Abstract
Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg’s salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg’s salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.
Introduction
Sperm intracellular Ca2+ plays a major role in the regulation of sperm function, such as capacitation, acrosome reaction and motility (Darszon et al. 2006). Ca2+ increase is crucial for the induction of each event and occurs through various species-dependent Ca2+-permeable channels and transporters. However, how the intracellular Ca2+ is maintained prior to these inductions is generally ignored since it is simply quite low without a specific stimulus in most cell types.
In the internal fertilization of the newt Cynops pyrrhogaster, sperm undergo acrosome reaction and initiate motility on the egg jelly in response to acrosome reaction-inducing substance (ARIS) and sperm motility-initiating substance (SMIS), respectively (Watanabe et al. 2009, 2010, Yokoe et al. 2016). Ca2+ influx into sperm occurs in both events through distinct Ca2+-permeable channels, such as the T-type voltage-dependent Ca2+ channel, transient receptor potential vanilloid 4 (TRPV4), and N-methyl D-Aspartate-type glutamate receptor (NMDAR) (Hiyoshi et al. 2007, Watanabe et al. 2011, Takayama-Watanabe et al. 2015, Endo et al. 2019). CatSper channels, which are essential for the regulation of sperm motility in mammals and echinoderms (Ren et al. 2001, Seifert et al. 2015), are suggested to be absent in amphibians including C. pyrrhogaster (Cai & Clapham 2008, Watanabe & Takayama-Watanabe 2014). A recent report in C. pyrrhogaster found that some sperm stored in the vas deferens spontaneously underwent the acrosome reaction during incubation in modified Steinberg’s salt solution (MST) when the Ca2+ concentration and pH were adjusted to those in the egg jelly (Kon et al. 2017). This phenomenon is influenced by extracellular Ca2+ and tends to occur after a longer storage period. Ectopic induction of the acrosome reaction severely decreased the fertilization efficiency (Takahashi et al. 2006), which suggests that the spontaneous acrosome reaction correlates with a decline in sperm quality for fertilization. Acrosome-reacted sperm are rarely seen in storage in the vas deferens of healthy males (Kon et al. 2017). Therefore, vas deferens secretions likely stabilize sperm intracellular Ca2+ before the onset of fertilization. Intracellular Ca2+ is localized to the midpiece of C. pyrrhogaster sperm (Watanabe et al. 2011). This observation is based on sperm stored in the vas deferens for a short time period, though we recently found that Ca2+ was sometimes localized to the principal piece in male newts that were captured at the end of their reproductive season (Fig. 1A and B). This suggests that sperm actually maintain their intracellular Ca2+ to localize to the midpiece in the vas deferens in correlation with sperm storage. However, it is unknown why and how intracellular Ca2+ is localized in C. pyrrhogaster sperm.
Localization patterns of intracellular Ca2+ in C. pyrrhogaster sperm. (A and B) Typical patterns of Ca2+ localization in sperm obtained from the vas deferens of males that were captured in early spring (A) and late autumn (B). (C, D, E, F and G) Ca2+ localization in sperm incubated in MST. (C) Sperm with Ca2+ localization to the midpiece. (D, E and F) Sperm with Ca2+ localization to the entire tail (midpiece and principal piece). (D, E, and F) Sperm with higher Ca2+ levels in the midpiece than in the principal piece, equal Ca2+ levels between the midpiece and principal piece, and lower Ca2+ levels in the midpiece than in the principal piece, respectively. (G) Sperm with Ca2+ localization to the principal piece. Arrows indicate the border between the midpiece and the principal piece. Bars: 50 µm (A and B) and 25 µm (C, D, E, F and G).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Localization patterns of intracellular Ca2+ in C. pyrrhogaster sperm. (A and B) Typical patterns of Ca2+ localization in sperm obtained from the vas deferens of males that were captured in early spring (A) and late autumn (B). (C, D, E, F and G) Ca2+ localization in sperm incubated in MST. (C) Sperm with Ca2+ localization to the midpiece. (D, E and F) Sperm with Ca2+ localization to the entire tail (midpiece and principal piece). (D, E, and F) Sperm with higher Ca2+ levels in the midpiece than in the principal piece, equal Ca2+ levels between the midpiece and principal piece, and lower Ca2+ levels in the midpiece than in the principal piece, respectively. (G) Sperm with Ca2+ localization to the principal piece. Arrows indicate the border between the midpiece and the principal piece. Bars: 50 µm (A and B) and 25 µm (C, D, E, F and G).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Localization patterns of intracellular Ca2+ in C. pyrrhogaster sperm. (A and B) Typical patterns of Ca2+ localization in sperm obtained from the vas deferens of males that were captured in early spring (A) and late autumn (B). (C, D, E, F and G) Ca2+ localization in sperm incubated in MST. (C) Sperm with Ca2+ localization to the midpiece. (D, E and F) Sperm with Ca2+ localization to the entire tail (midpiece and principal piece). (D, E, and F) Sperm with higher Ca2+ levels in the midpiece than in the principal piece, equal Ca2+ levels between the midpiece and principal piece, and lower Ca2+ levels in the midpiece than in the principal piece, respectively. (G) Sperm with Ca2+ localization to the principal piece. Arrows indicate the border between the midpiece and the principal piece. Bars: 50 µm (A and B) and 25 µm (C, D, E, F and G).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
In the present study, to show the significance of the localization of sperm intracellular Ca2+ for fertilization and the mechanism to maintain it in the vas deferens, we examined the changes in Ca2+ localization in sperm during incubation in MST using the Ca2+ indicator Fluo8H. The localization of intracellular Ca2+ time-dependently changed in MST from the midpiece to the principal piece. Similar changes also occurred in reconstructed vas deferens solution (RVDS), which includes ions and pH at equivalent levels to the vas deferens fluid. In egg jelly extract (JE) containing ARIS and SMIS, sperm with Ca2+ localization in the principal piece immediately underwent acrosome reaction but never initiated motility. On the other hand, chelation of extracellular Ca2+ or blockage of Ca2+ influx transiently delayed and reversed the change in Ca2+ localization. These results suggest that the localization of intracellular Ca2+ is maintained in the midpiece partly through Ca2+ transport in the vas deferens to allow sperm to respond to SMIS and initiate motility at fertilization.
Materials and methods
Gametes
The Committee for Animal Experiments of Yamagata University approved the experimental protocol (No. 29148). Newts were treated under the Guidelines for the Proper Conduct of Animal Experiments in Japan. Adult newts were captured in Yamagata prefecture, Japan in early spring or late autumn (i.e., at the beginning or the end of the reproductive season). Newts were maintained under hibernation conditions at 4°C. Female newts were injected three times daily with 300 IU human chorionic gonadotropin at room temperature to induce ovulation. Jellied eggs were obtained from the most posterior region of the oviduct termed the uterus. Sperm were collected from the vas deferens fluid by squeezing the vas deferens of mature males. Gametes were stored in the moist chamber.
