/. Embryo/, exp. Morph. Vol. 41, pp. 33-46, 1977
Printed in Great Britain © Company of Biologists Limited 1977
33
Events in the germ cell lineage after
entry of the primordial germ cells into the genital
ridges in normal and u.v.-irradiated Xenopus laevis
By BRIGITTA ZUST1 AND K. E. DIXON 2
From the School of Biological Sciences, Flinders University of
South Australia
SUMMARY
Approximately 20-25 primordial germ cells leave the endoderm between stages 38-41 and
localize in the dorsal root of the mesentery by stage 43/44. At this time all the cells contain
large quantities of yolk which is gradually resorbed. The cells begin dividing between stages
48-52. The number and size of the germ cells were measured in tadpoles between stages
48-54 of development. The results indicate that in females the germ cells divide more often
than in males. In both sexes the mitoses are grossly unequal, leading to the formation of a
new generation of germ cells which are considerably smaller (one-tenth to one-fifth) than the
size of the primordial germ cells at stage 48. The germ cells in male tadpoles at stage 54 are
larger than in female tadpoles at the same stage. In tadpoles which developed from eggs
irradiated in the vegetal hemisphere with u.v. light at the 2- to 4-cell-stage, primordial germ
cells migrate into the genital ridges much later (stage 46-48) than in unirradiated embryos.
They also differ morphologically from germ cells in control animals at this stage in that they
are approximately one-tenth the size, lacking yolk in the cytoplasm and have a more highly
lobed nucleus. Comparison of the results in unirradiated and irradiated animals suggests that
the germ cell lineage is composed of a series of ordered, predictable events, and serious
disruption of one of the events deranges later events.
INTRODUCTION
The role of early embryological events in cellular differentiation is commonly
ignored, an omission which is clearly due to the difficulty of identifying unequivocally the early stages in a cell lineage. The germ cells in anuran amphibians
provide an almost unrivalled opportunity to follow the events which lead up
to the formation of terminally differentiated cells. In the early stages of development, the germ cells lie among endoderm cells and can be recognized in
histological sections by their content of a cytoplasmic factor, germ plasm, which
can be visualized with appropriate staining techniques (Bounoure, 1934, 1964;
Blackler, 1958). Later in development, just before the young tadpoles begin to
1
Author's address: Zoologisches Institut der Universitat, CH1700 Fribourg, Switzerland.
Author's address: School of Biological Sciences, Flinders University of South Australia,
Bedford Park S.A. 5042, Australia.
2
34
B. ZUST AND K. E. DIXON
feed, the germ cells migrate out of the endoderm into the genital ridges where
study of their differentiation can continue since, although the germ plasm no
longer stains selectively, the germ cells can be readily distinguished because of
their characteristic morphology. In this paper the term primordial germ cells
will be applied to the germ cells in the genital ridges or in the sexually indifferent
gonads, in conformity with earlier usage (Nieuwkoop & Faber, 1967; Kerr &
Dixon, 1974; Whitington & Dixon, 1975).
The initial series of events in the germ cell lineage in Xenopus laevis embryos
have been described by Whitington & Dixon (1975) from measurements of the
number and size of the cells. This new study reported here is a continuation of
the earlier investigation, using the same general approach. The aim of this work
was to describe the patterns of proliferation and differentiation in the germ cell
lineage after the primordial germ cells settle in the genital ridges. Specifically,
changes in anatomy and histology of the gonads and in number and size of the
germ cells have been studied. This information, in addition to contributing
towards our understanding of the germ cell lineage, provides the standard of
normal development against which an evaluation can be made of the effects of
u.v. irradiation of the vegetal pole of 2- to 4-cell embryos, a procedure which is
reported to cause partial or total sterility (reviewed in Smith & Williams, 1975).
In a previous paper (Zlist & Dixon, 1975) we compared the endodermal phase
of the germ cell lineage in irradiated and normal embryos. The aim of the
experiments reported here was to examine irradiated embryos at later stages of
development, when the germ cells normally occupy the genital ridges, to determine whether disruption during an early phase of the cell lineage has any effect
later in development.
MATERIALS AND METHODS
Histological procedures
X. laevis tadpoles were staged according to the Normal Table of Nieuwkoop
& Faber (1967), fixed in Carnoy's fluid and embedded in paraffin. Sections
(5 jtim) were stained in Harris' haematoxylin or Mayer's haemalum and eosin.
