/. Embryo/, exp. Morph. Vol. 34, 1, pp. 209-220, 1975 Printed in Great Britain 209 The effect of u.v. irradiation of the vegetal pole of Xenopus laevis eggs on the presumptive primordial germ cells By BRIGITTA ZUST1 AND K. E. DIXON1 From the School of Biological Sciences, The Flinders University of South Australia SUMMARY The initial effect of u.v. irradiation of the vegetal pole was to inhibit cleavage in the vegetal hemisphere although karyokinesis was not substantially affected. In this way a syncytium formed in the vegetal hemisphere which broke down into individual cells some time between morula and late blastula. The movement of the germ plasm from the peripheral cortical regions into the interior of the egg was not appreciably delayed although aggregation of the germ plasm did not take place until the individual presumptive primordial germ cells were formed when the syncytium broke down. The method of segregation of the germ plasm and formation of the presumptive primordial germ cells was therefore very different in irradiated embryos from the normal orderly processes which depend on normal cleavage patterns. After neurula, the number of presumptive primordial germ cells declined rapidly and at stage 43/44, when the genital ridges in normal embryos contain primordial germ cells, the genital ridges in irradiated embryos were sterile. These results raise the question whether derangement of the segregation of the presumptive primordial germ cells is solely responsible for the later abnormalities in the cell lineage or whether u.v. irradiation affects the germ plasm and therefore indirectly the germ cells. INTRODUCTION Many anuran eggs and some insect eggs contain a microscopically identifiable cytoplasmic substance known as 'germ plasm' or 'pole plasm' respectively. These substances are believed to act as germ cell determinants because the cells which contain them ultimately give rise to the gametes (see reviews by Bounoure (1934, 1939, 1964) and Blackler (1965, 1966, 1970) on germ plasm; Mahowald, 1971 and Counce, 1973 on pole plasm). One of the major techniques used to demonstrate the role of germ plasm is to irradiate the region containing it with u.v. light. At particular wavelengths and at high enough doses, u.v. causes sterility in the adultswhich eventually develop (Geigy, 1931; Bounoure, 1937 a,b). Smith (1966) provided the most convincing evidence that germ plasm specifies germ cells when he reversed the sterility resulting from u.v. irradiation by 1 Authors' address: School of Biological Sciences, The Flinders University of S.A., Bedford Park, South Aust. 5042, Australia. 14-2 210 B. ZIJST AND K. E. DIXON transfer of vegetal pole cytoplasm, presumably containing germ plasm, from unirradiated eggs to the vegetal pole of irradiated eggs. He concluded that the u.v. directly affected the germ plasm, preventing it from performing its function. However, the precise role played by germ plasm in specifying germ cells remains unknown and the specific effects of u.v. which result in sterility have not been determined. The general aim of our work has been to identify the events in the germ cell lineage which are disrupted by u.v. treatment, in the expectation that this information could throw light on the role of the germ plasm. In a previous report from this laboratory, Whitington & Dixon (1975) described in detail the process of segregation of the presumptive primordial germ cells (i.e. cells which contain germ plasm but are still in the endoderm cf. primordial germ cells in the genital ridge or indifferent gonad) and also the events which take place in the endodermal phase of the cell lineage in Xenopus laevis. This study compares these processes in u.v. irradiated embryos. MATERIALS AND METHODS Adult female X. laevis were injected with chorionic gonadotrophin (Pregnyl or Chorulon, Organon) to produce eggs which were stripped manually (after Wolf & Hedrick, 1971) and fertilized with sperm from a macerated testis. In this way, synchronous development, at least during the early cleavage stages, was obtained. Embryos were staged according to the normal table of Nieuwkoop & Faber (1967). U.v. irradiation Twenty to fifty eggs, with jelly coats intact, were pipetted onto a quartz slide and oriented with the animal pole upwards, and as much water was removed as possible. The eggs were irradiated with u.v. light between the beginning of the first and the end of the second cleavage at 253-7 nm (98 %). Total u.v. doses, calculated from a dosemeter, ranged from 2000 to 33000 ergs/mm2. Most of the observations were made on eggs receiving either 11000 ergs/mm2, 18000 ergs/mm2 or 22000 ergs/mm2. Histological preparation Embryos (morula, blastula, gastrula, neurula and tail-bud) and early tadpole stages (to stage 46) were fixed in Smith's fixative (see Jones, 1954) after the jelly coat had been removed with cysteine/papain solution (2:1, pH 8-0). The fixed tissues were dehydrated and cleared in an ethanol/xylene series