Chemicals
Nifedipine (a specific blocker of L-type voltage-dependent Ca2+ channels; Furukawa et al. 2009), NiCl2 (a selective blocker of Cav3.2; Lee et al. 1999), RN1734 (a TRP channel blocker; Vincent et al. 2009), MK801 (a NMDAR blocker; Wong et al. 1986), diltiazem (a CNG channel blocker; Kolesnikov et al. 1990), and A23187 (a Ca2+ ionophore; Talbot et al. 1976) were used at final concentrations of 100 µmol/L (Nifedipine, NiCl, and RN1734), 500 µmol/L (MK801), 50 µmol/L (diltiazem), and 10 µmol/L (A23187). These chemicals were all purchased from Sigma-Aldrich. Ethylene glycol tetraacetic acid (EGTA; Dojindo Lab., Tokyo, Japan) was used at a final concentration of 10 mmol/L in MST (58.2 mmol/L NaCl, 0.67 mmol/L KCl, 6 mmol/L Ca(NO3)2, 0.83 mmol/L MgSO4, 10 mmol/L Tris–HCl: pH8.5) or 2 mmol/L in vas deferens fluid.
Ca2+ imaging
Sperm were obtained from males that were maintained in hibernation in the laboratory within 2 months or for more than 6 months. A Ca2+ indicator, Fluo8H-AM (5 µmol/L) (AAT Bioquest Inc., Sunnyvale, CA, USA), was loaded into sperm in the vas deferens fluid at 17°C for 2 h. In some experiments, a fluorescent dye for the mitochondria, MitoRed (20 µmol/L) (Dojindo Lab., Tokyo, Japan), was coloaded. Sperm were incubated in MST, RVDS (20 mmol/L NaCl, 6 mmol/L Na2SO4, 1 mmol/L KCl, 0.1 mmol/L Ca(NO3)2, 0.06 mmol/L MgSO4, 10 mmol/L HEPES–NaOH: pH6.9), or RVDS whose Ca2+ concentration and/or pH was raised to 6 mmol/L and/or 8.5 at room temperature for up to 24 h. The effect of a Ca2+ ionophore (A23187), EGTA or Ca2+-permeable channel blocker was examined by the addition of each chemical to a salt solution. The mixture of Ca2+-permeable channels was prepared by the addition of nifedipine, NiCl2, RN1734, MK801, and diltiazem to MST. In some experiments, a blocker mixture without containing one of the blockers was prepared. Ca2+ localization was observed using a confocal laser scanning microscope (488 (Fluo8H) and 561 (MitoRed) nm for excitation) (C2; Nikon Co.) or a fluorescence microscope (ECRIPSE Ti2-E/B, Nikon Co.). Localization of intracellular Ca2+ was evaluated in more than 100 sperm. The criteria were as follows: localization to the midpiece and to the entire tail with higher levels in the midpiece, equal levels between the midpiece and the principal piece, and lower levels in the midpiece, and to the principal piece.
Insemination assay
Insemination was performed according to Takahashi et al. (2013). Sperm were obtained from the vas deferens of males that were maintained under hibernation within 2 months or for more than 6 months in the laboratory (Kon et al. 2017). Sperm were suspended in a 1000-fold volume of MST, and 1 µL of the suspension (approximately 600 sperm) was placed on the surface of the jelly layer of a mature egg. In some experiments, sperm were preincubated in MST for 1 h. The inseminated egg was set in a moist chamber for 10–15 min and then immersed in distilled water. The egg was observed with binoculars 1 day after insemination. Fertilization was evaluated by the presence of a cleaving embryo.
Egg jelly extract
Egg jelly extract was prepared according to Ukita et al. (1999). Briefly, eggs were shaken in MST for 1 h. The supernatant was collected and centrifuged at 20,000 g at 4°C for 30 min. The supernatant was collected as the JE and stored at −25°C until experimental use.
Acrosome reaction
Fluo8H-loaded sperm were incubated in RVDS at room temperature for 1 or 24 h and then incubated in JE for 5 min. The absence of an acrosome was examined in live Fluo8H-loaded sperm simultaneously by clarifying the localization of intracellular Ca2+ using merged fluorescent images and differential interference contrast images under a confocal laser scanning microscope (C2; Nikon Co.). The acrosome reaction was evaluated in more than 50 sperm with Ca2+ localized in the midpiece, the principal piece, or the entire tail.
Sperm motility
Fluo8H-loaded sperm were incubated in RVDS at room temperature for 1 or 24 h and then incubated in JE for 5 min. Sperm motility was observed simultaneously with identifying the localization of intracellular Ca2+ using merged fluorescent images and differential interference contrast images under a confocal laser scanning microscope (C2; Nikon Co.). The motility of sperm with Ca2+ localized in the midpiece, the principal piece, or the entire tail was evaluated in more than 50 sperm. The criteria were as follows: motile-sperm moved forward; undulating (partial) and (whole)-sperm did not move forward, but the undulating membrane, which provides thrust for motility, was undulating in part of the tail or in the entire tail, respectively; and quiescent-sperm neither moved forward nor undulated the undulating membrane.
Statistical analysis
Significant differences were evaluated using Welch’s t-test (P < 0.01 or P < 0.05).
Results
Changes in the localization of sperm intracellular Ca2+ in MST
The calcium concentration (6 mmol/L) and pH (8.5) of MST used in the present study were adjusted to those in the egg jelly, which makes the sperm promote spontaneous acrosome reactions (Ukita et al. 1999, Kon et al. 2017). Sperm were obtained from the vas deferens of males that had shown reproductive behaviors in the field and were loaded with Fluo8H. When those sperm were incubated in MST, intracellular Ca2+ was localized to the midpiece in 77 ± 4.2% of the sperm at 5 min (Figs 1 and 2A), similarly to what was previously reported in Watanabe et al. (2011). Some sperm showed Ca2+ uniformly in the midpiece and principal piece or in the principal piece alone. Ca2+ distribution in part of the principal piece or midpiece in addition to the entire midpiece or principal piece, respectively, and abundantly in the midpiece or principal piece were also observed. Those distribution patterns were regarded as Ca2+ localization to the entire tail, with higher levels in the midpiece or principal piece. By observing the image under high magnification, intracellular Ca2+ detected by Fluo8H in the midpiece and principal piece was detected in the cytoplasm but not in the mitochondria and their localizing area (Fig. 3). At 1 h, 70 ± 16% of the sperm showed Ca2+ uniformly localized in the entire tail (i.e., the midpiece and principal piece) (Fig. 2B). Ca2+ localization to the midpiece was observed in only 8.6 ± 6.1% of sperm. At 24 h, 54 ± 22% and 23 ± 17% of the sperm showed Ca2+ localization uniformly in the entire tail and the principal piece, respectively. Intracellular Ca2+ was not localized in the midpiece in any sperm.
