/ . Embryo/, exp. Morph. Vol. 34, 1, pp. 209-220, 1975 2 0 9

Printed in Great Britain

The effect of u.v. irradiation ofthe 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 FlindersUniversity of South Australia

SUMMARY

The initial effect of u.v. irradiation of the vegetal pole was to inhibit cleavage in thevegetal hemisphere although karyokinesis was not substantially affected. In this way asyncytium formed in the vegetal hemisphere which broke down into individual cells sometime between morula and late blastula. The movement of the germ plasm from the peri-pheral cortical regions into the interior of the egg was not appreciably delayed althoughaggregation of the germ plasm did not take place until the individual presumptive primordialgerm cells were formed when the syncytium broke down. The method of segregation of thegerm plasm and formation of the presumptive primordial germ cells was therefore verydifferent in irradiated embryos from the normal orderly processes which depend on normalcleavage patterns. After neurula, the number of presumptive primordial germ cells declinedrapidly and at stage 43/44, when the genital ridges in normal embryos contain primordialgerm cells, the genital ridges in irradiated embryos were sterile. These results raise thequestion whether derangement of the segregation of the presumptive primordial germ cellsis solely responsible for the later abnormalities in the cell lineage or whether u.v. irradiationaffects the germ plasm and therefore indirectly the germ cells.

INTRODUCTION

Many anuran eggs and some insect eggs contain a microscopically identifiablecytoplasmic substance known as 'germ plasm' or 'pole plasm' respectively.These substances are believed to act as germ cell determinants because the cellswhich 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 todemonstrate the role of germ plasm is to irradiate the region containing it withu.v. light. At particular wavelengths and at high enough doses, u.v. causessterility in the adultswhich eventually develop (Geigy, 1931; Bounoure, 1937 a,b).Smith (1966) provided the most convincing evidence that germ plasm specifiesgerm 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., BedfordPark, South Aust. 5042, Australia.

14-2

210 B. ZIJST AND K. E. DIXON

transfer of vegetal pole cytoplasm, presumably containing germ plasm, fromunirradiated eggs to the vegetal pole of irradiated eggs. He concluded that theu.v. directly affected the germ plasm, preventing it from performing its function.However, the precise role played by germ plasm in specifying germ cells remainsunknown and the specific effects of u.v. which result in sterility have not beendetermined.

The general aim of our work has been to identify the events in the germ celllineage which are disrupted by u.v. treatment, in the expectation that thisinformation could throw light on the role of the germ plasm. In a previousreport from this laboratory, Whitington & Dixon (1975) described in detailthe process of segregation of the presumptive primordial germ cells (i.e. cellswhich contain germ plasm but are still in the endoderm cf. primordial germcells in the genital ridge or indifferent gonad) and also the events which takeplace in the endodermal phase of the cell lineage in Xenopus laevis. This studycompares these processes in u.v. irradiated embryos.

MATERIALS AND METHODS

Adult female X. laevis were injected with chorionic gonadotrophin (Pregnylor Chorulon, Organon) to produce eggs which were stripped manually (afterWolf & Hedrick, 1971) and fertilized with sperm from a macerated testis. Inthis 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 quartzslide and oriented with the animal pole upwards, and as much water was re-moved as possible. The eggs were irradiated with u.v. light between the begin-ning of the first and the end of the second cleavage at 253-7 nm (98 %). Totalu.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 tadpolestages (to stage 46) were fixed in Smith's fixative (see Jones, 1954) after the jellycoat had been removed with cysteine/papain solution (2:1, pH 8-0). The fixedtissues were dehydrated and cleared in an ethanol/xylene series or in dioxanand embedded in paraffin. Sections were cut at 5/«n and stained in eitherJanus green-neutral red (see Jones, 1954), Azan (after Gurr, 1962), acid fuchsin-azure II (Volkonsky, 1928), borax carmine-aniline blue-orange G (Boteren-

Effect ofu.v. on germ cells 211brood & Nieuwkoop, 1973), acid fuchsin-aniline blue-orange G (a combinationof the two previous procedures), Harris' haematoxylin or the Feulgen-Fastgreen sequence (Deitch, Wagner & Richart, 1968).

