An experimental study on sperm entry into the micropyle of the medaka egg was carried out in order to analyze the effect of micropylar size on fertilization. The eggs and micropyles of Oryzias latipes were larger than those of Oryzias melastigma. When eggs of O. latipes and O. melastigma were simultaneously inseminated by spermatozoa of O. latipes, O. melastigma or Aplocheilus panchax, O. melastigma spermatozoa rapidly entered the conspecies eggs as well as the large micropyles of O. latipes eggs, while most of the O. latipes and A. panchax spermatozoa failed to rapidly enter the small O. melastigma micropyles. Moreover, when O. latipes eggs with a small or a large vestibule were simultaneously inseminated by O. melastigma spermatozoa, the size of the micropylar vestibules (range of 17-23 μm in diameter) also affected rapid sperm entry into the egg. However, this effect was not recognized when the eggs were inseminated by conspecies spermatozoa. On the other hand, when O. latipes eggs were inseminated by a mixture of O. latipes and O. melastigma spermtozoa or O. latipes and A. panchax spermatozoa, the faster swimming spermatozoa fertilized significantly more frequently than the slower O. latipes spermatozoa. These results suggest that rapid sperm entry into the micropyle is not only affected by differences in the morphology and size of the micropyles, but also might be accelerated by the linear swimming velocity of the spermatozoa.
In teleostean eggs, there is a single narrow pore, the micropyle, at the animal pole side of a thick egg envelope (chorion). The micropyle functions to permit the entry of only one spermatozoon into the egg and to prevent superabundant or foreign spermatozoa and bacteria from entering the egg during fertilization. The spermatozoon which will penetrate into the ooplasm uses the micropyle to reach the surface of the egg plasma membrane. The egg and the spermatozoon lose their ability to fertilize a short time after they are released from the reproductive ducts into external water (Yamamoto, 1961; Ginsburg, 1972). Therefore, spermatozoa must rapidly find and efficiently enter the narrow micropylar canal before their fertilizability is lost. Substances secreted from the egg activate and prolong the movement of spermatozoa, which generally increases the probabilty of contact between gametes. So far, the presence of the following substances has been reported: a sperm attractant in the bitterling egg (Suzuki, 1958), a sperm activating substance in the herring egg (Morisawa et al., 1992; Yanagimachi et al., 1992; Pillai et al., 1993), and a sperm guiding substance in salmonid and herring eggs (Yanagimachi et al., 1992) and the medaka egg (Takano and Onitake, 1989; Iwamatsu et al., 1993, 1997). Various species-specific structures of the chorion surface and the micropyle have also been distinguished in teleostean fishes (Ginsburg, 1972; Riehl and Kock, 1989). According to Amanze and Lyengar (1990), Riehl and Patzner (1991) and Riehl and Kokoscha (1993), the chorions of Barbus conchonius, Sturisoma aureum and Luciocephalus sp. eggs, possess radially arranged grooves around the micropyle. The structures of the chorion and micropyle seem to play a role in assisting the substances that trap spermatozoa in the micropyle. This view point has not been experimentally verified in eggs of fishes (Hart, 1990), although fertilization between different genera or species of teleosts has been investigated (see Suzuki and Fukuda, 1971). To investigate the relationship between the micropylar structure and rapid sperm entry, we experimentally examined the effect of different micropylar sizes on rapid sperm entry. The results suggest that rapid sperm entry may be affected not only by the differences in morphology and size of micropyles, but also by the swimming velocity of the spermatozoa or other factors such as sperm guiding substances.
MATERIALS AND METHODS
Preparation of gametes
Mature Japanese medakas Oryzias latipes (orange-red type), Indian medakas O. melastigma and the killifish Aplocheilus panchax were bred in our laboratories. Unfertilized eggs of the Oryzias species were isolated in saline (6.5 g NaCl, 0.4 g KCl, 0.113 g CaCl2 and 0.15 g MgS04·7H20 in one liter of distilled water, adjusted to pH 7.3 with NaHC03) within 2 hr after ovulation. Females of these species spawned around the onset of light every day under light (14 hr)-and temperature (26-28°C)-controlled conditions. Sperm suspensions were prepared from isolated testes of mature males of all these species according to a routine procedure, then used within a few minutes.
