/ . Embryol. exp. Morph. Vol. 60, pp. 405-418, 1980
Printed in Great Britain © Company of Biologists Limited 1980
405
The control of trophoblastic growth
in the guinea pig
By E. B. ILGREN 1
From the Sir William Dunn School of Pathology and the Botany School,
University of Oxford
SUMMARY
The growth of mouse trophectoderm depends upon the presence of the inner cell mass.
Whether this applies to other species of mammals is not known. To investigate this problem,
the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast
resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig
trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig
blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly
equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm
and the other containing ICM tissue as well. Subsequently, the growth of these mural and
polar fragments was followed in vitro since numerous technical difficulties make an in vivo
analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural
trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig
polar fragments formed egg-cylinder-like structures. The latter contained regions structurally
similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and
extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal
growth may occur in a manner similar to the mouse and thus be under ICM control.
INTRODUCTION
There is evidence to suggest that an interaction occurs between the inner cell
mass (ICM) and the trophectoderm of the mouse blastocyst. Microsurgicallyisolated trophectodermal vesicles from 3-5-day mouse embryos not only fail to
proliferate but also form giant cells when transferred to the uteri of pseudopregnant hosts (Gardner, 1972). If, however, ICMs are inserted into such
vesicles prior to transfer, normal trophectodermal proliferation occurs (Gardner,
Papaioannou & Barton, 1973). Subsequent analyses have shown that the rate
of cellular proliferation within the trophectoderm overlying the ICM is higher
than that found in the mural trophectoderm away from the inner cell mass
(Copp, 1978). Together, these studies demonstrate that the mouse inner cell
mass is not only able to promote trophectodermal proliferation but can also
suppress the giant-cell transformation. Whether these findings are applicable to
the early development of other mammals is not known. The object of the present
study is to explore this problem in another species, namely the guinea pig. This
1
Author's address: Sir William Dunn School of Pathology, Oxford University, South
Parks Road, Oxford, 0X1 3RJB, U.K.
406
E. B. ILGREN
particular species was chosen for two reasons. First, placentation in the guinea
pig, in contrast to the mouse, occurs in a manner similar to that in man. Thus
implantation is interstitial and trophoblastic growth is highly invasive and syncitial (Boyd & Hamilton, 1970; Amoroso, 1952). Second, observations suggest
that the growth of guinea-pig trophectoderm may not be under ICM control
because cells accumulate at the abembryonic pole both in vivo (Sansom & Hill,
1931; Blandau, 1959; Enders & Schlafke, 1965) and in vitro (Blandau & Rumery,
1957; Amoroso, 1959) thereby forming a multilayered, partially syncitial
structure called the attachment cone. In addition, the proliferation of guinea-pig
trophectoderm has been observed to continue in culture under conditions in
which degeneration of the IGM was claimed to have occurred (Blandau, 1971).
However, an accumulation of cells in the abembryonic part of the blastocyst
does not prove that proliferation took place there especially since mouse mural
trophectoderm has been shown to grow by cell recruitment rather than cellular
division (Copp, 1979).
Furthermore, the observed 'ICMrindependent' proliferation of guinea-pig
trophectoderm (Blandau, 1971) could have been due to the presence of unrecognized ICM derivatives which either continued to proliferate or, if quiescent,
still retained the ability to promote trophectodermal growth. Therefore, the
initial aim of the present study was to cut the guinea-pig blastocyst so as to
produce two trophectodermal fragments of rougly equivalent size, one being
composed of pure trophectoderm whilst the other contained ICM tissue as well.
Thereafter, the subsequent growth of each fragment was followed in vitro.
However, without cell markers it is not possible to discriminate the derivatives
of polar trophectoderm from those of the inner cell mass making it somewhat
difficult to interpret this type of study critically. Still, the guinea-pig egg cylinder
is known to contain, by homology with the mouse, two presumptive polar
trophectodermal derivatives, namely extraembryonic ectoderm and an ectoplacental giant-cell region (Sansom & Hill, 1931; Kaufman &Davidoff, 1977).
