/. Embryol. exp. Morph. Vol. 64, pp. 133-147, 1981
133
Printed in Great Britain © Company of Biologists Limited 1981
Proliferation and migration of primordial
germ cells during compensatory growth
in mouse embryos
P. P. L. TAM AND M. H. L. SNOW1
From the MRC Mammalian Development Unit,
University College London
SUMMARY
Primitive-streak-stage mouse embryos were treated with Mitomycin C injected intraperitoneally into pregnant females at 6-75-7-0 days post coitum. The newborn mice developed
poorly and mortality was high during the suckling period. Many weaned survivors showed
impaired fertility and poor breeding performance. Histological examination revealed a
paucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduction of the primordial germ cell population in early embryonic life, which was not fully
compensated for during the compensatory growth phase of the Mitomycin C-treated embryo.
Also contributing to such impaired fertility were retarded migration of the primordial germ
cells into the genital ridges, poor development of the foetal gonad and secondary loss of the
germ cells during gametogenesis in males.
INTRODUCTION
We have recently shown that a single intraperitoneal injection of 100-120/*g
Mitomycin C (MMC) into pregnant mice does very extensive damage to
primitive-streak-stage embryos, reducing cell number to about 15% of normal
values, and resulting in severe developmental disturbance in the ensuing 48 h
of embryogenesis. Subsequently, accelerated growth and morphogenesis restores
gross morphology to normal by mid-organogenesis stages 3-4 days later.
Although embryonic mortality is low, post-natal development is poor and
fertility among offspring surviving to breeding age is low (Snow & Tam, 1979).
This report concerns the developmental events underlying the reduced fertility
following MMC-treatment and describes the origin, proliferation and migration
of primordial germ cells, and the formation of the foetal gonads in normal and
MMC-treated mice.
MATERIALS AND METHODS
Pregnant Q-strain mice received a single intraperitoneal injection of 100 ii%
Mitomycin C (Sigma, London) in 0-25 ml 0-9 % NaCl between 6-75 and 7-0 days
1
Authors'" address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London NW1 2HE, U.K.
134
P. P. L. TAM AND M. H. L. SNOW
Table 1. The viability and breeding performance of the offspring of Mitomycin
C-treated pregnant females
Viability
No. alive at
No.
litters
No.
newborn
Birth
7d
14 d
28 d
No.
weaned
15
99
93
78
59
36
29
Breeding performance
Class of mating
Sex
No.
No. mated
<J
§
18
11
18
9
Fertile
10
9
Fertile and sterile Sterile only
4
0
4
0
post coitum (p.c.) Embryos are in early- and mid-primitive-streak stages at
this time.
Initial observations were made on the offspring of such mice that survived to
breeding age. Each mouse was test-mated to a normal Q mouse of proven
fertility for sufficient time to allow the production of several litters. Subsequently
the animals were killed, both gonads removed and fixed in Bouin's fluid and
examined histologically.
The formation of gonads was studied in embryos between 8-5 day p.c. when
primordial germ cells (PGCs) are observable in the developing hindgut, and
13-5 days when colonisation of the genital ridge is complete. Embryos were
fixed in cold 80 % ethanol, dehydrated in absolute ethanol, cleared in chloroform
and embedded in a low melting-point (54 °C) wax. Serial sections were made at
8 or 10 ju,m and stained with Fast Red TR salt to detect alkaline phosphatase
according to the azo-dye coupling method of Gomori (Gabe, 1975). They were
mounted in glycerine. Complete undamaged and properly stained serial sections
were obtained from 57 normal embryos and 107 MMC-treated embryos, from
24 litters.
PGCs were identified by the high content of alkaline phosphatase in their
cytoplasm and on their membranes (Chiquoine, 1954; Ozdzenski, 1967; Jeon&
Kennedy, 1973). PGCs were scored on every section of 8-5 to 11-5-day embryos.
Abercrombie's formula (Abercrombie, 1946) was used to correct for cells
registered in both of two adjacent sections, thus giving a better estimate of cell
number. In 13-5-day embryos the number of PGCs in the genital ridges was
computed from the size of the ridge (gonadal volume) which was derived from
measurement of camera-lucida drawings and the number of PGCs per unit
tissue volume (cell density) determined from at least four sections per gonad.
PGCs lying outside the genital ridges were counted as in younger embryos.
