Investigations of the Germ-plasm in Relation to
Nuclear Transplantation
by MARIE A. DI BERARDINO1
From the Institute for Cancer Research, Philadelphia
WITH PLATE
INTRODUCTION
THE origin of the germ-cells has been extensively investigated in both invertebrates and vertebrates. In invertebrates it has been traced to the early cleavage
of the zygote, e.g. in such forms as Ascaris megalocephala (Boveri, 1887) and
Sciara (Metz, 1938). Within the vertebrate group the cases in which primordial
germ-cells have been detected in cleavage stages are very few. Eigenmann (1891)
identified gonocytes of Micrometrus aggregates in the late gastrula stage on the
basis of their large size and the uniform distribution of their chromatin. The first
clear demonstration of vertebrate primordial germ-cells appearing in a stage as
early as the blastula was made in the European frog, Rana temporaria (Bounoure,
1934). In this case germ-cells were found to be conspicuous because of a stainable cytoplasmic element, the germ-plasm. This germ-plasm first appears
shortly after fertilization in the form of islets concentrated in the vegetal pole
region of the egg (Bounoure, 1934,1939,1954). In these investigations Bounoure
traced the plasm-bearing cells to the blastula stage, at which time the germplasm is incorporated into cells near or on the floor of the blastocoele. In later
stages of embryonic development these primordial germ-cells were traced to the
genital folds. That these cells are directly ancestral to the mature germ-cells
was indicated by subjecting the vegetal pole of R. temporaria eggs to ultraviolet irradiation shortly after fertilization (Bounoure, 1939, 1954). Eggs treated
in this way developed into metamorphosed animals whose gonads, reduced in
size, possessed only a small quantity of germ-cells, and in a few cases were
devoid of germ-cells. More recently, Blackler (1958) has confirmed Bounoure's
studies in R. temporaria and has also made comparable studies on other Anuran
species. In Bufo bufo and Xenopus laevis, the presence of germinal cytoplasm
corresponded, in general, to that of R. temporaria; however, no germinal
cytoplasm was seen in gastrulae or neurulae of R. esculenta. Grafting experiments (Blackler, 1960) have demonstrated that the 'germ-cell region' of X. laevis
1
Author's address: Institute for Cancer Research, 7701 Burholme Avenue, Fox Chase, Philadelphia
11, Pa., U.S.A.
[J. Embryol. exp. Morph. Vol. 9, Part 3, pp. 507-513, September 1961]
508 M. A. DI BERARDINO—INVESTIGATIONS OF THE GERM-PLASM
neurulae is directly ancestral to the mature gametes. When the 'germ-cell
region' of a two-nucleoli Xenopus neurula was replaced with the 'germ-cell
region' of a neurula whose cells contain only one nucleolus, the resultant
mature animal produced germ-cells with only one nucleolus, and when the latter
was mated with a normal 2 N frog, it yielded in the F 2 generation a Mendelian
ratio of 1-nucleolar frogs.
Comparable information on the localization of primordial germ-cells in the
early developmental stages of R. pipiens has not been available. Since this species
is being used extensively in nuclear transplantation studies of blastula and early
gastrula stages (King, Briggs, & Di Berardino, 1959; Briggs & King, 1960),
it has become necessary to know whether or not germ-cells can be distinguished
from somatic cells and, if so, what their numbers and localizations are. The
present communication presents cytological studies on primordial germ-cells
in the mid-blastulae and early gastrulae of R. pipiens.
MATERIALS AND METHODS
R. pipiens blastulae, stage 8, and early gastrulae, stage 10 (Shumway, 1940),
were selected for study because these stages corresponded to the stages from
which donor nuclei were taken for nuclear transplantation studies. The eggs
were fixed and stained according to the techniques of Bounoure (1934) and
Blackler (1958). In general, the procedure consisted of a 5-minute hot neutral
formalin fixation followed by soaking in warm, saturated potassium dichromate
for 42 hours. Eggs were sectioned at 5/x, stained with the Altmann acid fuchsin
mitochondrial stain and counterstained with aqueous Azure A. Additional eggs
were handled with slight modifications; namely, some eggs were fixed in Smith's,
some were sectioned at 10/*, and in some cases acid fuchsin staining was
omitted. The use of these modifications simplifies the histological technique
and gives a satisfactory demonstration of the germ-plasm.
