Transfer of Primordial Germ-cells between Two
Subspecies of Xenopus laevis
by A. W. BLACKLER 1 ' 2
From the Embryology Laboratory, Department of Zoology, Oxford
WITH ONE PLATE
I N Anura the primordial germ-cells are discernible in the dorsal crest endoderm
of tail-bud stages of development and may be traced from this position throughout their migration into the undifferentiated gonadal rudiment. These facts have
been established by the descriptive studies of a number of workers (see review
by Johnston, 1951), the cells being recognizable by their large size, the retention
of yolk platelets long after their disappearance in neighbouring cells, the
sharply denned and often kidney-shaped nuclear membrane, and the poor
staining affinity of the nuclear contents.
By means of the application of the Altmann-Volkonsky staining technique,
Bounoure (1934) was able to demonstrate that germ-cells of the dorsal crest
endoderm are the lineal descendants of certain cells found in the ventral region
of the blastula. This discovery has been confirmed for Rana temporaria (the
species investigated by Bounoure) by Blackler (1958), and extended to other
Anuran species by Nieuwkoop (1956 a, b), Blackler (1958), and Di Berardino
(1961).
In a recent experimental study by Blackler (1960 a, b) and Blackler & Fischberg (1961), regions containing the so-called primordial germ-cells were grafted
from one Xenopus laevis embryo to another at the neurula stage. Graft cells
were distinguished from host cells with the help of a nuclear marker. The object
of this experiment was to ascertain if the stainable gonocytes of neurulae are
indeed primordial germ-cells by demonstrating that they give rise to definitive
gametes.
The results showed that true gonocytes occur in neurulae, since these grafts
contributed at least some of the functional gametes, although whether these
germ-cells are identical with those observed in histological studies remains a
matter awaiting proof.
This present paper describes experiments which follow up the success in
establishing a germ-cell transfer technique by exploring the possibilities of the
1
This paper is dedicated to Professor Louis Bounoure.
Author's address: Station de Zoologie Experimental, Route de Malagnou 154, Geneve, Switzerland.
2
[J. Embryol. exp. Morph. Vol. 10, Part 4, pp. 641-51, December 1962]
5584.4
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A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
technique for elucidating another long-standing problem of germ-cell studies
and in making a contribution to the study of oogenesis.
In female animals the maturation of the germ-cells takes place in the ovarian
environment and it is possible that ovarian influences manifest themselves in
the morphogenetic events that take place in an egg after its ovulation and
subsequent fertilization. Indeed, Raven (1958) has already suggested the
possibility that the follicle cells impart information which is stored in the
cortex of the egg. Normal females of the two subspecies employed in this present
analysis produce eggs which are distinctive in their size, colour, and later
development, so it was reasonable to assume that if the germ-cells of one subspecies were developing in the ovarian environment of the other subspecies, the
nature of the eggs finally produced would be some measure of the influence
of the host ovary on the growth of the graft oocyte. Moreover, the point at
issue is particularly relevant to the germ-line theory, i.e. that theory claiming the
mutual independence of the reproductive and somatic lines of cells.
Furthermore, there has long been controversy in germ-cell studies concerning
the opposing views that the primordial germ-cells give rise to all the definitive
gametes or that these cells give rise to only some gametes (the remainder deriving
from some secondary, somatic, source). The transfers of germ-cells between the
two subspecies were also based on the possibility that at least some experimental
animals might be obtained whose gametes were entirely of graft origin, in which
case the view of complete integrity of the germ-line would be upheld.
M A T E R I A L AND M E T H O D S
The two subspecies used were laboratory specimens of X. laevis laevis
(Daudin) and X. laevis victorianus (Ahl).
X. laevis laevis (hereafter referred to as XU) is the usual laboratory form of
X. laevis. Mature females lay eggs varying from 1 -3 to 1 -6 mm. in diameter, all
the eggs of any one female being of the same size and the smaller eggs being
usually laid by females shortly after gaining maturity (Plate, fig. A). The
animal hemisphere pigment is rather variable, but the commonest colour is
a rich chocolate brown (beautifully imitated in colour drawings published by
Bles, 1905).
