/ . Embryol. exp. Morph. Vol. 22, 2, pp. 229-51, September 1969 2 2 9

Printed in Great Britain

Observations on the origin of the ' germinalcytoplasm' in Xenopus laevis

By RENATA CZOLOWSKA1

From the Institute of Zoology, University of Warsaw, Poland,and the Hubrecht Laboratory, Utrecht, Holland

The early appearance of the 'germinal cytoplasm' and its behaviour duringthe formation of cells which are thought to represent the primordial germ cellshave been described in detail for Rana temporaria by Bounoure (1927, 1934,1939) and Bladder (1958). These observations were extended to Xenopus laevis(Nieuwkoop, 1956; Nieuwkoop & Faber, 1956; Blackler, 1958), Bufo bufo(Blackler, 1958), Ranapipiens (Berardino, 1961), Discoglossus pictus (Gipouloux,1962 a) and Rana esculenta (Hammer, cited by Blackler, 19656).

The above findings agree with respect to the earliest detection of the 'germinalcytoplasm'. Its first appearance was noticed as early as in the fertilized, unseg-mented egg, where small, distinctly staining islands of cytoplasm are localizedjust under the cell membrane in an area around the vegetative pole of the egg.

During cleavage the 'germinal cytoplasm' is distributed between the vege-tative blastomeres directly surrounding the vegetative pole. At the beginning ofgastrulation some cells containing 'germinal cytoplasm' are detectable in thepresumptive endoderm and are, from then on, considered as true primordialgerm cells, incapable of undergoing any somatic differentiation. They retaintheir embryonic character with respect to yolk and pigment content andrelatively large size. Subsequently, the primordial germ cells are graduallydisplaced in the endoderm by morphogenetic movements and afterwards,presumably, by active migration until they reach a position corresponding tothat of the future germinal ridges.

Making use of the localization of the 'germinal cytoplasm', Bounoure(1935a, b, 1937a, b, c, d, 1939, 1950), Aubry (1953) and Bounoure, Aubry &Huck (1954) have demonstrated that irradiation of the vegetative hemisphereof fertilized, unsegmented eggs of R. temporaria with ultraviolet light (u.v.) has avery definite effect on the future germinal line. After such treatment the gonadsof fully metamorphosed frogs are reduced in size and possess a markedlyreduced number of gonocytes, causing partial or complete sterility. Padoa (1963,1964) obtained similar results with R. esculenta. The effect of u.v. on the eggs

1 Author's address: Institute of Zoology, University of Warsaw, Warsaw 64, Poland.

230 R. CZOLOWSKA

of R.pipiens was extensively studied by Smith (1966). With a defined dose, wave-length and time of exposure he obtained complete sterilization in 100 % of thelarvae by irradiating the eggs from the vegetative side just before the first cleavage.Smith was also able to show that the germinal line is restored if vegetative plasmof a non-irradiated egg is injected into the vegetative half of an irradiated one.

These results allow us to conclude that a u.v.-sensitive material localizedaround the vegetative pole of the fertilized egg is responsible for germ-linedifferentiation.

Less clear results have been obtained with surgical methods. According toNieuwkoop & Suminski (1959) and Fischiarolo (1960) the removal of vegetativematerial in dividing eggs (4-cell stage) or early embryos (blastulae) of Xenopuslaevis and Discoglossus pictus respectively does not influence the future germinalline of the larvae. However, Monroy (1939) and Gipouloux (19626) reportedpositive results after removing the endoderm from neurulae, and Librera (1964)succeeded in obtaining sterile larvae when the cytoplasm lying nearest to thevegetative pole had been removed from uncleaved eggs.

Special attention has been paid to two basic problems: (1) whether cells whichcontain identifiable 'germinal cytoplasm' should be considered as true pri-mordial germ cells, i.e. whether they are transformed into the true gametesin the course of development, and (2) whether primordial germ cells representthe exclusive source of all gametes. In both cases a positive answer seemsto be justified when the results of Blackler (1960, 1961, 1962a, b, 1965tf, b) andBlackler & Fischberg (1961) are considered.

From morphological observations it is known that the 'germinal cytoplasm'is crowded with mitochondria, rich in pigment granules and very often associ-ated with small yolk platelets. Some RNA is present as was demonstrated by acytochemical test by Blackler (1958). This is consistent with the speculations ofPadoa (1964) and with the deduction of Smith (1966), who found that the range ofthe u.v. spectrum within which the germinal line is damaged corresponds to thatwithin which nucleic acids (both RNA and DNA) absorb and hence are affected.

In the present study an attempt is made to elucidate the origin of the 'germinalcytoplasm'.

MATERIAL AND METHODS

Fertilized eggs, unfertilized eggs, ovulated oocytes and ovarian oocytes ofXenopus laevis were used in the course of these studies, in total about 250specimens.

Fertilized and unfertilized eggs were obtained after injecting toads withgonadotrophic hormone (Physex, Leo Pharmaceutical Products), the males with40 i.u. on two successive days and the females with 200 i.u. on the second day.

Ovulated oocytes were taken from hormonally stimulated females which werekilled some time after they started to lay normal eggs. Material was collectedfrom the uterus, from different portions of the oviduct and from the body cavity.

Germinal cytoplasm in Xenopus 231

As a source of ovarian oocytes two kinds of females were used, thosestimulated by hormone injection and unstimulated ones. After dissection smallpieces of the ovary were fixed and individual oocytes were isolated withforceps.