Numbers of germ cells were determined directly from serial sections up to and
including stage 52, taking care to count all sections of the same germ cell as
one cell only. In stage-54 animals the number of germ cells was estimated
according to the formula:
total no. of sections
of germ cells counted
total no. of
10
sections of gonad
c
n
no. of germ cells =
-;
„ .
?
average no. of sections per germ cell
The total number of sections of germ cells counted was the number of germ
cell profiles in 10 sections selected from the serially sectioned embryo using a
Germ cell lineage in Xenopus
35
table of random numbers. In a separate study it was shown that the germ cells
are distributed at random along the gonad. The average number of sections
per germ cell was determined separately from the number of profiles of at least
12 germ cells. The volume of individual germ cells was calculated from measurements of the area of their largest section, making the assumption that the cell
was spherical.
U.v. irradiation
The irradiation procedure was as described in Ziist & Dixon (1975). Two
dose ranges were used: low, 2000-7300 ergs/mm2 and high, 11000-22000
ergs/mm2.
RESULTS
Observations on normal animals
Anatomical and histological changes. Between stages 38-41, the dorsal
mesentery develops, suspending the gut in the body cavity and between stages
41-46 it lengthens dorsoventrally. The median genital ridge is formed at the
dorsal root of the mesentery in the posterior half of the embryo (e.g. 800-900
fim from the anterior end of a tadpole about 1100/^m long in body excluding
tail).
At about stage 48, the lateral genital ridges develop, initially as thickenings
on either side of the dorsal mesentery, and later as folds which protrude into
the body cavity (Fig. 1). By stage 49/50, a few mesonephric blastema cells with
mesenchyme and pigment cells have penetrated into the genital ridges, forming
the medulla of the primitive gonad which is dilated at regular intervals along
its length by aggregations of the medullary tissues into nodular medullary
cords. At these stages, an antero-posterior gradient in development was
observed.
From about stage 50 onwards, the size of the gonads increases considerably,
due primarily to an increase in the number of somatic cells. After about stage
54, ovaries and testes can be identified on gross morphological criteria, with the
shorter unpigmented testis readily distinguishable, particularly in later stages,
from the scalloped, pigmented ovary. These observations are in substantial
agreement with earlier reports on the formation of the genital ridges and early
gonadogenesis in Xenopus and a number of other anuran amphibians (e.g.
Bouin, 1900; Allen, 1907; Kuschakewitsch, 1908; Witschi, 1914, 1929; Cheng,
1932; Witschi, 1956; Nieuwkoop & Faber, 1967; Witschi, 1971; Wylie &
Heasman, 1976; Wylie, Bancroft & Heasman, 1976).
Morphology of the germ cells. Between stages 38-41, the presumptive primordial germ cells were observed leaving the endoderm in the manner described
previously by Whitington & Dixon (1975), to become located primarily in the
dorsal root of the mesentery by stage 43/44 (Fig. 2) and form the median genital
ridge. Once in the genital ridge the germ cells are no longer considered
36
B. ZUST AND K. E. DIXON
presumptive. Sometimes in tadpoles up to stage 48, germ cells were visible embedded in the mesentery between the ventral and dorsal roots, suggesting that
if a presumptive primordial germ cell is not carried out of the endoderm by the
forming dorsal mesentery, it can continue migrating into the median genital
ridge. Wylie & Roos (1976) have reported that presumptive primordial germ
cells are capable of active migration.
Between stages 38-44, the germ plasm loses its ability to stain with the
standard sequences (Azan and Volkonsky) which can be used successfully
in earlier stages (Ziist & Dixon, 1975). The primordial germ cells can still be
recognized without difficulty however, because of their characteristic nuclear
morphology, their size, and to a lesser extent, and then only in earlier stages,
the presence of yolk granules in the cytoplasm. In stage-43/44 embryos, all the
primordial germ cells contain large quantities of yolk (Fig. 2); in tadpoles
from two batches of eggs, yolk was present in 155 of the 158 germ cells (98 %).
However, at stages 46-48, one batch of embryos had very few germ cells with
yolk (8 % of 346 cells) but in two other batches, most cells still contained yolk
(80% of 161 cells). These results suggest that yolk is gradually resorbed,
beginning at the latest at stage 43, but the rate of resorption is higher in some
batches than in others.
In sections of stage-43/44 embryos, the germ cell nucleus is approximately
bean-shaped, occasionally with 1-2 lobes (Fig. 3). The lobulation increases as
development proceeds, and at stage 48, individual lobes are often narrow and
finger-like (Fig. 4). The nuclei of germ cells in stage-54 tadpoles remain highly
lobed. At all stages, the nuclei appear strikingly 'empty' except for nucleoli.
