Relationship between the amount of the ' germinal plasm' and the number of primordial germ cells in Xenopus laevis

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1 /. Embryol. exp. Morph. Vol. 31, I, pp , 1974 go, Printed in Great Britain Relationship between the amount of the ' germinal plasm' and the number of primordial germ cells in Xenopus laevis ByKAZUYUKI TANAB^AND MINORU KOTANI 2 From the Department of Biology, Osaka City University, Japan SUMMARY Tadpoles of Xenopus laevis completely lacking primordial germ cells were obtained by irradiating the vegetal hemisphere of early 2-cell eggs with u.v. (wavelength, nm; dose, ca ergs/mm 2 ). An increasing number of primordial germ cells were observed as the stage at irradiation advanced from early 2-cell to early 4-cell stages. Furthermore, early 2-cell eggs irradiated with doses ranging from 750 to 6000 ergs/mm 2 grew into tadpoles carrying a decreasing number of primordial germ cells in accord with the increase of the dose. On the other hand, tadpoles developed from eggs irradiated immediately after being centrifuged at 150 g for 1 min at early 2-cell stage to displace the 'germinal plasm' deeper into the cytoplasm, carried a considerable number of primordial germ cells. These facts were interpreted to suggest the presence of u.v.-sensitive germ cell determinant in the 'germinal plasm'. It was revealed by varying the area of irradiation that the number of primordial germ cells decreased in direct proportion to the increase of the area irradiated. It was concluded that the amount of the u.v.-sensitive material(s) contained in the 'germinal plasm' determined the number of primordial germ cells in tadpoles. INTRODUCTION Bounoure (1934) first described in Rana temporaria a specially staining cytoplasm (the so-called 'germinal plasm') located in the peripheral cytoplasm around the vegetal pole of the fertilized eggs. Since then, several authors have studied the behaviour of the 'germinal plasm' during the normal course of embryonic development in histological sections of a wide variety of anuran eggs using various staining methods (Nieuwkoop & Faber, 1956; Blackler, 1958; DiBerardino, 1961; Gipouloux, 1962). This plasm becomes incorporated into some of the vegetal blastomeres during early developmental stages, and appears deep in the entoderm at the gastrula and neurula stages. These cells migrate later into the genital ridges, where they eventually differentiate into primordial germ cells (Blackler, 1958). 1 Author's address: Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545, Japan. 2 Author's address: Department of Biology, Faculty of Science, Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka 558, Japan.

2 90 K. TANABE AND M. KOTANI Bounoure, Aubry & Huck (1954), on the other hand, obtained animals without germ cells or partially sterile animals with a reduced number of germ cells by irradiating the vegetal hemisphere of fertilized eggs with ultraviolet light (u.v.). In 1966 Smith irradiated early 2-cell eggs with a larger dose of u.v. and found that all his experimental tadpoles (Rana pipiens) were completely lacking in primordial germ cells. He showed, further, that primordial germ cells were able to be reformed when irradiated eggs were injected with vegetal subcortical cytoplasm of unirradiated eggs. It was then concluded that some kind of u.v.-sensitive material(s) indispensable for differentiation of primordial germ cells was located near the vegetal pole. It was not known, however, whether this material(s) corresponded to the 'germinal plasm', since the 'germinal plasm' had not been demonstrated in early cleavage stages of Rana pipiens (DiBerardino, 1961; see Smith, 1966). On the other hand, the question whether the 'germinal plasm' really contained a germ cell determinant was investigated by Buehr & Blackler (1970), who removed the 'germinal plasm' together with some of the vegetal cytoplasm by pricking the egg near the vegetal pole. The result, that the proportion of embryos completely lacking 'germinal plasm' at blastula stage nearly equalled that of tadpoles lacking primordial germ cells, suggested a close relationship between the presence of the 'germinal plasm' and the formation of primordial germ cells. It was also suggested that partially sterile tadpoles were likely to have developed from operated eggs whose 'germinal plasm' was only partially removed. It is hard to define, however, what really was removed, quantitatively and qualitatively, through these microsurgical operations. In the present study, an advantage was taken of the fact that, with u.v., it should become much easier to inactivate a known proportion of the 'germinal plasm'. A close relation between the 'germinal plasm' and the u.v.-sensitive 'germinal determinant' was demonstrated by a combination of irradiation and centrifugation procedures. It was also examined, by varying the area of irradiation, whether a quantitative correlation existed between the amount of the intact 'germinal plasm' and the number of primordial germ cells formed. MATERIALS AND METHODS Fertilized eggs of Xenopus laevis were obtained by injecting human chorionic gonadotrophic hormone into mature toads (200 units for males, and 300 units for females). Embryos were staged after Nieuwkoop & Faber (1956). U.v. irradiation Eggs at stage 2~ (beginning of the first cleavage), 2 and 3~ (beginning of the second cleavage) and eggs centrifuged at stage 2~ were subjected to u.v. irradiation. In order to irradiate the vegetal hemisphere, eggs with jelly coats were placed on quartz slides without excess water, with the animal hemisphere up-

