Pholochembtry and Phoiobiology Vol. 45, No. 3, pp. 407412. 1987 Printed in Great Britain. All rights reserved

0031-8655187 $03.00+0.00 Copyright 0 1987 Pergamon Journals Ltd

PHOTOREVERSAL OF ABNORMAL MORPHOGENESIS

IRRADIATION IN SEA-URCHIN EMBRYOS CAUSED BY UV-

YOSHIHIRO AKIMOTO* and TSUGIO SHIROYA Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan

(Received 22 July 1986; accepted 7 August 1986)

Abstract-Morphological abnormalities induced by UV-irradiation of 8- or 16-cell-stage embryos of the sea-urchin, Hemicentrotus pufcherrimus, and their photoreversal were studied. UV-irradiation of the animal hemisphere of embryos caused the formation of exogastrulae, while that of the vegetal hemisphere caused the formation of permanent blastulae. These UV-induced morphological abnor- malities were photoreversed when the UV-irradiated embryos were subsequently illuminated with visible light, so that the UV-irradiated embryos developed into normal pluteus-larvae. When UV- irradiated embryos were illuminated with visible light up to the onset of the DNA-synthesis phase of the following cell cycle, the UV-induced morphological abnormalities were photoreversed almost completely. The effectiveness of an exposure to visible light declined thereafter and was subsequently completely lost.

INTRODUCTION

Embryonic development is drastically affected by UV irradiation. In the Chironomidae, two types of double malformation, a double cephalon and a double abdomen, occur by partial UV-irradiation of either the posterior or anterior half of the eggs, respectively (Yajima, 1964; Kalthoff, 1971). In the sea-urchin, UV-irradiation of the animal hemi- sphere of 8- or 16-cell embryos causes exo- gastrulation and that of the vegetal hemisphere causes the formation of permanent blastulae (Akim- oto et al., 1983).

In the present paper, we report the photoreversal of morphological abnormalities caused by UV- irradiation of the animal and vegetal hemispheres of 8- or 16-cell sea-urchin embryos, and also the stage dependency of the photoreversal. Eight- or 16-cell embryos were used as the test material, because the animal-vegetal axis can be easily dis- tinguished on the basis of the size and arrangement of blastomeres, for instance, blastomeres in the veg- etal hemisphere are smaller than those in the animal hemisphere at the 8-cell stage, and 8 mesomeres, 4 macromeres and 4 micromeres are arranged in 3 layers from the animal to the vegetal pole at the 16-cell stage.

MATERIALS AND METHODS

Sea-urchin. The sea-urchin used was Hemicentrotus pul- cherrirnus. Sperm and eggs were obtained after inducing spawning through 0.55 M KC1 injection. The eggs were washed 3 times with filtered seawater. The following pro- cedures were performed in a dark room, maintained at 20"C, under pure-yellow fluorescent lamps (Matsushita

*Present address: Department of Anatomy, Kyorin Uni- versity School of Medicine, Shinkawa, Mitaka, Tokyo 181, Japan. To whom correspondence should be addressed.

Electric; FL20Y-F) to avoid uncontrolled photoreversal. W-irradiation. Irradiation was done with a low-press-

ure germicidal lamp (Toshiba Electric; GL-10, 254 nm). The fluence rate was 8 W m-*, which was estimated by an UV monitor (Topcon; UVR-254).

For the study of changes in UV sensitivity of sea-urchin embryos, the embryos (lo3 me-l) were irradiated with UV light (fluence; 80 jm-.) at various phases during development. Forty-eight h after insemination aliquots were taken from the embryo suspension and fixed with 2% formalin in seawater. The pluteus-formation was cal- culated as percentage of the normal embryos in the 300 embryos of fixed samples.