At stage 43/44 there is usually one nucleolus and at stages 46-48, there are
Figures 1-6.
Fig. 1. Section through stage-48 X. laevis tadpole showing lateral genital ridge
either side of dorsal mesentery, each ridge with a primordial germ cell. Note the
narrow, finger-like nuclear lobes and the absence of yolk from the cytoplasm.
Fig. 2. Section through stage-43/44 X. laevis tadpole showing median genital ridge
containing a number of primordial germ cells (arrows), the cytoplasm of which contains large quantities of yolk.
Fig. 3. Section through primordial germ cell in stage-43/44 X. laevis tadpole, showing
slightly lobed, approximately bean-shaped nucleus with prominent nucleolus.
Fig. 4. Section through primordial germ cell in stage-48 X. laevis tadpole showing
nucleus with more numerous finger-like lobes and prominent nucleolus. One yolk
platelet is visible in the cytoplasm.
Fig. 5. Section through early gonad of stage-50-52 X. laevis tadpole showing germ
cells of two sizes, larger situated distally and smaller proximally (arrow). Nucleoli
in larger cell more prominent than in smaller cell.
Fig. 6. Section through genital ridges of stage-48 tadpole previously irradiated at
2- to 4-cell stage in vegetal hemisphere with 4800 ergs/mm2 u.v. Germ cells (arrows)
small, with highly lobed nucleus, prominent but small nucleolus, no yolk in
cytoplasm; compare to small germ cell in Fig. 5.
Germ cell lineage in Xenopus
37
38
B. ZUST AND K. E. DIXON
Table 1. Number of germ cells in untreated tadpoles ofX. laevis
Stage
Number of
animals
Number of germ cells
(mean ± S.E.)
43/44
46-48
50-52
54$
54c?
7
28
10
6
5
22-6 ±2-9
20-8 ±1-3
72-1 ±9-5
1908-8 ±422-5
364-4 ±93-5
frequently two. At stages 50-52, the nucleoli, up to four in number, become
larger and more basophilic (Fig. 5).
Number and size of the germ cells. The numbers of germ cells in tadpoles at
stages 43/44-54 are shown in Table 1. At stage 43/44, the number of primordial
germ cells in the median genital ridge indicates that two to three divisions of
the presumptive primordial germ cells have taken place in the endoderm since
segregation, confirming earlier observations (Whitington & Dixon, 1975; see
also Dziadek & Dixon, 1977). Between stages 43-48, their number does not
increase, thus indicating that no mitoses take place over this time. Mitosis
recommences between stages 48-52. At stages 50-52 the number of germ cells
in different individuals is quite variable as shown by the large standard errors.
This variability probably arises initially because mitosis begins at different
times in different animals and it is accentuated by the relatively fast rate of
division.
Although the number of tadpoles examined at stage 54 was relatively small,
there are obviously many more germ cells in female tadpoles than in males
(P < 0-05). Between stages 50-52 (5-11 days) the germ cells in females divide
about six to seven times while germ cells in males divide about four times.
The volumes of the germ cells at different stages of development are illustrated
in Fig. 7. The size of the germ cells which settle in the genital ridges at stage
43/44 is similar to that reported by Whitington & Dixon (1975). Their size
decreases up to stage 48 but this reduction can be attributed to yolk absorption
(see above).
At stages 50-52, the germ cells in the majority of animals are separable into
two distinct and relatively uniform size classes. In one class, the germ cells are
slightly smaller than germ cells at stages^ 46-48. In the other class, the volume
of the cells is reduced approximately tenfold. In stage-54 embryos, there are no
germ cells similar in size to those at stages 46-48, an observation which indicates
that the primordial germ cells are completely replaced by a new generation of
smaller germ cells. The cells of this new generation are of two sizes, each of
which is exclusive to a single animal. In tadpoles from stage 54 onwards, in
which the sex can be determined, the smaller of the two types is found exclusively
in ovaries whereas the larger type is found only in testes. The difference in size
Germ cell lineage in Xenopus
6 -
39
6 122/7
165,8
O
73/5
60/3
54/2
0 79/5
43 44
50-52
46-48
54
Stage
Fig. 7. Decrease in volume of individual germ cells with development stage 43/44stage 54. Figures beside each mean show number of cells measured and number of
tadpoles.
is significant (P < 0-05), and may be correlated with the different rates of division
observed in the two sexes.