Changes in the localization of sperm intracellular Ca2+ in saline. Fluo8H-loaded sperm were incubated in MST (A and B) or RVDS (C and D) for up to 24 h. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). (A and C) 5-min incubation and (B and D) 1−24-h incubation. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Changes in the localization of sperm intracellular Ca2+ in saline. Fluo8H-loaded sperm were incubated in MST (A and B) or RVDS (C and D) for up to 24 h. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). (A and C) 5-min incubation and (B and D) 1−24-h incubation. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Changes in the localization of sperm intracellular Ca2+ in saline. Fluo8H-loaded sperm were incubated in MST (A and B) or RVDS (C and D) for up to 24 h. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). (A and C) 5-min incubation and (B and D) 1−24-h incubation. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Distribution of intracellular Ca2+ detected by Fluo8H in the midpiece. (A) Schematic drawing of a cross section of the urodele sperm midpiece. Bold lines indicate the approximate positions observed in (B, C, D and E). (B and C) Merged, longitudinal images of the midpiece and the principal piece of sperm in which a Ca2+ indicator (Fluo8H) and a fluorescent dye for mitochondrion (MitoRed) were loaded. (D and E) Isolated images of (C) showing the distributions of Fluo8H-bound Ca2+ (D) and MitoRed-bound mitochondria (E). Green: Fluo8H, Red: MitoRed. Bars: 5 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Distribution of intracellular Ca2+ detected by Fluo8H in the midpiece. (A) Schematic drawing of a cross section of the urodele sperm midpiece. Bold lines indicate the approximate positions observed in (B, C, D and E). (B and C) Merged, longitudinal images of the midpiece and the principal piece of sperm in which a Ca2+ indicator (Fluo8H) and a fluorescent dye for mitochondrion (MitoRed) were loaded. (D and E) Isolated images of (C) showing the distributions of Fluo8H-bound Ca2+ (D) and MitoRed-bound mitochondria (E). Green: Fluo8H, Red: MitoRed. Bars: 5 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Distribution of intracellular Ca2+ detected by Fluo8H in the midpiece. (A) Schematic drawing of a cross section of the urodele sperm midpiece. Bold lines indicate the approximate positions observed in (B, C, D and E). (B and C) Merged, longitudinal images of the midpiece and the principal piece of sperm in which a Ca2+ indicator (Fluo8H) and a fluorescent dye for mitochondrion (MitoRed) were loaded. (D and E) Isolated images of (C) showing the distributions of Fluo8H-bound Ca2+ (D) and MitoRed-bound mitochondria (E). Green: Fluo8H, Red: MitoRed. Bars: 5 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Changes in the localization of sperm intracellular Ca2+ in RVDS
Reconstructed vas deferens solution is saline with pH and ion concentrations that are adjusted to those of the vas deferens fluid (Takayama-Watanabe et al. 2015). Notably, it contains 0.1 mmol/L Ca2+ at pH6.9, which are lower than those levels for MST. When Fluo8H-loaded sperm were incubated in RVDS, fluorescence from the Fluo8H was weaker than that in MST (Fig. 4). The fluorescence was at a similar strength to that in MST when the pH and Ca2+ concentration in RVDS were raised to those in MST. Additionally, the different pH between RVDS and MST hardly influenced the fluorescence intensity from the sperm when sperm intracellular Ca2+ was saturated by treatment with A23187 (Fig. 5). Thus, the weak fluorescence in RVDS was due to the relatively low pH and Ca2+ concentration surrounding sperm. Localization of Ca2+ to the midpiece was observed in 76 ± 9.6% of the sperm in RVDS at 5 min (Fig. 2C). This localization was still observed in 40 ± 7.4% of sperm at 1 h (Fig. 2D). However, sperm with Ca2+ localization to the midpiece decreased to 0.75 ± 0.92% at 4 hours, and 81 ± 8.1% of the sperm showed Ca2+ localization uniformly in the entire tail. At 24 h, 58 ± 5.1% and 15 ± 7.9% of the sperm showed Ca2+ localization uniformly in the entire tail and to the principal piece, respectively. These results indicate that a specific factor other than ions and pH is needed to maintain the localization of sperm intracellular Ca2+ in the vas deferens.
Effects of extracellular Ca2+ and pH on sperm intracellular Ca2+ levels. Fluo8H-loaded sperm were incubated for 5 min in MST (A), RVDS (B), RVDS in which the pH was raised to that in MST (C), RVDS in which the Ca2+ concentration was raised to that in MST (D), and RVDS in which both the pH and Ca2+ concentration were raised to those in MST (E). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Effects of extracellular Ca2+ and pH on sperm intracellular Ca2+ levels. Fluo8H-loaded sperm were incubated for 5 min in MST (A), RVDS (B), RVDS in which the pH was raised to that in MST (C), RVDS in which the Ca2+ concentration was raised to that in MST (D), and RVDS in which both the pH and Ca2+ concentration were raised to those in MST (E). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Effects of extracellular Ca2+ and pH on sperm intracellular Ca2+ levels. Fluo8H-loaded sperm were incubated for 5 min in MST (A), RVDS (B), RVDS in which the pH was raised to that in MST (C), RVDS in which the Ca2+ concentration was raised to that in MST (D), and RVDS in which both the pH and Ca2+ concentration were raised to those in MST (E). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Effect of extracellular pH on the fluorescent intensity from Fluo8H-loaded sperm. Fluo8H-loaded sperm were incubated for 5 min in A23187 (10 µmol/L) containing RVDS whose Ca2+ concentration was raised to 6 mmol/L (A) or Ca2+ concentration and pH were raised to 6 mmol/L and 8.5, respectively (B). Sperm were incubated in RVDS as a control (C). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Effect of extracellular pH on the fluorescent intensity from Fluo8H-loaded sperm. Fluo8H-loaded sperm were incubated for 5 min in A23187 (10 µmol/L) containing RVDS whose Ca2+ concentration was raised to 6 mmol/L (A) or Ca2+ concentration and pH were raised to 6 mmol/L and 8.5, respectively (B). Sperm were incubated in RVDS as a control (C). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Effect of extracellular pH on the fluorescent intensity from Fluo8H-loaded sperm. Fluo8H-loaded sperm were incubated for 5 min in A23187 (10 µmol/L) containing RVDS whose Ca2+ concentration was raised to 6 mmol/L (A) or Ca2+ concentration and pH were raised to 6 mmol/L and 8.5, respectively (B). Sperm were incubated in RVDS as a control (C). Each photograph was obtained under the same laser intensity and capturing conditions on a confocal laser scanning microscope.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Significance of the localization of sperm intracellular Ca2+ to the midpiece
It has been reported that sperm are not newly produced in newts during hibernation because of the cessation of spermatogenesis (Yazawa et al. 2003, Watanabe & Takayama-Watanabe 2014, Yokoe et al. 2014). The localization of sperm intracellular Ca2+ was examined in male newts that were maintained under hibernation for more than 6 months in the laboratory. Calcium localization uniformly in the entire tail was mostly observed in the sperm after 5 min incubation in MST (Table 1). However, fertilization was achieved at a high percentage when an aliquot of suspension that included approximately 600 sperm were used for insemination of an egg by natural fertilization (Takahashi et al. 2006) (Table 2). Because the sperm population stored for more than 6 months in the vas deferens included sperm with every pattern of intracellular Ca2+ localization (Table 1), we could not deny the possibility that eggs were fertilized with sperm that were in the population and had Ca2+ localization to the midpiece. Additionally, Ca2+ localization to the midpiece was recovered by treatment of the sperm with JE (Fig. 6), suggesting that a specific substance in the egg jelly promotes Ca2+ localization to the midpiece in the sperm and contributes to the success of fertilization in the insemination assay. Next, the responsiveness to extracellular triggers that act during fertilization was examined in sperm with different intracellular Ca2+ localization. When Fluo8H-loaded sperm were incubated in RVDS for 1 or 24 h and then suspended in JE containing ARIS and SMIS, most sperm underwent an acrosome reaction regardless of the intracellular Ca2+ localization (Fig. 7). In contrast, forward motility was decreased in sperm with Ca2+ localization uniformly in the entire tail (Fig. 8). Sperm with Ca2+ localization to the principal piece never showed motility. These results clearly indicate that localization of intracellular Ca2+ to the midpiece is needed for the initiation of sperm motility in response to SMIS. The fertilization rate decreased when eggs were inseminated with sperm that had been preincubated in MST for 1 h (Table 3), which may support the significance of Ca2+ in the midpiece for sperm fertilizability though an event other than the change of Ca2+ localization, such as spontaneous acrosome reaction should influence the fertilizability of preincubated sperm.
