Gametes

Matured newts, Cynops pyrrhogaster, were collected in Yamagata, Japan.

Ovulation was induced by two injections of gonadotropin (HCG; Teikoku Zoki Inc., Tokyo, Japan) at a dose of 100 IU daily. Mature eggs were surgically removed from the uterus. To obtain dry sperm, vas deferentia were removed and the contents pushed out from the duct with forceps. Eggs and dry sperm were stored in a moist chamber at room temperature before the experiments.

Insemination

Eggs were inseminated with 1 μl of dry sperm without immersing in solutions, then were incubated in a moist chamber at room temperature. Sperm entry points were checked at 5, 10, 15 and 30 min after the insemination. Once embryo had developed to the four-cell stage, they were transferred to Steinberg's salt solution (3 mM Hepes-NaOH, pH 7.8), and cultured.

Jelly extract

Egg-jellies were surgically removed from the mature eggs collectively shaken in Steinberg's salt solution (pH 7.8) for 1 hr at 4°C, and then centrifuged at 16000g for 30 min at 4°C. The supernatant was collected and stored at −35°C until use. For the heat treatment, jelly extract was heated at 100°C for 30 min and cooled at room temperature. For the protein digestion, jelly extract was diluted 1:4 with 4% trypsin-Steinberg's salt solution (pH 7.8), incubated for 60 min at 37°C, and then heated for 10 min at 100°C to stop the digestion.

Analysis of the ionic contents of egg-jelly

Egg-jellies were removed from the mature eggs and immediately frozen in liquid nitrogen. They were then freeze-dried and baked for 10 hr at 700°C. Next, they were dissolved in distilled water and filtrated with a millipore filter (0.45 μm; Toyo Roshi Inc. Tokyo, Japan). Ionic contents were analyzed by ICP luminescence analysis (SPS-7000; SEIKO, Japan). The presence of twenty six ions, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, Ti, V, Mo, W, Mn, Fe, Co, Ni, Pt, Cu, Ag, Au, Zn, Al, Si, Sn, and Pb, was investigated.

For the estimation of egg-jelly ion concentrations, 61 to 86 eggjellies were independently collected from the mature eggs obtained from each female of C. pyrrhogaster. The egg-jellies were immediately frozen in liquid nitrogen, then freeze-dried and baked for 10 hr at 700°C. Finally they were dissolved in distilled water of about 7.1 μl/egg, which was the approximate average volume of an egg-jelly as calculated from the average egg diameter, and the concentrations of sodium, potassium, calcium and magnesium ions were measured by ICP luminescence analysis.

Estimation of sperm motility

One-μl of dry sperm was added to 20 or 200 μl of test solution. Motility was observed with a microscope (IMT-2; Olympus, Tokyo, Japan) and time lapse video cassette recorder (AG-6720; Panasonic, Tokyo, Japan) at 1, 3, 5 and 10 min after the addition. Sperm waving the undulating membranes of their flagella were judged as motile. Three independent experiments using more than 80 sperm each were performed under each condition. The percentage of motile sperm to total sperm was calculated as sperm motility.

For analysis of the effect of osmolality on sperm motility, solutions of 240, 120 or 10 mOsm/kg were prepared with NaCl, mannitol (Nacalai Tesque Inc., Kyoto, Japan) and Steinberg's salt solution (58.2 mM NaCl, 0.67 mM KCl, 0.83 mM MgSO₄, 0.34 mM Ca(NO₃)₂, 3 mM Hepes-NaOH, pH 7.8).

To estimate the effect of ion on sperm motility, we first examined the seven cations using the solutions of 100 mM NaCl, 100 mM KCl, 67.5 mM CaCl₂, 67.5 mM MgCl₂, 67.5 mM BaCl₂, 67.5 mM SrCl₂ or 200 mM Mannitol as control. The dependencies of the activity on the ion concentration and pH were then examined using solutions of 1, 10, 30 and 60 mM of each cation at pH 7.8 or 8.5.

The effect of chloride ion was examined with choline chloride. In these all experiments, the final osmolality of each solution was prepared to 200 mOsm/kg with mannitol.

Estimation of egg-jelly pH

Egg-jellies were removed from the mature eggs without immersion in any solution. Each jelly was placed on the electrode of the pH meter (pH boy-2; Sindengen Inc., Japan) to measure its surface pH.