Oocytes obtained after ovulation was induced in vitro were an additionalsource of material. For this purpose small ovarian fragments were suspendedin Ringer solution (pH 7-2) containing 25 and 50 i.u. of Physex/ml. The Ringersolution used contained (g/1.): 6-5 g NaCl, 014 g KC1, 012gCaCJ2, 0-2 gNaHCO3, 005 g NaH2PO4.H2O. After 8h hormone treatment all oocyteslying freely on the bottom of the vials were collected and placed in Ringer for40 min or 13 h before fixation.

Short fixation (4-6 h) in neutral 4 % formol containing 0-5 % cetylpiridiniumchloride (the presence of cetylpiridinium chloride facilitates decapsulation) withsimultaneous decapsulation in the fixative was followed by standard dehydra-tion through alcohol and embedding in hard paraffin (M.P. 58 °C). Storage for48 h in isoamyl acetate, before embedding, was found to be helpful for obtaininggood sections.

Serial sections, 5-7 fi thick, were cut either parallel or perpendicular to theanimal-vegetative axis of the egg. The sections were stained using the Azanmethod (Romeis, 1948), but omitting differentiation with aniline/alcohol andtreatment with phosphotungstic acid. Staining in warm azocarmine for 15-30 swas followed by rinsing in distilled water and staining in orange G-anilineblue for 30 min.

As a specific method for RNA detection, staining with methyl green-pyroninewas used. The original formula of Unna (1913) was modified according toJ. Brachet (personal communication). The staining mixture consisted of 015 gof methyl green and 0-25 g of pyronine-Y dissolved in 100 ml of acetate buffer(pH 4-7). Serial sections were mounted alternately on slides. One slidefrom every sample was always treated with an aqueous solution of RNase(Worthington Biochemical Corporation), at a concentration 0-2 mg/ml, for1-2 h at 37-38 °C. As an additional control some slides were incubated indistilled water only. Subsequently, all slides were stained together for 1-2 h inthe staining mixture. Short rinses in distilled water and 96 % alcohol werefollowed by dehydration in absolute alcohol, clearing in xylol and mountingin DePeX.

RESULTS

Ovarian oocytes

Large oocytes isolated from the ovary are surrounded by a triple sheet con-sisting of peritoneum, connective tissue and follicular epithelium (Wartenberg& Schmidt, 1961; van Gansen, 1966). An underlying layer staining strongly withaniline blue represents the vitelline membrane (Hope, Humphries & Bourne,1963; Tokin & Gabayeva, 1963; Kemp & Istock, 1967).

232 R. CZOLOWSKA

Detailed descriptions of the amphibian oocyte with respect to yolk and pig-ment distribution, morphology of the nucleus and localization of the cytoplasmhave been given by Wittek (1952) and Kemp (1953, 1956).

An animal-vegetal 'gradient' in the distribution of the cytoplasm is wellmarked (Fig. 1 A). In the whole animal hemisphere large quantities of blue-stain-ing cytoplasm can be found. The pattern of its distribution is reminiscent of a

Germinal cytoplasm in Xenopus 233

y ^ r ..7 *i, • * , F - • • • • • . • • . • • . • * * " • V ! j " -I • • •*" I ,

VP

500//

Fig. 2. Distribution of cytoplasm in a fully grown oocyte isolated from the ovary.Meridional section. GV, Germinal vesicle; VP, vegetative pole region.

FIGURE 1

A. Meridional section of fully grown oocyte isolated from the ovary. Stained withAzan method. An accumulation of cytoplasmic material (AC) is seen on the vegeta-tive side of the germinal vesicle (G V). x 40.B. Nucleus of ovarian oocyte, meridional section. Azan staining. Cytoplasmicmaterial (AC) accumulated underneath the germinal vesicle (GV). x 97.C. Meridional section of vegetative pole region of ovarian oocyte under highmagnification. Stained with methyl green-pyronin. RNA-containing patches(PAT) are seen among the yolk platelets (YP) at the periphery of the oocyte.EV, Envelopes of the oocyte. x 910.D. Meridional section of ovulated oocyte collected from the body cavity. Stainedwith methyl green-pyronin. Germinal vesicle dissolved. Clear gradient of thedistribution of RNA with maximum in the animal hemisphere (AN). VEG, Vegeta-tive hemisphere. x41.E. Vegetative pole of an oocyte ovulated /// vitro. Meridional section, Azan staining.Islands of darkly staining cytoplasm (ISL) are seen at the periphery of the oocyte.x900.F. As E, stained with methyl green-pyronin. V1T, Vitelline membrane; ISL, islandsof darkly staining cytoplasm, x 850.

234 R. CZOLOWSKA

thick, ramifying, three dimensional network. The centre of the oocyte isoccupied by a dense network of fine granular cytoplasm which seems to origin-ate from the dispersion of material accumulated underneath the nucleus(Fig. 1A, B). The amount of cytoplasm decreases markedly towards thevegetative pole (Fig. 2).

The pigment around the vegetative pole forms a layer about 15-28 /-t thick;its granules are much less numerous and larger in diameter than those of theanimal hemisphere.