Scanning electron microscopic observations
A fresh sperm suspension was fixed with Karnovsky's fixative (4°C) for at least 12 hr. The samples were washed once with 0.1 M phosphate buffer (pH 7.3) and dehydrated in a graded series of ethanol solutions. After immersion in isoamyl acetate, they were dried in a critical point apparatus (Hitachi, HCP-2). These samples were then mounted on brass and coated with gold vapor using a fine coating apparatus (JOEL, JEC-1100 Fine Coat). Observations were performed using a scanning electron microscope (JOEL, JEM T-20)
Observation of sperm movement and entry into the micropyle
All experimental observations were performed at 23-26°C. Spermatozoa swimming in saline were observed on a television monitor. The swimming velocity of spermatozoa was measured from the image in the monitor.
Examination of fertilization rates
Insemination of unfertilized O. latipes or O. melastigma eggs was performed by adding a fresh suspension (final sperm concentration, 0.4-7.7 × 107 spermatozoa/ml) of O. latipes, O. melastigma or A. panchax spermatozoa, and was stopped after 1-300 sec by rinsing the eggs in 0.005% SDS-saline for 5-10 sec. The eggs were then transferred into fresh saline for development. Sixty minutes after insemination, the fertilization rate was calculated as the percentage of fertilized eggs among the total eggs inseminated. Fertilization of O. latipes eggs by O. melastigma or A. panchax spermatozoa was ascertained by the appearance of melanophores on the embryo.
In one experiment, O. latipes eggs with small or large micropylar vestibules were sorted by measuring the vestibule diameters under an ordinary light micro-scope (x 200). Only the eggs with a small micropylar vestibule were marked by cutting off the attaching filaments. Eggs of both types were mixed and inseminated together with a diluted sperm suspension (5.9-7.7 × 106 O. latipes spermatozoa/ml or 1.1-2.9 × 106 O. melastigma spermatozoa/ml). Each experiment was repeated 5 times.
The data were statistically analysed by the Student's t-test.
Morphology of gametes of O. latipes and O. melastigma
The eggs and micropyles of O. latipes were larger than those of O. melastigma (Table 1). The micropyle of O. melastigma eggs consisted of a small vestibule with a marginal ridge and a short canal (Fig. 1). In unfertilized eggs of this species, the average diameter of the vestibule was about 10 μm, and the overall length of the micropyle measured about 40 μm. The micropylar canal widened at the outer end where it joined the basal region of the vestibule.
Morphology of gametes of Oryzias latipes, O. melastigma and A. panchax
The overall length (ca. 22 μm) of O. latipes spermatozoa was less than that (ca. 26 μm) of O. melastigma spermatozoa. The width and length of the heads of O. latipes spermatozoa were significantly (p<0.005) greater than those of O. melastigma spermatozoa (Fig. 2, Table 1). These sperm measurements corresponded to the inner diameters of the micropylar canals in eggs of the two species.
Swimming velocity of O. latipes, O. melastigma and A. panchax spermatozoa
When spermatozoa of O. latipes, O. melastima and A. panchax were released from testes into saline, they began to actively swim straight forward. The maximum swimming velocity (26°C) of fresh O. melastigma spermatozoa was significantly greater than that of O. latipes or A. panchax spermatozoa (Table 1).
Fertilization rate in O. latipes eggs with different sizes of micropylar vestibules
O. latipes eggs with a large or a small micropylar vestibule (Fig. 3) were simultaneously inseminated for 10 sec by O. latipes or O. melastigma spermatozoa. The rate of fertilized eggs with a large micropylar vestibule was significantly (P<0.01) greater than that with a small micropylar vestibule upon insemination by O. melastigma spermatozoa (Fig. 4). However, when these eggs were inseminated by the conspecies spermatozoa, no significant difference in fertilization rates was recognized between the eggs with different sizes of micropylar vestibules.
Interspecific and intraspecific fertilization between O. latipes, O. melastigma and A panchax
Unfertilized eggs of O. latipes and O. melastigma were mixed and inseminated for 5-300 sec (23-25°C) using a sperm suspension from O. latipes (0.5–1.4 × 107 spermatozoa/ml), O. melastigma (0.4–1.0 × 107 spermatozoa/ml). or A. panchax (2.3–3.6 × 107 spermatozoa/ml). The eggs were quickly rinsed in saline containing 0.005% SDS, which has spermicidal action, and then transferred into fresh saline. The rates of cleaved eggs among the total eggs inseminated were calculated about 60 min after insemination. As presented in Figs. 5, 6 and 7, the results revealed that the thinner O. melastigma spermatozoa induced activation of both O. melastigma and O. latipes eggs within 10 sec after insemination, whereas the thicker spermatozoa of both O. latipes and A. panchax failed to induce activation of O. melastigma eggs. The vigorous motility of these spermatozoa gradually declined within 30 sec after insemination.