Measurements

The volume of germ plasm was calculated from measurements made withthe aid of a Zeiss integrating disc of the area of each patch in successive serialsections. The volume of individual germ cells was calculated from measurementsof 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, theeffects produced and the recovery from these effects varied somewhat betweeneggs of a single batch but the variation between different batches of eggs wasgreater, 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 ofu.v. irradiation is therefore generalized and is largely based on experimentsusing 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 regionof the vegetal pole. The first cleavage furrow, already apparent, frequentlyregressed in the irradiated region and subsequently new furrows were arrestedat about the equator of the egg (Fig. 1). The irradiated embryos developednormally as judged by the number and size of the animal pole blastomeres, butthe vegetal hemisphere had few, if any, cleavage furrows (Fig. 2) compared tocontrol embryos. There was no delay in the initiation of invagination at gastrulabut the highest mortality occurred at about this time, usually correlated withleakage of cytoplasm from the vegetal region, presumably through cell mem-branes 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 leastin early developmental stages and sometimes as late as blastula, no new cellmembranes had formed in the vegetal hemisphere (Figs. 3,4). However, in somemorula embryos, instead of one or two large cytoplasmic masses, a few smallermasses occupied the vegetal hemisphere. In blastula embryos, the vegetal halfwas occasionally composed of individual cells. The blastomeres near the equatorwere of roughly similar size to those in control embryos, but they were fre-quently connected to the large, uncleaved yolk mass through cytoplasmicbridges, 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 tohave regressed at the pole and the fourth cleavage planes (arrows) are arrested justpast the equator. Inset: unirradiated embryo at same developmental stage showingnormal cleavage pattern.Fig. 2. View of the vegetal pole of two stage-7 to stage-8 X. laevis embryosirradiated at the 2- to 4-cell stage showing cleavage in the vegetal hemisphere stillarrested although it has proceeded normally in the animal pole as shown in theembryo at the top left. Inset: unirradiated embryo at approximately the samedevelopmental stage showing normal cleavage pattern.

Fig. 3. Approximately median section through X. laevis blastula irradiated at thevegetal pole with u.v. at the 2- to 4-cell stage. The blastomeres in the animal hemi-sphere are normal but the vegetal hemisphere is practically uncleaved except for thefirst cleavage plane (arrow).

Fig. 4. Approximately median section through X. laevis blastula (slightly more devel-oped than Fig. 3) showing normal cleavage in the vegetal hemisphere for comparisonwith Fig. 3.

Effect ofu.v. on germ cells 213At the equator, the effect of the u.v. would presumably be considerably reduceddue to the curvature of the egg surface, but even so, complete separation of theblastomeres had been inhibited.

Although cytokinesis was delayed, nuclear division had continued. Examina-tion of sections showed that the vegetal hemisphere, although usually uncleaved,contained a number of nuclei and therefore constituted a syncytium. Fromcounts of Feulgen-treated sections of roughly equivalent volumes of uncleavedcytoplasm in irradiated embryos, and cleaved cytoplasm in control embryos,the number of nuclei was approximately the same, suggesting that the rate ofkaryokinesis in irradiated embryos was not greatly affected. Within a singlesyncytial mass, interphase nuclei and fully formed spindles were observed,suggesting that nuclear divisions were not synchronous. In about one-quarterof the gastrula embryos examined, a few of the nuclei, in both endodermal andpresumptive primordial germ cells, were very large, up to 22 times larger thanendoderm nuclei in control embryos. Enlarged nuclei were also observed atstages 15 and 29/30. This increase in size probably denotes an increase in DNAcontent, which could have arisen by repeated replication without division orthrough fusion of the nuclei (see Carroll & Van Deusen, 1973).

We conclude that the primary effect of the u.v. irradiation was to delaycytokinesis in the irradiated area without affecting karyokinesis substantially.The syncytia thus produced persisted for variable periods of time and cyto-kinesis was reinitiated at stages roughly between morula and late blastula. Itseems 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 germcells

Since segregation of the presumptive primordial germ cells depends on geo-metrical relationships between the position of the germ plasm and the cleavageplanes (Whitington & Dixon, 1975), retardation of cleavage could be expectedto affect their formation.

The germ plasm in all irradiated pre-blastula embryos was included in thesyncytial mass (see Fig. 5) except in some morulas, where part of the germ plasmwas sometimes contained within cells in the floor of the blastocoele comparablemorphologically to presumptive primordial germ cells in unirradiated embryos.The number of these cells was small and variable (0-2 ± 0-4) but had increasedby the blastula stage (2-9 ± 2-4). By early gastrula, when usually the syncytiumhad completely broken down, the number of individual presumptive primordialgerm cells rose to 9-4 + 5-4, about the same number (8-7 + 3-0) reported for thisstage by Whitington & Dixon (1975). These results reinforce our earlier inter-pretation that breakdown of the syncytia in some embryos begins about morulaand 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 (notinhibited) by the u.v. irradiation.

