Proliferation and migration of primordialgerm 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
SUMMARYPrimitive-streak-stage mouse embryos were treated with Mitomycin C injected intra-
peritoneally into pregnant females at 6-75-7-0 days post coitum. The newborn mice developedpoorly and mortality was high during the suckling period. Many weaned survivors showedimpaired fertility and poor breeding performance. Histological examination revealed apaucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduc-tion of the primordial germ cell population in early embryonic life, which was not fullycompensated for during the compensatory growth phase of the Mitomycin C-treated embryo.Also contributing to such impaired fertility were retarded migration of the primordial germcells into the genital ridges, poor development of the foetal gonad and secondary loss of thegerm cells during gametogenesis in males.
INTRODUCTION
We have recently shown that a single intraperitoneal injection of 100-120/*gMitomycin C (MMC) into pregnant mice does very extensive damage toprimitive-streak-stage embryos, reducing cell number to about 15% of normalvalues, and resulting in severe developmental disturbance in the ensuing 48 hof embryogenesis. Subsequently, accelerated growth and morphogenesis restoresgross morphology to normal by mid-organogenesis stages 3-4 days later.Although embryonic mortality is low, post-natal development is poor andfertility among offspring surviving to breeding age is low (Snow & Tam, 1979).This report concerns the developmental events underlying the reduced fertilityfollowing MMC-treatment and describes the origin, proliferation and migrationof primordial germ cells, and the formation of the foetal gonads in normal andMMC-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 StephensonWay, 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 MitomycinC-treated pregnant females
No.litters
15
No.newborn
99
Birth
93
ViabilityNo. alive at
7d 14 d
78 59
28 d
36
No.weaned
29
Breeding performanceClass of mating
Sex No. No. mated Fertile Fertile and sterile Sterile only
<J 18 18 10 4 4§ 11 9 9 0 0
post coitum (p.c.) Embryos are in early- and mid-primitive-streak stages atthis time.
Initial observations were made on the offspring of such mice that survived tobreeding age. Each mouse was test-mated to a normal Q mouse of provenfertility for sufficient time to allow the production of several litters. Subsequentlythe animals were killed, both gonads removed and fixed in Bouin's fluid andexamined histologically.
The formation of gonads was studied in embryos between 8-5 day p.c. whenprimordial germ cells (PGCs) are observable in the developing hindgut, and13-5 days when colonisation of the genital ridge is complete. Embryos werefixed in cold 80 % ethanol, dehydrated in absolute ethanol, cleared in chloroformand embedded in a low melting-point (54 °C) wax. Serial sections were made at8 or 10 ju,m and stained with Fast Red TR salt to detect alkaline phosphataseaccording to the azo-dye coupling method of Gomori (Gabe, 1975). They weremounted in glycerine. Complete undamaged and properly stained serial sectionswere obtained from 57 normal embryos and 107 MMC-treated embryos, from24 litters.
PGCs were identified by the high content of alkaline phosphatase in theircytoplasm 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 cellsregistered in both of two adjacent sections, thus giving a better estimate of cellnumber. In 13-5-day embryos the number of PGCs in the genital ridges wascomputed from the size of the ridge (gonadal volume) which was derived frommeasurement of camera-lucida drawings and the number of PGCs per unittissue 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 onlySterile onlyBoth types
? FertileNo litter
No.
1044
92
Empty
0(4), 2,22, 71,29, 40,
seminiferous tubules (%)
4, 5, 18, 22, 30100, 10054,61
Normal follicles (%)24, 42, 53, 77, 86, 86, 100 (2)47,50
Mean
87346
7449
The number of seminiferous tubules (fertile and 'empty') and the number of follicles of allstages of development were scored in 4-5 good sections of the testis and ovary respectively.
RESULTS
Table 1 shows the viability of offspring from MMC-treated mice and thebreeding performance of successfully weaned young. Two females nevermated; one developed ataxia which probably impaired her mating behaviour.Histological analysis of gonads shows a reduction in gonad size and of thenumber of germ cells (Table 2, Figs. 1, 2), particularly in the sub-fertile andsterile males where seminiferous tubules completely devoid of germ cells andcontaining only Sertoli cells were found (Fig. 1 b, c). In two of the sterile malesthe testes were completely devoid of germ cells (Fig. 1 d). In the females noovary was found to be devoid of follicles, the smallest having about 24 % of thenormal number of oocytes. There are more atretic follicles in MMC-treatedmice.