PAT

SUB

INT

Fig. 3. Tangential section of the vegetative pole region of a fully grown oocyteisolated from the ovary (distribution of the vegetative pigment not indicated).Boundary between peripheral zone and follicular epithelium not clearly visible.N, Nuclei of follicular epithelium and remaining envelopes; SUB, subperipheralzone crowded with small yolk platelets; INT, internal zone laden with large yolkplatelets; PAT, patches of compact RNA-rich cytoplasm. Patches in the subperi-pheral zone indicated with fine dotting.

The area around the vegetative pole of the oocyte contains material which isdistributed in three distinct zones. The peripheral zone appears directly under-neath the vitelline membrane as a thin cytoplasmic layer populated with smallyolk platelets. The underlying subperipheral zone is laden with small yolkplatelets which are packed very tightly together, forming a compact layer. Thecytoplasm occurs as delicate, dispersed patches. The internal mass is filled withlarge yolk platelets, very rarely interspersed by small yolk platelets. The cyto-plasm can be seen as compact patches situated among the platelets (Fig. 3).

Methyl green-pyronine staining shows an animal-vegetal 'gradient' of RNAcorresponding to the localization of the cytoplasm. Within this gradient thearea around the vegetative pole, although relatively poorer in RNA than theanimal hemisphere, nevertheless reacts strongly. It seems that some RNA-

Germinal cytoplasm in Xenopus 235containing material accumulates there, also in the zone corresponding to thevegetative pigment and the small yolk platelets (Fig. 1C).

No differences in any series were found in the fine structure of oocytes fromunstimulated as against hormonally induced females.

Oocytes collected from the body cavity

One of the characteristic features of ovulated oocytes is the presence of a singleenvelope, the vitelline membrane.

The cytoplasm is distributed along an animal-vegetal 'gradient' (Fig. ID).After the breakdown of the germinal vesicle the animal hemisphere appears tobe filled with fine granular cytoplasm and compact clusters of dark-stainingmaterial, originating from the disintegrated nucleus. The vegetative hemisphereseems much poorer in cytoplasm than the corresponding region of the non-ovulated oocyte. The cytoplasm forms a very fine network, highly dispersedamong the yolk platelets.

PER

SUB

Fig. 4. Schematic drawing illustrating a part of the vegetative pole region of anoocyte collected from the body cavity (vegetative pigment not indicated). PER,Peripheral zone composed of the strip of cytoplasm and yolk platelets with changedstaining properties; SUB, subperipheral zone; ISL, cytoplasmic island crowdedwith mitochondria.

In the vegetative pole region the three concentric zones of material are moredistinct. The peripheral zone, composed of the outermost strip of cytoplasm andpopulated with small yolk platelets, is underlain by a subperipheral zone whichappears to be crowded mainly with small and partly with large yolk platelets(Fig. 4). The internal mass, in sectioned material, is often separated from thesubperipheral zone by a crack (Fig. 1D). The crack itself is an artifact, but itspresence reflects true differences in composition and properties of both zoneswhich come to light during the histological procedure.

236 R. CZOLOWSKA

D

Germinal cytoplasm in Xenopus 237At the periphery around the vegetative pole a striking change in the staining

properties of the yolk platelets occurs (Fig. 4). This consists of a loss of affinityfor orange G and simultaneous increased staining with aniline blue. In a singleplatelet the change is seen, at first, at the outer margin and spreads subsequentlytowards its centre. The material of the platelet seems to be gradually consumed,a process leading finally to the disintegration of the platelet. The change instaining is confined mainly to small yolk platelets in the peripheral and sub-peripheral zones, but some large platelets are also affected. The pattern ofdistribution of the zone of altered yolk differs from oocyte to oocyte. In somethe change is visible over the whole vegetative periphery, up to the equator, butother oocytes show it only in a much more limited area.

One could suppose that the changes represent degenerative phenomenarelated to absorption of yolky material in overripe oocytes, as was demonstratedby a safranin staining method by Selman & Pawsey (1965). In order to checkthis possibility the proportion of successful fertilization obtained with eggscoming from the same female as ovulated oocytes collected afterwards from thebody cavity was determined. The percentage of successfully cleaving eggs wasestimated as 80 %. In the light of this high percentage it is rather unlikely thatthe ovulated oocytes studied were abnormal.

In many oocytes the region around the vegetative pole is marked by thepresence of blue cytoplasmic islands, distributed through the yolk (Fig. 5 A, B).These islands have always been detected only in the most vegetative sector andare localized exclusively within the peripheral and subperipheral zones. Thesize of the islands varies from 3 to 15 [i and their number varies from oocyte tooocyte. Under oil immersion their heavily stained cytoplasm is seen to becrowded with oblong granules, which are slightly larger than the pigmentgranules of the vegetative area (Fig. 4). In their size and appearance the granulesclosely resemble mitochondria. The localization of the islands often varies

FIGURE 5

A. A part of the vegetative pole region of an ovulated oocyte collected from thebody cavity. Meridional section, Azan staining. Three large islands of cytoplasmicmaterial (ISL) are situated at the boundary between subperipheral (SUB) andinternal zones (INT), x 640.B. Vegetative pole of an ovulated oocyteunder high magnification. Meridional section,Azan staining. Two large cytoplasmic islands (ISL) localized at the boundary betweensubperipheral (SUB) and internal zones (INT). VIT, Vitelline membrane, x 920.C. As B, stained with methyl green-pyronin. x 950.D. Tangential section of the vegetative pole region of an egg collected and fixed atthe time of insemination. Azan staining. Darkly staining cytoplasmic islands are dis-tributed throughout subperipheral zone, x 760.E. Tangential section of the vegetative pole region at the two-cell stage. Azanstaining. Islands of the 'germinal cytoplasm' (ISL) are distributed parallel to thecleavage furrow. SP, Space separating both blastomeres. x 520.