The size of the few individual presumptive primordial germ cells present atblastula (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 re-dressed by a single additional mitosis. At this stage in development of normalembryos cleavage is rapid, so that differences of the magnitude observed can beexpected. At late gastrula, the size of the presumptive primordial germ cells inthe irradiated embryos was similar to that in the control embryos. Furthermore,the presumptive primordial germ cells in irradiated embryos up to early gastrulawere more or less randomly distributed within the vegetal hemisphere as theyare in normal embryos.

Thus, in irradiated embryos, presumptive primordial germ cells similar innumber, size and position to those in unirradiated embryos are segregated byrandom processes instead of by the normal ordered and predictable sequenceof 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 upto gastrula, were similar to those in control embryos. The patches were granular,yolk-free, associated with a few pigment granules and surrounded by small yolkplatelets.

In unirradiated eggs, when cleavage commences, the germ plasm begins tomove towards the position of the cleavage furrow and to aggregate, a processwhich culminates in the formation of a small number of large patches of germplasm. Normally one patch occurs in each of the first four blastomeres, situatedclose 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 eachpresumptive primordial germ cell does not change until about the gastrulastage, when the germ plasm moves to a perinuclear position (Whitington &Dixon, 1975).

In early embryos developing from irradiated eggs, the variation in the numberof patches of germ plasm was wide (3-64 at morula, 8-61 at blastula), but inembryos of the same batch, it was narrower, providing another indication of thevariable sensitivity of different batches of eggs to u.v. irradiation. In gastrulae,the individual presumptive primordial germ cells each contained only one ortwo large patches, except for an occasional cell in which the germ plasm wasmore diffuse.

In morulae, 90 % of the patches lay in the interior of the syncytium close to acleavage membrane and the rest were immediately inside the surface about thevegetal pole. At later stages, the position of the individual patches was veryvariable 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 theregion 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 theindividual germ cells eventually moved to the nucleus in an apparently normalmanner.

These observations suggest the following interpretation. At the time of irradia-tion, the first cleavage division was either completed or still in progress. Hencethe germ plasm had begun to move into the first cleavage furrow and to aggre-gate in the normal way. After irradiation, the movement into the interior of theembryo apparently continued without obvious retardation, but aggregationwas delayed. Thus, the involution of cortical materials (Schechtman, 1934)invoked by Whitington & Dixon (1975) to explain the inward movement of thegerm plasm can apparently take place without the continuous formation ofcleavage membranes, but the membranes already present may direct the flowof 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/tm3x 102, compared to 380 ± 198 /*m3 x 102 in control embryos of thesame age. The differences between control and irradiated embryos were almostcertainly due to the greater difficulty in recognizing and accurately measuringa number of small patches of germ plasm. We conclude that the total amount ofgerm 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 nor-mally undergoes two or three divisions, and then at stages 38-41, they migrateout of the endoderm into the forming median genital ridge (Whitington &Dixon, 1975).

In irradiated gastrulae, as noted earlier, the number of presumptive primordialgerm cells was similar to that in unirradiated embryos. However, from neurulaonwards, the number of germ cells which could be recognized declined rapidly.Of 18 early tail-bud embryos, 7 had no presumptive primordial germ cells andthe others had a mean of 2-5 ±1-9 per embryo, while up to stage 46, presumptiveprimordial 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 ridgescontained easily recognizable primordial germ cells, in irradiated embryosreceiving 11000 ergs/mm2 or higher doses of u.v., no germ cells could be dis-tinguished in the genital ridges which were, however, developed to approxi-mately the normal extent.

Sections of irradiated embryos of stages 15-46 were processed simultaneouslywith sections of control embryos through either the Volkonsky staining sequence,Azan, the procedure of Boterenbrood & Nieuwkoop (1973), the combinationof these two or haematoxylin and eosin. However, either the presumptiveprimordial germ cells were not present in irradiated embryos or the stainingcharacteristics of the germ plasm had changed so that the germ cells, althoughpresent, could no longer be identified.

DISCUSSION

The first effect observed after u.v. irradiation of the vegetal pole of X. laeviseggs was a delay in cytokinesis in the vegetal hemisphere. However, nucleardivision continued so that a syncytium formed, which in early cleavage stagesoccupied almost all of the vegetal hemisphere. Inhibition or retardation ofcytokinesis 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 disruptedcleavage but the nature of this effect was not examined. In another study in ourlaboratory, Beal & Dixon (1975) have confirmed that u.v. irradiation of thevegetal pole of Xenopus eggs suspends cytokinesis. They suggested, after com-parison with the effects of cytochalasin B on X. laevis eggs (Bluemink, 1971;Hammer, Sheridan & Estensen, 1971) that the processes affected are adhesivenessof the blastomeres and the incorporation of new membrane into the cleavagefurrow.