X. laevis victorianus (hereafter referred to as Xlv) is the Uganda form of
X. laevis, smaller in adult size than XII and clearly recognizable by other
characters as well. Mature females lay eggs varying only from 1-00 to 1-09 mm.
in diameter, and the commonest egg colour is a very pale brown (Plate,
fig. B).
The subspecific status of the two forms, founded by Parker (1936), has been
experimentally tested by Blackler, Fischberg, & Newth (in preparation) who find
that XII and Xlv are mutually interfertile, as well as the offspring of such hybrid
crosses.
A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
643
The development of eggs of the two subspecies has also been subjected to
examination by Blackler, Fischberg, & Newth, and two characteristics of this
development are relevant to the present paper and demand some attention. The
first is that black melanophores appear on the rectal tube in Xlv at stages
49-50 of Nieuwkoop & Faber's (1956) table of Xenopus development (Plate,
fig. C) but do not manifest themselves until stage 57, i.e. just before the onset
of metamorphosis, in Xll (Plate, fig. D). Further to this, it is clear that the
genes affecting the time of appearance of these melanophores in Xlv are dominant over those of Xll, as revealed in the study of the reciprocal hybrids in which
all develop like Xlv in this respect. The significance of these data is commented
on below.
The technique of germ-cell grafting was almost the same as that described by
Blackler & Fischberg (1961). The primordial germ-cells in the piece of endoderm
grafted were later constituents of the experimental gonad, so it can be justifiably
assumed that they had migrated out of their graft milieu and come into contact
with the somatic cells of the host gonad. The slight variation in the technique
lay in transplanting between neurulae of stages 21-23 and not stage 26, since in
their discussion of graft failures Blackler & Fischberg (1961) drew attention to
the possibility that the germ-cells of this latter stage were no longer present in the
piece of tissue grafted from one embryo to another.
Grafts were made reciprocally with respect to both subspecies. The smaller
egg size of Xlv causes the neurulae to be smaller than neurulae of Xll at stage 23
(2-2 mm. as opposed to 2-5 mm.). This difference in size is small and yet is
sufficient to make the grafting of pieces of Xll neurulae into Xlv more difficult
than the reciprocal operation. One may add that this difficulty lies not in the
actual manipulation but in transferring a piece of Xll neurula tissue large enough
and likely to incorporate the primordial germ-cells.
In order to distinguish between graft and host cells, the nuclear marker
discovered by Fischberg (Elsdale, Fischberg, & Smith, 1958) has been used.
In theory, the removal of the host germ-cell region should result in host
sterility with respect to the host germ-cells, but in practice some of the host's
germ-cells can be left behind. The nuclear marker has been described so often in
publications from this laboratory (Elsdale, Fischberg, & Smith 1958; Fischberg,
Gurdon, & Elsdale, 1958; Gurdon, 1959; Elsdale, Gurdon, & Fischberg, 1960;
Gurdon, 1960 a, b; Fischberg & Wallace, 1960; Wallace, 1960; Blackler,
1960 a, b; Blackler & Fischberg, 1961) that a further repetition is hardly
justified. The reader is referred to Blackler & Fischberg (1961) for an account
of the use of the nuclear marker in the germ-cell transfer technique.
The results of the experiments performed have involved observations of
nuclei (for the marker), egg diameters and pigmentation, and the times of
appearance of the rectal tube melanophores.
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A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
RESULTS
Grafts
Forty-eight transfer operations were performed, of which 34 were successful
in producing metamorphosed frogs. The successful healing-in of grafts was
thus a little over 70 per cent, of all attempted transfers. This is an improvement
on the healing (40 per cent.) obtained in the development of the technique as
reported in Blackler & Fischberg (1961). Applications of the technique for other
purposes, not commented on here, have resulted in over 90 per cent, healing.
