy physiological needs during sperm maturation (Fig. 1A,B). The produced ATP was rearranged by sperm efficiently. Otherwise, abundant ATP in the cytosol might have been unfavorable for sperm maturation. Some ATP was stored in RGs (Figs 1C-E and 2A), although the mechanism remains unclear. Our data suggest that RGs are acidic organelles (Fig. 2A,B). To our knowledge, this is the first study demonstrating that RGs are ATP-containing vesicles. Some ATP was released into the extracellular space through innexin channels that were localized on the plasma membrane of sperm (Fig. 3). Therefore, nematode sperm are able to release ATP into the extracellular space. ATP release in astrocytes modulates depressive-like behavior in mice and is mediated by calcium and V-ATPase (Cao et al., 2013; Coco et al., 2003). Exogenous ATP treatment of human sperm improves in vitro fertilization (IVF) results by increasing sperm motility (Rodríguez-Miranda et al., 2008). In the nematode Ascaris suum, we have identified that ATP release plays a role in sperm activation. In some species, ATP is released through ATP channels and exocytosis (Taruno, 2018; Bao et al., 2004; Chekeni et al., 2010; Zhang et al., 2007). Pannexin in vertebrates and innexin in invertebrates mediate ATP release (Lu et al., 2015). Pannexin 3, which is one of three members of the pannexin family, is present in the epididymis of adult rats. It might play a role in the maturation and transport of sperm (Turmel et al., 2011). The function of INX-14 in the nematode C. elegans is essential for guiding spermatozoa to oocytes (Edmonds et al., 2011). We found that sperm of the nematode Ascaris suum were able to release ATP through Innexin-3 and Innexin-11. The mRNAs of innexin-3 and innexin-11 were expressed in Ascaris suum testis (Fig. 3A), and the Innexin-3 protein was localized on the plasma membrane of sperm (Fig. 3B,C). Carbenoxolone (CBX), which is a blocker of ATP channels, inhibited ATP release and attenuated sperm maturation (Fig. 3D and Fig. S2). This indicates that ATP is released via ATP channels and that this release is required for sperm function. Similar to RGs in Ascaris suum sperm, acrosomes in mammalian sperm are acidic organelles. Our results suggest that RGs are ATP storage sites in Ascaris suum sperm, which suggests that acrosomes may play similar roles in mammalian sperm. During the acrosome reaction, acrosomes fuse with the plasma membrane and expose their contents to facilitate sperm-egg recognition and fertilization. ATP stored in acrosomes may be released via exocytosis to regulate sperm motility and other activities of sperm to ensure successful fertilization.

Released ATP is not stable and is hydrolyzed by E-NTPDases (Lu and Insel, 2013). This process facilitates various cell functions. For example, extracellular ATP hydrolysis blocks synaptic transmission (Vroman et al., 2014). Ascaris spermatozoa show ecto-ATPase activity (Fig. 4B). In Ascaris suum, MIG-23 contributes to the ecto-ATPase activity of spermatozoa (Fig. 4D-F). Human membrane E-NTPDase contains an active site that is exposed to the extracellular space (Chiang and Knowles, 2008). Similar to human membrane E-NTPDase, the functional domain of MIG-23 is also exposed to the extracellular space. The ecto-ATPase activity of spermatozoa decreased partially when MIG-23 activity was blocked by MIG-23 antibody (Fig. 4F). Spermatozoa that were pretreated with MIG-23 antiserum showed migration defects because MSP filament assembly was slower than disassembly (Fig. 5, Movies 4-7). This led to depolymerization of the MSP filament cytoskeleton. Taken together, we conclude that MIG-23 is involved in sperm function by modulating extracellular ATP levels. MIG-23 is important for sperm migration and is a newly identified component of fertility pathways in Ascaris suum. How does MIG-23 regulate sperm motility via the activity of ecto-ATPase? What are the roles of ATP in sperm activation and sperm motility? Our results suggest that ATP produced in mitochondria is crucial for sperm activation (Fig. S1). The intracellular ATP level must be precisely regulated via ATP storage (Fig. 2) and release (Fig. 3) to modulate functions of sperm. Our data show that the volume of RGs dramatically decreases after sperm enter the female reproductive tract (Fig. 2C), which suggests that RGs may act as the arsenal of sperm and ATP stored in RGs may be used in sperm migration and fertilization. The released ATP can be hydrolyzed by MIG-23 and other E-NTPDases to produce ADP and AMP. It has been extensively reported that extracellular ATP, ADP and adenosine modulate a multiplicity of cellular functions by binding the purinergic receptors in the plasma membrane to trigger the downstream signaling cascade (Burnstock, 2018). The ligand-gated ion channel P2X2, an ATP receptor, has been found in mouse sperm. ATP-gated current mediated by P2X2 confers a selection advantage in sperm competition under frequent mating conditions (Navarro et al., 2011). However, the source of extracellular ATP that activates P2X2 and the intracellular signaling cascades mediated by P2X2 remains elusive. Our data imply that sperm can provide extracellular nucleotides in an autocrine manner. Mouse sperm might release ATP via exocytosis of acrosome and other channels in the plasma membrane to activate P2X2. In Ascaris suum sperm, the activities of MIG-23 and other E-NTPDases can produce ADP and AMP. Together with ATP and adenosine, these signaling molecules might bind to their receptors to regulate MSP cytoskeleton dynamics, thus modulating motility and other activities of sperm. Future studies to identify purinergic receptors in Ascaris suum sperm will extend understanding of the mechanisms of ATP- and MIG-23-regulated functions in sperm, and will contribute to studies in mammalian sperm and other cell types.