Proliferation
and migration of mouse primordial germ cells
135
Table 2. The reduction in germ cells in gonads of offspring from MMC-treated mice
Sex
Class of mating
61
Fertile only
Sterile only
Both types
No.
Empty seminiferous tubules (%) Mean
10 0(4), 2, 4, 5, 18, 22, 30
4 22, 71, 100, 100
4 29, 40, 54,61
8
73
46
Normal follicles (%)
9 24, 42, 53, 77, 86, 86, 100 (2)
74
49
2 47,50
The number of seminiferous tubules (fertile and 'empty') and the number of follicles of all
stages of development were scored in 4-5 good sections of the testis and ovary respectively.
?
Fertile
No litter
RESULTS
Table 1 shows the viability of offspring from MMC-treated mice and the
breeding performance of successfully weaned young. Two females never
mated; one developed ataxia which probably impaired her mating behaviour.
Histological analysis of gonads shows a reduction in gonad size and of the
number of germ cells (Table 2, Figs. 1, 2), particularly in the sub-fertile and
sterile males where seminiferous tubules completely devoid of germ cells and
containing only Sertoli cells were found (Fig. 1 b, c). In two of the sterile males
the testes were completely devoid of germ cells (Fig. 1 d). In the females no
ovary was found to be devoid of follicles, the smallest having about 24 % of the
normal number of oocytes. There are more atretic follicles in MMC-treated
mice.
Embryonic development
Figure 3 illustrates alkaline-phosphatase-positive PGCs in various sites in
their migration pathway. In Fig. 3 a PGCs are at the posterior end of the
primitive streak at the base of the allantois. This example is from an 8-5-day
MMC embryo which is retarded in development. In normal embryos this
developmental stage occurs at 7-75-8-0 daysp.c. In a normal 8-5-day embryo,
PGCs are found in the primary endoderm and early hindgut (Fig. 3b), by 9-5
days in the hindgut and just entering the mesentery (Fig. 3 c) and enter the
genital ridges at 10-5-11-5 days (Fig. 3d). The genital ridges are fully colonized
by 13-5 days (Fig. 4).
PGC number. Table 3 and Fig. 5 show the numbers of PGCs in normal and
MMC embryos according to gestational age. The lower PGC numbers in MMC
embryos do not simply reflect the retardation in overall development. Figure 6
illustrates graphically the relative development of the MMC embryos with
respect to PGC number, somite number and size, presomitic mesoderm length,
136
P. P. L. TAM AND M. H. L. SNOW
(B)
Fig. 1. (A) Normal testis showing prolific spermatogenic activity. Bar = 200 fim.
(B) Testis of sub-fertile male offspring from MMC-treated mice showing empty
seminiferous tubules. Bar = 200 fim. (C) Absence of germ cells in sterile tubules.
Bar = 50 /im. (D) Testis of sterile male which is totally devoid of germ cells.
Bar 200 /im.
Proliferation and migration of mouse primordial germ cells
137
(B)
Fig. (2). (A) Normal ovary showing follicles in various stages of development.
(B) Ovary from an offspring of a MMC-treated mouse showing many fewer follicles.
Bar = 200/tm.
axis length and foetal wet weight. Comparison of some of these growth parameters suggests they are under independent control (see later, and Snow, Tarn
& McLaren, 1981).
The PGC population doubling time in normal embryos is fairly uniform at
about 16 h between 8-5 and 13-5 days. A similar value is found in MMC
embryos between 10-5 and 13-5 days, but at the beginning of their migration
these PGCs divide very slowly (population doubling time 31 h), and between
9-5 and 10-5 days, very rapidly (doubling time 7 h) (Fig. 5). The period of
rapid proliferation coincides with the period of maximum compensatory
growth for other parts of the embryo but PGC number does not recover to
normal in treated embryos and when genital ridge differentiation commences
the gonads have about 50 % as many PGCs as normal (at 9-2- days there were
about 17% of normal values).
There is considerable variation between embryos, even within a single litter,
in the facility with which PGC number is restored. This variation is reflected in
the very much larger range of PGC numbers observed in 11-5-day MMC
138
P. P. L. TAM AND M. H. L. SNOW
Fig. 3. The location of primordial germ cells (arrows) in mouse embryos between
8-5 and 13-5 days p.c. (A) In the primitive streak and base of the allantois. Bar =
50 /tin. (B) In the hind-gut endoderm. Bar = 20 /tin. (C) In the hind-gut and dorsal
mesentery. Bar = 20/tin. (D) En route from mesentery to the genital ridges (GR).