This improvement has been obtained by culturing the experimental embryos in
0-1 Niu & Twitty solution until they reach the feeding stage. After an operation
the obliteration of the edges of the graft by ectodermal overgrowth is a poor
criterion of successful healing and the graft may break down at any time between
the neurula and swimming tadpole stages. Transfer of experimental embryos to
water too soon after operation increases the chance of graft breakdown.
Not all the 34 experimental frogs obtained have been tested for graft success.
Two were retarded in growth, 2 died shortly after metamorphosis, and 12 males
derived from operations in which the nuclear marker was not used were not
tested.
Transfer of 2-nucleolate germ-cells ofXlv to 1-micleolate XII recipients
All 9 experimental frogs of this combination developed into males and have
been mated with 2-nucleolate Xll females. Xll females were chosen since (a) it
is easier to mate Xll with Xll than Xll with Xlv because of adult size differences,
and (b) the graft success, as derived from observation of nuclei in tadpoles, can
be checked against the percentage graft success obtained from observations of
rectal tube melanophores (which, as explained above, are dictated in their time
of appearance by Xlv genes).
When a non-experimental 1-nucleolate (1 nu) frog1 is mated with a normal
2-nucleolate (2 nu) frog, approximately half the offspring are 1 nu and half 2 nu.
Thus, in the case of 2 nu-in-l nu experimental frogs mated to 2 nu normal frogs,
the percentage graft success may be calculated from the numerical excess of
2 nu over 1 nu offspring. Employing this principle, the graft success of the 9
males in this group was calculated from the nucleolarities of their offspring, as
recorded in Table 1.
The nucleolarities of the tadpoles in Table 1 were determined in phasecontrast preparations of the tail-tips. Other tadpole samples of the same matings
were kept and scored for the appearance of rectal tube melanophores as following the Xlv pattern (st. 50) or the Xll pattern (st. 57). Graft success, as calculated from these observations, is recorded in Table 2. It is apparent that very
1
In previous papers from this laboratory we have used the abbreviation N for nucleolarity. Since
this has sometimes been confused with n, or N used in studies of chromosome ploidy, it has been
decided to adopt the abbreviation nu in all future publications.
A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
645
TABLE 1
Transplantation of Xh germ-cells into Xll at the neurula stage, using the nuclear
marker
Results of mating the experimental animals (1 nu), possibly containing 2 nu grafted germ-cells,
with 2 nu Xll frogs. Percentage graft success is calculated from the excess of 2 nu over 1 nu
offspring. Nucleolarity derived from observations of phase-contrast preparations of tail-tips of
feeding tadpoles.
No. of
frog
Sex
1
2
3
4
S
<S
<J
<?
(?
<J
<J
3
$
5
6
7
8
9
No. of tail-tips
analysed (x)
200
150
100
90
100
100
66
100
100
No. of 2 nu No. of 1 nu
tadpoles (y) tadpoles (z)
Percentage graft
(y - z)100
X
141
150
100
81
100
59
0
0
9
41-0
1000
1000
0
1000
46
54
30
36
8
1
00
00
92
99
800
84-0
980
TABLE 2
Transplantation ofXlv germ-cells into Xll at the neurula stage
Results of mating the experimental animals (1 nu), possibly containing 2 nu graft germ-cells,
with 2 nu Xll frogs. Percentage graft success is calculated from observations of the time of
appearance of rectal tube melanophores. Frogs tested bear the same numbers as in Table 1.
No. of
frog
Sex
No. of tadpoles
examined
1
2
a<?
50
30
3
5
6
8
9
c?
<j
<J
<J
12
112
60
72
146
No. showing
melanophores
at st. 50
No. showing
melanophores
at st. 57
22
30
12
112
28
0
0
60
144
0
0
60
12
2
Percentage graft
success
44-0
1000
1000
1000
00
83-3
98-6
comparable values for graft successes are obtained by using the two different
methods of assessment.
Transfer of 1-nucleolate germ-cells of Xll neurulae to 2-nucleolate Xlv
recipients
Six experimental frogs of this combination have been obtained and mated
with 2-nucleolate Xlv frogs. Once again, the choice of the normal frog was
governed by the ease of mating, although in this case the observation of melanophore appearance cannot be used to determine graft success.
