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Microtubule-based movements during ooplasmic segregation in the medaka fish egg (Oryzias latipes).

Introduction

The optical clarity, size (diameter = 1.2 mm), and year-round availability of the medaka fish egg make it a favorable system in which to study ooplasmic segregation. Ooplasmic segregation in this egg consists of the bulk flow of ooplasm toward the animal pole of the egg, the movement of oil droplets toward the vegetal pole, and the saltatory movement of small ooplasmic inclusions toward the animal and vegetal poles of the egg (Iwamatsu, 1973; Abraham et al., 1993a), all of which take place more or less simultaneously. Formation of the blastodisc at the animal pole of zebrafish (Danio rerio) and loach (Misgurnus fossilis) eggs is inhibited by cytochalasin - cytochalasin B (CCB) in zebrafish (Katow, 1983) and cytochalasin D (CCD) in loach (Ivanenkov et al., 1987). This inhibition suggests that microfilaments are involved in the movement of ooplasm toward the animal pole of these eggs. However, there are no reports of the effects of CCD on streaming or on formation of the blastodisc in the medaka egg. Thus, one objective of the present study was to monitor the effects of CCD on these phenomena in the medaka egg.

The possible involvement of microtubules in segregation in the medaka egg has been explored more fully (Abraham et al., 1993a, 1993b). A network of microtubules is organized during the period of ooplasmic segregation in the medaka (Abraham et al., 1993b), and this network is absent from eggs incubated with a microtubule poison (Abraham et al., 1993b). Moreover, three microtubule poisons - colchicine, demecolcine, and nocodazole - inhibit the normal movement of oil droplets toward the vegetal pole, eliminate the saltatory motion of small inclusions, and slow the growth of the blastodisc: eggs treated with [Beta]-lumicolchicine, an inactive derivative of colchicine, segregated normally (Abraham et al., 1993a). The second objective of the present study was to determine whether the inhibitory effects of one of these poisons (demecolcine) on microtubules and ooplasmic segregation could be reversed by illuminating the eggs with ultraviolet light (360 nm), which photolyzes demecolcine, converting it to lumidemecolcine, a molecule that is not a microtubule poison (Aronson and Inoue, 1970). This "Colcemid-UV method" (Colcemid is a trade name for demecolcine) has been used by a number of investigators to study microtubule-mediated movements (Sluder, 1976; Hiramoto et al., 1984; Hamaguchi and Hiramoto, 1986; Ladrach and La Fountain, 1986).

The suggestion that microtubules are involved in the segregation of oil droplets toward the vegetal pole is inconsistent with the hypothesis that this segregation is caused simply by the bulk flow of ooplasm in the opposite direction, that is, toward the animal pole (Sakai, 1965). This hypothesis can be tested directly by determining whether oil droplets segregate toward the vegetal pole when there is no bulk flow of ooplasm toward the animal pole. Thus, another objective of the present study was to monitor the effects of CCD on the segregation of oil droplets toward the vegetal pole.

Sakai's hypothesis was based in part on the observation that when droplets of three oils - vegetable oil, liver oil, and mineral oil - were injected into the ooplasm of medaka eggs, they segregated toward the vegetal pole along with endogenous droplets (Sakai, 1965). However, it is possible that the injected droplets simply floated toward the vegetal pole, because the eggs in the experiments were all oriented with their vegetal pole uppermost. Thus, another objective of the present study was to monitor the movement of injected droplets of five fluids that differ widely in their chemical and physical properties; the eggs in the present study were oriented with either their animal pole or their vegetal pole uppermost.

A preliminary account of these findings has been published (Webb and Fluck, 1993).

Materials and Methods

Biological material

The dissection of gonads from breeding medaka, the preparation of gametes, and the in vitro fertilization of eggs have been previously described (Yamamoto, 1967; Abraham et al., 1993a). To indicate the relative temporal position of these events, we used a normalized time ([T.sub.n]) scale in which the time between fertilization and the beginning of cytokinesis is 1 unit.

