/. Embryol. exp. Morpli. Vol. 59, pp. 103-111, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Polyploidization of extraembryonic tissues during
mouse embryogenesis
By E. B. ILGREN 1
From the Botany School, Oxford and Sir William Dunn School of
Pathology, Oxford
SUMMARY
It has recently been shown that visceral yolk-sac endoderm is derived from the primitive
endoderm of the 4-5-day mouse blastocyst (Gardner & Papaioannou, 1975; Gardner &
Rossant, 1979). During development, primitive endodermal cells acquire nuclei with more
than four times the haploid amount of DNA. The finding of metaphases with multiple sets of
chromosomes suggests that the diploid precursors of such endodermal giant cells become
truly polyploid. Amniotic cells also contain giant nuclei but the mechanism by which these
arise is uncertain. The giant-cell transformation therefore appears to be a general feature of
mouse extraembryonic development rather than a phenomenon! restricted solely to trophoblast. The basis and significance of these findings are discussed in relation to the development
of other extraembryonic membranes both of plant and animal origin.
INTRODUCTION
The growth of mammalian extraembryonic membranes involves the proliferation of trophoblast as well as the development of a yolk sac and an amnion
(Amoroso, 1952; Gardner, 1975). Although the lineage (Gardner & Papaioannou
1975; Gardner & Rossant, 1979), cytology (Everett, 1935; Wislocki, Deane &
Dempsey, 1946; Klinger & Schwarzacher, 1958) and biochemistry (Adamson
& Gardner, 1979; Graham, 1977) of extraembryonic tissues have been studied,
giant nuclei have not been reported in any normal, mouse foetal membrane
other than trophoblast (Sherman, McLaren & Walker, 1972). This paper,
however, documents the presence of nuclei containing more than four times the
haploid amount of DNA (4c) within both the visceral yolk sac (VYS) and the
amnion of the mouse.
MATERIALS AND METHODS
Embryonic tissues were dissected from the pregnant uteri of CFLP mice
(Anglia Laboratory Animals, Ltd.) on the 8th, 9th, 16th, and 17th days of
gestation in PB-1 medium (Whittingham & Wales, 1969) containing 10 % foetal
calf serum (Flow). Mating was assumed to occur at the midpoint of the dark
1
Author's address: Sir William Dunn School of Pathology, South Parks Road, Oxford,
OXI 3RE, U.K.
104
E. B. ILGREN
period and was ascertained by the presence of a copulation plug. The day a plug
was found was called day ' 0 ' of pregnancy. 15^-day VYS endoderm and its
presumptive precursors (Gardner & Rossant, 1979) 7-^-day and 8^-day visceral
extraembryonic endoderm (VEE), were removed from embryos using the method
of Levak-Svajger, Svajger & Skreb (1969).
For cytophotometry, separated 8^-day as well as 15^-day VYS endoderm and
mesoderm and also 16-^-day amnion were spread onto acid-cleaned slides, dried,
and prepared according to a standardized Feulgen technique. 7-^-day VEE was
dissociated in TVP enzyme solution (Bernstein, Hooper, Grandchamps &
Ephrussi, 1973) on glass slides, dried, and then fixed and stained (Ugren in
preparation). At the time of preparation, liver smears were added to slides to
provide standard, known DNA contents for analysis. All DNA measurements
were made with a Vicker's M.85 microdensitometer (A = 565 nm).
Cytological analysis was performed on isolated tissue layers cultured in the
alpha-modification of Eagle's medium (Flow) containing 10 % foetal calf serum
(Flow) in the presence of 0-04/*g/ml of colcemid (Grand Island Biological) for
2 h. Tissues were then exposed to 1 % sodium citrate (pH 8-45, 26 °C, 22 min)
and prepared for chromosome counts using a method developed by Evans,
Burtenshawe & Ford (1972). The level of binucleation within the VYS endoderm
was determined on tissues rinsed in Ca2± and Mg 2± free Hanks' balanced salt
solution (HBSS) dissociated in a mixture of 0-1 % collagenase (Worthington,
N.J.), 0-1 % trypsin (Difco, 1:250), and 10 % chick serum (Flow) made up in
HBSS (pH 7-6, 37 °C, 10 min), dried for 15 min on acid-cleaned slides, and then
stained with a 10 % Giemsa- and 1 % May-Grunwald solution for 3 min.