RESULTS
The Altmann mitochondrial stain, acid fuchsin, stains the ground cytoplasm,
yolk platelets, and mitochondria red; the counterstain, Azure A, colours the
germ-plasm purple. The affinity of the germ-plasm for Azure A would be
expected, since Azure A is a good stain for revealing nucleic acids, and Blackler
(1958) established that the germ-plasm contains RNA. The germ-plasm exists
in the form of a distinct purple islet(s) in the cytoplasm and, depending on the
stage of development, may be situated near the cell membrane or close to the
nucleus (Plate, figs. A, C). Occasionally, the germ-plasm may be seen in the form
of a ring (Plate, figs A, B).
Stage 8
All of the thirteen blastulae studied contained germ-plasm. The number of
cells possessing the germinal cytoplasm varies among the individual blastulae;
M. A. DI BERARDINO—INVESTIGATIONS OF THE GERM-PLASM 509
this range extends from 5 to 14 cells (Table 1). The primordial germ-cells are
situated in the vegetal hemisphere, and at this stage 84 per cent, are found in
the upper third. This area corresponds to the floor of the blastocoele and
approximately the second and third layers subjacent to the blastocoele cavity.
The germ-plasm is usually situated near the cell membrane (54 per cent, of the
cells) or it may be near the nucleus; however, it is rarely seen at this stage adjoining the nucleus (Table 1).
TABLE 1
Germ-plasm in Rana pipiens {stage 8)
Location in vegetal
hemisphere
Egg
1
2
3
4
5
6
Number of
cells
containing
germplasm
5
5
6
7
8
9
7
8
9
10
11
12
13
9
9
9
10
11
12
14
Total no.
Percentage
114
100
Upper
third
3
6
6
8
9
9
6
8
8
10
Middle
third
Lower
third
3
2
—
1
—
—
2
—
—
—
—
—
—
—
—
2
—
2
—
14
3
1
—
1
1
—
96
841
12
10-5
9
6
5-3
Location in cell
Near
cell
membrane
5
1
4
2
—
5
4
3
2
9
8
7
11
61
53-5
Half-way
between cell
membrane
and
Near
nucleus
nucleus
2
2
3
—
1
1
3
4
1
3
5
2
27
23-7
2
—
1
8
3
4
3
3
—
—
—
—
24
210
Adjoining
nucleus
—
—
1
—
—
—
—
—.
—t.
—
1
2
1-8
Stage 10
Eleven out of twelve gastrulae contained cells with germ-plasm, and the
number of cells displaying the plasm ranges from 4 to 15 cells (Table 2). As in
stage 8 the majority of stage-10 cells containing germ-plasm is also present in
the upper third of the vegetal hemisphere; however, the percentage is lower (64
per cent, for stage 10 compared with 84 per cent, for stage 8). Table 2 illustrates
that a slight shift of plasm-bearing cells to the middle-third of the vegetal
hemisphere has occurred in the early gastrula. The most striking difference
between stage 8 and stage 10 plasm-bearing cells is in the intracellular location
of the germ-plasm. At stage 10, 84 per cent, of the primordial germ-cells
contain the germ-plasm in a juxtanuclear position (Table 2). In stage 8 the
germ-plasm is situated near the cell membrane (53-5 per cent, of cells) or
at intermediate locations (44-7 per cent.), and is only rarely found directly
surrounding the nucleus (1-8 per cent.).
510
M. A. DI BERARDINO—INVESTIGATIONS OF THE GERM-PLASM
TABLE 2
Germ-plasm in Rana pipiens {stage 10)
Location in vegetal
hemisphere
Egg
1
2
3
4
5
6
7
8
Number of
cells
containing
germUpper Middle
plasm
third
third
0
4
4
—
5
—
5
6
1
5
7
6
1
9
1
7
9
7
2
11
9
2
9
10
11
12
11
12
14
15
7
9
11
11
3
2
3
4
Total no.