or in dioxan and embedded in paraffin. Sections were cut at 5/«n and stained in either Janus green-neutral red (see Jones, 1954), Azan (after Gurr, 1962), acid fuchsinazure II (Volkonsky, 1928), borax carmine-aniline blue-orange G (Boteren- Effect ofu.v. on germ cells 211 brood & Nieuwkoop, 1973), acid fuchsin-aniline blue-orange G (a combination of the two previous procedures), Harris' haematoxylin or the Feulgen-Fast green sequence (Deitch, Wagner & Richart, 1968). Measurements The volume of germ plasm was calculated from measurements made with the aid of a Zeiss integrating disc of the area of each patch in successive serial sections. The volume of individual germ cells was calculated from measurements of their largest section, making the assumption that the cell was spherical. RESULTS U.v. irradiation produced an effect at all doses, but at the lower doses ( ^ 4400 ergs/mm2), not all eggs were affected. From 11000 to 22000 ergs/mm2, the effects produced and the recovery from these effects varied somewhat between eggs of a single batch but the variation between different batches of eggs was greater, in agreement with earlier reports (Bounoure, Aubry & Huck, 1954; Gurdon, 1960; Padoa, 1963; Smith, 1966; Burgess, 1967; Grant & Wacaster, 1972; McAvoy, Dixon & Marshall, 1975). The description of the effects of u.v. irradiation is therefore generalized and is largely based on experiments using doses of 11000-22000 ergs/mm2. Effect ofu.v. irradiation on cleavage The initial effect of irradiation with u.v. was to inhibit cleavage in the region of the vegetal pole. The first cleavage furrow, already apparent, frequently regressed in the irradiated region and subsequently new furrows were arrested at about the equator of the egg (Fig. 1). The irradiated embryos developed normally as judged by the number and size of the animal pole blastomeres, but the vegetal hemisphere had few, if any, cleavage furrows (Fig. 2) compared to control embryos. There was no delay in the initiation of invagination at gastrula but the highest mortality occurred at about this time, usually correlated with leakage of cytoplasm from the vegetal region, presumably through cell membranes weakened by the u.v. irradiation. When gastrulation proceeded normally, the yolk plug appeared cellular. Examination of serial sections of irradiated embryos confirmed that at least in early developmental stages and sometimes as late as blastula, no new cell membranes had formed in the vegetal hemisphere (Figs. 3,4). However, in some morula embryos, instead of one or two large cytoplasmic masses, a few smaller masses occupied the vegetal hemisphere. In blastula embryos, the vegetal half was occasionally composed of individual cells. The blastomeres near the equator were of roughly similar size to those in control embryos, but they were frequently connected to the large, uncleaved yolk mass through cytoplasmic bridges, making it difficult to determine the upper limits of the uncleaved portion. 212 B. ZUST AND K. E. DIXON Fig. 1. View of the vegetal pole of an X. laevis egg irradiated at the 2-4 cell stage. The first cleavage plane (*) appears normal, the second cleavage plane appears to have regressed at the pole and the fourth cleavage planes (arrows) are arrested just past the equator. Inset: unirradiated embryo at same developmental stage showing normal cleavage pattern. Fig. 2. View of the vegetal pole of two stage-7 to stage-8 X. laevis embryos irradiated at the 2- to 4-cell stage showing cleavage in the vegetal hemisphere still arrested although it has proceeded normally in the animal pole as shown in the embryo at the top left. Inset: unirradiated embryo at approximately the same developmental stage showing normal cleavage pattern. Fig. 3. Approximately median section through X. laevis blastula irradiated at the vegetal pole with u.v. at the 2- to 4-cell stage. The blastomeres in the animal hemisphere are normal but the vegetal hemisphere is practically uncleaved except for the first cleavage plane (arrow). Fig. 4. Approximately median section through X. laevis blastula (slightly more developed than Fig. 3) showing normal cleavage in the vegetal hemisphere for comparison with Fig. 3. Effect ofu.v. on germ cells 213 At the equator, the effect of the u.v. would presumably be considerably reduced due to the curvature of the egg surface, but even so, complete separation of the blastomeres had been inhibited. Although cytokinesis was delayed, nuclear division had continued. Examination of sections showed that the vegetal hemisphere, although usually uncleaved, contained a number of nuclei and therefore constituted a syncytium. From counts of Feulgen-treated sections of roughly equivalent volumes of uncleaved cytoplasm in irradiated embryos, and cleaved cytoplasm in control embryos, the number of nuclei was approximately the same, suggesting that the rate of karyokinesis in irradiated embryos was not greatly affected. Within a single syncytial mass, interphase