Embryonic development
Figure 3 illustrates alkaline-phosphatase-positive PGCs in various sites intheir migration pathway. In Fig. 3 a PGCs are at the posterior end of theprimitive streak at the base of the allantois. This example is from an 8-5-dayMMC embryo which is retarded in development. In normal embryos thisdevelopmental 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 9-5days in the hindgut and just entering the mesentery (Fig. 3 c) and enter thegenital ridges at 10-5-11-5 days (Fig. 3d). The genital ridges are fully colonizedby 13-5 days (Fig. 4).
PGC number. Table 3 and Fig. 5 show the numbers of PGCs in normal andMMC embryos according to gestational age. The lower PGC numbers in MMCembryos do not simply reflect the retardation in overall development. Figure 6illustrates graphically the relative development of the MMC embryos withrespect 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 emptyseminiferous 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 para-meters suggests they are under independent control (see later, and Snow, Tarn& McLaren, 1981).
The PGC population doubling time in normal embryos is fairly uniform atabout 16 h between 8-5 and 13-5 days. A similar value is found in MMCembryos between 10-5 and 13-5 days, but at the beginning of their migrationthese PGCs divide very slowly (population doubling time 31 h), and between9-5 and 10-5 days, very rapidly (doubling time 7 h) (Fig. 5). The period ofrapid proliferation coincides with the period of maximum compensatorygrowth for other parts of the embryo but PGC number does not recover tonormal in treated embryos and when genital ridge differentiation commencesthe gonads have about 50 % as many PGCs as normal (at 9-2- days there wereabout 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 inthe 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 between8-5 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 dorsalmesentery. 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 populationin MMC embryos is fairly uniformly depleted and no embryo falls within thenormal range; at 11-5 days however, while many MMC embryos show severelyreduced PGC numbers some 35 % could be classified as normal, and thus fullyrecovered.
No embryos were found to be without germ cells but 5 (33 %) were recordedwith less than 20 at 8-5 days. In some MMC 13-5-day male genital ridges therewere apparently germ-cell-free patches, suggesting incomplete or non-randomcolonization 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 mouseembryos
Age (day p.c.)
8-5
9-5
10-5
11-5
13-5
Group
NormalMMC
NormalMMC
NormalMMC
NormalMMC
NormalMMC
No. litters
12
13
22
34
24
No. embryos
716
822
1314
1525
1430
Mean PGC No.(±1 S.E.)
145±1734±6
364 ±3258 ±8
1012 ±69611 ±52
2999 ±1841595 ±200
25791 ±227613906 + 732
104 -
103
E 102
10
1 L8-5 13-59-5 10-5 11-5 12-5
Age of embryos (days p. c.)
Fig. 5. The increase in number of PGCs in mouse embryos between 8-5 and13-5 days p.c. • = Normal, O = MMC-treated.
normal or MMC-treated, that could reasonably account for the more severepost-natal effect seen in males (Tables 1, 2). Table 4 shows PGC number inembryos of 10-5 to 13-5 days, and Table 5 gives the gonadal volume in 13-5-dayembryos. (The 13-5-day embryos were sexed by gonad histology and youngerembryos from chromosome preparations made from fetal membranes. Some ofthese preparations were inadequate for confident sexing and hence not allembryos 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 L8-5 9-5 10-5 11-5 12-5 13-5 14-5
Age (days/J.C. )
Fig. 6. The relative development of MMC-treated embryos with respect to pre-somitic 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 . 1200
9-5 d
. O , o500
10 5 d
• 8 « , 8 X . o t . I . .1500
11-5 d
• 8 . 8 .JlJ.ol.olJ,4000
13-5 d
0
o, o ,
40000Number 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
Statistics
10-5 d11-513-5
Age (day
10-5
11-5
13-5
; Student
p.c.) Group
NormalMMCNormalMMCNormalMMC
/-test.male vs. female
At \
Normal MMCn.s.n.s.n.s.
n.s.n.s.n.s.