Effect ofu.v. on germ cells 217The 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 leaston rather general morphological criteria. However, the orderly processes whichnormally result in the segregation of the presumptive primordial germ cells(Bounoure, 1934; Whitington & Dixon, 1975) were deranged by the retardationof cleavage. If a cell lineage is the result of a causal sequence of events, it seemspossible that disturbance of the processes normally responsible for the initiationof the germ cell lineage may have repercussions at later stages of the lineage.

In unirradiated embryos, the presumptive primordial germ cells segregatedat the blastula stage undergo two or three divisions between early gastrula andthe time (stage 41) they leave the endoderm (Whitington & Dixon, 1975).Using [3H]thymidine autoradiography Dziadek & Dixon (1975) have confirmedthat the presumptive primordial germ cells synthesize DNA and divide fromgastrula onwards. In contrast, in post-neurula irradiated embryos the number ofpresumptive primordial germ cells which could be recognized declined rapidly,and at stages 43-46 when the genital ridges would normally be populated byeasily identifiable primordial germ cells, none were present. Previous reportsconcerning u.v. irradiation of the vegetal pole of early anuran embryos agreethat practically no primordial germ cells could be detected at stages when theywere normally easily identified, and the animals were therefore consideredsterile (Bounoure et al. 1954; Padoa, 1963, 1964; Smith, 1966; Buehr, 1969;Ikenishi, Kotani & Tanabe, 1974; Tanabe & Kotani, 1974). Our observationsagree, in that at doses of 4000 ergs/mm2 or less, some primordial germ cells ofnormal 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 determinehow this sterility is produced. Our results show that the presumptive primordialgerm cells cannot be detected at stages later than neurula, even though aconcerted effort was made, using many different staining sequences, to dis-tinguish the germ plasm, the single reliable criterion by which presumptiveprimordial germ cells can be identified. Two possible explanations can beadvanced 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 thegerm plasm changes so that it can no longer be recognized with the normalstaining sequences.

Three observations suggest that the latter explanation is more likely to becorrect. First, the presumptive primordial germ cells in irradiated embryoscontinue to synthesize DNA and divide up to late neurula (Dziadek & Dixon,1975). This pattern is not consistent with (but it does not exclude) the hypothesisof cell death or loss of determined state. Secondly, in u.v.-treated embryos inour experiments, primordial germ cells appear in the genital ridges about stage48, 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 theendoderm although their migration into the genital ridges may have been delayed.Thirdly, the germ plasm normally loses its stainability when the primordial germcells enter the median genital ridge (Bladder, 1958; Whitington &Dixon, 1975);the inability to stain the germ plasm in post-neurula irradiated embryos mayrepresent a precocious expression of the normal process.

In summary, the results reported here show that u.v. irradiation of the vegetalpole of X. laevis eggs causes a substantial delay in cleavage of the vegetalhemisphere and thus affects the segregation of the presumptive primordial germcells and their subsequent behaviour in the endoderm after the neurula stage.

Other investigators have attempted to show that u.v. directly affects the germplasm. Smith (1966) has shown that transfer of vegetal pole cytoplasm fromunirradiated eggs reversed the effect of u.v. and germ cells migrated into thegenital ridges at the normal time. Ikenishi et ah (1974) have described changesin the germ plasm and associated mitochondria in u.v. irradiated eggs. Tanabe& Kotani (1974) centrifuged X. laevis eggs and thereby displaced the germplasm into the interior of the egg; subsequent irradiation of the vegetal polereduced the number of primordial germ cells in the genital ridges compared tounirradiated embryos, but in uncentrifuged irradiated embryos no primordialgerm cells were detected. However, in studies of the action of u.v. irradiationof 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 theformation of pole cells. The latter authors question whether the primary effectof u.v. is against the pole plasm or against the processes by which the pole cellsare formed. Our observations reported here show that the latter effect is import-ant in irradiated X. laevis embryos. Whether derangement of the segregationof the presumptive primordial germ cells is solely responsible for the laterabnormalities in the cell lineage remains to be determined.

Our thanks are due to Dr P. Janssens, The Australian National University and to MrH. A. Clair, University of Western Australia, for assistance in setting up our Xenopuscolony. Mrs E. Harrland gave valuable technical assistance. B. Ziist was supported by aFlinders University Research Scholarship.

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{Received 4 February 1975, revised 19 March 1975)