Preparation of reconstructed ionic solution

Reconstructed ionic solution (RIS) of egg-jelly was prepared according to the concentration of four major ions (Table 3). It contained 20 mM NaCl, 2.66 mM KCl, 5.06 mM CaCl₂, 0.39 mM MgCl₂ and 10 mM Tris-HCl, and was adjusted to pH 8.5.

Effect of osmolality on sperm motility

Decrease of osmolality around a sperm results in the initiation of motility in amphibian sperm as well as in the sperm of other species (Morisawa, 1994). To examine the effect of osmolality in C. pyrrhogaster, sperm motility was observed in the solutions of 10, 120 and 240 mOsm/kg of NaCl, mannitol or Steinberg's salt solution. In 240 and 120 mOsm/kg solutions, no or faint motility was observed (Table 1). No significant difference in sperm motility was seen among the solutes of any of the three types. However, vigorous motility was observed in distilled water or 10 mOsm/kg solutions. These results indicate that sperm motility of C. pyrrhogaster is induced by a decrease of osmolality.

Table 1

Effect of osmolarity on sperm motility in C. pyrrhogaster

Insemination of the egg with dry sperm

The mature eggs were inseminated with dry sperm in the absence of solution. Ten min after the insemination, sperm entry points were observed in 44.3% of eggs (Figs. 1B, 2). At 30 min, the percentage had increased to 90.2%, and the eggs had developed to the four-cell stage in the moist chamber within 1day (Fig. 1C). After the transfer into water, embryos developed normally to the tail-bud stage (Fig. 3). These results indicate that sperm can fertilize eggs in the absence of a change of osmolality.

Fig. 1

Development of C. pyrrhogaster without immersion in any solutions. Eggs were inseminated with dry sperm and cultured in a moist chamber. A: An unfertilized egg. B: An egg 30 minutes after the insemination. Sperm entry points were seen on the surface. C. A 4-cell stage embryo. A view from the animal hemisphere is shown. Bar=0.5 mm.

Fig. 2

The percentage of eggs with sperm entry points of C. pyrrhogaster after insemination with dry sperm. Eggs were cultured in a moist chamber and the number of eggs with sperm entry points was counted with binoculars.

Fig. 3

Development of C. pyrrhogaster after placing the 4-cell stage embryo in Steinberg's salt solution. The 4-cell stage embryo, which developed without immersion in solution, was placed in Steinberg's salt solution and cultured. A: Morula. B: A tail-bud stage embryo. Bar=5 mm.

The observation that dry sperm penetrated directly into egg-jelly suggested that sperm migrated forward by the waving of the undulating membrane in egg-jelly when dry sperm were directly inseminated (Fig. 4). The tail of motile sperm was straight, and the migration speed was 16 to 32 μm/sec (Table 2).

Fig. 4

Sperm of C. pyrrhogaster migrating in egg-jelly. Egg-jelly was removed and placed on a slide glass. Dry sperm was inseminated, and its motility was observed with the microscope and recorded with a time lapse video cassette recorder. A: A view of the time-lapse video. B and C: The same view observed ten and twenty seconds after A. Sperm indicated by arrow heads in (A) and arrow in (B) are the same as those in (B) and (C) respectively. They migrated forward with a straight morphology. Bar=100 μm.

Table 2

Speed of motile sperm in egg jelly of C. pyrrhogaster

Table 3

Cation concentrations in egg-jelly of Cynops pyrrhogaster (mM)

Effect of egg-jelly extract on sperm motility

During fertilization, the first contact between sperm and egg occurs in the egg-jelly. To examine the effect of egg-jelly on sperm motility, dry sperm was treated with egg-jelly extract. 52.3% of total sperm were motile in 1 min (Figs. 5, 6A). The percentage had rapidly increased to 98.5% at 3 min and remained high at 10 min. In Steinberg's salt solution, 3.52% of total sperm were motile in 1 min, and the percentage did not increase further. This result indicates that jelly extract contains the substances responsible for initiation of sperm motility.

Fig. 5

Observation of sperm of C. pyrrhogaster in solution by phase contrast microscope. A: Immotile sperm. The undulating membrane was clearly observed (arrow heads). B: Motile sperm. The undulating membrane was moving too vigorously to be detected. Arrows indicate the head of sperm. Bar=25 μm.