When a 1 HM-in-2 nu experimental frog is mated with a normal 2 nu frog,
any 1 nu tadpoles among the offspring must have been derived from graft cells.
Moreover, a proportion of the 2 nu offspring (numerically equal to the 1 nu
646
A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
tadpoles) are also of graft origin although these cannot be distinguished from
those offspring derived from host cells. The percentage graft success may,
therefore, be calculated from the number of 1 nu tadpoles. Graft success in the
6 frogs of this group, using this principle, was calculated from the data set out in
Table 3.
TABLE 3
Transplantation of Xll germ-cells into Xlv at the neurula stage, using the
nuclear marker
Results of mating the experimental animals (2 nu), possibly containing 1 nu graft germ-cells,
with 2 nu Xlv frogs. Percentage graft success is calculated from the number of 1 nu offspring.
Nucleolarity derived from observations of phase-contrast preparations of tail-tips of feeding
tadpoles.
Percentage graft
10
11
12
13
14
15
Sex
?
9
•to
No. of
frog
a
No. of tail-tips
analysed (x)
52
122
65
121
60
44
No. of 1 nu No. of 2 nu
tadpoles
tadpoles (y)
0
60
0
3
0
0
52
62
65
118
60
44
, (2J01OO
X
00
98-3
00
4-9
00
00
1
The graft success is calculated on the assumption that the offspring of a mating 1 nu X 2 nU are in
the ratio of 50 1 nu: 50 2 nu per 100 embryos. Actually, there are always slightly more 2 nu offspring
than 1 nu; thus the figures quoted for graft success are slightly lower than the correct percentage
(e.g. for frog 11, the graft success is 100 per cent.)
Three of the 6 frogs were females, and it became clear that observation of
the type of egg laid could serve as a check on graft success as calculated from
nucleolarity. In Table 3, $$ 10 and .12, both negative for graft success as calculated from the nucleolarity of their offspring, laid eggs absolutely typical of
Xlv—that is, with a mean diameter of 1 mm. and pale grey-brown pigment,
$11, on the other hand, demonstrated the graft origin of her oocytes by laying
typically Xll eggs, with a mean diameter of 1-32 mm. and coloured chocolate
brown. The eggs of $11 are presented in Plate, fig. A. Every egg laid by this
female was of Xll type and nothing could be observed, either in the eggs or their
mode of development, that indicated qualities in them intermediate between
Xlv and Xll that could be ascribed to the ovarian environment. For $11, observations of the nucleolarity of offspring and the type of eggs from which they
developed indicated total graft success.
Transfers of germ-cells between Xlv and Xll in which the nuclear marker
was not employed
(a) Xll germ-cells transferred to Xlv. Seven frogs were obtained from operations in this group, but all were male and have not been tested. Graft success
A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
647
could have been determined from observations of melanophores in offspring
derived from matings with Xll frogs, but such crosses are obtained only with
difficulty and it was unlikely that any information further to that obtained from
transfers of this kind in which the nuclear marker had been used would be
forthcoming.
(b) Xlv germ-cells transferred to Xll. Eight frogs were obtained from operations in this group. Of these, 5 were male and have not been tested, although
graft success could have been determined from melanophore observations. The
object in testing the females was to obtain further data on the type of eggs laid.
The 3 female frogs in this group were mated to normal Xll males. The results
of mating these females are recorded in Table 4 and it can be readily appreciated
that all three operations were successful. The great majority of eggs laid were of
Xlv type and hence these females are the reciprocals of $11 (vide supra) in
respect of egg type. Again, no indications of qualities that could be ascribed to
the ovarian environment were observable, and the melanophore observations
on offspring support the egg data.
TABLE 4
Transplantation of Xlv germ-cells into Xll at the neurula stage
Results of mating the experimental animals with Xll male frogs. Percentage graft success is calculated from egg type and time of appearance of rectal tube melanophores.