Percentage
103
100
66
640
34
330
Lowe/third
Location in cell
Half-way
between cell
membrane
Near
and
cell
Near
Adjoining
nucleus nucleus nucleus
membrane
—
—
—
—
1
—
—
—
—
—
1
1
—
—
—
—
2
—
1
1
—
—
—
2
—
—
2
1
—
3
2-9
4
3-9
5
4-9
—
—
—
—
5
2
—
—
1
—
—
8
7-7
4
5
6
7
1
6
11
11
7
13
15
86
83-5
DISCUSSION
The present study of germ-plasm in R. pipiens blastulae and gastrulae closely
parallels that reported by Bladder (1958) for comparable stages in R. temporaria, B. bufo, and X. laevis. In R. pipiens mid-blastulae and early gastrulae
a limited number of cells (from 4 to 15) contain a distinctive cytoplasmic component, the germ-plasm, which stains purple after Azure A staining. All of these
cells are confined to the presumptive endoderm. Within these cells the germplasm occupies a more or less peripheral position at the blastula stage, then at
the beginning of gastrulation the germ-plasm surrounds the nucleus.
Since the primordial germ-cells of R. pipiens can be localized in situ, some
correlation can be made with the in vivo nuclear transplantation studies. King
et ah (1959) and Briggs & King (1960) tested nuclei from different areas of the
animal and vegetable hemispheres of the early gastrula as well as animal hemisphere nuclei from the mid-blastula. Since no primordial germ-cells are present
in the animal hemisphere, and very small numbers of gonocytes are present in
the vegetable hemisphere, these nuclear transplantation studies as well as
previous ones involving these stages of R. pipiens (see Briggs & King, 1959, for
references to the earlier work) must have involved entirely or almost entirely
somatic nuclei.
What appears most significant is that somatic nuclei can promote the formation not only of somatic cells, but also germ-cells. Briggs & King (1960) found
that somatic cell nuclei, which still retain the capacity to promote the formation
M. A. DI BERARDINO—INVESTIGATIONS OF THE GERM-PLASM 511
of the normal array of somatic cells, also retain the capacity to promote germcell formation. However, among larvae derived from blastula animal hemisphere
nuclei, they found that fewer than expected possessed ovaries with growing
oocytes. The interpretation of this result remains uncertain until a microscopic
analysis of the gonads is performed. Nevertheless, some somatic nuclei appear
to promote germ-cell formation. Similarly, Gurdon et dl. (1958) have obtained
sexually mature individuals from transplantation of Xenopus endoderm nuclei;
these endoderm donor nuclei are derived from areas not containing primordial
germ-cells. The explanation in the cases of R. pipiens and X. laevis may be that,
since the germinal cytoplasm is a normal constituent of the unfertilized egg,
activation of the host egg by the transplantation technique may be sufficient to
'organize' the germinal cytoplasm in the unfertilized egg. Blackler (1958) has
shown that R. temporaria parthenogenetic blastulae contain cells with germplasm, and he has suggested that the appearance of germ-plasm in the recently
fertilized egg may be part of the cortical reaction that follows fertilization.
The question arises whether germ-cell nuclei can promote the formation not
only of germ-cells, but also of somatic cells. Such a test requires definite identification of living germ-cells in various stages of development. Preliminary
attempts to identify living gonocytes by means of vital staining were made by
Blackler (1959). Vital staining carried out by us has revealed that neutral red
'selectively' stains a few cells on the floor of the blastocoele. Whether or not
these stained cells do in fact contain germ-plasm has not yet been determined.
SUMMARY
1. Primordial germ-cells in R. pipiens mid-blastula and early gastrula
stages were detected in serial sections, because these cells contained a stainable
component, the germ-plasm.
2. Their location was of prime interest in determining whether or not the
nuclei of primordial germ-cells had been used in previous transplantation
studies (King et ah, 1959; Briggs & King, 1960).
3. In the mid-blastula and early gastrula stages the primordial germ-cells
were confined to the vegetal hemisphere. The majority of these cells was
situated in the upper-third of the vegetal hemisphere.
4. An intracellular displacement of the germ-plasm occurred between midblastula and early gastrula stages.
5. The number and location of the primordial germ-cells suggest that only in
rare cases could a nucleus from one of these cells have been transplanted in the
previous studies, and such cases would probably have been undetectable. What
appears most significant is that nuclei derived from cells not containing germplasm are capable of giving rise to larvae whose gonads contain numerous
auxocytes.
512 M. A. DI BERARDINO—INVESTIGATIONS OF THE GERM-PLASM
RESUME
Recherches sur le plasme germinal, en liaison avec les experiences
de transplantation nucleaire
1. On a decouvert les cellules germinales de Rana pipiens sur les coupes
seriees des stades de la blastula moyenne et de la jeune gastrula, grace au fait
que ces cellules renferment un composant colorable, le plasme germinal.