Bar = 20 /tm.
Proliferation and migration of mouse primordial germ cells
139
Fig. 4. The genital ridges of 13-5-day mouse embryos. (A) male and (B) female.
Bar = 50 /tm.
embryos than at other times (Fig. 7). At 8-5 and 9-5 days the PGC population
in MMC embryos is fairly uniformly depleted and no embryo falls within the
normal range; at 11-5 days however, while many MMC embryos show severely
reduced PGC numbers some 35 % could be classified as normal, and thus fully
recovered.
No embryos were found to be without germ cells but 5 (33 %) were recorded
with less than 20 at 8-5 days. In some MMC 13-5-day male genital ridges there
were apparently germ-cell-free patches, suggesting incomplete or non-random
colonization of the gonad.
There was no difference observed between male and female embryos, either
140
P. P. L. TAM AND M. H. L. SNOW
Table 3. The numbers of primordial germ cells (PGCs) in 8-5- to 13-5-day mouse
embryos
Age (day p.c.)
Group
No. litters
No. embryos
Mean PGC No.
( ± 1 S.E.)
8-5
Normal
MMC
Normal
MMC
Normal
MMC
Normal
MMC
Normal
MMC
1
2
1
3
2
2
3
4
2
4
7
16
8
22
13
14
15
25
14
30
145±17
34±6
364 ±32
58 ±8
1012 ±69
611 ±52
2999 ±184
1595 ±200
25791 ±2276
13906 + 732
9-5
10-5
11-5
13-5
10 4 -
103
E 102
10
1 L
8-5
9-5
10-5
11-5 12-5
Age of embryos (days p. c.)
13-5
Fig. 5. The increase in number of PGCs in mouse embryos between 8-5 and
13-5 days p.c. • = Normal, O = MMC-treated.
normal or MMC-treated, that could reasonably account for the more severe
post-natal effect seen in males (Tables 1, 2). Table 4 shows PGC number in
embryos of 10-5 to 13-5 days, and Table 5 gives the gonadal volume in 13-5-day
embryos. (The 13-5-day embryos were sexed by gonad histology and younger
embryos from chromosome preparations made from fetal membranes. Some of
these preparations were inadequate for confident sexing and hence not all
embryos are included in Table 4). Although there is a clear difference between
Proliferation and migration of mouse primordial germ cells
141
100 -
75
50
25
J
8-5
L
9-5
10-5
11-5
12-5
Age (days/J.C. )
13-5
14-5
Fig. 6. The relative development of MMC-treated embryos with respect to presomitic mesoderm length ( • ) , size of newly formed somite (+), somite number (#),
axial length (A), foetal wet weight (O), and PGC number ( • ) .
8-5 d
•8.8.8.1
200
9-5 d
. O , o
500
10 5 d
•8«,8X.ot.I.
.
1500
11-5 d
• 8 . 8 .JlJ.ol.olJ,
4000
13-5 d
o
, o ,
40000
0
Number of PGCs
Fig. 7. The range in numbers of PGCs in embryos of 8 5 to 13-5 days. • = Normal,
O == MMC-treated, Note the large variation in number in 11 -5 day MMC-embryos.
142
P. P. L. TAM AND M. H. L. SNOW
Table 4. Comparison of PGC numbers between male and female mouse embryos
Number of PGCs
Age (day p.c.) Group
10-5
Normal
MMC
Normal
MMC
Normal
MMC
11-5
13-5
Male (n)
Female (n)
1074±120(4)
642 ±62 (8)
3316 ±279 (6)
1646 ±204 (10)
27123 ±3020 (10)
13056±937 (13)
977 ±163 (8)
569 ±93 (6)
2854 ±414 (2)
1538 ±223 (7)
22463 ±2230 (4)
14556±1074 (17)
Statistics ; Student /-test.
male vs. female
norma 1 vs. MMC
A
A
t
10-5 d
11-5
13-5
\
Normal
n.s.
n.s.
n.s.
MMC
n.s.
n.s.
n.s.
n.s., no
Male
t(10) = 3.6, P < 001
t(14) = 4-9, P < 0001
t(21) = 6-4, P < 0001
significant difference.