The size of the germ cells therefore changes between stages 48-54 according
to a rather complex pattern which can be explained in the following way. The
first mitoses appear to be grossly unequal, leading to the formation of germ cells
which are considerably smaller than the primordial germ cells. As mitosis
continues, the primordial germ cells are completely replaced by a new generation of germ cells distinguishable because of their smaller size, tentatively
designated gonial cells.
40
B. ZUST AND K. E. DIXON
Experiments with u.v.-irradiated embryos
The results obtained varied somewhat with the dose of u.v. and with the
batch of eggs. Some batches were more sensitive to u.v. irradiation than others
and hence the effects of irradiation were greater. Differential sensitivity of
batches of eggs has been previously reported (Bounoure, Aubry & Huck, 1954;
Gurdon, 1960; Padoa, 1963; Smith, 1966; Malacinski, Benford & Chung, 1975;
McAvoy, Dixon & Marshall, 1975).
Numbers of germ cells. The numbers of germ cells in u.v.-irradiated tadpoles
at stages 41-52 are shown in Table 2. No germ cells were found in 14 tadpoles
examined between stages 41-46, although the median genital ridges, dorsal
crests of the endoderm and dorsal mesenteries were closely searched. In two
other tadpoles examined, one had one germ cell and the other had three.
However, in tadpoles examined at stage 48 the genital ridges contained germ
cells. We conclude therefore that migration of the germ cells from the endoderm
into the genital ridge is delayed in irradiated animals compared to normal
animals in which it takes place between stages 38-41 (Whitington & Dixon,
1975).
At stage 48, the number of germ cells in the genital ridges depended on the
dose of u.v. which the early embryos received; at high doses there were significantly fewer germ cells (P < 0-01) and at low dose rates, the numbers were
equivalent to those found in non-irradiated animals (although this equivalence
is not significant).
By stages 50-52, the number of germ cells increased indicating that between
stages 48-50 the cells began to divide. Animals which received a low dose of
u.v. contained more germ cells than those receiving a high dose of u.v.
Size of germ cells. The germ cells in irradiated tadpoles were of two sizes,
called here for convenience, large and small (Table 3), the difference in volume
between the two classes being highly significant (P < 0001). The large germ
cells resemble those in control tadpoles at the same stage of development, but
the small cells differ, in addition to their being only approximately one-tenth
the size, in having a more highly lobed nucleus and no yolk in the cytoplasm
(Fig. 6). The relative proportions of the two classes depends on the dose of
u.v. the early embryos received. In stage-48 tadpoles developing from embryos
exposed to low doses of u.v., approximately one-third of the cells were large
but in those which received high doses, all the cells were small. Germ cells in
tadpoles examined at stages 50-52 showed the same dose dependency but the
proportion of large cells was reduced relative to stage-48 tadpoles. By stage 54
(not shown in Table 3), large germ cells could not be found in nine tadpoles which
contained a total of 2550 germ cells.
The data on the number and size of germ cells in irradiated animals can be
summarized in the following way. The migration of the germ cells out of the
endoderm was delayed and in tadpoles which received high doses of u.v., fewer
Germ cell lineage in Xenopus
41
Table 2. Number of germ cells in u.v.-irradiated tadpoles o/X. laevis
Developmental
stage
Dose rate
(ergs/mm2)
No. of animals
examined
No. of germ cells
(mean ± S.E.)
41-46
15000-18000
2000- 7300
11000-22000
2000- 7300
11000-22000
16
12
11
8
11
0-25 ±0-3
32-7±2-3
17-6 ±2-2
700 ± 9 0
49-6 ±4-3
48
48
50-52
50-52
Table 3. Size distribution of germ cells in u.v.-irradiated tadpoles of
X. laevis
No. germ
No. of large
germ cells
No. of small
germ cells
A.
Developmental
stage
Dose rate
(ergs/mm2)
animals
examined
46-48
Control
2000-7300
11000-22000
Control
2000-7300
11000-22000
28
12
11
10
8
11
48
50-52
(mean ±
(mean ±
S.E.)
S.E.)
%
20-8 ±1-3 20-8 ±1-3 100
32-7 ±2-3 11-8 ±2-9 36
17-6±2-2 0
0
72-1 ±9-5 33-4±6-3 46
6
700 ± 9 0 4-1 ±1-5
0
49-6 ±4-3 0
(mean ±
S.E.)