2. Leur localisation presentait un grand interet pour determiner si les noyaux
des gonocytes primordiaux ont ete ou non utilises dans des experiences anterieures de transplantation (King et ah, 1959; Briggs & King, 1960).
3. Dans la blastula moyenne et la jeune gastrula, les gonocytes primordiaux
sont confines a rhemisphere vegetatif. La majorite d'entre eux est situee dans
le tiers superieur de cet hemisphere.
4. Un deplacement intracellulaire du plasme germinal survient entre les
stades blastula et jeune gastrula.
5. Le nombre et la localisation des gonocytes primordiaux font penser qu'un
de leurs noyaux n'aurait pu etre transplants que rarement au cours des recherches anterieures, et que de tels cas n'auraient probablement pas ete decelables. II est par contre ties significatif que des noyaux provenant de cellules qui
ne renferment pas de plasme germinal soient capables de donner naissance a des
larves dont les gonades contiennent de nombreux auxocytes.
ACKNOWLEDGEMENTS
The author wishes to express her appreciation to Dr. Thomas J. King and
Dr. Robert Briggs for suggesting this problem and for their helpful criticisms of
the manuscript. This investigation was supported by a Public Health Service
Predoctoral Fellowship (CF-8911 and -Cl) of the National Cancer Institute.
REFERENCES
BLACKLER, A. W. (1958). Contribution to the study of germ-cells in the Anura. / . Embryol. exp.
Morph. 6, 491-503.
(1959). Personal communication.
(I960). International Institute of Embryology Symposium, Pallanza, Italy. In press.
BOUNOURE, L. (1934). Recherches sur lalignee germinale chez la grenouille rousse aux premiers stades
du developpement. Ann. Sci. nat. 17, 67-248.
(1939). Lorigine des cellules reproductrices et leprobleme de la ligne'egerminate. Paris: GauthierVillars.
AUBRY, R., & HUCK, M. (1954). Nouvelles recherches experimentales sur les origines de la
lignee reproductrice chez la Grenouille rousse. / . Embryol. exp. Morph. 2, 245-63.
BOVERI, T. (1887). Ueber Differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris
megalocephala. Anat. Anz. 2, 688-93.
BRIGGS, R., & KING, T. J. (1959). Nucleocytoplasmic interactions in eggs and embryos. The Cell:
Biochemistry, Physiology, Morphology, Vol. I, 537-617, ed. J. Brachet & A. E. Mirsky, New York
and London: Academic Press.
(1960). Nuclear transplantation studies on the early gastrula (Rana pipiens). I, Nuclei of
presumptive endoderm. Dev. Biol. 2, 252-70.
EIGENMANN, C. H. (1891). On the precocious segregation of the sex cells in Micrometrus aggregatus,
Gibbons. / . Morph. 5, 481-93.
GURDON, J. B., ELSDALE, T. R., & FISCHBERG, M. (1958). Sexually mature individuals of Xenopus
laevis from the transplantation of single somatic nuclei. Nature, Lond. 182, 64-65.
J. Embryol. exp. Morph.
Vol. 9, Part 3
M. A. DI BERARDINO
M. A. D I B E R A R D I N O — I N V E S T I G A T I O N S OF T H E G E R M - P L A S M
513
KINO, T. J., BRIGGS, R., & Di BERARDINO, M. A. (1959). Nuclear transplantation studies on the early
gastrula (Rana pipiens). I. Comparison of nuclei from different regions of the early gastrula.
Anat. Rec. 134, 594.
METZ, C. W. (1938). Chromosome behavior, inheritance and sex determination in Sciara. Amer.
Nat. 72, 485-520.
SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. I. External form. Anat.
Rec. 78, 139-47.
E X P L A N A T I O N OF PLATE
FIG. A. TWO cells from a mid-blastula stage of R. pipiens. Germ-plasm is present in the form of a
ring in the cell on the right. The germ-plasm in the adjacent cell is located at the periphery of the cell
in the form of an islet, x 666.
FIG. B. Cell from a mid-blastula stage of R. pipiens. x 666.
FIG. C. Cell from an early gastrula stage of R. pipiens. The germ-plasm is in close association with
the nucleus and forms a cap around the nucleus, x 666.
G, germ-plasm; P, pigment; N, nucleus.
{Manuscript received 30:i:61)