3 Primordial germ cells in Xenopus Egg with jelly coat 91. Quartz slide mini u.v. filter Germicidal lamp wavelength nm Aluminum adhesive tape O Q&r m. : -: Quartz slide. 3,, - Proportion of vegetal hemisphere irradiated Fig. 1. Schematic representation of u.v. irradiation. (A) Method of u.v. irradiation (side view). (B) Irradiation of proportions of the vegetal hemisphere (upper view). wards. Then the slides were laid on the band-path filter (Corning Glass Works, 7-54) fixed horizontally just above the light source, Nikko Sekiei Works' Super- Light' LS-D1 (Fig. 1 A). The dose rate at a wavelength of nm was about 150ergs/mm 2 /sec (1 erg = 10-7 J) when measured on the surface of the slide. A total sterility was obtained with a dose of about 6000 ergs/mm 2. In order to examine the relationship between the amount of the intact (unirradiated) 'germinal plasm' and the number of primordial germ cells, known proportions of the vegetal hemisphere of stage 2~ eggs were irradiated by locating eggs at various sites on the edges of aluminium adhesive tape, which was ascertained beforehand to cut u.v. off completely (Fig. 1B). Centrifugation To displace the 'germinal plasm' without disturbing subsequent development, eggs at stage 2~ with jelly coats were placed, animal pole upwards, at the bottom of a 10 ml centrifuge tube filled with distilled water and centrifuged at 150 g for 60 sec. Eggs which completed the first cleavage during centrifugation procedures were discarded. A number of the centrifuged eggs were killed immediately after centrifugation to see the location of the' germinal plasm'. The rest, with or without subsequent irradiation, were allowed to develop into feeding tadpoles, where the degree of formation of primordial germ cells was examined.