Fig. 6

Effect of jelly extract on sperm motility in C. pyrrhogaster. Dry sperm was added in jelly extract (JE) and sperm motility was observed with a phase contrast microscope. A: More than 50% of sperm began to move within 1 min after the JE treatment (open square). Few sperm was moved after treatment with Steinberg's salt solution (ST) used as a control (solid square). B: JE was treated with trypsin at 37°C for 60 min and heated at 100°C for 10 minutes. The activity initiating sperm motility disappeared in the trypsin-digested JE (solid circle). It remained in JE treated in the same manner without the trypsin digestion (open circle). Open and solid squares show the activity in JE and ST.

To characterize these substances, jelly extract was boiled for 30 min. 63.0% of total sperm were motile in 1 min (Fig. 6B). The percentage had increased to 98.1% at 3 min and remained high at 10 min. This result indicates that the substances were heat-stable. When jelly extract was treated with trypsin, 2.9% of total sperm were motile in 1 min (Fig. 6B). The percentage did not increase further. In jelly extract prepared in the same manner except for trypsin treatment, 50% of total sperm were motile in 1 min and most sperm were motile by 3 min (Fig. 6B). These results suggest that a proteinacious molecule is significant for the initiation of sperm motility.

Estimation of ions in egg-jelly

Ions in egg-jelly were identified by ICP luminescence analysis. An assay was made for 26 kinds of ions, and sodium, potassium, magnesium, calcium, barium and strontium ions were detected. The former four were major ions, while the latter two were present in very low numbers. In order to estimate the concentrations of these four major cations in egg-jelly, eggs were collected from 11 females and the ion concentrations were independently measured. The concentrations of sodium, potassium, magnesium and calcium ions were approximately 23.83, 2.66, 5.06 and 0.39 mM (Table 3).

Effect of ions on sperm motility

Sperm of C. pyrrhogaster is inseminated with eggs in the cloaca of the female, where the osmolality around sperm is thought to be similar to that of body fluid and does not change during the process of fertilization. It has been reported that the osmolality is about 200 mOsm/kg in the red spotted newt (Hardy and Dent, 1986). In the present study, dry sperm were treated with solutions containing 100 mM of monovalent ions or 67.5 mM of divalent ions. In sodium-ion solution at pH 7.8, 5.6% of total sperm were motile in 1 min. The percentage had gradually increased to 54.7% by 10 min. In potassium-ion solution, 27.1% of total sperm were motile in 1 min, and the percentage had rapidly increased to 94.1% by 3 min (Fig. 7A). When dry sperm were treated with the sodium-ion solution at pH 8.5, 19.3% of total sperm were motile in 1 min, and the percentage had rapidly increased to 80.1% by 3 min. In potassium-ion solution, 83.8% of total sperm were motile as early as 1 min (Fig. 7B). No sperm was actually motile for 10 min in the solutions of calcium or magnesium ions (Fig. 7), or in those of barium or strontium ions (data not shown) at either pH 7.8 or 8.5. These results suggest that both sodium and potassium ions may independently initiate sperm motility without a change of osmolality.

Fig. 7

Effect of ionic solutions on sperm motility in C. pyrrhogaster. Dry sperm was added in ionic solution and sperm motility was observed with a phase contrast microscope. The solutions of 100 mM NaCl (open square), 100 mM KCl (solid square), 100 mM CaCl₂ (open circle), 100 mM MgCl₂ (solid circle), and 200 mM Mannitol (open triangle) were tested in 3 mM Hepes-NaOH, pH 7.8 (A) and pH 8.5 (B).

Next, the dependency on the concentration and pH was examined in these two monovalent cations. In sodium-ion solution, the activity for the initiation of sperm motility was not detected in 1 to 60 mM NaCl at pH 7.8 (Fig. 8A). At pH 8.5, when dry sperm were treated with 60 mM NaCl, 17.8% of total sperm were motile by 3 min (Figs. 7B, 8B). The percentage of motile sperm gradually increased to 80.7% at 10 min. In potassium-ion solution, when dry sperm were treated with 60 mM KCl at pH 7.8, 15.9% of total sperm were motile in 3 min (Fig. 8C), and the percentage gradually increased to 53.5% at 10 min. At pH 8.5, when dry sperm were treated with 60 mM KCl, 18.7% of total sperm were motile in 1 min. This percentage had rapidly increased to 87.2% at 3 min and remained high at 10 min (Fig. 8D). To estimate the effect of chloride ion on sperm motility, choline chloride was used. No sperm was actually motile for 10 min in the solutions of 60 mM choline chloride at pH 7.8 or 8.5 (data not shown). These results indicate that the activity for the initiation of sperm motility was induced only in solutions of sodium or potassium ion at concentrations of greater than 60 mM. Furthermore, this activity was enhanced by high pH.