RESULTS
Histograms were constructed from analyses of 7^-day VEE (twelve embryos,
two litters), 15^-day VYS (four embryos, two litters), and 16^-day amnion (four
embryos, two litters) nuclei. In addition, VYS endoderm nuclei from six 8£-day
embryos were also scanned.
Cytophotometric measurements show 7^-day VEE nuclei have only diploid
(2-4c) DNA values (see Fig. Id). By 8£ days post coitum (p.c), occasional
(< 1 %) nuclei with nuclear DNA contents greater than 4c appear within VYS
endoderm cells (five out of six embryos). At 15^-days (p.c), the nuclear DNA
contents of 5-20 % of VYS endoderm cells are greater than 4c (see Fig. 1 b).
Although it was obvious from scanning a considerable number of these 15^-day
VYS endoderm nuclei that the distribution of those nuclei with higher DNA
contents was non-uniform, it could not be determined, due to the extensive
ingrowth of mesoderm and the large numbers of cells present, whether these
nuclei were more frequent about vessels or more common in certain areas of the
VYS than in other regions. In contrast to 15^-day VYS mesoderm which did not
contain nuclei with DNA contents greater than 4c (see Fig. lc) or metaphases
Polyploidization during mouse embryogenesis
105
ifl)
7 day visceral extraembryonic
Endoderm
i
9
i
i
15
i
i
21
i
i
27
i
i
33
(b)
15-5 day visceral yolk sac
Endoderm
32C
-i—r
64C
128C
r i—r-*i—i—i—i-
40 >< 60
120
180
15-5 day visceral yolk sac
Mesoderm
1
6
J
1
12
1
r
39
1
1
1
1
18
24
Arbitrary DNA-index units
1
30
1
240
(c)
1
36
Fig. 1. DNA contents of nuclei within (a) 7^-day visceral extraembryonic endoderm
(n = 105), (b) 15|-day visceral yolk-sac endoderm (n = 672), and (c) 15^-day
visceral yolk-sac mesoderm (n = 233). Columns in black refer to nuclei which fall
cleanly into a polyploid resting peak.
with more than 40 chromosomes, between 1-2 % of all VYS endoderm metaphases contained 80 (see Fig. 2 a) and 160 chromosomes (see Fig. 2 c) with
corresponding DNA values (see Fig. 3). To determine whether metaphases with
large numbers of chromosomes could have been formed by overlapping mitoses,
parallel chromsome preparations of 7^-day embryonic ectoderm were made. In
all cases, ectoderm controls, prepared at a density comparable to VYS endoderm
(8000-10000 nuclei per slide) and having a mitotic index (the number of metaphases per 100 nuclei counted) greater than VYS endoderm (10-9 % compared
106
E. B. I L G R E N
Fig. 2. (a) Late metaphase - early anaphase with 160 chromatids (80 chromosomes)
in 15i-day visceral yolk-sac endoderm. Toluidine blue (x 1250). (b) Binucleate cell
within 15^-day visceral yolk-sac endoderm. Giemsa-May-Grunwald (XI250). (c)
Metaphase with 160 chromosomes in 15^-day visceral yolk-sac endoderm. Toludine
blue (x 1250).
107
Polyploidization during mouse embryogenesis
4C
8C
16C
8
(a)
6 -
r
4 -
J
2 _
0
i
i
n
i
i
i
4C
-
i
i
i
I
1
1
8C
8
>f r.uclei
f
-
1n
16C
(b)
o 4 _
"1
12
:
0
n
••
•
2C
1
I
4C
1
l
I
l
1
1
1
i
nII
8C
8
6 _
4
2
n
I
:
I
n
200
300
r
I
I
100
I
(c)
I
400
Arbitrary DNA-index units
1
500
600
Fig. 3. Distribution of DNA values in Feulgen-stained mitotic figures of 15^-d.ay
VYS endoderm (51 nuclei); (a) prophase, (b) metaphase, and (c) one-half of each
anaphase nucleus.
with 4 %), never contained 160 chromosome metaphases. Furthermore, 15^-day
VYS endoderm contained up to 6 % binucleate cells (see Fig. 2b) as well as
occasional multinucleates whereas cells with two or more nuclei were rarely
found within 15^-day VYS mesoderm.