Changes in Ca2+ localization in the egg jelly extract. Fluo8H-loaded sperm were incubated in MST (black column) or JE (white column) for 5 min. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). Data were obtained using sperm populations in which the initial intracellular Ca2+ distribution was mostly observed in the midpiece (A) or the principal piece (B). Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Changes in Ca2+ localization in the egg jelly extract. Fluo8H-loaded sperm were incubated in MST (black column) or JE (white column) for 5 min. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). Data were obtained using sperm populations in which the initial intracellular Ca2+ distribution was mostly observed in the midpiece (A) or the principal piece (B). Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Changes in Ca2+ localization in the egg jelly extract. Fluo8H-loaded sperm were incubated in MST (black column) or JE (white column) for 5 min. The localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). Data were obtained using sperm populations in which the initial intracellular Ca2+ distribution was mostly observed in the midpiece (A) or the principal piece (B). Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
ARIS-inducing acrosome reaction in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in RVDS for 1 or 24 h. Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 minutes. The acrosome reaction was evaluated in more than 100 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. The percentage of acrosome reaction was calculated in sperm with Ca2+ localization to the midpiece (M), the entire tail with equal levels between the midpiece and the principal piece (M = P), or the principal piece (P). All data are presented from three independent experiments. Asterisks indicate significant differences (**P < 0.01).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
ARIS-inducing acrosome reaction in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in RVDS for 1 or 24 h. Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 minutes. The acrosome reaction was evaluated in more than 100 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. The percentage of acrosome reaction was calculated in sperm with Ca2+ localization to the midpiece (M), the entire tail with equal levels between the midpiece and the principal piece (M = P), or the principal piece (P). All data are presented from three independent experiments. Asterisks indicate significant differences (**P < 0.01).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
ARIS-inducing acrosome reaction in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in RVDS for 1 or 24 h. Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 minutes. The acrosome reaction was evaluated in more than 100 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. The percentage of acrosome reaction was calculated in sperm with Ca2+ localization to the midpiece (M), the entire tail with equal levels between the midpiece and the principal piece (M = P), or the principal piece (P). All data are presented from three independent experiments. Asterisks indicate significant differences (**P < 0.01).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
SMIS-inducing motility in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in MST for 1 (A and B) or 24 h (C and D). Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 min. Sperm motility was observed in more than 50 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. (A) Intracellular Ca2+ was localized to the midpiece (M). (B and C) Intracellular Ca2+ was localized to the entire tail and its level was equal between the midpiece and the principal piece (M = P). (D) Intracellular Ca2+ was localized to the principal piece (P). Motile: sperm was moving forward. Undulating (partial) and (whole): sperm did not move forward, but the undulating membrane that provides thrust for motility was undulating in part of the tail and in the entire tail, respectively. Quiescent: sperm neither moved forward nor showed undulation of the undulating membrane. All data are presented from three independent experiments. Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
SMIS-inducing motility in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in MST for 1 (A and B) or 24 h (C and D). Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 min. Sperm motility was observed in more than 50 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. (A) Intracellular Ca2+ was localized to the midpiece (M). (B and C) Intracellular Ca2+ was localized to the entire tail and its level was equal between the midpiece and the principal piece (M = P). (D) Intracellular Ca2+ was localized to the principal piece (P). Motile: sperm was moving forward. Undulating (partial) and (whole): sperm did not move forward, but the undulating membrane that provides thrust for motility was undulating in part of the tail and in the entire tail, respectively. Quiescent: sperm neither moved forward nor showed undulation of the undulating membrane. All data are presented from three independent experiments. Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
SMIS-inducing motility in sperm with different intracellular Ca2+ localization. Fluo8H-loaded sperm were incubated in MST for 1 (A and B) or 24 h (C and D). Egg jelly extract was added to an aliquot of sperm suspension and incubated for 5 min. Sperm motility was observed in more than 50 sperm under a confocal laser microscope, and the localization of intracellular Ca2+ was assessed. (A) Intracellular Ca2+ was localized to the midpiece (M). (B and C) Intracellular Ca2+ was localized to the entire tail and its level was equal between the midpiece and the principal piece (M = P). (D) Intracellular Ca2+ was localized to the principal piece (P). Motile: sperm was moving forward. Undulating (partial) and (whole): sperm did not move forward, but the undulating membrane that provides thrust for motility was undulating in part of the tail and in the entire tail, respectively. Quiescent: sperm neither moved forward nor showed undulation of the undulating membrane. All data are presented from three independent experiments. Asterisks indicate significant differences (P < 0.01** and 0.05*).
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Percentages of sperm with different localization patterns of intracellular Ca2+ in correlation to the storage duration in the vas deferens.
| M | M > P | M = P | M < P | P | |
|---|---|---|---|---|---|
| Exp. 1* | 69 ± 4.4 | 14 ± 6.8 | 23 ± 2.4 | 0.70 ± 0.90 | 0 |
| Exp. 2# | 13 ± 9.7 | 4.7 ± 3.7 | 54 ± 1.6 | 15 ± 7.5 | 17 ± 8.9 |
Sperm were stored in the vas deferens within 2 months (*) and more than 6 months (#) before experiments.