For UV-irradiation of either the animal or vegetal hemi- sphere, embryo was submerged in the seawater on a hol- low slide-glass and covered with a quartz cover-glass. By sliding the quartz cover-glass on the hollow slide, the animal pole or vegetal pole of the embryo was turned upward and irradiated through the quartz cover-glass (Fig. 1). The direction of the incident radiation was per- pendicular to the hollow slide-glass. The irradiation was carried out at 8-cell- or 16-cell-stage of the embryo, since at these stages the animal pole or vegetal pole can be distinguished easily by both the size and the arrangement of blastomeres. The embryos were subjected to UV- irradiation at the early phase of the 8- or 16-cell-stage

quartz slide /

I l l 1 1 / ,hollow slide

\ 'embryo sea water

Figure 1. Diagram of standard arrangement for UV- irradiation of the embryos. For further explanations see

text.

407

408 YOSHIHIRO AKIMOTO and TSUGIO SHIROYA

(just after the S phase) when the embryos were most sensitive to UV, as shown in Fig. 2. After irradiation the embryos were transferred into a Petri dish (15 mm diam.) and incubated at 20°C under the dark condition. At 48 h after fertilization when normal embryo reached pluteus stage, the effects of UV-irradiation on the morphology of the embryos were examined microscopically.

Scanning electron microscopic observation. Embryos were fixed with 1% OsO, in 0.6 M sucrose-0.4 M sodium cacodylate buffer (pH 7.4), and then subjected to dehy- dration through immersion in a series of graded ethanol solutions. Finally, the embryos were dried by the critical- point drying method using liquid CO, as the transitional fluid, glued to a stage and then cut manually with a glass knife under a binocular microscope. The bisected embryos were then coated with gold (Eiko IB-3) and observed under a Hitachi HHS-2R scanning electron microscope. About 500 embryos were collected for one sample.

Photoreversal. After UV-irradiation, the eggs not tre- ated with visible light were kept in the dark at 20°C. while those treated with visible light were placed under a white fluorescent lamp (Matsushita Electric; 20 W-NL) for 1 h and kept at the same temperature. The intensity of the visible light was 11 W m-*. as determined with a radio- meter (YSI-Kettering; Model 65).

Observation of the VV effect and photoreversal. The effect of UV and its photoreversal were examined mor- phologically and expressed as per cent normal pluteus formation at 48 h after fertilization (Ejima and Shiroya, 1982a). The per cent normal phteus formation is cal- culated on the basis of developing embryos. For the obser- vation of chromosome, the embryos were fixed in ethanol-acetic acid (3:1), washed twice with 45% acetic acid, and stained with 2% acetic-orcein according to Ejima and Shiroya (1982b).

Estimation of the DNA-synthesis phase. The incor- poration of [?H]thymidine into the acid-insoluble fraction was measured according to Hinegardner et al. (1964).

180 200 220 Time of Irradiation after Inserninatton (min)

Figure 2. Changes in UV-sensitivity of sea-urchin embryos during the 8- and 16-cell-stages. Top: the pluteus for- mation rates for developing sea-urchin embryos irradiated with 80 J m-z of UV light at various phases in the cleavage cycle are plotted. 4s and 5 s denote the fourth and fifth DNA-synthetic periods, respectively. Bottom: the per- centages of embryos in the third (3C) and fourth (4C)

cytokinesis stages are plotted.

RESULTS

Changes in UV-sensitivity of sea-urchin embryos

UV-irradiation of sea-urchin embryos resulted in the formation of abnormal embryos which failed to become pluteus-larvae. In Fig. 2, the frequency of normal pluteus formation as well as the percentage of cleavage are plotted against the irradiation time of UV, with a fluence of 80 J mp2 (irradiation period: 10 s). UV sensitivity of embryos fluctuated during development, and embryos were most sen- sitive just after the S phase. Under the present culture conditions, the cell cycles of the embryos proceeded highly synchronously until the fifth division, and the fourth (4s) and fifth (5s) S phases corresponded to the third (3C) and fourth (4C) cytokinesis stages of cell division, respectively.