Finally, from 1-2 % of all 16^-day amnion nuclei contained more than 4c
DNA contents and up to 2 % of all amnion cells of this same age were binucleate.
DISCUSSION
These observations show that nuclei with greater than 4c DNA contents exist
within both the derivatives of the primitive endoderm and, to a lesser extent, the
amnion. Both the nuclei with more than 4c DNA contents as well as the multi-
108
E. B. ILGREN
2
4 T
400
Arbitrary DNA-index units
Fig. 4. DNA contents of nuclei within 16^-day amnion (n = 208).
nucleate cells within the VYS endoderm could have been formed by cell fusion.
No evidence of GP1-1AB heteropolymer was ever found, however, in 15-^-day
VYS endoderm chimaeric for the two electrophoretic variants of the enzyme
glucose phosphate isomerase (GPI) and derived from injections of 3^-day inner
cell mass cells and 4-^-day primitive endoderm cells into blastocysts (Gardner &
Rossant, 1979). Cell fusion, if it does occur in VYS endoderm, is therefore
probably a rare event especially since the percentage of VYS endoderm nuclei
with DNA contents greater than 4c (5-20 %) is considerably above the sensitivity
of the GPI assay (1-3 %) (Chapman, Ansell & McLaren, 1972; Chapman,
Whitten & Ruddle, 1971). The presence of nuclei with multiple sets of chromosomes demonstrates that at least some primitive endoderm cells are truly polyploid. In addition, the finding of cells with two, and occasionally more than two,
nuclei suggests that one mechanism by which primitive endodermal polyploidization occurs is by acytokinesis either followed by continuous rounds of
DNA replication and/or occasionally by mitosis, a process similar to that found
in the liver (Brodskii & Uryvaeva, 1977). The failure to find polytenic structures
in endoderm such as those reported to occur in giant trophoblastic nuclei
(Snow & Ansell, 1974) is also consistent with this hypothesis. Two findings
argue against the idea that the 160 chromosome metaphases found in the VYS
were actually overlapping nuclei. First, single metaphases with multiples of the
diploid numbers of chromosomes were never found in preparations of rapidly
dividing embryonic ectoderm even though this tissue was prepared in a manner
similar to, and at a density comparable to, the 15^-day VYS endoderm. Furthermore, the chromosomes within large endoderm metaphases were morphologically uniform in contrast to the variable appearance of those found within
superimposed mitoses.
Since only 1-2 % of the nuclei within 16-^-day amnion cells had nuclear DNA
contents greater than 4c, a figure approaching the maximum sensitivity of the
GPI assay, and amniotic metaphases never had as many as 160 chromosomes,
Polyploidization during mouse embryogenesis
109
the mechanism by which this tissue replicates its DNA is less certain than
endoderm. In addition, the overall number of binucleate cells in both primitive
endoderm and amnion is usually rather low (6 % and 2 % respectively). For this
reason, polyploidy may not always be associated with binucleation and DNA
replication may proceed in the absence of either nuclear or cytoplasmic division.
Also, both amnion and endoderm nuclei were always regular in shape which
would suggest that polyploidization had not occurred via restitution cycles
which usually give rise to highly misshapen nuclei (Paulus, 1968). It is also
unlikely that contamination by other polyploid tissues such as foetal liver
(Ilgren, Evans & Burtenshaw, in preparation), bladder epithelium (Walker, 1958),
trophoblast, or decidua (Ansell, Barlow & McLaren, 1974) could account for
these findings since 15^-day VYS and 16^-day amnion were always dissected
well away from the foetus and their placental insertion.