238 R. CZOLOWSKA

within one and the same specimen. Some of them can be detected in very closeproximity to the cytoplasm of the peripheral zone. Others seem to be slightlyseparated from the peripheral cytoplasm. Finally some are completely isolatedfrom the peripheral zone, lying deep among the yolk, at the boundary betweenthe subperipheral and the internal zones.

The general distribution of the pyronine-staining material corresponds tothat of the cytoplasm. The animal half appears to be extremely rich in RNA inboth the granular cytoplasm and the clusters. There is a very sharp decline inamount of RNA in the cytoplasm filling the oocyte below the equator. Thevegetative pole region is, as a rule, very poor in RNA, and pink-stainingmaterial forming a dispersed network in the yolk can barely be seen there.Nevertheless, some sections show RNA-rich islands occupying the most vegeta-tive region and corresponding in their position and appearance to the bluecytoplasmic islands described above (Fig. 5C). Sections have also been foundwhich illustrate the closeness, of the RNA-rich islands to the peripheral cyto-plasm.

Oocytes ovulated in vitro

Oocytes from two experimental groups have been used for preliminarystudies. Group I consists of oocytes which were collected after 8 h of hormonetreatment and kept for 40 min in Ringer solution before fixation. Group IIconsists of oocytes collected after 8 h hormone administration and culturedsubsequently for 13 h in Ringer solution before fixation. Oocytes obtained inthese ways are always coated with a vitelline membrane.

The pattern of distribution of yolk and cytoplasm resembles that of oocytesovulated in vivo. Group I oocytes are characterized by an accumulation of cyto-plasm in the centre of the upper hemisphere, while those of group II reveal amore uniform distribution of material in the upper half of the egg. In bothgroups granular cytoplasm and cytoplasmic clusters are always present.

In the region around the vegetative pole the yolky material of the subperi-pheral zone and the internal mass is apparently condensed in oocytes of group I,but more loosely distributed in group II oocytes. The same is observed withrespect to pigment localization. The layer of vegetative pigment in oocytes ofgroup I is 15—19 /* thick and increases in group II to 18-56 /i.

The peculiar changes in staining properties of the yolk noticed in oocytesobtained from the body cavity have never been observed in oocytes ovulatedin vitro.

Cytoplasmic islands around the vegetative pole are rather scarce and barelydiscernible in group I oocytes, but they become clearly visible in the peripheraland subperipheral zones of oocytes belonging to group II. Islands localizedin the peripheral zone are flattened and about 5—15 /̂ long; they are continuouswith the peripheral cytoplasm and distributed at irregular intervals along thewhole vegetative side (Fig. 1E). In some regions islands can be found associated

Germinal cytoplasm in Xenopus 239

with small protrusions of the peripheral zone into the subperipheral layer.Many other islands occupy a more internal position in the subperipheral zoneand even in the internal mass. An array of oblong granules seems to be a charac-teristic of the internal composition of these islands (Fig. 6).

Cytochemical tests made on samples from group II reveal a general animal-vegetative gradient of distribution of RNA. In the region around the vegetativepole numerous islands extremely rich in RNA occupy a position just underneaththe peripheral cytoplasm. RNA-rich islands occur also in the subperipheralzone and even in the internal mass (Fig. 1F). After RNase treatment some of theislands still retain a weak pinkish colour.

PERVIT

SUB

25//

Fig. 6. Schematic drawing illustrating a part of the vegetative pole region of anoocyte ovulated in vitro (vegetative pigment not indicated). VIT, Vitelline mem-brane; PER, peripheral zone; SUB, subperipheral zone; ISL, cytoplasmic islandscrowded with mitochondria and rich in RNA.

Oocytes collected from the oviduct

Oocytes from different parts of the oviduct were classified into four groups.(I) Oocytes collected from the uppermost part of the tube, in the neighbour-

hood of the ostium, surrounded only by the vitelline membrane.(II) Oocytes collected from the upper part of the tube, also surrounded only

by the vitelline membrane.(III) Oocytes collected from the middle part of the tube, surrounded by the

vitelline membrane and partially by jelly.(IV) Oocytes collected in the lowest part of the tube, near the uterus,

surrounded by the vitelline membrane and fully coated with jelly.Except for group 1, all samples were cut tangentially to the vegetative pole.In all the four groups studied a progressive change is observed in the peri-

pheral zone near the vegetative pole, which becomes more compact and distinctas oocytes are transported down the oviduct. Yolk platelets with altered staining

240 R. CZOLOWSKA

properties can only be found peripherally in the vegetative region of group Iand II oocytes (Fig. 7).

The subperipheral zone and internal mass are characterized by a rather com-pact appearance. Some variations are observed with respect to the distributionof the large yolk platelets. In some cases they appear to be absent from theperipheral and subperipheral zones, in others they approach quite closely to theperiphery of the oocyte.

SUB

Fig. 7. Schematic drawing illustrating a part of the vegetative pole region of anoocyte collected from the uppermost part of the oviduct (vegetative pigment notindicated). For the abbreviations see Fig. 4.

In oocytes of all four groups intensely stained bodies gradually appear in theperipheral and subperipheral zones. These spots look like vacuoles and theirnumber, shape and size vary from sample to sample.