Effects of cytochalasin D on segregation

Stock solutions of CCD (Sigma) in dimethylsulfoxide (DMSO, Fisher) were diluted 100-fold to make working solutions. Four eggs were placed in a small Syracuse watch glass containing 500 [[micro]liter] of either 1% DMSO in buffered saline solution (BSS: 111 mM NaCl; 5.37 mM KCl; 1.0 mM Ca[Cl.sub.2]; 0.6 mM MgS[O.sub.4]; 5 mM HEPES, pH 7.3) or 10 [[micro]gram] [ml.sup.-1] CCD/1% DMSO in BSS and incubated for 1 h at room temperature (24-25 [degrees] C). The eggs were fertilized by pipetting 50 [[micro]liter] of a fresh sperm suspension, prepared by mincing testis in BSS, onto the eggs and stirring quickly. Fertilized eggs were transferred to a microscope slide on which a cover glass was supported by four pillars of petroleum jelly (Abraham et al., 1993a). The volume of the blastodisc was measured with an image analysis program (Microcomp Planar Morphometry, Southern Micro Instruments; Abraham et al., 1993a). To monitor cytoplasmic streaming, we oriented the eggs with their animal pole-vegetal pole axis parallel to the surface of the slide, placed the slide on the stage of a compound microscope (Nikon Optiphot), and used time-lapse video microscopy via a 40X phase-contrast objective and a Dage/MTI Newvicon camera to record ooplasmic movements. On playback, the positions of five particles in each egg at [T.sub.n] [approximately equal to] 0.35 were mapped onto acetate sheets (Abraham et al., 1993a). At the end of recording, the eggs were washed twice with embryo reading medium (17 mM NaCl; 0.4 mM KCl; 0.3 mM Ca[Cl.sub.2]; 0.67 mM MgS[O.sub.4]; 0.001 g/l methylene blue) and transferred to a petri dish containing 3 ml of embryo rearing medium. Subsequent development of the eggs was monitored for several days. A total of 111 eggs from six females were used in these experiments; we monitored ooplasmic segregation closely in 30 eggs.

Effects of demecolcine on segregation

Working solutions of demecolcine (N-deacetyl-N-methyl-colchicine; Sigma) were prepared by diluting (at least 100X) an aqueous stock solution of 0.35 mM demecolcine. Eggs were placed in BSS containing the appropriate concentration of demecolcine and incubated for 1 h at room temperature in dim light. Typically three eggs were incubated in 375 [[micro]liter] or four eggs in 500 [[micro]liter] of medium. To fertilize the eggs, we minced a portion of testis in 200 [[micro]liter] of BSS and pipetied 50 [[micro]liter] of sperm suspension onto the eggs in the appropriate (drug-containing) medium. Eggs were then transferred to a coverglass-slide assembly for microscopy. To monitor ooplasmic segregation in these eggs, we transilluminated the egg with light from a quartz-halogen lamp, using a heat filter (KG5) and a 486 DF32 filter (Omega Optical). Movements were recorded by time-lapse video microscopy with a Dage/MTI SIT camera.

In some experiments, we treated eggs with both CCD (10 [[micro]gram] [ml.sup.-1]) and demecolcine (0.35 [[micro]molar]).

Irradiating eggs with ultraviolet light

To irradiate eggs with UV light, we used an Osram 100 W mercury arc lamp. The light from the lamp was passed through a custom filter cube (Omega Optical) containing a 360 DF 40 filter and a DC 405 dichroic mirror. An octagonal diaphragm was used to control the size of the light beam. To reduce the light intensity in some experiments, we used either an ND16 filter (Nikon, which reduced the intensity [approximately equal to] 94%) or an ND 1.5 filter (Omega Optical, which reduced the intensity [approximately equal to] 97%). The light was projected onto the egg via one of three objective lenses (all from Nikon): Plan 4X, N.A. = 0.1; Fluor/Ph 2 DL 10X; N.A. = 0.5; Fluor/Ph 3 DL 20X, N.A. = 0.75. Specific regions of the egg - animal pole, vegetal pole, equatorial region - were illuminated either en face or en profil. In most of the experiments described herein, we used the 10X objective lens and irradiated an octagonal region having a diameter of 475 [[micro]meter] and an area of 1.9 x [10.sup.5] [[[micro]meter].sup.2]. Light intensity was measured with a UVX radiometer with a long wave sensor (UVP, Inc.). Given a light intensity of 523 [Mu]W [cm.sup.-2] and assuming that all the UV light was of wavelength 360 nm, we calculated an incident light intensity of 4.9 x [10.sup.9] quanta [s.sup.-1] [[[micro]meter].sup.-2] (10x objective lens; no neutral density filter). To monitor the subsequent development of eggs, we washed them twice with embryo rearing medium and transferred them to a petri dish containing 3 ml of embryo rearing medium. A total of 292 eggs from 18 females were used in these experiments; we monitored ooplasmic segregation closely in 96 eggs.