Female
t(8) = 2.4, P < 005
t(7, = 2-8, P < 005
t(l9) = 3-2, P < 001
Table 5. The size of the genital ridges of 13-5-day mouse embryos
Gonadal volume (x
Group
Male (± S.E. («))
Female (± S.E. («))
Normal
653 ±43 (10)
420 ±34 (4)
MMC
459 ±18 (13)
329±10 (17)
* Means of the average volume of the two gonads in each embryo.
Statistics; Student t-Test.
Normal S
MMC?
t(12)
tU9)
Normal $
= 3-2, P < 001
= 3-4, P < 001
MMC<?
t(2D = 4-5, P < 0001
t(28) = 6'6, P < 0001
the sexes with respect to gonadal volume, and male gonads surfer a greater size
reduction in response to MMC treatment, the magnitude of the difference
seems insufficient to account for, and difficult to relate to, the totally germ-cellfree testes found in sterile males.
Examination of seven post-natal mice up to 5 days of age gives no further
clue to the manner in which the 'empty' testes arise. All testes examined,
although small, showed no evidence of the empty seminiferous tubules observed
later.
PGC migration. Figure 8 illustrates the proportions of PGCs found in various
sites between 8-5 and 13-5 days and suggests migration is slightly retarded with
respect to time in MMC embryos. However, since the whole embryo is somewhat
Group
No. of
embryos
,
PS
ALYS
HGN
A
MCW
Distribution of PGCs: mean no (%)
GR
Total
PGC no.
1-10
Normal
0
115(83-8)
0
6
22 (16-2)
137
0
MMC
0
39 (78-6)
0
12
50
3 (5-3)
8 06-1)
11-20
Normal
0
0
261 (91-5)
21 (7-2)
3
4(1-3)
286
0
0
2 (3-7)
MMC
58 (961)
15
61
1 (0-2)
Normal
273 (671)
21-30
7
0
7(17)
122 (29-9)
407
5(1-3)
MMC
4(1-2)
0
47 (14-9)
266 (83-6)
8
318
1 (0-3)
11
Normal
0
83 (7-7)
69 (6-4)
934 (85-9)
0
31-3
1086
554 (890)
622
MMC
0
38 (61)
30 (4-9)
10
0
PS, primitive streak; ALYS , allantoic base and yolk-sac endoderm; HGN, hind-gut endoderm; MCW, mesentery and coelomic wall; GR, genital
ridge.
Somite no.
of embryos
Table 6. The location of primordial germ cells in mouse embryos at 1- to 36-somite stages, equivalent to 8-5-10-5 days p.c.
Hi
i
a'
§"
-t
<^
S"
5'
s
§
3
fa
Proi
144
P. P. L. TAM AND M. H. L. SNOW
100
8-5 d
n rk
0
100
9-5 d
CO
t 100
o
10-5 d
o
0
"100
0
100
n
11-5 d
i—k
13-5 d
.n.
Primitive Yolk sac Gut Mesentery Genital
streak allantois
ridges
Fig. 8. The migration of PGCs from the primitive streak to the genital ridge,
• = Normal, • = MMC-treated.
retarded it would seem more meaningful to assess PGC migration with respect
to developmental stage. Somite number can be used as an index of developmental status but may be misleading (Snow & Tarn, 1979; and in preparation).
Nevertheless in Table 6 the distribution of germ cells is given with respect to
somite number. Migration still appears retarded for early somite stages, but
then appears to accelerate such that in MMC embryos the PGCs seem further
along their migration path than controls in embryos of 21-30 somites. Beyond
10-5 days (33 somites) there is no discrepancy in somite numbers between
control and MMC embryos but the entry of PGCs into the genital ridge is
delayed in MMC embryos (Table 7).
DISCUSSION
The PGC numbers reported here for normal embryos are in very close
agreement with the figures given by Mintz & Russell (1957) in a study of 8- to
Proliferation and migration of mouse primordial germ cells 145
Table 7. The entry of primordial germ cells into genital ridges in normal and
MMC-treated embryos between 10-5 and 13-5 days p.c.
Mean no. ofPGCsf%)
Group
Normal
Age
(d)
105
Sex
6
9
11-5
<J
9
13 5
<J
9
MMC
105
<J
9
11-5
c?