nuclei and fully formed spindles were observed, suggesting that nuclear divisions were not synchronous. In about one-quarter of the gastrula embryos examined, a few of the nuclei, in both endodermal and presumptive primordial germ cells, were very large, up to 22 times larger than endoderm nuclei in control embryos. Enlarged nuclei were also observed at stages 15 and 29/30. This increase in size probably denotes an increase in DNA content, which could have arisen by repeated replication without division or through fusion of the nuclei (see Carroll & Van Deusen, 1973). We conclude that the primary effect of the u.v. irradiation was to delay cytokinesis in the irradiated area without affecting karyokinesis substantially. The syncytia thus produced persisted for variable periods of time and cytokinesis was reinitiated at stages roughly between morula and late blastula. It seems likely that the rate of breakdown of the syncytia was also very variable, although it is not possible to deduce this from our evidence. Effect of cleavage inhibition on segregation of the presumptive primordial germ cells Since segregation of the presumptive primordial germ cells depends on geometrical relationships between the position of the germ plasm and the cleavage planes (Whitington & Dixon, 1975), retardation of cleavage could be expected to affect their formation. The germ plasm in all irradiated pre-blastula embryos was included in the syncytial mass (see Fig. 5) except in some morulas, where part of the germ plasm was sometimes contained within cells in the floor of the blastocoele comparable morphologically to presumptive primordial germ cells in unirradiated embryos. The number of these cells was small and variable (0-2 ± 0-4) but had increased by the blastula stage (2-9 ± 2-4). By early gastrula, when usually the syncytium had completely broken down, the number of individual presumptive primordial germ cells rose to 9-4 + 5-4, about the same number (8-7 + 3-0) reported for this stage by Whitington & Dixon (1975). These results reinforce our earlier interpretation that breakdown of the syncytia in some embryos begins about morula and continues up to late blastula, and that the formation of the individual cells 214 B. ZUST AND K. E. DIXON (somatic and germ) is due to division processes which have been delayed (not inhibited) by the u.v. irradiation. The size of the few individual presumptive primordial germ cells present at blastula (806-3 ± 583 /<m3 x 104) was about twice that recorded by Whitington & Dixon (1975), but this difference is without significance since it can be redressed by a single additional mitosis. At this stage in development of normal embryos cleavage is rapid, so that differences of the magnitude observed can be expected. At late gastrula, the size of the presumptive primordial germ cells in the irradiated embryos was similar to that in the control embryos. Furthermore, the presumptive primordial germ cells in irradiated embryos up to early gastrula were more or less randomly distributed within the vegetal hemisphere as they are in normal embryos. Thus, in irradiated embryos, presumptive primordial germ cells similar in number, size and position to those in unirradiated embryos are segregated by random processes instead of by the normal ordered and predictable sequence of events. Effect of u.v. irradiation on germ plasm In stained sections of irradiated embryos, viewed in the light microscope, the general appearance and staining properties of the germ plasm, at least up to gastrula, were similar to those in control embryos. The patches were granular, yolk-free, associated with a few pigment granules and surrounded by small yolk platelets. In unirradiated eggs, when cleavage commences, the germ plasm begins to move towards the position of the cleavage furrow and to aggregate, a process which culminates in the formation of a small number of large patches of germ plasm. Normally one patch occurs in each of the first four blastomeres, situated close to the cleavage membrane some distance internally from the egg surface. The number of patches of germ plasm in the embryo and their position in each presumptive primordial germ cell does not change until about the gastrula stage, when the germ plasm moves to a perinuclear position (Whitington & Dixon, 1975). In early embryos developing from irradiated eggs, the variation in the number of patches of germ plasm was wide (3-64 at morula, 8-61 at blastula), but in embryos of the same batch, it was narrower, providing another indication of the variable sensitivity of different batches of eggs to u.v. irradiation. In gastrulae, the individual presumptive primordial germ cells each contained only one or two large patches, except for an occasional cell in which the germ plasm was more diffuse. In morulae, 90 % of the patches lay in the interior of the syncytium close to a cleavage membrane and the rest were immediately inside the surface about the vegetal pole. At