Indirect immunofluorescence

The methods for fixing eggs and performing indirect immunofluorescence assays for alpha-tubulin have been described previously (Gard, 1991; Abraham et al., 1993b). Briefly, the eggs were fixed for 4 h in a mixture of formaldehyde (Electron Microscopy Sciences) and glutaraldehyde (Electron Microscopy Sciences) at room temperature and then in cold (-20 [degrees] C) methanol; treated with sodium borohydride; incubated with a monoclonal mouse anti-alpha-tubulin antibody (ICN, DM1A: diluted 1:250 with TBS (155 mM NaCl: 10 mM Tris-Cl, pH 7.4; 0.1% Nonidet P-40) containing 2% bovine serum albumin; and incubated with a secondary antibody (Organon Teknika, rhodamine-conjugated goat anti-mouse IgG, diluted 1:25 with TBS containing 2% bovine serum albumin). Control eggs were not incubated with the primary antibody. Eggs that were in a solution of demecolcine and being irradiated with UV light were fixed on the stage of the microscope for 5 min before being transferred to a vial of fixative. To stain nuclei, we incubated the eggs for 30 min in a solution of Hoechst 33258 (Sigma, 10 [[micro]gram] [ml.sup.-1]) in TBS. A total of 68 eggs from 11 females were used in these experiments.

Microinjecting fluids into medaka eggs

Using methods described previously (Fluck et al., 1991, 1992, 1994), droplets of fluid were microinjected into the ooplasm between 35 [degrees] and 90 [degrees] arc from the animal pole. Of the 25 eggs used in these experiments, 21 were parthenogenetically activated by the injection needle, and four others were fertilized after injection. Similar results were obtained with parthenogenetically activated and fertilized eggs. Droplets of five fluids were injected: mineral oil, density = 0.84 g [ml.sup.-1]; vegetable oil, density = 0.9 g [ml.sup.-1]; silicone fluid (Sigma), density = 1.05 g [ml.sup.-1]; and two fluorinated aliphatic compounds, FC-77 and FC-3275 (3M), density = 1.78 g [ml.sup.-1]. Although droplets of silicone fluid and the fluorinated compounds could be distinguished visually from endogenous oil droplets, we stained the vegetable oil and mineral oil with either Sudan black B or Sudan Ill to identify them.

To monitor the movement of the injected droplets, we used one of two techniques. In the first, we placed an egg on a small mound of Dow Corning high vacuum grease in the bottom of a rectangular plastic spectrophotometer cuvette and used two video cameras and two time-lapse videocassette recorders to record the movement of the droplets simultaneously from two perspectives: a polar view and a side view. In the second technique, we glued the egg to the bottom of a petri dish (diam., 35 mm) with Cel-Tak (Bio-Polymers, Inc.) and placed a right-angle prism (Melles-Griot) next to the egg. The petri dish was then placed on the stage of an inverted microscope (Nikon Diaphot) and viewed either directly (polar view) or indirectly via the prism (side view). To view the egg indirectly, we illuminated it from the side, using a fiber optic cable.