Worm harvest and sperm preparation

Ascaris suum male worms were collected from slaughterhouse and were kept in worm buffer [PBS containing 100 mM NaHCO3 (pH 7.0)] at 37°C. Male worms were dissected to release sperm (spermatids) from seminal vesicles into HKB buffer [50 mM HEPES, 70 mM KCl and 10 mM NaHCO3 (pH 7.6 for stock, adjust pH to 7.1 with CO2 before use)].

Sperm activation

Spermatids were activated in HKB buffer at 37°C for 10 min with the addition of the SASs. Pseudopod extension and fusion of membranous organelles with the plasma membrane were considered as hallmarks of sperm activation. SASs were extracted from the male vas deferens. The tails of male worms were dissected and the glandular vas deferens was collected into HKB buffer and homogenized with Douncer on ice. The homogenate was centrifuged at 12,000 g at 4°C for 30 min to remove cell debris and the remaining supernatant was the SASs.

Measurement of MMP

The MMP of sperm was measured with JC-1 kit (Beyotime). Sperm were stained with JC-1 (5 μg/ml) at 37°C for 20 min and then washed twice with washing buffer in the kit. Sperm were analyzed in a flow cytometer. Fluorescence intensities were measured using the standard emission filters for green fluorescence (FL1 channel) and red fluorescence (FL2 channel). The parameters were set as λex 488 nm and λem 530 nm for JC-1 monomer, and λex 525 nm and λem 590 nm for JC-1 aggregates. A total of 30,000 sperm from each sample were measured in a FACSCalibur flowcytometer (BD Biosciences) and the acquired data were analyzed with the FCS Express 5 software (De Novo Software).

Measurement of ATP concentrations

ATP concentration was measured using ATP assay kit (Beyotime). In this assay, luciferin oxidation was catalyzed by the firefly luciferase in the presence of ATP. Luminescence emitted was detected with a Sirius luminometer (Berthold Detection Systems). In measurements of extracellular ATP, spermatids were washed twice with HKB buffer. At the indicated time points, 50 μl of sperm suspensions were collected and centrifuged at 12,000 g for 1 min to separate sperm from medium. The concentrations of extracellular ATP in medium could be measured immediately. To detect cytosolic ATP concentration, sperm were lysed with lysis buffer in the kit and centrifuged at 13,000 g for 10 min at 4°C to remove cell debris. The supernatant was collected for cytosolic ATP measurement. The protein concentrations in the sperm lysate were determined using BCA protein assay kit (Beyotime) and were used for normalization.