later stages, the position of the individual patches was very variable between embryos but the majority occupied the region of the syncytium Effect ofu.v. on germ cells 215 Fig. 5. Diagram of section through irradiated X. laevis early blastula showing the region of the syncytium through which the patches of germ plasm are scattered. shown in Fig. 5. After the syncytium had broken down, the germ plasm in the individual germ cells eventually moved to the nucleus in an apparently normal manner. These observations suggest the following interpretation. At the time of irradiation, the first cleavage division was either completed or still in progress. Hence the germ plasm had begun to move into the first cleavage furrow and to aggregate in the normal way. After irradiation, the movement into the interior of the embryo apparently continued without obvious retardation, but aggregation was delayed. Thus, the involution of cortical materials (Schechtman, 1934) invoked by Whitington & Dixon (1975) to explain the inward movement of the germ plasm can apparently take place without the continuous formation of cleavage membranes, but the membranes already present may direct the flow of materials. Aggregation, which is much less efficient in irradiated embryos, may depend on membrane formation. The mean volume of germ plasm in ten irradiated blastulas was 207-4 + 98-2/tm 3 x 102, compared to 380 ± 198 /*m3 x 102 in control embryos of the same age. The differences between control and irradiated embryos were almost certainly due to the greater difficulty in recognizing and accurately measuring a number of small patches of germ plasm. We conclude that the total amount of germ plasm was probably not significantly altered by irradiation. 216 B. ZUST AND K. E. DIXON Effect of u.v. irradiation on mitosis in presumptive primordial germ cells Between gastrula and stage 38, each presumptive primordial germ cell normally undergoes two or three divisions, and then at stages 38-41, they migrate out of the endoderm into the forming median genital ridge (Whitington & Dixon, 1975). In irradiated gastrulae, as noted earlier, the number of presumptive primordial germ cells was similar to that in unirradiated embryos. However, from neurula onwards, the number of germ cells which could be recognized declined rapidly. Of 18 early tail-bud embryos, 7 had no presumptive primordial germ cells and the others had a mean of 2-5 ±1-9 per embryo, while up to stage 46, presumptive primordial germ cells were recognized in only 5 of 30 embryos examined (2-2 ± 1-3 per embryo). At stages 43-46, when in control embryos the genital ridges contained easily recognizable primordial germ cells, in irradiated embryos receiving 11000 ergs/mm2 or higher doses of u.v., no germ cells could be distinguished in the genital ridges which were, however, developed to approximately the normal extent. Sections of irradiated embryos of stages 15-46 were processed simultaneously with sections of control embryos through either the Volkonsky staining sequence, Azan, the procedure of Boterenbrood & Nieuwkoop (1973), the combination of these two or haematoxylin and eosin. However, either the presumptive primordial germ cells were not present in irradiated embryos or the staining characteristics of the germ plasm had changed so that the germ cells, although present, could no longer be identified. DISCUSSION The first effect observed after u.v. irradiation of the vegetal pole of X. laevis eggs was a delay in cytokinesis in the vegetal hemisphere. However, nuclear division continued so that a syncytium formed, which in early cleavage stages occupied almost all of the vegetal hemisphere. Inhibition or retardation of cytokinesis or mitosis by u.v. irradiation has been reported in sea-urchin eggs (Rustad, 1971), cultured mammalian cells (reviewed in Carlson, 1954; Giese, 1969; Painter, 1970), some invertebrate eggs and Protozoa (Kimball, 1955) and in Drosophila embryos (Geigy, 1931; Okada, Kleinman & Schneiderman, 1974). In previous experiments with amphibian eggs, Grant & Wacaster (1972) noted that u.v. irradiation of the vegetal region of Rana pipiens eggs disrupted cleavage but the nature of this effect was not examined. In another study in our laboratory, Beal & Dixon (1975) have confirmed that u.v. irradiation of the vegetal pole of Xenopus eggs suspends cytokinesis. They suggested, after comparison with the effects of cytochalasin B on X. laevis eggs (Bluemink, 1971; Hammer, Sheridan & Estensen, 1971) that the processes affected are adhesiveness of the blastomeres and the incorporation of new membrane into the cleavage furrow. Effect ofu.v. on germ cells 217 The effect of the u.v., even at the highest doses, was to delay cytokinesis, not to inhibit it, since the syncytium eventually broke down about gastrulation. The cells which were formed were indistinguishable from normal cells, at least on rather general morphological criteria. However, the orderly processes which normally result