Results

Effects of cytochalasin D and demecolcine on ooplasmic segregation and development

Eggs treated with 1% DMSO formed a blastodisc (volume at [T.sub.n] [approximately equal to] 0.85-1.0 = 15.4 [+ or -] 2.0 nl, [Mathematical Expression Omitted], N = 7 eggs; [ILLUSTRATION FOR FIGURE 1A OMITTED]), displayed cytoplasmic streaming (speed of parcels toward the animal pole = 9.00 [+ or -] 5.52 [[micro]meter] [min.sup.-1], [Mathematical Expression Omitted], N = 7 eggs), and developed normally. In contrast, eggs treated with CCD (10 [[micro]gram] [ml.sup.-1]) had essentially no cytoplasmic streaming (velocity = -0.66 [+ or -] 0.84 [[micro]meter] [min.sup.-1], [Mathematical Expression Omitted], N = 5 eggs), did not form a blastodisc [ILLUSTRATION FOR FIGURE 1B OMITTED], and did not undergo cell division. CCD had no apparent effect on the segregation of oil droplets toward the vegetal pole [ILLUSTRATION FOR FIGURE 1B OMITTED] or on the saltatory movement of particles toward either the animal or vegetal pole.

The effects of demecolcine on the eggs were those previously described by Abraham et al. (1993a): (1) oil droplets did not segregate to the vegetal pole but instead floated to the portion of the egg that was uppermost during the experiment - equator [ILLUSTRATION FOR FIGURE 1C OMITTED], animal pole [ILLUSTRATION FOR FIGURE 2C AND D OMITTED], or vegetal pole; (2) a smaller-than-normal blastodisc formed [ILLUSTRATION FOR FIGURE 1C OMITTED]; (3) saltatory motion was absent; and (4) the eggs did not cleave.

In eggs treated with both CCD and demecolcine, we saw no saltatory movement, no cytoplasmic streaming, and no movement of oil droplets toward the vegetal pole [ILLUSTRATION FOR FIGURE 1D OMITTED]. These eggs also did not cleave (Table I).

Reversal of the inhibitory effects of demecolcine on ooplasmic segregation and embryonic development

When demecolcine-treated eggs were irradiated with UV light, the inhibitory effects of demecolcine were reversed: oil droplet movement and saltatory movement were restored; and the eggs subsequently cleaved, formed an embryonic axis, and hatched. The effects of UV irradiation were less pronounced at higher concentrations of demecolcine. For example, of 11 eggs treated with 0.35 [[micro]molar] demecolcine and irradiated en face at the animal pole (2.8 x [10.sup.8] quanta [s.sup.-1] [[[micro]meter].sup.-2]), vegetal pole, or equator, all 11 cleaved and developed an embryonic axis; while of three eggs treated with 3.5 [[micro]molar] demecolcine and irradiated under similar conditions, all three cleaved but none developed an embryonic axis. The effects of UV irradiation were also less pronounced when we irradiated the eggs en profil instead of en face. For example, of five eggs treated with 0.35 [[micro]molar] or 1.0 [[micro]molar] demecolcine and irradiated en face near the equator (2.8 x [10.sup.8] quanta [s.sup.-1] [[[micro]meter].sup.-2]), all five cleaved and developed an embryonic axis. However, of three eggs treated similarly but irradiated en profil at the equator, none cleaved.

Apparently normal oil droplet movement was restored when we irradiated the animal pole or equatorial region en face, with oil droplets near the animal pole or equator moving away from the animal pole and toward the vegetal pole more or less along meridian lines [ILLUSTRATION FOR FIGURE 2A-F OMITTED]. Under appropriate experimental conditions (1.0 [[micro]molar] demecolcine; irradiation with low intensities of UV light), this reversal of the effects of demecolcine on oil droplet movement was restricted to the portion of the egg that was irradiated. In such eggs, oil droplets outside the irradiated region floated to the top of the egg and accumulated at the edge of the irradiated region, while droplets within the irradiated region moved out of the irradiated region and toward the vegetal pole [ILLUSTRATION FOR FIGURE 2G AND H OMITTED].

When we irradiated an equatorial region en profil, oil droplets in the irradiated region moved toward the vegetal pole, while oil droplets in the rest of the egg either floated to the top of the egg or did not move at all [ILLUSTRATION FOR FIGURE 2I AND J OMITTED]. In eggs irradiated en profil at the equator, we also saw a small accumulation of ooplasm at the animal-pole side of the irradiated region [ILLUSTRATION FOR FIGURE 2I AND J OMITTED].