In exogenous ATP degradation assay, 5 μM ATP was added into spermatozoa suspensions (HKB buffer as control). At the indicated time, 50 μl of samples were collected and centrifuged at 12,000 g for 1 min. Concentrations of ATP in supernatants were measured to indicate the amount of remaining exogenous ATP in extracellular medium. To test the effects of ecto-ATPase inhibitors, spermatozoa were pre-incubated with inhibitors of ecto-ATPase (ZnCl2 250 μM, DIDS 5 μM, RB-2 50 μM, suramin 10 μM) at 37°C for 10 min (for 20 min for MIG-23 antiserum and preimmune serum), then 5 μM exogenous ATP was added into sperm suspensions. 50 μl of samples were collected at serial time points and then centrifuged at 12,000 g for 1 min. Concentrations of ATP in supernatants were measured to indicate the amount of remaining exogenous ATP in extracellular medium.

Blockage of sperm activation triggered by SASs

In HKB buffer, spermatids were pre-incubated with inhibitors (CBX 100 μM, CCCP 10 μM, antimycin A 10 μM, oligomycin 10 μM) at 37°C for 10 min (for 20 min for MIG-23 antiserum and preimmune serum diluted at 1:100). SASs were then added to activate sperm. Sperm were stained with FM 1-43 (5 μg/ml) for 5 min and then observed under an FV1200 confocal microscope equipped with a 60×/1.35 NA oil immersion objective (Olympus) in the GFP channel to detect MO fusion. FM 1-43 showed punctate signal in activated sperm. When sperm activation was blocked, the signal of FM 1-43 was evenly distributed on the surface of sperm, as it was in spermatids.

Subcellular fractionation of sperm

Sub-cellular fractionation was performed according to previous studies (Ren et al., 2006). Sperm were washed twice with HKB buffer then were resuspended in 5 ml lysis buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 1×protease inhibitor cocktail and 250 mM sucrose) and incubated on ice for 30 min. After 30 strokes of Dounce homogenization, the homogenate was layered onto 5 ml lysis buffer (1 M sucrose) and centrifuged at 6000 g for 10 min at 4°C. Upper phase (cytoplasmic fraction) was transferred and centrifuged twice at 9800 g for 10 min at 4°C. Then the supernatant was collected and centrifuged at 100,000 g for 30 min at 4°C. The supernatant obtained was S-100 media (cytosol without mitochondria) and the pellet was plasma membrane.

RT-PCR assay

cDNAs were synthesized from total RNA in testis of Ascaris suum. To extract total RNA from testis, Testis and seminal vesicle were collected from dissected male worms and were immediately put into liquid nitrogen before storage at −80°C. Total RNAs were prepared using TRIzol reagent (Invitrogen). cDNAs were synthesized using SuperScrip III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions.

Primers used for PCR were: innexin-3 F, 5′-CTCGAGATGTTCCTCGGTATCCCACAACTGA-3′; innexin-3 R, 5′-GGTACCACGCGGTAACTCTTTCATCGGCAGTG-3′; innexin-6 F, 5′-AAGCTTATGAGTTCACAAATTGGCGCAATCG-3′; innexin-6 R, 5′-GGATCCGACGGCTTTATTTCCTTTCTGGAGC-3′; innexin-10 F, 5′-AAGCTTATGGTGCTCACAACGGTCCTTTCAA-3′; innexin-10 R, 5′-GGATCCGATGATAAGATTTGGGACCTTCTTTGGCG-3′; innexin-11 F, 5′-CTCGAGATGATGATCGAAAGTCTCATGGCGA-3′; innexin-11 R, 5′-GGTACCGTCATCGGAAGGTGTTTTCGGTGAG-3′; mig-23 F, 5′-GCGGCCGCATGGTGCGAGGCATGCTGAG-3′; mig-23 R, 5′-GGATCCGAAAAGCTTGGTATATTGTAAT-3′; actin F, 5′-AATCAAAGCGAGGTATCCTCAC-3′; and actin R, 5′-TGGGTCATCTTTTCTCTGTTTG-3′. Annealing temperatures and PCR cycle numbers were 56°C, 28 cycles for innexins and actin, and 58°C, 30 cycles for mig-23.