Some of the samples studied contain characteristic cytoplasmic islands (Fig. 7).These have been found distributed within all three regions, in variable size andnumber. The islands appear less regular in shape, more compact and moreheavily stained than corresponding ones from previous stages. Only verydetailed observation reveals their granular composition. Cytoplasmic islandsare tightly associated with small yolk platelets. Pigment granules associatedwith the cytoplasmic islands are present in the same concentration as in thesurrounding regions.

Cytochemical tests made on samples from group II reveal the presence ofislands which are significantly richer in RNA than the very diffuse stainingregions of the peripheral cytoplasm at the vegetative side. The size and localiza-tion of the RNA-rich islands correspond to that of the blue cytoplasmic islandsobserved after Azan staining.

Germinal cytoplasm in Xenopus 241

Eggs collected from the uterus and unfertilized eggs

If serial tangential sections of the vegetative pole region are studied, the threezones are more or less clearly distinguishable. In all cases the peripheral zoneas well as the outermost region of the subperipheral zone are marked by thepresence of bodies, which are already observed in the previous stage, but havenow increased in number and size. These bodies very likely represent thecortical vacuoles already described by many authors.

SUB

Fig. 8. Tangential sections of the vegetative pole region of unfertilized eggs (distri-bution of the vegetative pigment not indicated). A, Distinct separation of all threezones. B, Obliterated pattern of yolk distribution. SUB, subperipheral zone; INT,internal zone; ISL, islands of RNA-rich cytoplasm.

Variations are observed in the general distribution of the yolky materialwhich seem to be related to some unknown individual differences between eggscoming from different females. According to the type of variations two maingroups of eggs can be distinguished.

The first group represents unfertilized eggs where a very distinct separationof all three zones is observed (Fig. 8 A). The subperipheral zone is pronounced,being crowded with small yolk platelets, while only a few large yolk granules arefound in that region. Cytoplasmic islands have been observed mainly in thesubperipheral zone and at the boundary between the subperipheral zone andthe internal mass, very obviously separated from the peripheral layer. The

16 ]EEM 22

242 R. CZOLOWSKA

islands are easily distinguishable due to their heavy staining, large size (largestreaching 16/0 ar)d abundance.

In eggs of the second group taken from the uterus as well as in some un-fertilized eggs the pattern of yolk distribution is obliterated (Fig. 8B). The clearsegregation of fine and coarse yolk platelets is not observed; the large plateletsare distributed over the whole section and approach the peripheral layer veryclosely (Fig. 5D). The cytoplasmic islands occupy a less fixed position. Generallydiminished in size and number in comparison with the first group, they aredispersed not only in the subperipheral zone and even into the internal mass butalso often in the peripheral zone, where they are barely recognizable among thecortical vacuoles.

In both groups the cytoplasmic islands are found along the curvature of thevegetal cap of the egg varying from 40 to 216 /«• in width. The cytoplasmicislands seem to be associated with small yolk platelets (Fig. 5 D) and have a finegranular appearance.

After staining with methyl green-pyronine the cytoplasm of the vegetativeregion is barely visible, being dispersed among the yolk platelets. Contrastingwith this background, RNA-rich islands have been found, corresponding in sizeand appearance to the cytoplasmic islands described above.

Fertilized eggs at the time of first furrow formation

The same three zones can be easily distinguished in serial tangential sectionsof the vegetative pole region. Cortical vacuoles have disappeared from the peri-pheral zone. The peripheral and subperipheral zones are characterized by thepresence of irregular cytoplasmic islands which differ in size, number and distribu-tion from egg to egg. These islands are found only in a region directly surround-ing the vegetative pole covering an area varying from 96 to 174/t in width. Bycomparison with previous stages it seems that the cytoplasmic islands which areoriginally dispersed over a rather extensive circular area undergo some processof concentration as soon as the first furrow approaches the vegetative pole.

Eggs with two separated blastomeres show compact islands of materiallocalized in the neighbourhood of the cleavage furrow (Fig. 5E). Islands areoften grouped lying in a row, like a string of beads, parallel to the egg membrane.Their size varies from 1 to 22 jn across.

Each cytoplasmic area is associated with groups of small yolk platelets(Fig. 5E) and contains fine oblong granules.

The highly dispersed cytoplasm in the vegetative half of the egg stains weaklywith methyl green-pyronine. However, islands extremely rich in RNA arealways present, distributed within a definite region. The appearance and sizeof these islands seems to be identical with those of the blue cytoplasmic islandsafter Azan staining.

Germinal cytoplasm in Xenopus 243

DISCUSSION

Unfertilized and fertilized eggs of Amphibia both show an unequal distribu-tion of yolk concentrated predominantly in the vegetative hemisphere. Conse-quently there is an apparent decrease in cytoplasm along the animal-vegetativeaxis. In the most vegetative region of the egg a cytoplasmic network can barelybe seen. In the previous section, this unequal distribution has been called ananimal-vegetative gradient. Here the term 'gradient' only indicates the pro-nounced differences in cytoplasm and yolk content along the primary axis, anddoes not indicate that a smooth, continuous decline or increase of these com-ponents occurs in animal-vegetative direction.