No. of
frog Sex
OfOfOf
16
17
18
No. of tadpoles Percentage
No. of eggs No. of eggs Percentage No. of tadpoles
examined examined
showing melano- showing melano- graft
graft
Xll type
Xlv type
phores at st. 50 phores at st. 57
success
success
120
22
200
3
0
0
97-5
1000
1000
99
22
80
2
0
0
980
1000
1000
DISCUSSION
Grafting technique
In their 1961 study, Blackler & Fischberg were unable to obtain female frogs
showing graft success. However, in the present analysis females as well as males
have been obtained which show graft success. Thus there is no proof that
transfers of primordial germ-cells into female recipient embryos are less likely
to be successful than transfers into males. The numbers of experimental frogs
obtained and tested have been small, but, nevertheless, it does seem that more
successful transfers result when neurulae not later than stage 23 are used.
Egg type
Female frogs of one subspecies showing graft success have laid eggs typical
of the other subspecies. These eggs were the direct descendants of those
primordial germ-cells originally present in the graft and which have migrated
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A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
from it into the 'foreign' somatic environment of the host ovary. The results are
limited in that the eggs are defined as of graft type on only two characters—
dimension and colour, and involve only subspecific grafts. Nevertheless, it is
clear that the size and colour of an egg is, in this graft combination, determined
by the egg itself and not by the ovarian environment in which it develops.
Although the egg acquires much of its substance from the host via the somatic
cells of the ovary, it seems that the organization of this substance is specified by
the egg itself. Once again, the independence of the germ-line is manifest.
In conclusion, one could go further and cite this control of egg type as being
centred in the egg nucleus. Gurdon (1961) has been able to show, by means of
nuclear transplantations between the two subspecies used in this study, that the
kind of frog produced, and the type of eggs laid by female transplant-frogs, is
determined by the subspecific nature of the nuclei transplanted.
Reproductive metaplasia
The primordial germ-cell grafting technique, employing the nuclear marker,
has shown that primordial germ-cells exist in neurula stages and that these cells
give rise directly to cells which, in turn, form the definitive gametes. However, the
cases of complete graft success (100 per cent.) can be cited as evidence against
the hypothesis of a secondary, somatic, origin of some definitive sex-cells
(reproductive metaplasia). This hypothesis originated in a paper by Butcher
(1929) on the germ-cells of the lamprey, in which the gradual transition of
peritoneal cells into primordial germ-cells was described. The issue has remained
a controversial topic in germ-cell studies, and evidence for the existence of such
reproductive metaplasia, derived entirely from descriptive studies, permeates
the literature (particularly the literature concerned with the germ-line of
mammals).
Experimental approaches to the problem of reproductive metaplasia have been
few. In birds, Benoit (1930) and Dulbecco (1946) by irradiation methods, and
Dantschakova (1931) by cauterization, have shown that removal of early
embryonic extra-gonadal germ-cells leads to the production of embryonic
sterile gonads. However, the design of these experiments does not eliminate the
possibility that such gonads might subsequently acquire a complement of sexcells from a somatic source. The experiment of Domm (1929) does, by contrast,
point to the incapacity of the post-embryonic bird ovary to produce gonocytes
after destruction of the primordial germ-cells. Briefly outlined, Domm found
that extirpation of the left ovary is followed by hypertrophy of the normally
atrophying right ovary to form a testis. This testis is only fertile, however, if the
extirpation of the left ovary is performed while the primordial germ-cells of the
right ovary have not degenerated. This degeneration is complete 3 weeks after
hatching of the chick and operations result only in the production of sterile
right testes thereafter.
A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
649
In mammals, Mintz & Russell (1957) have worked on the problem of sterility
in mice caused by the presence of certain mutated genes. These workers find
that the primordial germ-cells are segregated normally in the embryos but that
they degenerate in the course of their migration to the gonadal sites. The
analyses of Mintz & Russell strongly deny the existence of reproductive metaplasia in the mouse, although it should be borne in mind that their mice were
abnormal in ways other than sterility, and the abnormal genotype might affect
the expression of metaplasia adversely.