Fig. 8

Effect of sodium and potassium ions on sperm motility in C. pyrrhogaster. Dry sperm was added in the solution of NaCl (A, B) or KCl (C, D) ion with 10 mM Tris-HCl at pH 7.8 (A, C) or 8.5 (B, D). Sperm motility was observed with a phase contrast microscope. The concentration of each test solution was 60 mM (open triangle), 30 mM (solid circle), 10 mM (open circle) or 1 mM (solid square). Steinberg's salt solution was used as a control (open square). Motile sperm were actually observed in 60 mM NaCl at pH 8.5 (B) and in 60 mM KCl at pH 7.8 (C) and 8.5 (D).

Calcium ion solution did not, by itself, induce the initiation of sperm motility (Fig. 7). However, when dry sperm were treated with the solution of 60 mM NaCl and 6 mM CaCl₂ at pH 8.5, 85.2% of total sperm were motile by 3 min (Fig. 9A). The percentage remained high at 10 min. In the solution of 60 mM KCl and 6 mM CaCl₂ at pH 8.5, 52.9% of total sperm were motile in 1 min (Fig. 9B). This percentage had rapidly increased to 91.8% by 3 min and remained high at 10 min. These results indicate that, although calcium ion did not in itself have the ability to initiate sperm motility, it strengthened the activity caused by sodium or potassium ion.

Fig. 9

Co-effect of monovalent cation with calcium ion on sperm motility in C. pyrrhogaster. Dry sperm was added in the solutions of NaCl (A) or KCl (B) containing 6 mM CaCl₂ with 10 mM Tris-HCl at pH 8.5, and sperm motility was observed with a phase contrast microscope. The concentration of monovalent cation in each test solution was 60 mM (solid circle), 30 mM (open circle), 10 mM (solid square) or 1 mM (open square). Most sperm were motile in three minutes after the treatment of 60 mM NaCl, 6 mM CaCl₂ (A) or KCl, 6 mM CaCl₂ (B).

pH in egg-jelly

Egg-jellies without the immersion of any solution were used. Five mature eggs were collected from the uterus of each of 10 females and the pH of fifty egg-jellies were measured. The mean pH of the eggjellies was 8.5±0.02.

Effect of reconstructed ionic solution on sperm motility

Reconstructed ionic solution (RIS) was prepared according to the concentration of sodium, potassium, calcium and magnesium ions (Table 3) and pH in egg-jelly. When dry sperm was treated with RIS, 20.3% of total sperm were motile by 1 min (Fig. 10). However, this percentage had not increased at 10 min. The activity was quite low compared to that of jelly extract, suggesting that these ions cannot initiate sperm motility in egg-jelly by themselves.

Fig. 10

Effect of reconstructed ionic solution on sperm motility in C. pyrrhogaster. Reconstructed ionic solution (RIS) was prepared according to the ion concentration in egg-jelly shown in Table 3. Dry sperm was added in RIS, and sperm motility was observed with a phase contrast microscope. In RIS (open circle), some sperm were motile in one minute but the percentage of motile sperm remained low at ten minutes. Open and solid squares indicate JE and ST.

Substances for the initiation of sperm motility in egg-jelly

The present study demonstrated that the substances for the initiation of sperm motility exist in the egg-jelly of C. pyrrhogaster (Figs. 4, 5). One of the substances was stable under heat treatment and sensitive to trypsin digestion (Fig. 6), suggesting that a proteinaceous factor is involved in the initiation of sperm motility. Non-ionic factors for the regulation of sperm motility have been reported in the sea urchin (Suzuki et al., 1981; Garbers et al., 1982; Suzuki, 1990), the Horseshoe crab (Clapper and Brown, 1980a, b; Clapper and Epel, 1982a, b), Ciona (Yoshida et al., 1993; Yoshida et al., 1994) and the Herring (Morisawa et al., 1992; Oda et al., 1994; Pillai et al., 1993). We recently identified a molecule of 50 kDa in molecular weight in jelly extract as an active substance for the initiation of sperm motility (Mizuno et al., submitted).