Cytoplasmic islands, apparently different from the dispersed cytoplasm, havebeen observed near the vegetative pole of the fertilized eggs of Xenopus laevis(Nieuwkoop, 1956; Nieuwkoop & Faber, 1956; Blackler, 1958) and identifiedas the 'germinal cytoplasm'. These observations have been confirmed in thepresent studies. 'Germinal cytoplasm' was found close to the cell membrane inthe most vegetative region of Xenopus eggs fixed at the time of formation of thefirst furrow. The islands are easily detectable due to their strong staining withaniline blue and characteristic internal granules, corresponding in size to mito-chondria. The presence of a crowded population of mitochondria as a constantcomponent of the 'germinal cytoplasm' has been demonstrated previously byspecific staining with Altman's acid fuchsin (Bounoure, 1934; Blackler, 1958;Berardino, 1961).

The exact time of the initial appearance of the 'germinal cytoplasm' and themechanism of its formation has still remained obscure. According to the avail-able data in the literature the 'germinal cytoplasm' appears in the egg shortlyafter fertilization; its appearance seems to be correlated with the activation ofthe egg rather than with the presence of sperm (Blackler, 1958). Blackler couldnot detect the 'germinal cytoplasm' in unfertilized eggs and oocytes of R.temporaria. Although he does not make any definative statement he mentionsthat '"germ plasm" was seen in the process of formation in the vegetal corticalregion', thus implying a cortical origin. However, Bounoure (1964) holds theopinion that the 'germinal cytoplasm' ... 'prend evidemment naissance au coursde l'ovogenese, dans cette phase obscure du grand accroissement, ou se fixentles structures et les potentialites de l'ovule'.

With Bounoure's idea in mind, unfertilized eggs of X. laevis as well asovulated oocytes were investigated. Cytoplasmic islands in the most vegetativeregion were found in a high percentage of specimens in every group. The follow-ing criteria have been selected for their identification and comparison: (1) strongstainability with aniline blue, (2) localization restricted to a limited sector of theegg around the vegetative pole, (3) the presence of an internal granulationresembling mitochondria, and (4) rich in RNA.

The distribution of the islands, as well as their number and size, varies from

lfi-2

244 R. CZOLOWSKA

sample to sample and from female to female; however, they are usually situatedin the subperipheral zone which is laden with small yolk platelets and rich inpigment granules.

Bladder (1958) has reported the presence of RNA in the 'germinal cytoplasm'of fertilized, uncleaved eggs of R. temporaria. In the course of the present studiesthe same cytochemical test was used for X. laevis eggs. Eggs studied when thefirst cleavage furrow is formed revealed the presence of RNA within the'germinal cytoplasm'. In the present studies unfertilized eggs and ovulatedoocytes likewise showed a positive reaction for RNA within the cytoplasmicislands distributed around the vegetative pole. These isolated accumulationsof RNA are distinguishable from the general animal-vegetative 'gradient' ofRNA, originally described by Brachet (1940).

Although conclusions drawn from static observations have serious limitations,it seems reasonable to suppose that the islands observed in groups of eggs takensequentially do represent the same material. This material is segregated in adefinite region of the cell in the ovulated oocyte and stored there during itsfurther development. In its appearance and composition the material resemblesclosely the cytoplasmic islands described by Bounoure. It is therefore suggestedthat we are dealing throughout with the 'germinal cytoplasm' or, at least, withits precursor.

On the basis of our present knowledge of the synthesis of nucleic acids allRNA found in the cell can be considered as a product of the activity of thenucleus. Therefore, the RNA component of the 'germinal cytoplasm' is thoughtto be exclusively of nuclear origin. On the other hand, an extranuclear originof the second component of the 'germinal cytoplasm', the mitochondria, seemsplausible.

The characteristic appearance of the 'germinal cytoplasm' could not be recog-nized earlier than in the ovulated oocyte. This may mean that either (1) the time ofits actual formation corresponds to the ovulation period, or (2) that the time of itscharacteristic manifestation coincides with ovulation, the 'germinal cytoplasm',however, existing already in some other form in the non-ovulated oocyte.

1. Is the 'germinal cytoplasm' formed during maturation?

Ovulation itself is only one of numerous phenomena occurring during matura-tion of the oocyte. According to Dettlaff, Nikitina & Stroeva (1964) and Briggs& Cassens (1966) the significance of maturation for the further fate of the egg,even for processes as remote as cleavage and post-gastrulation, becomes moreand more evident.

It is known that by the end of oogenesis a pronounced inhibition of thesynthesis of nucleic acids and proteins occurs (Monroy, 1967). During matura-tion the general pattern of synthesis changes.

According to Dettlaff (1966) the initiation of maturation by hormonalstimulus in R. temporaria acts through the induction of the synthesis of specific

Germinal cytoplasm in Xenopus 245

m-RNA; actinomycin, when applied in the hormone-dependent period, inhibitsmaturation. On the contrary, Brachet (1967a) reports that all stages of matura-tion of the X. laevis oocyte can be inhibited by actinomycin. Protein synthesis,tested by using pyromycin, seems to be involved in all steps of the maturationprocess (Dettlaff, 1966; Brachet, 1967a).

Biochemical and autoradiographical data indicate that small amounts ofheterogeneous, rapidly labelled RNA (probably m-RNA) is indeed synthesizedat the time of maturation (Ficq, 1964; Brown, 1966; Brown & Littna, 1964a,1966 a). Monroy (1967) reports detectable synthesis of s-RNA and Smith,Ecker & Subtelny (1966) active synthesis of proteins during maturation.