Male (n)
1074±120(4)642 ±62 (8)
3316 ±279 (6)1646 ±204 (10)
27123 ±3020 (10)13056±937 (13)
norma
Malet(10) = 3.6, P < 001t(14) = 4-9, P < 0001t(21) = 6-4, P < 0001
Female (n)
977 ±163 (8)569 ±93 (6)
2854 ±414 (2)1538 ±223 (7)
22463 ±2230 (4)14556±1074 (17)
1 vs. MMCA
Femalet(8) = 2.4, P < 005t(7, = 2-8, P < 005t(l9) = 3-2, P < 001
n.s., no significant difference.
Table 5. The size of the genital ridges of 13-5-day mouse embryos
Gonadal volume (x
Group Male (± S.E. («)) Female (± S.E. («))
NormalMMC
653 ±43 (10)459 ±18 (13)
* Means of the average volume of the two gonads
Statistics; Student t-Test.
Normal SMMC?
Normal $t(12) = 3-2, P < 001tU9) = 3-4, P < 001
420 ±34 (4)329±10 (17)
in each embryo.
MMC<?t(2D = 4-5, P <t(28) = 6'6, P <
00010001
the sexes with respect to gonadal volume, and male gonads surfer a greater sizereduction in response to MMC treatment, the magnitude of the differenceseems insufficient to account for, and difficult to relate to, the totally germ-cell-free testes found in sterile males.
Examination of seven post-natal mice up to 5 days of age gives no furtherclue to the manner in which the 'empty' testes arise. All testes examined,although small, showed no evidence of the empty seminiferous tubules observedlater.
PGC migration. Figure 8 illustrates the proportions of PGCs found in varioussites between 8-5 and 13-5 days and suggests migration is slightly retarded withrespect to time in MMC embryos. However, since the whole embryo is somewhat
Tab
le 6
. T
he lo
cati
on o
f pri
mor
dial
ger
m c
ells
Som
ite
no
.of
em
bryo
s
1-10
11-2
0
21-3
0
31-3
PS
, pr
imit
ive
stre
ak;
AL
YS
ridg
e.
Gro
up
No
rmal
MM
C
No
rmal
MM
C
No
rmal
MM
C
No
rmal
MM
C
No.
of
,em
bryo
s
6 12 3 15 7 8 11 10
in m
ouse
PS
0 3 (5
-3)
0 0 0 0 0 0
embr
yos
at
Dis
trib
utio
n
AL
YS
22 (
16-2
)8
06
-1)
4(1
-3)
1 (0
-2)
5(1-
3)1
(0-3
)
0 0
1- t
o 36
-som
ite
stag
es,
of P
GC
s: m
ean
no (
%)
A
HG
N
115(
83-8
)39
(78
-6)
261
(91-
5)58
(96
1)
273
(671
)47
(14
-9)
69 (
6-4)
30 (
4-9)
MC
W
0 0 21 (
7-2)
2 (3
-7)
122
(29-
9)26
6 (8
3-6)
934
(85-
9)55
4 (8
90
)
equi
vale
nt
GR
0 0 0 0
7(1
7)
4(1-
2)
83 (
7-7)
38 (
61
)
to 8
-5-1
0-5
days
p.c
.
Tot
alP
GC
no.
137 50 286 61 407
318
1086 622
, al
lant
oic
base
and
yol
k-sa
c en
dode
rm;
HG
N,
hind
-gut
end
oder
m;
MC
W,
mes
ente
ry a
nd c
oelo
mic
wal
l; G
R,
geni
tal
Proi f a 5' s § 3 S" <̂ -t §" a' i Hi
144 P. P. L. TAM AND M. H. L. SNOW
100
0100
CO
t 100o
o 0" 1 0 0
0100
8-5 d
n rk9-5 d
10-5 d
n11-5 d
i—k. n .13-5 d
Primitive Yolk sac Gut Mesentery Genitalstreak 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 respectto developmental stage. Somite number can be used as an index of develop-mental status but may be misleading (Snow & Tarn, 1979; and in preparation).Nevertheless in Table 6 the distribution of germ cells is given with respect tosomite number. Migration still appears retarded for early somite stages, butthen appears to accelerate such that in MMC embryos the PGCs seem furtheralong their migration path than controls in embryos of 21-30 somites. Beyond10-5 days (33 somites) there is no discrepancy in somite numbers betweencontrol and MMC embryos but the entry of PGCs into the genital ridge isdelayed in MMC embryos (Table 7).