Reconstitution (Gardner & Papaioannou, 1975) and injection (Rossant, Gardner
& Alexandre, 1978) analyses of early mouse development show definitively that
these tissues come from the polar trophectoderm. Unfortunately, in the guinea
pig, such experimental studies are impracticable due to the lack of readily
available enzyme polymorphisms, the low number of embryos per litter, and
the non-expansive behaviour of trophectodermal vesicles. Nevertheless, the
trophectodermal origin of extraembryonic ectoderm and the ectoplacental cone
is also supported by the results of in vitro experiments in which tissues were
isolated from the mouse egg cylinder (Rossant & Offer, 1977). Thus, it may be
particularly instructive to isolate these same tissue layers from the guinea-pig
egg cylinder and compare their nuclear DNA contents, mitotic indices, and cell
numbers with those of the mouse both before and after growth in vitro. Since
only blastomere-derived (Sherman, 1975) and thymidine-induced (Snow, 1973)
trophectodermal vesicles have been studied previously in culture, the in vitro
Trophoblastic growth in the guinea pig
407
development of microsurgically sectioned mouse blastocysts has also been
followed in the present study. The findings obtained suggest that guinea-pig,
pre- and post-implantation, trophoblastic tissues grow in vitro in a manner
similar to those of the mouse. However, until reconstitution experiments are
carried out with the guinea-pig blastocyst in vivo these findings will remain
suggestive.
MATERIALS AND METHODS
(1) Animals. All animals were maintained on a regime of 12 h light: 12 h
dark. For mice, ovulation was assumed to occur at the midpoint of the dark
period (1.00 a.m.). The post-coital ages of mouse embryos were then estimated
from the time of assumed ovulation (Time ' 0 ' of pregnancy). For guinea pigs,
ovulation was assumed to occur 10 h after the start of oestrous (Ediger, 1976),
but the age of embryos was taken from the actual time of mating. In either
species, oestrous females were placed with males and mating ascertained by the
presence of a copulation plug.
(2) Recovery of embryos and dissection of tissues. All embryos were recovered
from uteri dissected free of fat and debris in PBl medium (Whittingham &
Wales, 1969) plus 10% foetal calf serum. In all fifty-one 5-5-day blastocysts,
twelve 10-5-day primitive-streak-stage egg cylinders, and four 17-5-day neuraltube-closure-stage embryos were recovered from the uteri of 59, random-bred,
guinea pigs (Nuffield Inst. Med. Res., Oxford) killed with ether. Ninety-five
3-5-day blastocysts, 44 7-5-day primitive-streak-stage egg cylinders, and four
neural-tube-closure-stage embryos were recovered from the uteri of 22 randombred, CFLP mice (Anglia Lab.) killed by cervical dislocation.
For the recovery of blastocysts, the methods described by Copp (1978) and
Blandau (1971) were used. Immediately after recovery, blastocysts were transferred to pre-equilibrated tissue-culture medium and then microsurgically
sectioned no more than 2 h later. Microsurgery was carried out using the
methods of Gardner (1972). Thus, blastocysts were initially held at the abembryonic pole by means of a suction pipette. They were then cut equitorially with
siliconized, glass microneedles parallel to the surface of the inner cell mass so
that each 'mural' and 'polar' fragment contained roughly equal numbers of
trophectodermal cells. Both fragments isolated from the same embryo were
then grown as parallel cultures.
All egg cylinders and later-stage embryos were dissected in PBl medium plus
serum using glass microneedles made as described by Diacumakos (1973).
Seven and one-half-day mouse extraembryonic ecoderm was isolated as described
by Rossant & Offer (1977). Guinea-pig extraembryonic ectoderm was also
removed from the embryo using 'three cuts' (see Fig. 1). Endoderm was then
stripped away from the extraembryonic ectoderm using microneedle tips.