The effects of UV irradiation on oil droplet movement and other phenomena clearly extended beyond the irradiated region when we used a lower concentration of demecolcine or a higher intensity of UV light. For example, eggs irradiated en face with a high intensity of UV light near their equator often underwent cleavage and formed an embryonic axis. In addition, oil droplets in such eggs moved beyond the edge of the irradiated region [ILLUSTRATION FOR FIGURE 2K OMITTED].

The movements of droplets within the irradiated regions were very similar to those in control (not treated with demecolcine) eggs; that, is roughly along meridian lines and away from the animal pole [ILLUSTRATION FOR FIGURE 3 OMITTED].

Segregation appeared normal in medaka eggs that were irradiated with UV light intensities [less than or equal to] about 530 [Mu]W [cm.sup.-2] (4.9 x [10.sup.9] quanta [s.sup.-1] [[[micro]meter].sup.-2]) but not treated with demecolcine. However, in preliminary experiments, we found that higher light intensities ([greater than or equal to] 1100 [Mu]W [cm.sup.-2], 1.0 x [10.sup.10] quanta [s.sup.-1] [[[micro]meter].sup.-2]) inhibited both the growth of the blastodisc and the movements of oil droplets.

Microtubule regeneration by ultraviolet irradiation

We saw no microtubules in eggs that were treated with demecolcine but not irradiated with UV light before fixation [ILLUSTRATION FOR FIGURE 4A OMITTED]. Moreover, we saw no or very few microtubules outside the irradiated region of eggs that were irradiated with UV light [ILLUSTRATION FOR FIGURE 4B AND C OMITTED]. However, the UV-irradiated regions contained microtubules that in number and orientation were very similar to those in control eggs that had not been treated with demecolcine [ILLUSTRATION FOR FIGURE 5 OMITTED].

The movement of droplets of injected fluids

Droplets of mineral oil were injected into five eggs, which were oriented with their animal pole uppermost. [TABULAR DATA FOR TABLE 1 OMITTED] In two of them, the injected droplets fused with native droplets, and the hybrid droplets moved toward the vegetal pole. In two eggs in which the injected droplets were not observed to fuse with native droplets, the injected droplets moved up, toward the animal pole [ILLUSTRATION FOR FIGURE 6A OMITTED]; and in another egg, the droplet of mineral oil did not move at all, even though nearby native droplets did move toward the vegetal pole. The eggs into which we injected FC-77 (four eggs), FC-3275 (one egg), and silicone oil (four eggs), were oriented with their vegetal pole uppermost. In all these eggs, the droplets of injected fluids moved toward the animal pole and came to rest in the blastodisc [ILLUSTRATION FOR FIGURE 6B-D OMITTED].

We injected vegetable oil into 11 eggs and oriented them with their animal pole uppermost. In eight of these eggs, the injected droplet fused with endogenous oil droplets, and these hybrid droplets segregated toward the vegetal pole. However, in one egg in which the injected droplet was not observed to fuse with endogenous droplets, the injected droplet moved up, toward the animal pole, and came to rest near the blastodisc, while nearby endogenous droplets moved by it in the opposite direction on their way to the vegetal pole. In two cases in which we injected vegetable oil, the injected droplets appeared to follow larger endogenous droplets that they were touching.

In the case of every fluid - vegetable oil, mineral oil, FC77, FC3275, and silicone oil - the movements of nearby or immediately adjacent endogenous oil droplets appeared to be unaffected by the presence of the injected fluid. In other words, endogenous droplets, moving toward the vegetal pole, passed by the droplets of injected fluid, which were either stationary or moving toward the animal pole.

Discussion

The inhibition of cytoplasmic streaming and formation of the blastodisc by CCD in medaka eggs is consistent with data obtained from other teleost eggs (Katow, 1983; Ivanenkov et al., 1987) and from eggs of other organisms, including ascidians (Sawada and Osanai, 1981; Jeffery, 1984) and an oligochaete (Shimizu, 1982). The inhibition suggests that the formation of the blastodisc in the medaka egg is the result of the streaming of ooplasm to the animal pole and that microfilaments are involved in this streaming. This suggestion should be pursued by staining eggs with fluorescently labeled phalloidin during segregation.