To these experimental approaches in birds and mammals, the present
contribution for the frog Xenopus may be added. In this study certain experimental frogs, absolutely normal in their phenotype but containing germ-cells of
graft origin, produce normal eggs and sperm entirely of the graft type. The
conclusion must be that the existence of reproductive metaplasia in vertebrates
is not only unproven, but is, to say the least, highly improbable. The germ-line
is not only distinct from the somatic line in adults (shown unequivocally for the
guinea-pig in an experimental study by Castle & Phillips, 1911) but in embryos
as well.
SUMMARY
1. Transplantations of primordial germ-cells have been carried out between
two subspecies of X. laevis at the neurula stage according to the technique of
Blackler & Fischberg (1961). Adult experimental frogs have been tested for
graft success by matings with normal frogs, and graft success quantitatively
assessed with the help of a nuclear marker, rectal tube melanophores, and eggtype.
2. Some experimental frogs only produced gametes of graft origin. This
finding is adduced as strong evidence against the theory which claims that some
normal gametes originate from sources other than the primordial germ-cells.
3. After successful grafting, female frogs of one subspecies laid eggs in size
and colour identical with those of the other subspecies and no intermediate
characters were observed. It is concluded that for the two subspecies used, the
oocyte specifies these egg characters itself and independently of the ovarian
environment in which it develops.
RESUME
1. Les transplantations des cellules germinales primordiales ontetepratiquees
entre les deux sous-especes de Xenopus laevis au stade neurula, selon la technique
de Blackler & Fischberg (1961). Pour le controle du succes des greffes, les
grenouilles experimentales adultes ont ete accouplees avec des grenouilles
normales et le resultat quantitatif des greffes a ete evalue par les signes
nucleaires, les melanophores du tube rectal et par le type de l'oeuf.
2. Certaines grenouilles experimentales ont produit seulement des gametes
d'origine provenant de la greffe. Cette constation est une preuve evidente contre
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A. W. BLACKLER—TRANSFER OF PRIMORDIAL GERM-CELLS
la theorie qui pretend que certains gametes normaux ont comme origine d'autres
sources que les cellules germinatives primordiales.
3. Parmi les greffes reussies, les grenouilles femelles d'une des sous-especes ont
pondu des oeufs identiques, et de taille et de couleur, a l'autre sous-espece. Des
caracteres intermediaries n'ont pas ete observes. II a ete conclu que dans les
deux sous-especes experimentees, seules les oocytes sont responsables de ces
caracteres de l'oeuf et cela independamment de l'environnement de l'ovaire dans
lequel il se developpe.
ACKNOWLEDGEMENTS
It is a pleasure to acknowledge the advice afforded by my colleague Prof. M.
Fischberg and the technical assistance of Miss J. D. McConnell. The author
wishes also to thank Mme P. Ahmad-Zadeh for some help with the manuscript
and the British Empire Cancer Campaign for a grant to support work of which
this study forms a part.
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Vol. 10, Part 4
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EXPLANATION OF PLATE
FIG. A. Photomicrograph to show the appearance of the eggs of X. laevis laevis (blastula stage).
The eggs were actually laid by a female of X. laevis victorianus but were indistinguishable from
normal Xll eggs.
FIG. B. Photomicrograph to show the appearance of the eggs of Xlv (blastula stage). The eggs
were actually laid by a young female of Xll but were indistinguishable from normal Xlv eggs. The
photograph is presented at the same magnification as that of fig. A for direct comparison.
FIG. C. Photograph to show the rectal tube region of a stage 50 tadpole of Xlv (the shape of the
limb bud gives the stage number). Melanophores are visible at this stage in the wall of the rectal tube.
FIG. D. Photograph to show the rectal tube region of a stage 54 tadpole of Xll (the shape of the
foot gives the stage number). Melanophores are not visible at this stage in the wall of the rectal tube.
HLB, hind limb-bud; HLF, hind limb-foot; VTF, ventral tail fin; LL, lateral line; RT, rectal tube; M,
melanophores.
{Manuscript received 16: v:62)