DISCUSSION
The PGC numbers reported here for normal embryos are in very closeagreement 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 andMMC-treated embryos between 10-5 and 13-5 days p.c.
12-day-old embryos with a C37BL/6 genetic background. Their study did notextend to full genital ridge colonization but the increase from 40 PGCs at8 days to some 4000 at 12 days represents a population doubling time of around14 h (compared to our 16 h) and would suggest that by 13-5 days their miceshould have about 24000 PGCs in their genital ridges.
It is clear that the reduced fertility in mice exposed to MMC during primitive-streak-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 reductionin primordial germ cells early in embryonic life which is not wholly compensatedfor. In males the finding of sterile testes totally devoid of germinal tissue indicatesa secondary loss of germ cells since no embryo was seen without substantialnumbers of germ cells at the time of onset of gonadal differentiation. Even if it isassumed that the empty testes are derived from those genital ridges containingthe fewest PGCs in 13-5-day embryos then a testis with some 30% of normalnumbers of germ cells would be expected. No ovary entirely devoid of germ cellshas been found so it would appear perhaps that in females a functional gonadresults from a similar severely depleted 13-5-day genital ridge although itwould perhaps be expected that such females would have a shorter reproductivelife than normal mice.
The mechanism of the secondary loss of germ cells in males is not known butit is probably brought about by degeneration of the tissue rather than loss byemigration from the testis or by differentiation of the entire population intosperm which were then shed. Firstly although emigration of germ cells from thetestis 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 mitoticcompetence and it seems improbable that all the cells of the mitotic stem lineshould embark upon terminal differentiation into sperm at an early age andthus deplete the entire germ cell population.
It seemed likely that the loss would occur when mitotic proliferation resumedafter the gonocyte growth phase, since extensive degeneration of germ cells isseen in the normal rat testis at this time (Roosen-Runge & Leik, 1968; Hilscheret al. 1974). In the rat the atresia is maximal at 4-6 days and declines rapidlythereafter (Beaumont & Mandl, 1962, 1963). Up to 50% of the gonocyte popu-lation may fail to resume mitosis and die (Clermont & Perey, 1957; Novi &Saba, 1968). In the mouse there is no evidence of degeneration in early post-natal males (Snow & Tarn, unpublished observations; P. S. Burgoyne, personalcommunication), but considerable atresia is seen in testes 1 or 2 days before birth(A. McLaren, personal communication). The healthy appearance of the testesin young MMC males suggests that they survive the resumption of mitosis andthat the secondary loss of germ cells occurs later than 7 days post partum.
The PGC population in MMC-treated embryos is only partially restored afterthe initial depletion but other tissues and organs appear to recover to full size by13-5-14-5 daysp.c. (Fig. 6; Snow & Tarn, 1979; Tarn, in preparation). The failureto restore full numbers of PGCs is due to the fact that a raised proliferation rateis only achieved between 9-5 and 10-5 daysp.c. rather than over the whole periodof development from 7-5 to 13-5 days, as happens with other organ systems Thisfact has an important bearing on the assessment of the rate of migration of PGCsin MMC-treated embryos. The results in Table 6 suggest a slightly retarded mig-ration during early somite stages, more rapid passage through hindgut and themesentery, but delayed entry into the genital ridge. Since the period of maximumproliferation of PGCs in MMC-treated embryos, 9-5 to 10-5 days or 22- to 32-somite stage, coincides with the time the cells are in the mesentery, it seems morelikely 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 ratherthan immigration from the hindgut.
P. P. L. Tam was supported by a British Commonwealth Scholarship.
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{Received 17 September 1980, revised 1 March 1981)