Embryos of O. latipes lack melanophores, while those of O. melastigma and A. panchax possess melanophores. When eggs of O. latipes were fertilized by spermatozoa of O. melastigma or A. panchax, they developed to form interspecific hybrid embryos identified by the presence of melanophores. In order to examine the difference in rates of rapid sperm entry, O. latipes eggs were inseminated by a mixture of sperm suspensions from O. latipes (3.6 × 107 spermatozoa/ml) and O. melastigma (7.7 × 106 spermatozoa/ml) for 5 or 10 sec. The number of the hybrids with melanophores was significantly greater than the number developing into embryos without melanophores (Fig. 8). When the insemination mixture contained O. latipes (2.3-3.4 × 107 spermatozoa/ml) and A. panchax (3.1-6.1 × 107 spermatozoa/ml) spermatozoa, 74% of fertilized eggs developed to normal embryos without melanophores.
The present experiments show that O. latipes spermatozoa enter quickly into large micropyles of conspecific eggs, but not into the smaller and morphologically different micropyles of O. melastigma eggs. The same result was also obtained on the entry of spermatozoa from a different genus of fish, A. panchax, into O. latipes and O. melastigma eggs. Also, O. melastigma spermatozoa could enter more rapidly into O. latipes eggs with a large micropylar vestibule than into those with a small micropylar vestibule. These data on interspecific and intergeneric fertilizations seem to indicate that rapid sperm entry into the micropyle may be affected by the size and morphology of the vestibules. On the contrary, another experiment on intraspecific fertilization in O. latipes failed to reveal that rapid sperm entry was affected by different micropylar sizes. This contradictory finding suggests that in O. latipes, factors other than the diameter of the micropylar vestibule within the range of 17-23 μm may facilitate the rapid sperm entry into the micropyle. Such factors may include sperm guiding substances. Recently, we found sperm guiding substances (chorionic glycoproteins) that were distributed as a diluted mucous area (DMA) on the chorion surface and within the micropyle of O. latipes eggs (Iwamatsu et al., 1997). The O. melastigma eggs may also have species-specific substances to guide spermatozoa since O. melastigma spermatozoa can rapidly enter the small micropyles of conspecific eggs. O. melastigma spermatozoa, which have a small head and a long tail, may be capable of effecting rapid entry into the micropyle due to their high swimming speed.
Spermatozoa of the different fishes used in the present experiment swim in saline with the velocity of O. melastigma > O. latipes > A. panchax. The swimming velocity of O. latipes spermatozoa in the present data agrees with the velocities reported previously (Ishijima et al., 1993; Iwamatsu et al., 1993). When O. latipes eggs were inseminated by a mixture of O. latipes and O. melastigma spermatozoa, O. melastigma spermatozoa entered the micropyle faster than O. latipes spermatozoa. On the other hand, when O. latipes eggs were inseminated by a mixture of spermatozoa from O. latipes and A, panchax, the former spermatozoa entered the micropyles faster than the latter spermatozoa. These results suggest that high swimming velocity may be important for rapid entry of spermatozoa into the micropyle if the diameter of the vestibule is more than 10 μm. In addition to activators (substances and temperature) of sperm movement, sperm guiding substances, and morphological characteristics of the egg surface during fertilization, the concentration of spermatozoa also plays a role. If a less concentrated suspension of spermatozoa is applied, the time required for fertilization of all the eggs is greatly prolonged (Iwamatsu et al., 1991). The time required for all unfertilized eggs to make contact with spermatozoa is 3-6 sec in the trout and 10-15 sec in the sturgeon when the sperm concentration is more than 107 spermatozoa/ml (cf. Ginsburg, 1972). In order to eliminate sperm concentration as a factor in the present experiments, high concentrations of spermatozoa (107/ml) were used with the result that most eggs were fertilized within 12 sec (Iwamatsu et al., 1991). Therefore, this effect seems to be unimportant in the present data.
Thus, the present data indicate that in Oryzias latipes eggs, rapid sperm entry into the micropyle seems to be accelerated by a larger micropylar vestibule and by a high velocity of straight forward swimming by the spermatozoa, in addition to such chemical factors as sperm guiding substances (Iwamatsu et al., 1997).