Brachet (1965, 19676, c) describes certain very interesting phenomena corre-lated with maturation. Hormone treatment of the isolated oocyte for 3-4 hleads to changes in the germinal vesicle. The nuclear membrane on the vegetativeside shows numerous invaginations, extremely rich in RNA. The nuclear sapbecomes very basophilic, corresponding to an increase in ribosome content(unpublished observations of van Gansen, cited by Brachet, 1967 c). The fateof this particular population of ribosomes is not known, the possibility of theireventual occurrence in the 'germinal cytoplasm' remaining open.

Unfortunately the ultrastructure of the 'germinal cytoplasm' in Anura hasnot yet been intensively studied. Balinsky (1966) has presented the single electronmicrograph demonstrating the vegetative region of the fertilized egg of Phryno-batrachus natalensis. Besides large groups of mitochondria occupying thisregion he found 'peculiar rounded bodies about 018 [i in diameter. These bodiesare electron-dense and appear to consist of aggregations of particles which aresmaller than the ribosomes seen in the same area'. According to his suggestion'these [structures] may represent the basophilic areas of cytoplasm...known as"germinal cytoplasm"'.

J. Brachet (personal communication) holds the opinion that rapidly labelledheterogenous RNA may have something to do with the formation ofthe 'germinal cytoplasm'. At present this hypothesis is being studied in hislaboratory.

Only ribosomal RNA can be detected with cytochemical methods. In thelight of the present results the participation of r-RNA in the 'germinal cyto-plasm' is therefore strongly indicated.

Unfortunately the cytological observations do not provide convincing evi-dence for the nuclear origin of the RNA component of the 'germinal cytoplasm'.The author has, indeed, seen sections which suggest a possible transport ofanimal material towards the vegetative pole. At the time of ovulation the massof vegetative yolk becomes very loose and streams of cytoplasm seem to becomeintermingled with yolk platelets. This cytoplasm is always seen as a fibrous,dispersed network. Material resembling the compact islands of the 'germinalcytoplasm' has, however, never been observed within the mass of yolk plateletsfilling the vegetative hemisphere.

246 R. CZOLOWSKA

There is some doubt how to interpret observations on the oocytes ovulatedin vitro.

Ovulation in vitro occurs under the influence of a high dose of gonadotrophichormone. This rather brutal treatment evokes phenomena which develop moreintensively and more rapidly than in the natural ovulation process (Dettlaff,1966). It remains open how far oocytes ovulated in vitro are a model systemillustrating processes which are normally known to occur in a masked way.

Observations on the oocytes ovulated in vitro reveal a few interesting detailswith respect to the 'germinal cytoplasm'. The number of islands as well as theregion of their distribution are significantly greater than those of oocytesovulated in vivo. The islands are extremely rich in RNA. The time factor seemsto play an important role in their manifestation and distribution. A high numberof inclusions always remain in close contact with the outermost cytoplasmiclayer of the peripheral zone.

Some sections suggest a possible displacement of the islands within thevegetative area. The layer containing the vegetative pigment and the small yolkplatelets increases in thickness in the course of maturation. This phenomenonas well as a general loosening of the yolk could be due to rapid hydration of theovulated oocyte (Tchou-Su & Yen Pai-Hu, 1950), resulting from a change inpermeability of the cell membrane (Morrill, 1965; Morrill, Rosenthal &Watson, 1966). In addition a very sharp increase in the contractility of theegg surface occurs at the time of the breakdown of the germinal vesicle (Dettlaff,1966).

Although we are dealing with static pictures the evidence presented abovesuggests that in oocytes ovulated in vitro some RNA-rich material, closelyresembling the 'germinal cytoplasm', is localized initially in the peripheral zoneat the vegetative side. This material becomes more clearly visible in the courseof maturation and migrates from the periphery towards deeper layers of theoocyte.

2. Is the 'germinal cytoplasm' formed in the growing oocyte?

The period of growth of the amphibian oocyte is marked by numerousmorphological phenomena (Wittek, 1952; Wartenberg & Schmidt, 1961;Balinsky & Devis, 1963; Kemp & Istock, 1967).

A very high rate of synthesis of nucleic acids and proteins occurs in the oocyteduring the growth period. Most of the RNA synthesized at this period consistsof ribosomal RNA which seems to be gradually accumulated during oogenesis(Brown, 1964, 1966; Brown & Gurdon, 1964; Brown & Littna, 19646; David-son, Allfrey & Mirsky, 1964; Ficq, 1964). The synthesis of s-RNA proceeds at alower level (Brown, 1964; Brown & Littna, 19646, 19666; Ficq, 1964; Monroy,1967). There is strong evidence that the synthesis and storage of small quantitiesof m-RNA also occurs during the growth period (Ficq, 1964; Davidson, Crippa,Kramer & Mirsky, 1966; Crippa, Davidson & Mirsky, 1967).

Germinal cytoplasm in Xenopus 247

In view of these findings the possibility cannot be excluded that particularRNA components of the 'germinal cytoplasm' may already be formed in theperiod preceding maturation.

Large oocytes isolated from the ovary demonstrate the presence of RNA incytoplasmic patches at the vegetative pole region. It is probable that this materialrepresents a dispersed state of the 'germinal cytoplasm'. In the course of matura-tion the RNA-containing material would undergo a process of condensation.