Fertilization rate in inseminating sperm after the storage in the vas deferens.
| Exp. | Eggs (n) | Fertilized n (%) |
|---|---|---|
| 1-1* | 17 | 10 (59) |
| 1-2* | 12 | 9 (75) |
| 1-3* | 26 | 20 (77) |
| Total | 55 | 39 (71) |
| 2-1# | 16 | 4 (25) |
| 2-2# | 21 | 16 (76) |
| 2-3# | 34 | 28 (82) |
| Total | 71 | 48 (68) |
Sperm were stored in the vas deferens for less than 2 months (*) and 6 months (#) before experiments.
Fertilization rates in the insemination of the sperm after the incubation in MST.
| Exp. | Unincubated | 1 h in MST | ||
|---|---|---|---|---|
| Fertilized (n.)/total (n.) | % | Fertilized (n.)/total (n.) | % | |
| 1 | 7/11 | 64 | 0/11 | 0 |
| 2 | 6/9 | 67 | 0/13 | 0 |
| 3 | 4/7 | 57 | 0/11 | 0 |
| 4 | 5/13 | 38 | 0/12 | 0 |
| 5 | 8/8 | 100 | 5/8 | 63 |
| Mean | 69 | Mean | 13* | |
*P < 0.01 by Student’s t-test.
Delay in the change in sperm intracellular Ca2+ by suppression of Ca2+ influx
To estimate the contribution of extracellular Ca2+ to the changes in Ca2+ localization, Fluo8H-loaded sperm were observed in the vas deferens fluid. Fluorescence from Fluo8H was observed in the tail of many sperm, although the amount of Fluo8H incorporated was highly varied among the sperm (Fig. 9). When EGTA was added to the vas deferens fluid, the fluorescence was severely decreased within 5 min. This result suggests that Ca2+ influx into sperm maintains the intracellular Ca2+ concentration in the vas deferens. However, it was difficult to precisely determine the localization of Ca2+ in those sperm because of the high sperm concentration in the vas deferens fluid. Then, sperm were incubated in MST containing EGTA. EGTA also weakened the fluorescence from Fluo8H in each sperm, suggesting that the intracellular Ca2+ level was decreased. Intracellular Ca2+ was still localized to the midpiece in 58 ± 24% and 41 ± 17% of the sperm at 1 and 2 h, respectively (Fig. 10A). However, sperm with Ca2+ localization to the midpiece decreased to 7.5 ± 4.0% at 4 h, and those with Ca2+ localization uniformly in the entire tail increased to 84 ± 6.4%. At 24 h, 57 ± 12% and 20 ± 7.8% of the sperm showed Ca2+ localization uniformly in the entire tail and to the principal piece, respectively. Next, the participation of Ca2+-permeable channels in the changes in the localization of intracellular Ca2+ was pharmacologically examined. Ni2+, RN1734, nifedipine, MK801, or diltiazem alone had no effect (Fig. 10B), but a mixture of all the blockers delayed the change in the localization of intracellular Ca2+ (Fig. 10C). The blocker mixture did not weaken the fluorescence. These results suggest that multiple types of Ca2+-permeable channels participate in the initial change in the localization of intracellular Ca2+ in the sperm.
Decrease in intracellular Ca2+ in sperm by chelating Ca2+ in the vas deferens fluid. EGTA was added to the vas deferens fluid including Fluo8H-loaded sperm, and the fluorescence from the Ca2+ indicator was observed after 5 min using a fluorescence microscope. (A) and (B) present the sperm in the vas deferens fluid without (A) or with (B) EGTA. The fluorescence in (A) and (B) was optimized for the sperm with strong fluorescence, although fluorescence was observed in all sperm. Bars: 25 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Decrease in intracellular Ca2+ in sperm by chelating Ca2+ in the vas deferens fluid. EGTA was added to the vas deferens fluid including Fluo8H-loaded sperm, and the fluorescence from the Ca2+ indicator was observed after 5 min using a fluorescence microscope. (A) and (B) present the sperm in the vas deferens fluid without (A) or with (B) EGTA. The fluorescence in (A) and (B) was optimized for the sperm with strong fluorescence, although fluorescence was observed in all sperm. Bars: 25 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Decrease in intracellular Ca2+ in sperm by chelating Ca2+ in the vas deferens fluid. EGTA was added to the vas deferens fluid including Fluo8H-loaded sperm, and the fluorescence from the Ca2+ indicator was observed after 5 min using a fluorescence microscope. (A) and (B) present the sperm in the vas deferens fluid without (A) or with (B) EGTA. The fluorescence in (A) and (B) was optimized for the sperm with strong fluorescence, although fluorescence was observed in all sperm. Bars: 25 µm.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Involvement of Ca2+ influx in the changes in the localization of sperm intracellular Ca2+. Fluo8H-loaded sperm were incubated for up to 24 h in MST containing EGTA (A), a Ca2+ channel blocker (B), or the mixture of Ca2+ channel blockers (C). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Involvement of Ca2+ influx in the changes in the localization of sperm intracellular Ca2+. Fluo8H-loaded sperm were incubated for up to 24 h in MST containing EGTA (A), a Ca2+ channel blocker (B), or the mixture of Ca2+ channel blockers (C). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Involvement of Ca2+ influx in the changes in the localization of sperm intracellular Ca2+. Fluo8H-loaded sperm were incubated for up to 24 h in MST containing EGTA (A), a Ca2+ channel blocker (B), or the mixture of Ca2+ channel blockers (C). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Reversibility of the change of sperm intracellular Ca2+ localization
To examine the reversibility of the changes in sperm Ca2+ localization, sperm were incubated in MST for 1 h before EGTA was added to the MST. One hour after EGTA addition, 28 ± 2.1% of the sperm showed Ca2+ localization to the midpiece in contrast to the 2.6 ± 2.0% of sperm without the addition of EGTA (Fig. 11A). This localization was also seen in 45 ± 13% of sperm at 2 h, but it decreased to 2.5 ± 3.6% at 4 hours. Intracellular Ca2+ was uniformly localized in the entire tail in 78 ± 12% of the sperm at 4 h. At 24 h, 60 ± 3.9% and 18 ± 6.5% of the sperm showed Ca2+ localization uniformly in the entire tail and to the principal piece, respectively. These results suggest that the intracellular Ca2+ that once appeared in the principal piece when the sperm were in MST was transiently decreased by the depletion of extracellular Ca2+. However, the mixture of Ca2+ channel blockers did not reverse the change in Ca2+ localization (Fig. 11B). This suggests that an additional channel or transporter should be blocked to reverse the change.