4 92 K. TANABE AND M. KOTANI Histology Sterility or fertility of experimental animals was checked as follows. As aliquots of irradiated and unirradiated batches of eggs derived from the same mother were allowed to develop into feeding tadpoles (stage 46), those showing an abnormal appearance were discarded eventually so that only those looking normal throughout development were fixed with Bouin's fixative. Tadpoles derived from centrifugation experiments were treated similarly. Serial paraffin sections, 5 fim thick, were stained with Mayer's hematoxylin and eosin. Primordial germ cells lodged in the genital ridges and dorsal mesentery were identified and counted on characteristics such as the large cell size, cytoplasm rich in yolk platelets, and large, highly lobed, weakly staining nucleus. To see the location of the 'germinal plasm', eggs at stages 2~, 2 and 3~ and centrifuged eggs were fixed with 10 % formalin in 1/15 M phosphate buffer, ph 7-3. Fixed materials were sectioned serially parallel to the animal-vegetal axis at thickness of 5 jam, and stained with Heidenhain's azan (Czolowska, 1969). The distance from the vegetal surface to the centre of the islets of ' germinal plasm' was measured, in every fifth section, parallel to the main axis of the egg, under an ocular micrometer, x240 (minimum unit, 0-24 /tm) islets per egg were measured. RESULTS Although completely sterile tadpoles were rarely found in the normal populations, the average number of primordial germ cells per tadpole varied from batch to batch and the actual number carried by an individual also varied considerably within a batch. U.v. irradiation with various doses Stage 2~ eggs were irradiated with doses ranging from 750 to 6000 ergs/mm 2 by varying irradiation time from 5 to 40 sec, respectively. The average number of primordial germ cells of tadpoles per dose is shown in Fig. 2, in which it is seen clearly that the number of primordial germ cells decreases along with the increase of the dose and that it reaches zero around 30-sec irradiation. Location of the 'germinal plasm' and the effect of u.v. It is shown in Table 1 that 6000 ergs/mm 2 of u.v., which drives all stage 2~ eggs into totally sterile tadpoles, drives greater proportions of stage 2 and 3" eggs only into partially sterile tadpoles. What is more, a greater number of primordial germ cells are formed as the stage at irradiation advances. Microscopic observations, on the other hand, of the interior of stage 2~ eggs reveal that islets of 'germinal plasm' are located in the subcortical cytoplasm of the vegetal hemisphere and are distributed uniformly around the vegetal pole

5 Primordial germ cells in Xenopus (30) (30) (30) (23) (30) (10) (30) Duration of irradiation (sec) o- 40 (30) Fig. 2. Average number of primordial germ cells (PGCs) per tadpole, with standard error. Eggs were irradiated at stage 2~ for various durations, and allowed to develop into feeding tadpoles (stage 46). Dose rate was about 150 ergs/mm 2 /sec. Number of tadpoles examined is given in parentheses. All eggs were obtained from one batch. Table 1. Location of the 'germinal plasm' at early cleavage stages and effect ofu.v. irradiation on the formation of primordial germ cells (PGCs)* Egg Stage 2- Stage 2 Stage 3 Un irradiated No. islets examined 995 (7)t 940 (9) 530 (9) Average distance of the islets r from the vegetal surface (/*m) 3-3 ± ± ±2-3 * All eggs were obtained from one batch. t No. of eggs examined. \ Mean value with standard error. Stage No. animals Stage With germ cells Average no. PGCs per tadpole (60-7 %) 3-5±O-8J 25 (89-3 %) (100%) 33-5 ±6-2

6 94 K. TANABE AND M. KOTANI Fig. 3. Location of the 'germinal plasm' at early cleavage stages. x270. Sections, 5/itn thick, were stained with Azan. (A) stage 2~; islets (arrows) of the 'germinal plasm' are scattered in the vegetal subcortical cytoplasm. 9 islets are seen in this figure. (B) stage 2; islets (arrows) are located more to the interior and nearer to the cleavage furrow. (C) stage 3~; islets (arrows) are located farther from the egg surface and coalesced along the cleavage furrow. (D) centrifuged egg at stage 2 ; islets (arrows) are located much deeper in the cytoplasm. VP\ vegetal pole. within an area of about a half of the diameter of the egg (Fig. 3 A). These islets coalesce gradually during stages 2 and 3~ while at the same time migrating interiorly along the cleavage furrow (Fig. 3B, C). The distances from the vegetal surface to the centre of the islets are summarized in Table 1. Centrifugation experiment Histological observations of centrifuged eggs reveal that a considerable number of islets of 'germinal plasm' has been displaced deeper into the cytoplasm (Fig. 3D). The average distances of the 'germinal plasm' from the vegetal surface are shown in Table 2. The number of primordial germ cells was counted in feeding tadpoles which had descended from normal eggs, from eggs either irradiated or centrifuged and from those irradiated immediately after centrifugation. The average number of primordial germ cells per animal is shown in Table 2, which demonstrates that tadpoles belonging to the last group possess a considerable number of