(3) Tissue culture. Following microdissection, all pre- and postimplantation
tissues were grown in the alpha-modification of Eagle's medium (Flow) with
408
E. B. ILGREN
EPGC
Ex
EPC
ExC
Fig. 1. Primitive streak stage embryos of the guinea pig and the mouse. Schematic
diagrams depict (a) 7-5-day mouse (modified from Snell & Stevens, 1966) and (b)
10-5-day guinea pig (compare with Fig. 1D, Ptyler & Strasser (1925) and Sansom &
Hill (1931) Fig. 33) egg cylinders. Three-cut (
) dissection method used to isolate
trophectoderm-derived ( • ) extraembryonic ectodermal, (Ex) primitive endodermal
(@), and embryonic ectodermal (M) tissues. Primitive mesodermal ('m') cells
partially line the guinea-pig exocoelomic cavity (ExC) whilst, in the mouse, mesoderm
is more abundant forming a small allantoic bud ('al'). Guinea pig does not have an
ectoplacental cone (EPC) but only a rim of mesometrially-situated ectoplacental
giant cells (EPGC).
10% foetal calf serum (Flow), nucleosides ( 3 X 1 0 ~ 5 M ) , and antibiotics. Preimplantation tissues were explanted in 30 mm plastic tissue-culture dishes
(Sterilin) with 2-5 ml of medium and postimplantation tissues cultured in 50 mm
Petri dishes (Sterilin) with 5-0 ml of medium. Both were then incubated in 5%
CO 2 in air (37 °C). No more than one tissue fragment was explanted in a single
Petri dish. Also, cultures were not disturbed for 18 h following explantation and
the medium was not changed during the experimental period.
(4) Cytophotometry
(a) Recovery of tissues. All attached explants were mechanically removed
Trophoblastic growth in the guinea pig
409
Table 1. Procedure used to fix andfeulgen-strain tissues for cytophotomeiry
Fixation
Post-fixation
Drying
Hydrolysis
Distilled water
Feulgen-stain*
Sulphorous acid
Tap water
Dehydration
Mount
3 absolute alcohol A.R.: 1 acetic acid A.R. (5 min)
85 absolute alcohol A.R.: 10 formalin: 5 acetic acid A.R. (60 min)
(35 min)
5 normal HC1 = 144 ml HC1:106 ml distilled water (55 min, 26 °C)
(5 min)
(2 h)
1 (1 %) K+ metabisulphite (BDH): 1 (01 normal) HC1 (3 changes of
10 min each)
(5 min)
Graded alcohol series through xylene
DPX (Gurr)
* The procedure for preparing Feulgen stain is from Dr F. A. L. Clowes, Botany School,
Oxford. Briefly, 40 g of pararosaniline acetate (Hopkins & Williams) are mixed with 800 ml
of boiling distilled water. This solution is then allowed to cool for 30 min and 12 g K + metabisulphite plus 120 ml of (1 normal) HC1 were added. The following day the solution is
decolorized with activated charcoal and filtered into dark-glass bottles. The stain is always
freshly prepared several days prior to an experiment, never used twice nor kept for more
than one week. It is stored at 4 °C and discarded if pink or crystalline.
from the surface of the Petri dish with the edge of a siliconized Pasteur pipette.
For 5-day polar trophectoderm-ICM cultures, the attached trophectodermal
base was initially separated from the organized ICM derivatives with a glass
microneedle before being removed from the Petri dish.
(b) Dissociation of tissues. Preimplantation tissues, either freshly dissected or
following culture, were dissociated using two different methods. The first was a
modification of a technique developed by Evans, Burtenshaw & Ford (1972)
whereby each tissue was initially transferred to hypotonic solution (1 % Na
citrate; 15-60 sec; pH 8-45; 26 °C), fixed (three methanol/acetic acid), and then
dissociated on acid-cleaned slides in several drops of 60 % acetic acid (30-60 sec;
26 °C). Comparable results were obtained when fresh tissues were placed
directly into several drops of TVP (Bernstein, Hooper, Grandchamp & Ephrussi,
1973) enzyme solution on glass slides (30-60 sec; pH 7-5; 26 °C).
Postimplantation tissues, analysed immediately after dissection, were incubated in TVP (5-15 mm; pH 7-5; 26 °C) and then transferred to slides in enzyme.