Reversibility of the changes in the distribution of intracellular Ca2+ in sperm. Fluo8H-loaded sperm were incubated in MST for 1 h, and EGTA (A) or the mixture of Ca2+ channel blockers (B) was added to the MST. The sperm were further incubated for up to 24 h. Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the localization patterns of Ca2+ as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). The time after the addition of EGTA or the mixture of Ca2+ channel blockers is expressed in the graph. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Reversibility of the changes in the distribution of intracellular Ca2+ in sperm. Fluo8H-loaded sperm were incubated in MST for 1 h, and EGTA (A) or the mixture of Ca2+ channel blockers (B) was added to the MST. The sperm were further incubated for up to 24 h. Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the localization patterns of Ca2+ as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). The time after the addition of EGTA or the mixture of Ca2+ channel blockers is expressed in the graph. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Reversibility of the changes in the distribution of intracellular Ca2+ in sperm. Fluo8H-loaded sperm were incubated in MST for 1 h, and EGTA (A) or the mixture of Ca2+ channel blockers (B) was added to the MST. The sperm were further incubated for up to 24 h. Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the localization patterns of Ca2+ as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). The time after the addition of EGTA or the mixture of Ca2+ channel blockers is expressed in the graph. All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Characterization of Ca2+-permeable channels that change Ca2+ localization
To further characterize the participation of Ca2+-permeable channels in the changes in Ca2+ localization, we selected a sperm population that mostly showed strong Ca2+ distribution to the midpiece (Fig. 12). The change in Ca2+ localization was also delayed in those sperm by the Ca2+ channel blocker mixture (Fig. 12A and C). When diltiazem was removed from the blocker mixture, the change in the Ca2+ localization was promoted within 5 min (Fig. 12A). After 1 h, the suppression of the change in Ca2+ localization completely disappeared by the removal of Ni2+ (Fig. 12C). Finally, the change in the Ca2+ localization was suppressed by the copresence of diltiazem and Ni2+ (Fig. 12D). Although diltiazem blocks L-type voltage-dependent Ca2+ channels (Kraus et al. 1998), nifedipine had no effect. Nickel ion also blocks other voltage-dependent Ca2+ channels (Adachi-Akahane & Nagao 2000). However, only L-type and T-type voltage-dependent Ca2+ channels are suggested to be present in the C. pyrrhogaster sperm (Watanabe & Takayama-Watanabe 2014). Thus, the results of the present study suggest that T-type voltage-dependent Ca2+ channels and CNG channels are major participants in changing the Ca2+ localization from the midpiece to the principal piece. However, reversing the change in Ca2+ localization was not observed by Ca2+ channel blocker mixture alone (Fig. 12B and D). This result supports that an additional channel or transporter is needed to reverse the change.
Characterization of Ca2+-permeable channels in sperm participating in the change in Ca2+ localization. Fluo8H-loaded sperm were incubated in MST containing the mixture of Ca2+ channel blockers without including one of the blockers (A and C), EGTA (B and D), or Ni2+ and diltiazem (B and D) for 5 min (A and B) and 1 h (C and D). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Characterization of Ca2+-permeable channels in sperm participating in the change in Ca2+ localization. Fluo8H-loaded sperm were incubated in MST containing the mixture of Ca2+ channel blockers without including one of the blockers (A and C), EGTA (B and D), or Ni2+ and diltiazem (B and D) for 5 min (A and B) and 1 h (C and D). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Characterization of Ca2+-permeable channels in sperm participating in the change in Ca2+ localization. Fluo8H-loaded sperm were incubated in MST containing the mixture of Ca2+ channel blockers without including one of the blockers (A and C), EGTA (B and D), or Ni2+ and diltiazem (B and D) for 5 min (A and B) and 1 h (C and D). Localization of intracellular Ca2+ was observed using a fluorescence microscope. More than 100 sperm were observed in each experiment and divided into 5 groups according to the Ca2+ localization patterns as follows. Intracellular Ca2+ was localized to the midpiece (M); the entire tail with higher levels in the midpiece (M > P), equal levels between the midpiece and the principal piece (M = P), or lower levels in the midpiece (M < P); and the principal piece (P). All data are presented from three independent experiments.
Citation: Reproduction 159, 3; 10.1530/REP-19-0252
Discussion
Sperm storage allows fertilization to be achieved independently of the timing of spermatogenesis and ensures the success of a specific species-dependent reproductive strategy (Neubaum & Wolfner 1999). In C. pyrrhogaster, sperm are stored in the vas deferens for months during hibernation. The long storage results in a decline in sperm quality, which is characterized by the increasing potential of spontaneous acrosome reaction observed by 1–24-h incubation in MST (Kon et al. 2017). In the present study, localization of intracellular Ca2+ changed from the midpiece to the principal piece during the 1–24-h incubation in MST (Fig. 2). The change in Ca2+ localization also occurred in RVDS but was rarely seen in sperm stored in the vas deferens for a relatively short time period (Table 1). These results suggest that Ca2+ localization in sperm is maintained in the midpiece in the vas deferens. In the internal fertilization of C. pyrrhogaster, sperm stored in the sperm reservoir of females are inseminated on the jelly layer of ovulated eggs, responding to ARIS and SMIS for the induction of acrosome reaction and the initiation of motility, and penetrating the egg jelly. It takes 5–10 min for inseminated sperm to initiate motility and enter the jelly layer (Watanabe et al. 2010, Takahashi et al. 2013). Although Ca2+ remained localized to the midpiece in many sperm in saline for at least 5 min (Fig. 2), the activity recovering Ca2+ localization to the midpiece was also present in the egg jelly (Fig. 6). These results suggest the significance of maintaining intracellular Ca2+ localization prior to the onset of fertilization in C. pyrrhogaster. Actually, sperm that did not maintain the localization of intracellular Ca2+ in the midpiece completely lost responsiveness to SMIS (Fig. 8). SMIS is a key ligand protein for achieving internal fertilization in C. pyrrhogaster (Watanabe et al. 2010, Takayama-Watanabe et al. 2014). Its receptor is suggested to be localized to the midpiece (Yokoe et al. 2016, Sato et al. 2017), and the SMIS-induced initiation of sperm motility is delayed by the decrease in intracellular Ca2+ (Watanabe et al. 2011). Thus, maintaining the role of intracellular Ca2+ in the midpiece allows sperm to efficiently initiate motility during the fertilization process.
Although the localization of intracellular Ca2+ did not influence the induction of the acrosome reaction by ARIS (Fig. 7), the change in Ca2+ localization showed some similarities to the spontaneous acrosome reaction in occurring in MST (Fig. 2), the quickness of the reaction in MST compared to RVDS (Fig. 2), and the dependency on extracellular Ca2+ (Fig. 10) (Kon et al. 2017). T-type voltage-dependent Ca2+ channels and CNG channels were involved in the changes in Ca2+ localization (Fig. 12), and the gating of these channels may lead to spontaneous acrosome reaction. Actually, Ni2+ and diltiazem independently inhibited the spontaneous acrosome reaction while nifedipine did not (Kon et al. 2020). However, the spontaneous acrosome reaction occurs only in a part of the sperm after 1- and 24-h incubation in MST (Kon et al. 2017), while most sperm changed their localization of intracellular Ca2+ within 1 h (Fig. 2). This suggests that the change in Ca2+ localization occurs through normal responses of the Ca2+-permeable channels to a change in extracellular conditions, which leads to the spontaneous acrosome reaction in some sperm that exhibit a decrease in quality for fertilization (Kon et al. 2017). Therefore, the maintenance of Ca2+ localization additionally suppresses the spontaneous acrosome reaction, which decreases the fertilization efficiency (Takahashi et al. 2006).