It may be concluded that these cytological observations suggest a multipleorigin of the 'germinal cytoplasm'. On the one hand they emphasize the signifi-cance of the period of oogenesis in which the actual formation of the 'germinalcytoplasm' seems to take place. The large ovarian oocytes, mainly, show thepresence of condensations of RNA-containing material before the onset ofmaturation. Since all RNA can be considered as a nuclear product, this RNAcomponent is evidently formed during oogenesis. The constant association ofthe 'germinal cytoplasm' with mitochondria may point towards a relationshipwith the Balbiani body, which is thought to give rise to the mitochondrialpopulation of the oocyte (Wittek, 1952; Balinsky & Devis, 1963). It seemspossible therefore that the structures participating in the formation of the'germinal cytoplasm' have their origin partially in early as well as in later phasesof oogenesis. It also seems likely from these studies that an 'informative' com-ponent is added after the breakdown of the germinal vesicle, leading to theultimate differentiation of the 'germinal cytoplasm'. The actual time of syn-thesis of this RNA component may precede considerably its movement towardsthe vegetative pole of the egg.

Without elaborating the theoretical implications the author wishes to pointout the possible significance of these observations for the problem of nuclearversus cytoplasmic inheritance, as this will in part depend upon the character ofthe nuclear and cytoplasmic components of the 'germinal cytoplasm' and theirtime and mode of formation.

SUMMARY

1. Fertilized eggs, unfertilized eggs, oocytes ovulated in vivo and in vitro aswell as ovarian oocytes were investigated in order to elucidate the origin of the'germinal cytoplasm'. For its cytological identification meridional and tangen-tial sections were stained by the Azan method and the methyl green-pyronineof Unna.

2. A high percentage of specimens in all groups of eggs and ovulated oocytessequentially studied has shown the presence of cytoplasmic islands in the mostvegetative region of the cell. The islands are located in the peripheral and sub-peripheral zones of a limited sector of the vegetative pole region. They are ladenwith small yolk platelets and rich in pigment. The presence of internal granula-tion resembling mitochondria seems to be one of the characteristic features ofthe islands studied.

248 R. CZOLOWSKA

3. In large ovarian oocytes the cytoplasmic patches are scattered in thevegetative pole region. These patches are apparently smaller, more dispersedand less numerous than the corresponding cytoplasmic islands in ovulatedoocytes.

4. It has been shown cytochemically that the cytoplasmic islands in all thegroups studied contain RNA. These isolated accumulations of RNA at thevegetative pole region are additional to the general animal-vegetative 'gradient'of distribution of RNA in the oocyte and in the egg.

5. On the basis of the present results it is suggested that the cytoplasmicpatches localized at the vegetative pole region of ovarian oocytes represent the'germinal cytoplasm', or its precursor, in the unfertilized and fertilized egg.The nuclear and extranuclear origin of the various components of the 'germinalcytoplasm' is discussed as well as the possible time of their formation.

RESUME

Observations sur Vorigine du 'cytoplasme germinal* chezXenopus laevis

1. Pour elucider l'origine du cytoplasme germinal, on a examine des oeufsfecondes, des oeufs vierges, des oocytes ovules in vivo et in vitro, ainsi que desoocytes ovariens. Pour le mettre en evidence cytologiquement, des coupestangentielles et meridiennes ont ete colorees a l'Azan et au vert de methyle-pyronine d'Unna.

2. Un fort pourcentage d'echantillons dans les groupes d'ceufs et d'oocytesovules, etudies en coupes seriees, ont montre la presence d'ilots de cytoplasmedans la region la plus vegetative de la cellule. Ces ilots sont localises dans leszones peripheriques et subperipheriques d'un secteur limite de la region dupole vegetatif. Us sont charges de petites plaquettes vitellines et riches en pig-ment. La presence de granulations internes ressemblant a des mitochondriessemble etre une des caracteristiques des ilots etudies.

3. Dans les gros oocytes ovariens, les taches cytoplasmiques sont disperseesdans la region du pole vegetatif. Ces taches sont apparemment plus petites, plusdispersees et moins nombreuses que les ilots cytoplasmiques correspondantdes oocytes ovules.

4. On a montre cytochimiquement que les ilots cytoplasmiques dans tous lesgroupes etudies contiennent du RNA. Ces accumulations isolees de RNA dansla region du pole vegetatif se situent en dehors du 'gradient' general, animal-vegetatif, de repartition du RNA dans l'oocyte et l'ceuf.

5. Sur la base des resultats obtenus, on suggere que les taches cytoplasmiqueslocalisees dans la region du pole vegetatif des oocytes ovariens representent le'cytoplasme germinal' (ou son precurseur) de Foeuf non feconde et feconde.L'origine nucleaire et extranucleaire des divers composants du 'cytoplasmegerminal' est discutee, ainsi que le moment possible de leur formation.

Germinal cytoplasm in Xenopus 249

This project was suggested by Professor P. D. Nieuwkoop, Hubrecht Laboratory, towhom I would like to express my deep gratitude for his lively interest, stimulating advice, andencouragement during the course of this work.

I am greatly indebted to Dr G. A. Ubbels for her friendly help in the histological procedureand would like to thank Dr E. C. Boterenbrood for her help in obtaining the material.

1 wish to thank Dr K. A. Lawson for her criticisms and suggestions made during thecorrection of the English manuscript.

The author is grateful to Mrs N. Pulle, Central Embryological Library, for her help insupplying literature.

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{Manuscript received 8 October 1968)