7 Primordial germ cells in Xenopus 95 Table 2. Location of the 'germinal plasm' after centrifugation and effect ofu.v. irradiation on the formation of primordial germ cells (PGCs)* Egg Normal Irradiated Centrifuged Irradiated after centrifugation No. islets examined 1305 (9)t 437 (8) Average distance of the islets from the vegetal surface (/*m) 5-3 ± 0-6J 99-4 ±260 * All eggs were obtained from one batch. t No. of eggs examined. X Mean value with standard error. f No. animals Stage Stage Average no. PGCs Dcr tadpole $ ±2-5 ll-5±2-2 Table 3. Effect of irradiating half the vegetal hemisphere on the formation of primordial germ cells (PGCs)* Average no. of PGCs Area irradiated No. of eggs No. of tadpoles at stage 8 at stage 46 per tadpole with standard error 0 (Unirradiated) 45, f Either one of the blastomeres 49 i \ A half of both blastomeres 45 Whole of the vegetal hemisphere 42 * All eggs were obtained from one batch ± ± ±2-2 primordial germ cells, in spite of the fact that they have received a dose strong enough to cause complete sterility. U.v. irradiation on known proportions of the vegetal hemisphere Two series of experiments were performed to examine the relationship between the amount of 'germinal plasm' and the number of primordial germ cells. In the first experiment, a half of the vegetal hemisphere of early 2-cell eggs, including either only one of the two blastomeres or a half of both, was subjected to irradiation. The number of primordial germ cells found in the descendant tadpoles is summarized in Table 3. It can be seen from these results that tadpoles derived from either of the two groups carry nearly half the number of primordial germ cells possessed by the control animals. In the second experiment, one to four quarters of the vegetal hemisphere of stage 2~ eggs were irradiated. It is clearly seen in Fig. 4 that the average number of primordial germ cells decreases in direct proportion to the increase of the area irradiated. 0

8 96 K. TANABE AND M. KOTANI (37) (37) (33) (40) (36) Area (%) of vegetal half irradiated Fig. 4. Average number of primordial cells (PGCs) per tadpole, with standard error. Different areas of the vegetal half were irradiated. Number of tadpoles examined is given in parentheses. DISCUSSION Since more primordial germ cells are found in the descendant tadpoles as u.v. dose decreases (Fig. 2) and u.v. hardly penetrates an anuran egg (Grant & Wacaster, 1972), an apparent drop in sensitivity of an egg to u.v. associated with the inward migration of the 'germinal plasm' during early cleavage stages (Table 1) suggests that the islets of 'germinal plasm' located deeper in the vegetal cytoplasm do not receive a sufficient dose to cause complete sterility in tadpoles. This is further supported by the results from the centrifugation experiments. These show that tadpoles derived from irradiated eggs carry a considerable number of primordial germ cells and that centrifuged eggs possess the 'germinal plasm' located much deeper in the vegetal cytoplasm (Table 2). Hence it is highly probable that the apparent drop of sensitivity to u.v. observed in eggs beyond stage 2~ is not due to a gradual loss of sensitivity to u.v. of the 'germinal plasm' with age, but to a decrease of dose reaching the 'germinal plasm'. It is concluded from these evidences that a u.v.-sensitive germ-cell determinant is present in the 'germinal plasm'. It has been revealed by a recent study that the fine structure of the 'germinal plasm' in Xenopus laevis is essentially the same as that of Ranapipiens (Czolowska, 1972). Comparison at ultrastructural and at light microscope levels of