9
13-5
6
9
No. of
embryos
4
8
6
2
10
4
8
6
10
7
13
17
Extragonadal
sites
1030 (95)
900 (92)
221 (7)
193 (7)
337(1)
332 (1)
611 (95)
542 (95)
641 (39)
604 (39)
120(1)
114(1)
Genital ridge
44(5)
77(8)
3096 (93)
2662 (93)
26785 (99)
22130 (99)
31(5)
27(5)
1005 (61)
934(61)
12936 (99)
14442 (99)
Total
1074
977
3317
2855
27122
22462
642
569
1646
1538
13056
14556
12-day-old embryos with a C37BL/6 genetic background. Their study did not
extend to full genital ridge colonization but the increase from 40 PGCs at
8 days to some 4000 at 12 days represents a population doubling time of around
14 h (compared to our 16 h) and would suggest that by 13-5 days their mice
should have about 24000 PGCs in their genital ridges.
It is clear that the reduced fertility in mice exposed to MMC during primitivestreak-stages of embryogenesis is the result of germ cell deficiency in the gonads.
In females the paucity of germ cells can be accounted for by a severe reduction
in primordial germ cells early in embryonic life which is not wholly compensated
for. In males the finding of sterile testes totally devoid of germinal tissue indicates
a secondary loss of germ cells since no embryo was seen without substantial
numbers of germ cells at the time of onset of gonadal differentiation. Even if it is
assumed that the empty testes are derived from those genital ridges containing
the fewest PGCs in 13-5-day embryos then a testis with some 30% of normal
numbers of germ cells would be expected. No ovary entirely devoid of germ cells
has been found so it would appear perhaps that in females a functional gonad
results from a similar severely depleted 13-5-day genital ridge although it
would perhaps be expected that such females would have a shorter reproductive
life than normal mice.
The mechanism of the secondary loss of germ cells in males is not known but
it is probably brought about by degeneration of the tissue rather than loss by
emigration from the testis or by differentiation of the entire population into
sperm which were then shed. Firstly although emigration of germ cells from the
testis tubule has been reported in the rabbit (Gould & Haddad, 1978) it is not
146
P. P. L. TAM AND M. H. L. SNOW
extensive and is probably rare. Secondly, the male PGCs are of proven mitotic
competence and it seems improbable that all the cells of the mitotic stem line
should embark upon terminal differentiation into sperm at an early age and
thus deplete the entire germ cell population.
It seemed likely that the loss would occur when mitotic proliferation resumed
after the gonocyte growth phase, since extensive degeneration of germ cells is
seen in the normal rat testis at this time (Roosen-Runge & Leik, 1968; Hilscher
et al. 1974). In the rat the atresia is maximal at 4-6 days and declines rapidly
thereafter (Beaumont & Mandl, 1962, 1963). Up to 50% of the gonocyte population may fail to resume mitosis and die (Clermont & Perey, 1957; Novi &
Saba, 1968). In the mouse there is no evidence of degeneration in early postnatal males (Snow & Tarn, unpublished observations; P. S. Burgoyne, personal
communication), but considerable atresia is seen in testes 1 or 2 days before birth
(A. McLaren, personal communication). The healthy appearance of the testes
in young MMC males suggests that they survive the resumption of mitosis and
that the secondary loss of germ cells occurs later than 7 days post partum.
The PGC population in MMC-treated embryos is only partially restored after
the initial depletion but other tissues and organs appear to recover to full size by
13-5-14-5 daysp.c. (Fig. 6; Snow & Tarn, 1979; Tarn, in preparation). The failure
to restore full numbers of PGCs is due to the fact that a raised proliferation rate
is only achieved between 9-5 and 10-5 daysp.c. rather than over the whole period
of development from 7-5 to 13-5 days, as happens with other organ systems This
fact has an important bearing on the assessment of the rate of migration of PGCs
in MMC-treated embryos. The results in Table 6 suggest a slightly retarded migration during early somite stages, more rapid passage through hindgut and the
mesentery, but delayed entry into the genital ridge. Since the period of maximum
proliferation of PGCs in MMC-treated embryos, 9-5 to 10-5 days or 22- to 32somite stage, coincides with the time the cells are in the mesentery, it seems more
likely that the increased proportion of PGCs in the mesentery at this time
(Fig. 8 and Table 6) is the result of a population increase by cell division rather
than immigration from the hindgut.
P. P. L. Tam was supported by a British Commonwealth Scholarship.
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