Sodium or potassium ion independently showed the ability to initiate sperm motility (Fig. 7). In salmonids, a decrease in the concentration of potassium ion has been shown to initiate sperm motility (Morisawa, 1994; Morisawa and Suzuki, 1980). This is unlikely to be the case in the newt, C. pyrrhogaster, because the decrease in ion concentration does not occur in time for the fertilization. In this study, the activity for the initiation of sperm motility was low in both the monovalent cations compared to the jelly extract (Figs. 6A, 7A, C). Furthermore, the activity was actually detected in the sodium and potassium ion solutions of more than 60 mM in concentration (Fig. 8), while the concentration of sodium and potassium ions in egg-jelly was approximately 23.28 mM and 2.66 mM respectively (Table 3). These results indicate that although monovalent cations have the ability to initiate sperm motility, they cannot act as the main factor to induce sperm motility during egg insemination. It has been reported that external potassium ion maintains or activates sperm motility in some species (Morisawa, 1983; Mita and Yasumasu, 1984; Kobayashi, 1993). Monovalent cations may therefore cooperate with other substances in the initiation of sperm motility in egg-jelly.

Calcium ion is known to be essential for sperm motility. In many species, the influx of calcium ion into sperm is necessary for the initiation and the activation of sperm motility (Morisawa, 1994). In this study, 5.06 mM of calcium ion was detected in egg-jelly (Table 3). Although no activity for the initiation of sperm motility was observed by calcium ion alone (Fig. 7), 6 mM calcium ion strengthened the activity for the initiation of sperm motility of sodium or potassium ion (Fig. 9). These results suggest that calcium ion may co-operate with monovalent cations to induce sperm motility in egg-jelly of C. pyrrhogaster.

The activity for the initiation of sperm motility was stronger in the solutions of monovalent cations at pH 8.5 than pH 7.8 (Fig. 7), while it was not observed in the solutions of sodium ion at 240 mOsm/kg or 120 mOsm/kg (Table 1). The pH of those solutions was below 7.0. These results suggest that sperm motility is sensitive to higher pH, and are thus incompatible with a previous report that the external pH does not affect the initiation of sperm motility in Notophthalmus viridescens (Hardy and Dent, 1986). However, these previous experiments investigated pH change over a range of pH 6.5 to 7.4, while in the present study, a decrease in osmolality, rather than a change in pH, was thought to affect the initiation of sperm motility at the low pH range. The egg-jelly of C. pyrrhogaster was at pH 8.5, indicating that egg-jelly is in a suitable pH range for sperm to initiate motility.

In reconstructed ionic solution that was prepared according to the concentration of four major cations and pH in eggjelly, the activity for the initiation of sperm motility was detected but was weak compared to that in jelly extract (Fig. 10). This result suggests that these ions are effective but not sufficient for the initiation of sperm motility in egg-jelly; this would in turn suggest that the proteinacious factor in jelly extract and the cations in egg-jelly may co-operate to initiate sperm motility in egg-jelly of C. pyrrhogaster during fertilization.

In C. pyrrhogaster, a female picks up a spermatophore and the sperm in it were stored in the sperm reservoirs (Tsutsui, 1931). Fertilization is achieved in the cloaca of the female with the stored sperm. Several regulating factors are involved in the initiation of sperm motility. One acts as the sperm moves to spermatheca in the cloaca of the female. At that time, sperm may be initiated to move probably by the decrease of osmolality, because the spermatophore is immersed in water. Others act without the change of osmolality as the sperm touches the egg-jelly. In order to inseminate sperm with an egg, sperm must be transported to the egg surface from spermatheca. Probably, the myoepitherial cells around spermatheca contract and push the sperm out to the eggs (Dent, 1970).

In this study, motile sperm migrated in egg-jelly at a speed of 16 to 32 μm/sec (Fig. 4, Table 2). It has been reported that it takes 5 minutes for the female of C. pyrrhogaster to spawn an egg (Street, 1940). This is enough time for motile sperm to reach the vitelline envelope and achieve fertilization before spawning, since the egg-jelly of C. pyrrhogaster is approximately 500 μm thick. Therefore, C. pyrrhogaster is suitable for the investigation of not only the initiation of sperm motility but the fertilization process in egg-jelly of amphibians.