Western blotting and immunostaining assay

Both Innexin-3 polypeptide antibody and MIG-23 polypeptide antibody were raised in rabbit. The peptide sequence of Innexin-3 was PDEISRPLSALQGDPIDD. Peptide sequence of MIG-23 was RDVQRRYLLDKRR. Both antibodies were purified. Western blotting was performed according to standard methods. Both Innexin-3 antibody and MIG-23 antibody were diluted at 1:500. In the immunofluorescence assay, sperm were fixed with 1.25% glutaraldehyde in HKB buffer and then were permeabilized with 0.5% Triton X-100 or not permeabilized. Sperm were incubated with blocking solution (2% BSA in PBS) for 4 h at room temperature and were then incubated with primary antibody (anti-Innexin-3, 1:50, Hanlin Biotechnology; anti-MIG-23, 1:50, Hanlin Biotechnology) overnight at 4°C. After a PBS wash, secondary antibodies (anti-rabbit IgG-Alexa Fluor 488, 1:400, Abcam, ab15007) were incubated with sperm for 2 h at room temperature. After PBS wash, the coverslips with sperm were sealed and observed under a confocal microscope (63×/NA 1.4 oil objective, Zeiss) in the Alexa Fluor 488 channel.

Transmission electron microscopy

All the sperm were fixed with GTS-Fixative (2.5% glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin in HKB) for 40 min on a Thermanox plastic coverslip (electron microscopy, EM), followed by washing in HKB buffer and then water. Samples were post-fixed in 1% osmium tetroxide for 30 min, dehydrated in a graded series of ethanol followed by propylene oxide, and then infiltrated and embedded with EMbed-812 resin (EM). Ultrathin sections (80 nm) were cut on a Leica UC6 ultramicrotome, collected on formvar-coated copper grids, and stained with uranyl acetate and lead citrate. TEM images were captured using an FEI Spirit 120 kV electron microscope operated at 100 kV.

Preparation of sperm extracts for MSP fiber assay in vitro

Spermatids were treated with preimmune serum or MIG-23 antiserum (1:100 dilution) at 37°C for 20 min. SASs together with preimmune serum or MIG-23 antiserum were added to activate sperm. Sperm were harvested after 10 min and were centrifuged at 12,000 g for 1 min. After removing the supernatant, the cell pellets were frozen at −80°C overnight and then thawed on ice. Sperm were centrifuged at 23,000 g for 15 min at 4°C and then the supernatant was collected and centrifuged at 100,000 g for 1 h at 4°C. The supernatant (S100) was used for the fiber assay. In this assay, S100 was diluted at 1:5 in KPM buffer [0.5 mM MgCl2 and 10 mM potassium phosphate (pH 6.8)] and 1 mM ATP was added. After 10 min, the solutions were pipetted into glass chambers and examined on an Axio Imager M2 microscope (Carl Zeiss) equipped with a 40×/NA 0.95 Ph3 Korr objective. Images were acquired using a Zeiss Axiocam digital camera and were analyzed using the MetaMorph software.

We thank Drs Long Miao and Tao Jiang for providing comments on the manuscript. We thank the staff members at the Center for Biological Imaging and Flow Cytometry Core Facility in Institute of Biophysics, Chinese Academy of Sciences for data collection.

Author contributions

Methodology: R.H., J.S.; Software: Q.Z.; Validation: R.H.; Formal analysis: Q.W.; Investigation: Q.W., R.H., J.S.; Resources: Q.Z.; Data curation: Q.Z.; Writing - original draft: Q.W.; Writing - review & editing: X.W.; Visualization: L.C.; Supervision: X.W.; Project administration: X.W., P.W., Y.Z.; Funding acquisition: Y.Z.

Funding

This work was supported by grants from the Ministry of Science and Technology of the People's Republic of China (2017YFA0503502), the National Key Research and Development Program of China (2018YFC1003500) and the National Natural Science Foundation of China (32070694, 31872822 and 31571436).

The peer review history is available online at https://journals.biologists.com/dev/article-lookup/doi/10.1242/dev.200478.

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Competing interests

The authors declare no competing or financial interests.

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