9 Primordial germ cells in Xenopus 97 u.v.-irradiated and unirradiated 'germinal plasm' of Xenopus Jaevis has demonstrated that a sterilizing dose (6000 ergs/mm 2 ) of u.v. on the vegetal hemisphere of stage 2~ eggs causes swelling and vacuolation of mitochondria and fragmentation of electron-dense germinal granules without diminishing the stainability in the histological sections (Ikenishi, Kotani & Tanabe, 1974). These facts suggest that the intactness of subcellular organelles such as mitochondria and germinal granules is essential for differentiation of primordial germ cells, and also support the conclusion from the centrifugation experiments. That u.v. irradiation of half the vegetal hemisphere reduced the average number of primordial germ cells to nearly half may be interpreted as indicating two features of the 'germinal plasm' (Table 3). One is that the irradiated 'germinal plasm' does not damage a certain function of the intact plasm in the formation of primordial germ cells, because the descendant tadpoles were able to form primordial germ cells up to half the normal number. The other is that, when such tadpoles are found to carry only half the normal number of primordial germ cells, the intact plasm does not compensate for injured function of the irradiated plasm. In other words, a definite amount (half) of the intact 'germinal plasm' is considered to be responsible for the formation of only a limited number (half) of the primordial germ cells. The latter suggestion is further confirmed by the result from u.v. irradiation on known proportions of the vegetal hemisphere. The result - that the number of primordial germ cells formed in tadpoles increases in direct proportion to the increase of the area unirradiated - leads us to conclude that the number of the primordial germ cell found in tadpoles is determined by the amount of the u.v.-sensitive material(s) present in the 'germin alplasm'. This conclusion justifies the suggestion that eggs bearing the reduced amount of 'germinal plasm' grow into partially sterile tadpoles (Buehr & Blackler, 1970). We are grateful to Drs Marina Dan Sohkawa and Mitsuru Furusawa for critical readings of the manuscript, and Dr Seiichiro Kamisaka for his useful suggestions. Thanks are also due to Mr Taizo Kimura for his help in photographic preparations. REFERENCES BLACKLER, A. E. (1958). Contribution to the study of germ cells in the Anura. /. Embryol. exp. Morph. 6, BOUNOURE, L. (1934). Recherches sur la lignee germinale chez la grenouille rousse aux premiers stades du developpement. Annls Sci. nat. 10 e ser. 17, BOUNOURE, L., AUBRY, R. & HUCK, M.-L. (1954). Nouvelles recherches experimentales sur les origines de la lignee reproductrice chez la grenouille rousse. J. Embryol. exp. Morph. 2, BUEHR, M. L. & BLACKLER, A. W. (1970). Sterility and partial sterility in the South African clawed toad following the pricking of the egg. /. Embryol. exp. Morph. 23, CZOLOWSKA, R. (1969). Observation on the origin of the 'germinal cytoplasm' in Xenopus laevis. J. Embryol. exp. Morph. 22, E M B 3 I

10 98 K. TANABE AND M. KOTANI CZOLOWSKA, R. (1972). Thefinestructure of the' germinal cytoplasm' in the eggs of Xenopus laevis. Wilhelm Roux Arch. EntwMech. Org. 169, DIBERARDINO, M. (1961). Investigation of the germ-plasm in relation to nuclear transplantation. /. Embryol. exp. Morph. 9, GIPOULOUX, J.-D. (1962). Mise en evidence du 'cytoplasme germinal" dans l'oeuf et l'embryon du Discoglosse: Discoglossus pictus Otth. C. r. hebd. Seanc. Acad. Sci., Paris GRANT, P. & WACASTER, J. F. (1972). The amphibian gray crescent region. A site of developmental information? Devi Biol. 28, IKENISHI, K., KOTANI, M. & TANABE, K. (1974). Ultrastructural changes associated with u.v. irradiation in the 'germinal plasm' of Xenopus laevis. Devi Biol. 36 (in the Press). NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/xenopus laevis (Daud.). Amsterdam: North-Holland Publishing Co. SMITH, L. D. (1966). The role of a 'germinal plasm' in the formation of primordial germ cells in Rana pipiens. Devi Biol. 14, {Received 1 May 1973, revised 1 July 1973)