0
0/
/o
0
20-8 ±4-2 64
17-6 ±2-2 100
38-7 + 5-3 54
65-9 + 9-2 94
49-6 ±4-3 100
germ cells entered the genital ridges. In embryos exposed to low doses of u.v.,
the germ cells were initially of two sizes but as these tadpoles developed to
stage 54, the larger cells were eliminated. In embryos exposed to high doses of
u.v., no large cells were seen at any stage of development.
We interpret these results as indicating that u.v. irradiation of the early
embryo disrupts events in the endodermal phase of the germ cell lineage,
resulting in division patterns which produce very small cells which migrate less
efficiently and therefore later than normal.
DISCUSSION
One of the aims of this study was to describe the patterns of proliferation of
the primordial germ cells from the time they settle in the genital ridges. Our
observations have confirmed earlier reports that the presumptive primordial
germ cells leave the endoderm between stages 38-41 (Whitington & Dixon,
1975) and settle in the lateral genital ridges by stage 46. The primordial germ
cells, as they can then be termed, do not divide until about stage 50, although
the time mitosis begins varies between individual animals. These results are in
substantial agreement with earlier reports (Kalt & Gall, 1974; Ijiri & Egami,
1975). There is therefore a period of mitotic inactivity of the order of 12 days
\
42
B. ZUST AND K. E. DIXON
between the last of the so-called cloning divisions (Whitington & Dixon, 1975)
which take place about stage 36-37 (Dziadek & Dixon, 1977) and the initiation
of a new series of divisions. In other words, proliferation of the germ cells is
not continuous between gastrulation and stage 54 but is separable into two
distinct phases.
A second aim of this investigation was to determine the patterns of differentiation of the germ cells. Proliferation results in the formation of a new generation of germ cells which are much smaller and replace completely the original
population of primordial germ cells. A reduction in size of the germ cells at
this stage in development has been previously reported in Xenopus and in a
number of species of Rana. In X. laevis, Kalt (1973) observed that spermatogonia measure 12-20 ju,m in diameter compared to approximately 25 /tm in the
primordial germ cells. Reed & Stanley's (1972) measurements of spermatogonia
(8-5-12-5/*m in diameter) when compared to those of Kalt (1973) and those
reported in this paper, confirm that the primordial germ cells are considerably
larger than later generations of cells. Similarly, in R. fusca, Nussbaum (1880)
reported that the diameter of the germ cells decreases from 35-40 /tm to about
30 fim. In R. temporaria, Bouin (1900) observed that the primordial germ cells
are five to six times larger than germ cells at later stages, an estimate confirmed
by comparison of Bounoure's (1934) measurements of the diameter of primordial germ cells (31 fim) with those of gonial cells (16-20 fim) by Di Berardino
& Hoffner (1971). The results of Kuschakewitsch (1908) show that in R. esculenta, the diameter of the germ cells decreases from 47 to 14-5 jim. over this
period in development. Thus, in anuran embryos, the germ cells are commonly
if not always reduced in size between the time they enter the genital ridges and
later stages, a decrease which is associated with proliferation. We consider that
a reduction in cell size of this order is a criterion for differentiation, and in this
case, marks the production of a different generation of germ cells. We tentatively call these cells gonial cells but the validity of this designation depends
on whether they persist in the mature gonad as spermatogonia or oogonia.
The patterns of proliferation and differentiation of the germ cells differ in
males and females. The rate of division at least up until stage 54 is higher in
females than in males and the gonial cells in males are larger than in females, a
difference which is maintained at least until metamorphosis (Ziist & Dixon,
unpublished results). Histological changes in the gonads have often been reported as the first indications of sexual differentiation in anuran tadpoles
(e.g. Witschi, 1929; Cheng, 1932) but recently Kalt (1973) noted ultrastructural
differences between germ cells before histological differences became apparent
in X. laevis. Ijiri & Egami (1975) have reported that germ cells are more
numerous and smaller in ovaries than in testes. The observations reported here
represent the earliest indications of sexual differentiation in anurans yet reported.
In several animal species, different rates of division of the germ cells have
been implicated in sexual differentiation. In the coleopteran Leptinotarsa, germ
Germ cell lineage in Xenopus
43
ells in the male divide more often (Richard-Mercier, 1972) but in the teleosts
Platypoedlus (Wolf, 1931) and Oryzias (Satoh & Egami, 1972) and in the chick
(Van Limborgh, 1968) germ cells divide more often in females. In a number of
other animals, the gonads of one sex contain more germ cells than the gonads
of the other sex. In Drosophila, the testes contain more germ cells than the
ovaries (Kerkis, 1931; Sonnenblick, 1950) and similar differences have also
been reported in salmonids, viviparous cyprinodonts, the black-mouthed bass
(reviewed in Hardisty, 1967) and in rats (Beaumont & Mandl, 1961, 1963;
Baker, 1972).