Regional patterns of polyploidization can potentially offer clues to the manner
in which polyploid nuclei arise (Nagl, 1978). For example, polyploid nuclei in
mouse liver are frequently found near incoming vessels (Carriere, 1969) and
mouse secondary trophoblastic giant cells commonly attain their maximum size
at the abembryonic pole (Chew & Sherman, 1975). Even though the precise
pattern of polyploidy within VYS endoderm could not be determined at this
time, further study may show the regional variation of endoderm nuclei to be
similar to that found in liver or trophoblast.
The functional importance of polyploid nuclei within mammalian extraembryonic tissues is poorly understood. Polyploid yolk sac and trophoblastic
cells display an increase in total surface area which may, in turn, enhance their
ability to absorb as well as phagocytose nutrients which can then be transferred
to the developing embryo (Beck & Lloyd, 1966; Faulk & Gailbraith, 1979). A
similar situation may be seen in some species of higher plants. For instance,
suspensor tissue may not only become polyploid but, in a manner similar to
trophoblast, also anchor embryonic to maternal tissues, provide channels for
nutrients, and synthesize hormones (reviewed by Nagl, 1978). Suspensor may,
in some instances, even' aggressively penetrate the vascular strands of the ovule'
(from Nagl, 1978, but also see Crete, 1963 on the Crassulaceae) in a manner
similar to the chorioallantoic invasion of maternal decidua (Amoroso, 1952). In
those species of angiosperm 'where the suspensor is either absent or largely
diploid, the endosperm is often highly developed containing polyploid nuclei
throughout or within special haustoria' (from Nagl, 1978; also see Johansen,
1950; Davis, 1966; and Chopra & Sachar, 1963 on certain Euphorbiaceae,
Cucurbitaceae, Balsaminaceae, and Scrophulariceae species). Such endosperm
haustoria may also acquire a suspensor-like capacity to invade the adjacent
integumentary and/or nucellar tissues of the ovule (Chopra & Sachar, 1963). To
a certain extent this situation resembles that found in mammalian, extraembryonic membranes in which significant numbers of polyploid endoderm nuclei
first appear after secondary trophoblastic giant cells have degenerated either^by
8
EMB
59
110
E. B. ILGREN
the end of the second week of normal mouse development (Amoroso, 1952) or
even earlier in certain mutant strains of mice (Vankin & Caspari, 1979) and in
other rodents such as the guinea-pig (Ilgren, in preparation). Conversely, in
those angiosperms with massive suspensors, e.g. the Trapaceae and the Orchidaceae, the endosperm may be absent although, less often it may actually be
haustorial, e.g. in certain Papilionaceae and Rosaceae (Johansen, 1950; Chopra
& Sachar, 1963; Crete, 1963; Davis, 1966). Furthermore, endosperm, in a
manner similar to yolk sac endoderm, may also influence embryonic development, possess bi- and multinucleate cells, and contain nuclei with multiple sets
of chromosomes distributed in a non-random manner throughout the plant
embryo sac (D'Amato, 1952; Chopra & Sachar, 1963).
It would therefore appear that new patterns of DNA synthesis may be
initiated within the extraembryonic tissues of certain animal or plant species,
e.g. primitive endoderm or endosperm, following the dysfunction and/or loss
of a nearby supportive tissue layer such as trophoblast or suspensor.
I should like to thank Dr A. J. Copp, Dr P. W. Barlow, Prof. R. L. Gardner, Dr Anne
McLaren, Prof. Armin C. Braun, Dr C. F. Graham, Dr E. P. Evans, Dr M. Burtenshawe.
Dr F. A. L. Clowes, and my laboratory colleagues for comments, criticisms, and support.
This work was initiated and completed whilst E.B.I, was a fellow of the International Agency
for Research on Cancer, the World Health Organization IACR/R.882, the American Cancer
Society SPF-14, and the National Institutes of Health, the National Institute for Child Health
and Human Development IF32 HD 05592-01X1.1 am also most grateful for the kindness and
generous support given by the Department of Zoology where this work was begun.
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(Received 21 January 1980, revised 20 March 1980)