The solation-contraction coupling hypothesis (Janson and Taylor, 1993) provides a model for how this streaming, which is likely driven by the interaction of actin and myosin, could be organized and driven by the zone of elevated cytosolic [Ca.sup.2+] present at the animal pole throughout the period of segregation (Fluck et al., 1992). Dissipation of this zone of elevated [Ca.sup.2+] inhibits formation of the blastodisc (Fluck et al., 1992, 1994).

The experiments with CCD directly test the hypothesis that the movement of oil droplets toward the vegetal pole is caused by the movement of ooplasm in the opposite direction - toward the animal pole. The results are not consistent with this hypothesis, because oil droplets moved toward the vegetal pole even when we did not observe any cytoplasm streaming to the animal pole. Other data from the present study also conflict with the hypothesis. Specifically, in demecolcine-treated eggs oriented with their animal pole uppermost, oil droplets collected at the animal pole even as ooplasm was also streaming in that direction.

Moreover, the failure of injected droplets of several other fluids - fluorinated aliphatic compounds, silicone fluid, mineral oil, and vegetable oil - consistently to move away from the animal pole and toward the vegetal pole during segregation is inconsistent with the hypothesis. This failure was probably not due to a toxic effect of the fluids on nearby cell components, because the movements of adjacent endogenous droplets appeared to be unaffected by proximity to a droplet of injected fluid. The behavior of the injected droplets also suggests a specificity of the mechanism responsible for the movement of endogenous oil droplets.

We have previously suggested that microtubules are required for the segregation of oil droplets to the vegetal pole of the medaka egg (Abraham et al., 1993a). In the present study, we explored the connection between microtubules and oil droplet movement further by using the "Colcemid-UV" method to photolyze demecolcine in specific regions of the egg and thus presumably create conditions that might support microtubule polymerization (Dustin, 1984, ch. 5; Bray, 1992, p. 207). We found that irradiation of the egg with UV light regenerated both microtubules and apparently normal oil droplet movement within the irradiated region of the egg. These results are consistent with a role for microtubules in the movement of oil droplets. To determine whether the microtubule-based motor protein, kinesin, has any role in oil droplet movement, we are planning experiments in which we will microinject anti-kinesin antibodies (Ingold et al., 1988) into medaka eggs.

Microtubule poisons also disrupt intermediate filaments in a number of cells (Knapp et al., 1983; Hunt and Davis, 1990; Pasdar et al., 1992), so it is possible that some of the effects we observed were caused by such disruption in the medaka egg. We are investigating whether intermediate filaments are present in the medaka egg and can be disrupted by microtubule poisons.

Although the effects of UV irradiation on oil droplet movement could be localized under appropriate experimental conditions, the effects of irradiation sometimes clearly extended beyond the irradiated region. For example, eggs that were irradiated en face at their equator usually cleaved and formed an embryonic axis. These results suggest that UV irradiation at the equator lowered the concentration of demecolcine in the animal pole region (where cell division occurs) enough to permit the polymerization of tubulin. Moreover, oil droplets often moved beyond the edge of the UV-irradiated region, suggesting that microtubules that formed in the irradiated region subsequently extended beyond this region. Irradiation of the egg en face likely photolyzed demecolcine in the ooplasm on both the near side of the egg and its antipode as well as in the portion of the yolk vacuole that is in the light path. As molecules of demecolcine were inactivated within the irradiated region and the product diffused away from it, molecules of demecolcine in adjacent, unirradiated regions diffused into the irradiated region. In other words, the irradiated region acted as a demecolcine sink, and eventually the concentration of demecolcine decreased throughout the egg. The spatio-temporal pattern of the change in demecolcine concentration in an irradiated egg presumably depends on at least three variables: (a) the initial concentration of demecolcine in the egg, (b) the intensity of the UV light, and (c) the volume of the irradiated region. Our results demonstrate that all three variables affected the extent to which UV irradiation reversed the effects of demecolcine on oil droplet movement, cleavage, and subsequent development.