The observations reported here contribute to our understanding of the events
in the germ cell lineage in X. laevis. Previous work in this laboratory (Whitington
& Dixon, 1975; Dziadek & Dixon, 1975, 1977) has shown that the initial
clone of four presumptive primordial germ cells is segregated during cleavage,
and immediately enters into the first proliferative phase in which there are two
to three divisions of each cell. At the end of this phase, the germ cells migrate
out of the endoderm and settle into the genital ridges. This study has shown
that a second proliferative phase then ensues resulting in the formation of a
generation of gonial cells. The number of divisions and the size of the gonial
cells are characteristic for each sex. The overall picture of the germ cell lineage
which is beginning to emerge is one of a series of ordered, predictable events.
A third aim of this investigation was to compare the differentiation of germ
cells in u.v.-irradiated and normal embryos. After u.v. irradiation of the
vegetal hemisphere of 2- to 4-cell embryos, segregation during cleavage of the
initial clone of cells is disrupted (see also Beal & Dixon, 1975) and subsequently,
after neurula, the number of germ cells in the endoderm declines rapidly; only
an occasional germ cell, if any, could be detected and then only in embryos
irradiated with low u.v. doses (Ziist & Dixon, 1975). The results reported here
show that germ cells eventually migrate out of the endoderm but their arrival
in the genital ridges is much later than in normal embryos. An additional
abnormal feature is that they differ morphologically from germ cells in control
animals at this stage in development, most obviously in their smaller size, but
also in nuclear morphology and absence of yolk from the cytoplasm. Indeed,
in morphology and size they closely resemble the germ cells seen in normal
tadpoles at stage 54. This comparison prompts the hypothesis that, in u.v.irradiated animals, the germ cells, while still in the endoderm, enter into the
phase of proliferation which in normal tadpoles begins after they have entered
the genital ridges - i.e. the series of unequal mitoses which constitute the
second phase of proliferation. If this hypothesis is correct several predictions
can be made:
(i) Small germ cells would appear in the genital ridges of stage-48 irradiated
animals, perhaps in less affected (e.g. low dose) animals in company with some
normal sized germ cells, which would however be replaced as the second
proliferative phase continued.
44
B. ZUST AND K. E. DIXON
(ii) The germ plasm would lose its affinity for the standard staining sequences
(as it does in normal animals just before the second proliferative phase begins)
while the germ cells are still in the endoderm, with the result that they would
no longer be detectable (see Ziist & Dixon, 1975).
(iii) The germ cells which result from precocious initiation of the second
proliferative phase might be less able to migrate than the normal cells and therefore their arrival in the genital ridge would not only be delayed but fewer might
successfully complete the migration.
(iv) The first proliferative phase, in which divisions normally occur about
stages 22-24 and 35-36 (Dziadek & Dixon, 1977), would be disrupted, resulting
in fewer germ cells entering the genital ridges.
Examination of the data presented here and earlier (Ziist & Dixon, 1975)
shows that all these predictions are fulfilled to a greater or lesser extent and
therefore the hypothesis must be seriously entertained. However, the hypothesis
clearly stands or falls on the question of the equivalence of the small germ cells
in irradiated embryos at stage 48 with those in normal embryos at stage 54.
Work on this question is proceeding.
Nevertheless, whether this hypothesis is correct or not, a general conclusion
is obvious: although a primary effect of u.v. irradiation of the 2- to 4-cell
embryos is to disrupt segregation, other events later in development are also
deranged. This conclusion is reinforced by our unpublished observations of
irradiated animals which have metamorphosed. In a proportion of these
adolescent frogs (up to 6 months post-metamorphosis) growth of the gonads
and meiosis are delayed, and the number of gonial cells is greatly reduced.
Earlier in this report, we concluded that the germ cell lineage is apparently
composed of a series of ordered, predictable events. An additional conclusion
might now be possible: that serious disruption of one of these events deranges
later events. That is, the sequence of events is not only ordered but, at least to
some extent, casual.
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SONNENBLICK,
(Received 5 January 1977, revised 4 February 1977)