in the segregation of the presumptive primordial germ cells (Bounoure, 1934; Whitington & Dixon, 1975) were deranged by the retardation of cleavage. If a cell lineage is the result of a causal sequence of events, it seems possible that disturbance of the processes normally responsible for the initiation of the germ cell lineage may have repercussions at later stages of the lineage. In unirradiated embryos, the presumptive primordial germ cells segregated at the blastula stage undergo two or three divisions between early gastrula and the time (stage 41) they leave the endoderm (Whitington & Dixon, 1975). Using [3H]thymidine autoradiography Dziadek & Dixon (1975) have confirmed that the presumptive primordial germ cells synthesize DNA and divide from gastrula onwards. In contrast, in post-neurula irradiated embryos the number of presumptive primordial germ cells which could be recognized declined rapidly, and at stages 43-46 when the genital ridges would normally be populated by easily identifiable primordial germ cells, none were present. Previous reports concerning u.v. irradiation of the vegetal pole of early anuran embryos agree that practically no primordial germ cells could be detected at stages when they were normally easily identified, and the animals were therefore considered sterile (Bounoure et al. 1954; Padoa, 1963, 1964; Smith, 1966; Buehr, 1969; Ikenishi, Kotani & Tanabe, 1974; Tanabe & Kotani, 1974). Our observations agree, in that at doses of 4000 ergs/mm2 or less, some primordial germ cells of normal appearance were visible in the genital ridges of tadpoles at stages 43-46, but at higher doses, no primordial germ cells could be identified. However, in all previous work there has not been any attempt to determine how this sterility is produced. Our results show that the presumptive primordial germ cells cannot be detected at stages later than neurula, even though a concerted effort was made, using many different staining sequences, to distinguish the germ plasm, the single reliable criterion by which presumptive primordial germ cells can be identified. Two possible explanations can be advanced to account for the inability to detect germ cells: either they are lost (actually, by cell death or effectively, by loss of their determined state) or the germ plasm changes so that it can no longer be recognized with the normal staining sequences. Three observations suggest that the latter explanation is more likely to be correct. First, the presumptive primordial germ cells in irradiated embryos continue to synthesize DNA and divide up to late neurula (Dziadek & Dixon, 1975). This pattern is not consistent with (but it does not exclude) the hypothesis of cell death or loss of determined state. Secondly, in u.v.-treated embryos in our experiments, primordial germ cells appear in the genital ridges about stage 48, that is much later than normal (Ziist & Dixon, in preparation), thus 218 B. ZUST AND K. E. DIXON demonstrating that the presumptive primordial germ cells were present in the endoderm although their migration into the genital ridges may have been delayed. Thirdly, the germ plasm normally loses its stainability when the primordial germ cells enter the median genital ridge (Bladder, 1958; Whitington &Dixon, 1975); the inability to stain the germ plasm in post-neurula irradiated embryos may represent a precocious expression of the normal process. In summary, the results reported here show that u.v. irradiation of the vegetal pole of X. laevis eggs causes a substantial delay in cleavage of the vegetal hemisphere and thus affects the segregation of the presumptive primordial germ cells and their subsequent behaviour in the endoderm after the neurula stage. Other investigators have attempted to show that u.v. directly affects the germ plasm. Smith (1966) has shown that transfer of vegetal pole cytoplasm from unirradiated eggs reversed the effect of u.v. and germ cells migrated into the genital ridges at the normal time. Ikenishi et ah (1974) have described changes in the germ plasm and associated mitochondria in u.v. irradiated eggs. Tanabe & Kotani (1974) centrifuged X. laevis eggs and thereby displaced the germ plasm into the interior of the egg; subsequent irradiation of the vegetal pole reduced the number of primordial germ cells in the genital ridges compared to unirradiated embryos, but in uncentrifuged irradiated embryos no primordial germ cells were detected. However, in studies of the action of u.v. irradiation of Drosophila eggs on subsequent fertility, Geigy (1931) and Okada et ah (1974) have reported that u.v. irradiation of the polar region of the egg prevents the formation of pole cells. The latter authors question whether the primary effect of u.v. is against the pole plasm or against the processes by which the pole cells are formed. Our observations reported here show that the latter effect is important in irradiated X. laevis embryos. 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