Those analysed following culture were initially incubated in Ca2+- and Mg2+-free
Hank's balanced salt solution (HBSS, 30 min, 37 °C), placed into CTC (0-1 %
collagenase, Worthington, N.J.; 0-1% trypsin, Difco 1:250; and 10% chick
serum (Flow), made up in deficient HBSS - 15 to 30 min, pH 7-6, gassed) solution, and then transferred on to slides in several drops of CTC. Afterwards,
tissues in either CTC or TVP on glass slides were drawn through siliconized
Pasteur pipettes of different bore sizes until a single cell suspension was produced.
Slides were then dried beneath an air jet.
(c) Fixation and staining. The method used to fix and stain slides for cytophotometry is outlined in Table 1.
410
E. B. ILGREN
(d) Method of scanning. To reduce the risk of fading, slides were stored in the
dark and not used for DNA measurements if more than 3 months had elapsed
from the time of preparation. Nuclei selected for analysis were stained an intense
magenta red and generally had well-delineated nuclear membranes. Although
every nucleus in each chosen field was measured, usually not every field was
scanned. Prior to staining, slides were smeared with fresh liver slices to provide
known DNA contents. All measurements were carried out with a Vicker's M-85
microdensitometer (A = 585 nm).
(5) Histology. Pre- and postimplantation tissues were initially fixed in Bouin's
solution, dehydrated, cleared in cedar-wood oil, embedded in paraffin wax
(56 °C), serially sectioned (5 jum), and then stained with haematoxylin and eosin.
(6) Determination of cell numbers and mitotic indices. Since it was usually not
possible to see cell outlines, counts were frequently made by determining the
number of nuclei on slides prepared for either cytophotometric or cytological
study. Although it was assumed that changes in nuclear numbers reflected variations in the number of cells present, binucleates and multinucleates were occasionally seen in both guinea-pig and mouse tissues. However, the dissociation
procedure used frequently disrupted cytoplasmic margins. Therefore, the precise
number of uni, bi- and multinucleates in each preparation could not be determined. Approximate cell numbers were also estimated on serially-sectioned
embryos. In the case of in vivo and in vitro, mouse and guinea-pig egg cylinders,
all cells with clearly-defined nuclear membranes were counted in alternate serial
sections. For the guinea pig, these were then assigned to the following anatomic
regions: endoderm, see Ptyler & Strasser (1925); embryonic and extraembryonic
ectoderm as well as the ectoplacental giant cell region, see Sansom & Hill (1931)
and Mossman (1937); and extraembryonic mesoderm, see Kaufman & Davidoff
(1976). The methods used to determine the mitotic indices (total number mitoses,
total number cells scored x 100) of pre- and post-implantation guinea pig and
mouse tissues are described beneath Tables 2 and 3.
RESULTS
(1) Trophoblastic nature of the presumptive trophectodermal derivatives of the
guinea pig
At the primitive-streak stage, the 7-5-day mouse embryo is similar to the
10-5-day guinea-pig egg cylinder in several ways. Structurally, the presumptive
embryonic and extraembryonic ectodermal regions of the 10-5-day guinea-pig
embryo closely resemble those of the 7-5-day mouse egg cylinder (Fig. 1). Also,
immediately after isolation from either the guinea pig or mouse embryo, these
embryonic and extraembryonic tissues are totally diploid (2-4 c), contain considerable numbers of mitoses, and have approximately the same number of cells
(Table 2 A). However, after 72 h in culture guinea-pig and mouse extraembryonic
ectoderm not only stop dividing as indicated by a fall in mitotic index but also
Trophoblastic growth in the guinea pig
411
Table 2. Trophoblastic nature of the presumptive trophectodermal
derivatives of the guinea pig
Table 2 A
0 h in vitro
A.