UV-induced morphological abnormalities

Normal embryos hatched 12 h after fertilization, and the primary mesenchyme cells ingressed into the blastocoel of blastulae at 13 h (Fig. 3A) and gastrulation occurred at 16 h (Fig. 3C). These embryos became pluteus-larvae by 48 h after fer- tilization. UV-irradiation (200 J m-’, irradiation period: 25 s) of the vegetal hemisphere of 8- or 16-cell embryos inhibited gastrulation and skeleton formation. which led to the formation of permanent blastulae (Fig. 3B). UV-irradiation (the same flu- ence) of the animal hemisphere of either embryos disturbed the normal development at the gastrula stage and caused the formation of exogastrulae (Fig. 3D). In contrast, while UV-irradiation of the animal or vegetal hemisphere of 1-cell to 4-cell embryos inhibited the formation of normal pluteus-larvae, it did not induce the formation of either exogastrulae or permanent blastulae. We assume that almost all the UV-energy absorbed by the embryos remained in the hemisphere irradiated, because both a cleav- age delay and a chromosomal abnormality were observed frequently in the blastomeres of the UV- irradiated hemisphere (Figs. 4 and 5) . Scanning electron microscopic observation revealed that cellular fragmentation at the basal side of the blast- ular wall began 3 h after UV-irradiation, and giant cells appeared and were incorporated into the blastocoel.

Photoreversal of the morphological abnormalities and chromosomal abnormality

The UV-induced morphological abnormalities were prevented by exposure of embryos to visible light (11 W m-2) for 1 h immediately after UV- irradiation (Table 1). Following the treatment, almost all of the embryos developed normally. The treatment of the embryos with visible light immedi- ately after UV-irradiation also prevented the chro- mosomal abnormality. The treatment with visible light before UV-irradiation had n o effect on the

Photoreversal in embryos 405

Figure 3. Scanning electron micrograph of normal embryos and UV-induced abnormal embryos. (A) A normal mesenchyme blastula embryo bisected along the animal-vegetal axis 14 h after fertilization. pm: primary mesenchyme cell. (B) UV-induced permanent blastula embryo bisected along the animal-vegetal axis 48 h after fertilization. ( C ) Normal gastrula embryo bisected along the animal-vegetal axis 17.5 h after fertilization. a; archenteron, pm: primary mesenchyme cell, sm: secondary mesenchyme cell. (D) UV-induced exogastrula embryo 48 h after fertilization. i: intestine, s: stomach, e: esophagus. Bars, 10 pm. In each figure, the animal pole is at the top and the vegetal

pole at the bottom.

reversal of both morphological and chromosomal abnormality. The photoreversal was only achieved during the 20-30 min after UV-irradiation (Fig. 6). Illumination with visible light 60 min after UV- irradiation had little effect on the abnormal devel- opment.

DISCUSSION

UV-irradiation of the animal hemisphere of 8- or 16-cell embryos inhibited the normal development

of the epithelium and caused the formation of exogastrulae, while that of the vegetal hemisphere inhibited skeleton formation and gastrulation, caus- ing the formation of permanent blastulae (Akimoto et al., 1983).

The UV-induced morphological abnormalities were prevented by exposure of the embryos to vis- ible light during a 30-rnin period. This period is the time before S phase. Of the embryos, 50--100% developed normally with response to the cell cycle, which at these stages takes 40-50 min at 20°C.

410 YOSHIHIRO AKIMOTO and TSUGIO SHIROYA

Figure 4. Cell division and the mitotic figure. (A) A normal blastula embryo (11 h after fertilization). (B) The mitotic figure of a normal blastula. (C) An embryo the vegetal hemisphere of which was UV- irradiated at the 16-cell-stage (11 h after fertilization). (D) The mitotic figure of a UV-irradiated embryo. AP: animal pole, VP: vegetal pole. Small arrows indicate giant cells and large arrows indicate

the direction of UV-irradiation. Bar, 50 pm.