In the present study, Ca2+ visualized by Fluo8H was not in the mitochondria but in the cytoplasm (Fig. 3). That Ca2+ appears to be largely influxed because a low Ca2+ concentration in the RVDS (Fig. 4) or chelating of extracellular Ca2+ remarkably decreased intracellular Ca2+. Efflux of Ca2+ is also significant for promoting Ca2+ localization to the midpiece since reversal can be caused by the depletion of extracellular Ca2+. The mitochondria may also contribute to Ca2+ localization, which should be examined in future studies.
Sperm intracellular Ca2+ was decreased by EGTA even in the vas deferens fluid (Fig. 9), suggesting that sperm promote Ca2+ transport through the plasma membrane in the vas deferens. In mammals, the sperm membrane lipid content is changed to influence membrane fluidity in the epididymis (Gervasi & Visconti 2017). This phenomenon may be essential for the suppression of the acrosome reaction until sperm reach the oviduct. In aves, high concentrations of lactic acid cause cytoplasmic acidification, making sperm quiescent in the sperm storage tubules before the activation of motility (Matsuzaki et al. 2015). Sperm storage in the vas deferens of C. pyrrhogaster is unique in that sperm maintain their fertilizability by the ion transport through the plasma membrane. The mechanism for maintaining fertilizability appears to consist of two steps. One step is functional for several hours in MST by the modulation of Ca2+ transport through multiple types of channels/transporters (Figs 10, 11 and 12). In this step, the distribution of intracellular Ca2+ changes between the midpiece and the principal piece. Results of the present study revealed that blockage of Ca2+-permeable channels, including T-type voltage-dependent Ca2+ channels and CNG channels, was effective for the transient maintenance of Ca2+ localization in MST (Fig. 12). Participation of CNG channels may be reasonable because the cAMP levels in C. pyrrhogaster sperm are increased by a 5-min incubation in MST (Kon et al. 2017). The increase in cAMP can promote the gating of T-type voltage-dependent Ca2+ channels (Kim et al. 2006). Additionally, an unknown channel/transporter is required for the reversal in Ca2+ localization (Figs 11 and 12). We have reported that cysteine-rich secretory protein 2 (CRISP2) was expressed in the vas deferens of C. pyrrhogaster (Kon et al. 2017) as well as in the oviduct that secretes egg jelly components (Watanabe & Takayama-Watanabe 2014). CRISP family proteins possess an ion channel regulatory domain and potentially modulate Ca2+ influx through multiple types of Ca2+ channels (Roberts et al. 2007, Burnett et al. 2008). In mammalian sperm, another member of the C risp family proteins, C risp1, affects hyperactivated motility by blocking the CatSper channel (Ernesto et al. 2015). C risp2 is a candidate for the modulation of Ca2+ influx into the C. pyrrhogaster sperm to maintain intracellular Ca2+ localization in the vas deferens as well as in the egg jelly. The other step may be fundamentally needed for the modulation of Ca2+ transport in the vas deferens, as Ca2+ localization finally changed to the principal piece even in the absence of extracellular Ca2+ (Fig. 10). A mechanism other than the blockage of Ca2+ transport appears to work in this step.
The maintenance of sperm Ca2+ localization in the vas deferens of C. pyrrhogaster is a novel phenomenon in sperm storage sites. Surprisingly, it can sustain sperm fertilizability for months. Further study focused on the identification of molecules involved in this process will reveal the mechanism to maintain sperm quality for fertilization and may be applicable to fertilizing sperm of other vertebrates as related to sperm storage.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
N M and N S performed experiments and analyzed data. E T-W and A W conceived the study and wrote the paper.
References
Adachi-Akahane S & Nagao T 2000 Ca2+ channel antagonists and agonists. In Pharmacology of Ionic Channel Function. Eds. Endo M, Kurachi Y & Mishima M. Berlin: Springer, pp. 119–154.
Burnett LA, Xiang X, Bieber AL & Chandler DE 2008 Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals. International Journal of Developmental Biology 489–501. (https://doi.org/10.1387/ijdb.072545lb)
Cai X & Clapham DE 2008 Evolutionary genomics reveals lineage-specific gene loss and rapid evolution of a sperm-specific ion channel complex: CatSpers and CatSper beta. PLoS ONE e3569. (https://doi.org/10.1371/journal.pone.0003569)
Darszon A, Acevedo JJ, Galindo BE, Hernández-González EO, Nishigaki T, Treviño CL, Wood C & Beltrán C 2006 Sperm channel diversity and functional multiplicity. Reproduction 977–988. (https://doi.org/10.1530/rep.1.00612)
Endo D, Kon S, Sato T, Toyama F, Katsura Y, Nakauchi Y, Takayama-Watanabe E & Watanabe A 2019 NMDA-type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm. Molecular Reproduction and Development 1106–1115. (https://doi.org/10.1002/mrd.23225)
Ernesto JI, Weigel Muñoz M, Battistone MA, Vasen G, Martínez-López P, Orta G, Figueiras-Fierro D, De la Vega-Beltran JL, Moreno IA & Guidobaldi HA et al. 2015 CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization. Journal of Cell Biology 1213–1224. (https://doi.org/10.1083/jcb.201412041)
Furukawa T, Nukada T, Namiki Y, Miyashita Y, Hatsuno K, Ueno Y, Yamakawa T & Isshiki T 2009 Five different profiles of dihydropyridines in blocking T- type Ca2+ channel subtypes (Cav 3.1 (1G), Cav 3.2 (1H), and Cav3.3 (1I)) expressed in Xenopus oocytes. European Journal of Pharmacology 100–107. (https://doi.org/10.1016/j.ejphar.2009.04.036)
Gervasi MG & Visconti PE 2017 Molecular changes and signaling events occuring in spermatozoa during epididymal maturation. Andrology 204–218. (https://doi.org/10.1111/andr.12320)
Hiyoshi W, Sasaki T, Takayama-Watanabe E, Takai H, Watanabe A & Onitake K 2007 Egg-jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope. Journal of Experimental Zoology 301–311. (https://doi.org/10.1002/jez.376)
Kim JA, Park JY, Kang HW, Huh SU, Jeong SW & Lee JH 2006 Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A. Journal of Pharmacology and Experimental Therapeutics 230–237. (https://doi.org/10.1124/jpet.106.101402)
Kolesnikov SS, Zhainazarov AB & Kosolapov AV 1990 Cyclic nucleotide-activated channels in the frog olfactory receptor plasma membrane. FEBS Letters 96–98. (https://doi.org/10.1016/0014-5793(90)81515-p)
Kon S, Sato T, Endo D, Takahashi T, Takaku A, Nakauchi Y, Toyama F, Meyer-Rochow VB, Takayama-Watanabe E & Watanabe A 2017 Sperm storage influences the potential for spontaneous acrosome reaction of the sperm in the newt Cynops pyrrhogaster. Molecular Reproduction and Development 1314–1322. (https://doi.org/10.1002/mrd.22932)
Kon S, Takaku A, Toyama F, Takayama-Watanabe E & Watanabe A 2020 Acrosome reaction inducing substance triggers two different pathways of sperm intracellular signaling in newt fertilization. International Journal of Developmental Biology In press.