Irradiation of control medaka eggs with UV light (360 nm) at intensities adequate to regenerate microtubules and normal movement of oil droplets in poisoned eggs had no apparent effect on ooplasmic segregation. These results are consistent with those of other studies in which UV light has been used to modify amphibian development (Grant and Wacaster, 1972; Scharf and Gerhart, 1980; Holwill et al., 1987). In all these studies, the predominant wavelength emitted by the lamps was about 254 nm. Moreover, an action spectrum of the effects of UV irradiation on the inactivation of components required for neural induction in the amphibian egg showed a peak at 280 nm and a decline toward both 250 and 320 nm (Youn and Malacinski, 1980). However, irradiation of cells with high-intensity near-UV light (360 nm, 2.5 mW [cm.sup.-2], about 5-fold higher than that used in the present study) disrupts f-actin in epithelial cells (Rafferty et al., 1993). Although we saw an inhibition of segregation at such a high UV light intensity in preliminary experiments, UV light intensities sufficient to regenerate microtubules and normal oil droplet movement had no apparent effect on ooplasmic segregation in the medaka egg.

That we could regenerate microtubules and oil droplet movement near the animal pole by irradiating this region en face is not surprising, given the apparent presence of a microtubule-organizing center there (Abraham et al., 1993b). More interesting is the observation that we could regenerate microtubules and oil droplet movement near the equator region and could regenerate microtubules at the vegetal pole. This result is consistent with Sakai's observation (1964) that oil droplets will segregate toward the vegetal pole in vegetal halves of eggs that have been surgically separated from their animal halves. This result suggests the presence of microtubule-organizing centers outside the animal pole region of the medaka egg. In Xenopus laevis eggs, the biochemical basis for such an ability may be the presence of [Gamma]-tubulin in cortical cytoplasm in the vegetal hemisphere of the egg (Gard, 1994). We are currently pursuing this question.

Finally, we note that when we irradiated an equatorial region of an egg en profil, ooplasm accumulated at the animal pole side of the irradiated region, suggesting that microtubules are involved not only in the movement of oil droplets toward the vegetal pole but also in the movement of components of the ooplasm toward the animal pole. Though the growth of the medaka blastodisc is slowed by microtubule poisons (Abraham et al., 1993a), no specific effect of these poisons on the movement of the ooplasm or its constituents toward the animal pole has been reported. However, here we have described the saltatory movement of subcellular parcels toward the animal pole and its inhibition by demecolcine. This movement was especially apparent in eggs treated with CCD, which displayed no cytoplasmic streaming; this streaming obscures the component of saltatory movement directed toward the animal pole. Ooplasm in and near these irradiated interpolar regions appeared to segregate along the same axis as that of the entire egg: oil droplets moved toward the vegetal pole and certain other components of the ooplasm toward the animal pole. These results suggest that a local mechanism, operating with a predetermined polarity, is responsible for the segregation of ooplasm in the medaka egg. This suggestion is consistent with results obtained by Gilkey (1983), who achieved local activation of medaka eggs by injecting them with Ca/EGTA mixtures. He reported that oil droplets in such eggs moved to the vegetal-pole side of the activated region and a small amount of ooplasm accumulated at the animal pole side. We are seeking to determine whether these localized regions of segregation in the medaka egg are accompanied by polar zones of elevated cytosolic [[Ca.sup.2+]] like those present at the poles of the egg during segregation (Fluck et al., 1992).

Acknowledgments

Supported by NSF DCB-9017210 and NSF MCB-9316125 to RAF. Franklin and Marshall College's Hackman Scholar Program, and a grant from GTE to Franklin and Marshall College. We thank Shinya Inoue for suggesting the use of the Colcemid-UV method and for his advice in the selection of fluids for microinjection into the eggs, Vivek Abraham for helpful discussions. Richard Elinson for telling us about the paper by Youn and Malacinski, and Valerie Stone for improving the text of the manuscript.

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Author:Webb, Tamika A.; Kowalski, Wendy J.; Fluck, Richard A.
Publication:The Biological Bulletin
Date:Apr 1, 1995
Words:5754
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