Illumination with visible light after 40 min had little effect. Less than 20% were photoreversed. The data imply that effectiveness of photoreversal is closely correlated with DNA synthesis after U V irradiation. This observation is consistent with earlier reports that the photoreversibility of the cleavage delay or morphological abnormalities induced by UV- irradiation of sperm (Shioda and Shiroya, 1974; Ejima and Shiroya, 1982a) or unfertilized eggs (Cook and Rieck, 1962) decreased with the time after fertilization and was completely lost after the completion of DNA synthesis. The lesion respon- sible for these developmental abnormalities would appear to be “fixed” during the S phase of the next cell cycle following UV-irradiation. This may occur in several ways. First, as the primary UV lesion, which may be most probably pyrimidine dimers of nuclear DNA, causes some kind of structural change of newly synthesized DNA, the pyrimidine dimers may not be photorepaired (Ciarrocchi and Suther- land, 1983). Second, as mismatched bases are paired to pyrimidine dimers during the S phase, while pyrimidine dimers may be photorepaired when UV-

irradiated embryos are exposed to visible light, mis- matched bases may not. Third, the enzyme activity involved in photoreversal may change during the cell cycle. However, such enzyme activity is constant throughout the development of the sea-urchin embryo from the 1-cell t o the blastula stage (unpub-

Table 1 . Photoreversal of UV-induced inhibition of plu- teus formation

“/o pluteus formation Irradiated

Animal Vegetal Unirradiated hemisphere hemisphere

~ ~

Dark 100 3 0

Light 100 100 93

Sixteen-cell embryos were irradiated with UV (200 J m-*), and then either kept subsequently in the dark OK exposed to white light for 1 h under a fluorescent lamp (11 W m-z) immediately after UV-irradiation.

A total of about 30 embryos were used for each sample.

Photoreversal in embryos

Figure 5. Mitotic figures of morula embryos 6 h after fertilization. One embryo was irradiated at the 16-cell-stage at the UV fluence of 200 J r r 2 . Chromosomes of normal (A) and UV-irradiated (B)

embryos. Arrows indicate chromosomal bridges. Bar, 10 pm.

411

412 YOSHIHIRO AKIMOTO and TSUGIO SHIROYA

Time of starting PR treatment (min)

Figure 6. Effects of visible light treatment at various times after UV-irradiation on the UV-induced inhibition of plu- teus formation. UV-irradiation was conducted at the early phase of the 16-cell-stage. The UV fluence was 200 J m-2. The embryos were exposed to visible light (11 W m-2) for 1 h at different times after UV-irradiation. The rate of pluteus formation is plotted (0: irradiation of the animal hemisphere, .: irradiation of the vegetal hemisphere). 0: the incorporation of [3H]thymidine into the acid-insoluble

fraction in the embryos.

lished data). It has also been shown that a pho- toreactivating enzyme activity is present in eggs of Hemicentrotus pulcherrimus (Shiroya et al., 1984). We have measured the pyrimidine dimers in the DNA of isolated nuclei in sea-urchin embryos after UV-irradiation. Pyrimidine dimers were formed in a dose-dependent manner and were photoreversed completely.

Furthermore, the chromosomal abnormality (anaphase bridges) observed in the blastomeres of the UV-irradiated hemisphere, indicates that the morphological abnormality is a result of nuclear damage (Fig. 5 ) . Chromosomal bridges were also found at the anaphase of the mitotic division in the case of sea-urchin eggs fertilized with UV-irradiated sperm (Ejima and Shiroya, 1982b). Thus we con- sider that DNA rather than RNA is the primary target of the UV-effect. However, as RNA may be the primary target in the case of the double malformation in the Chironomidae, and as the pyrimidine dimers on RNA can be photoreactivated (Jackle and Kalthoff, 1978, 1980; Kalthoff et a/ . , 1978), we cannot neglect an effect of UV on RNA. Indeed, protein synthesis is largely supported by the stored maternal mRNA in sea-urchin embryos until the blastula stage (Gross and Cousineau, 1963; Gross et a/ . , 1964; Terman and Gross, 1965).

Acknowledgements-We wish to thank Dr. Nobuo Egami, The National Institute for Environmental Studies, for the valuable discussion in the course of the work, Dr. Shonan Amemiya, Misaki Marine Biological Station of University of Tokyo, for his technical advice as to scanning electron microscopy, Misaki Marine Biological Station of Uni- versity of Tokyo and Fukushima-ken Fisheries Exper- imental Station, for supplying the experimental material. This work was supported partly by Grant-in-Aid (No. 59540450) from the Ministry of Education, Science and Culture of Jaoan.

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