Kraus RL, Hering S, Grabner M, Ostler D & Striessnig J 1998 Molecular mechanism of diltiazem interaction with L-type Ca2+ channels. Journal of Biological Chemistry 27205–27212. (https://doi.org/10.1074/jbc.273.42.27205)
Lee JH, Gomora JC, Cribbs LL & Perez-Reyes E 1999 Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha-1H. Biophysical Journal 3034–3042. (https://doi.org/10.1016/S0006-3495(99)77134-1)
Matsuzaki M, Mizushima S, Hiyama G, Hirohashi N, Shiba K, Inaba K, Suzuki T, Dohra H, Ohnishi T & Sato Y et al. 2015 Lactic acid is a sperm motility inactivation factor in the sperm storage tubules. Scientific Reports 17643. doi:10.1038/srep17643
Neubaum DM & Wolfner MF 1999 Wise, winsome, or weird? Mechanisms of sperm storage in female animals. Current Topics in Developmental Biology 67–97. (https://doi.org/10.1016/s0070-2153(08)60270-7)
Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, Tilly JL & Clapham DE 2001 A sperm ion channel required for sperm motility and male fertility. Nature 603–609. (https://doi.org/10.1038/35098027)
Roberts KP, Johnston DS, Nolan MA, Wooters JL, Waxmonsky NC, Piehl LB, Ensrud-Bolin KM & Hamilton DW 2007 Structure and function of epididymal protein cysteine-rich secretory protein-1. Asian Journal of Andrology 508–514. (https://doi.org/10.1111/j.1745-7262.2007.00318.x)
Sato T, Yokoe M, Endo D, Morita M, Toyama F, Kawamura Y, Nakauchi Y, Takayama-Watanabe E & Watanabe A 2017 Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm. Molecular Reproduction and Development 686–692. (https://doi.org/10.1002/mrd.22849)
Seifert R, Flick M, Bönigk W, Alvarez L, Trötschel C, Poetsch A, Müller A, Goodwin N, Pelzer P & Kashikar ND et al. 2015 The CatSper channel controls chemosensation in sea urchin sperm. EMBO Journal 379–392. (https://doi.org/10.15252/embj.201489376)
Takahashi S, Nakazawa H, Watanabe A & Onitake K 2006 The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster. Journal of Experimental Zoology 1010–1017. (https://doi.org/10.1002/jez.a.295)
Takahashi T, Kutsuzawa M, Shiba K, Takayama-Watanabe E, Inaba K & Watanabe A 2013 Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt, Cynops pyrrhogaster. Development, Growth and Differentiation 657–667. (https://doi.org/10.1111/dgd.12073)
Takayama-Watanabe E, Takahashi T, Yokoe M & Watanabe A 2014 Acrosome reaction-mediated motility initiation that is critical for the internal fertilization of urodele amphibians. In Sexual Reproduction in Animals and Plants. Eds. Sawada H, Inoue N & Iwano M. Tokyo: Springer, pp. 97–103.
Takayama-Watanabe E, Ochiai H, Tanino S & Watanabe A 2015 Contribution of different Ca2+ channels to the acrosome reaction-mediated initiation of sperm motility in the newt Cynops pyrrhogaster. Zygote 342–351. (https://doi.org/10.1017/S0967199413000609)
Talbot P, Summers RG, Hylander BL, Keough EM & Franklin LE 1976 The role of calcium in the acrosome reaction: an analysis using ionophore A23187. Journal of Experimental Zoology 383–392. (https://doi.org/10.1002/jez.1401980312)
Ukita M, Itoh T, Watanabe T, Watanabe A & Onitake K 1999 Substances for the initiation of sperm motility in egg-jelly of the Japanese newt, Cynops pyrrhogaster. Zoological Science 793–802. (https://doi.org/10.2108/zsj.16.793)
Vincent F, Acevedo A, Nguyen MT, Dourado M, DeFalco J, Gustafson A, Spiro P, Emerling DE, Kelly MG & Duncton MAJ 2009 Identification and characterization of novel TRPV4 modulators. Biochemical and Biophysical Research Communications 490–494. (https://doi.org/10.1016/j.bbrc.2009.09.007)
Watanabe A & Takayama-Watanabe E 2014 In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster. International Journal of Developmental Biology 873–879. (https://doi.org/10.1387/ijdb.140193aw)
Watanabe A, Fukutomi K, Kubo H, Ohta M, Takayama-Watanabe E & Onitake K 2009 Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster. Molecular Reproduction and Development 399–406. (https://doi.org/10.1002/mrd.20959)
Watanabe T, Kubo H, Takeshima S, Nakagawa M, Ohta M, Kamimura S, Takayama-Watanabe E, Watanabe A & Onitake K 2010 Identification of the sperm motility-initiating substance in the newt, Cynops pyrrhogaster, and its possible relationship with the acrosome reaction during internal fertilization. International Journal of Developmental Biology 591–597. (https://doi.org/10.1387/ijdb.092894tw)
Watanabe A, Takayama-Watanabe E, Vines CA & Cherr GN 2011 Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels. Development, Growth and Differentiation 9–17. (https://doi.org/10.1111/j.1440-169X.2010.01216.x)
Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN & Iversen LL 1986 The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. PNAS 7104–7108. (https://doi.org/10.1073/pnas.83.18.7104)
Yazawa T, Nakayama Y, Fujimoto K, Matsuda Y, Abe K, Kitano T, Abe S & Yamamoto T 2003 Abnormal spermatogenesis at low temperatures in the Japanese red-bellied newt, Cynops pyrrhogaster: possible biological significance of the cessation of spermatocytogenesis. Molecular Reproduction and Development 60–66. (https://doi.org/10.1002/mrd.10328)
Yokoe M, Sano M, Shibata H, Shibata D, Takayama-Watanabe E, Inaba K & Watanabe A 2014 Sperm proteases that may be involved in the initiation of sperm motility in the newt, Cynops pyrrhogaster. International Journal of Molecular Sciences 15210–15224. (https://doi.org/10.3390/ijms150915210)
Yokoe M, Takayama-Watanabe E, Saito Y, Kutsuzawa M, Fujita K, Ochi H, Nakauchi Y & Watanabe A 2016 A novel cysteine knot protein for enhancing sperm motility that might facilitate the evolution of internal fertilization in amphibians. PLoS ONE e0160445. (https://doi.org/10.1371/journal.pone.0160445)