Cell
number
Extra embryonic
ectoderm
Guinea pig
Mouse
Embryonic
ectoderm
Guinea pig
Mouse
1863 + 684
*(8)
2051±1094
•(17)
2209 + 520
*(3)
1976 + 455
•(11)
Nuclear DNA
content (a)
99-6% (2-4 c)
*(6)
100% (2-4 c)
*(12)
100% (2-4 c)
•(4)
100% (2-4 c)
•(12)
Mitotic
index (6)
6-1 ±2-5%
•(7)(*)
10-6+1-2%
*(7)(/)
2-6 + 0-2%
*(2)fc)
7-4 + 0-5%
*(W)
Table 2 B
72 h in vitro
A
Cell
number
Extra embryonic
ectoderm
Guinea pig
Mouse
Embryonic
Guinea pig
Mouse
307 + 21
•(11)
4568 + 826
*(5)
ND
6000 + 455
*(5)
Nuclear DNA
content (c)
Mitotic
index (d)
52-2% (> 4 c)
*(6)
57-8%(> 4c)
•(14)
100% (2-4 c)
•(5)
100% (2-4 c)
*(6).
o-i%
*(11) W
008%
*(5)(/)
7-5±5-3%(/i)
*(3) (e)
2-4 ±0-4%
•(7) CO
Sample size for (a), n = 107-682; (b), n = 5326-22,798; (c), n = 350-2746; and (d),
n = 2456-22,398 except for (It) where n = 460. Mitotic indices determined on (<?), noncolcemid-treated tissues originally prepared for cytophotometry, (/) colcemid (Grand Is.
Biol.; 0-4/^g/cc; 75 min) treated tissues prepared after Evans, Burtenshaw & Ford (1972)
where exposure to hypotonic was 2-3 min and acetic acid 10-15 min (26 °C), and (g) alternate
histological sections of non-colcemid treated in vivo egg cylinders as well as cytophotometry
preparations. *( ) represents number of embryos.
acquire a large number of nuclei with DNA contents greater than (4 c) (Table
2B and Fig. 2). In addition, guinea-pig extraembryonic ectodermal cell numbers
fall over the 72 h time course whereas those of the mouse increase (Table 2B).
In contrast to extraembryonic ectoderm, mouse and guinea-pig embryonic
ectodern not only remain diploid (2-4 c) after 72 h in culture but also continue
to divide (Table 2B).
At the neural-tube-closure stage, there are numerous trophoblastic giant cells
in the trophoblastic tissues of the 10-5-day mouse embryo (Fig. 3 a). At a comparable stage of development, there are also many giant cells in the placental
412
E. B. ILGREN
(h)
(a)
4c
8c
16c
32 c
c
-60
32 c
16c
-40 Z
- 2 0 XI
«^i
•-•
^-P-T_
f.
rv_
I
60
0
Arbitrary DNA-index units
60
Fig. 2. Nuclear DNA contents of (a) 7-5-day mouse and (6) 10-5-day guinea
extraembryonic ectoderm after 72 h in vitro.
0
150
I
1000
0 20
Arbitrary DNA-index units
80
pig
140
Fig. 3. Nuclear DNA contents of (a) 10-5-day mouse secondary and (b) 17-5-day
guinea-pig placental giant cells.
disc of the 17-5-day guinea-pig embryo but their nuclear DNA contents are
frequently less than those of the mouse (Fig. 3 b).
(2) Development of isolated mural trophectoderm and polar trophectoderm-lCM
fragments in vitro
The 5-5-day guinea-pig blastocyst resembles the 3-5-day mouse embryo in
several ways. Morphologically, the blastocysts of both species are quite similar
except for a thickening of the guinea-pig's abembryonic trophectoderm (Fig. 4).
Also, shortly after microsurgical isolation, the trophectodermal constituents of
both the guinea-pig and mouse blastocyst are almost totally diploid (2-4 c) and
contain approximately the same number of cells (Table 3 A). However, after
5 days growth in vitro, guinea-pig and mouse mural trophectoderm always
acquire nuclei with DNA contents greater than (4 c), never have included mitotic
figures, and do not increase in cell number (Table 3B and Fig. 5). In contrast to
mural tissues, polar trophectoderm-ICM fragments form structures which
resemble 10-5-day guinea-pig (14/17 cases) and 7-5-day mouse (4/30 cases) egg
Trophoblastic growth in the guinea pig
413
Fig. 4. 5-5-day guinea-pig blastocyst (live) with abembryonic thickening (AbT) and
Zona pellucida (ZP) penetrated by several pseudopodia. (
) Represents plane
of microsurgical section. Inverted phase, (x 1000).
cylinders following five days in culture. These, in turn, contain two presumptive
polar trophectodermal derivatives namely, the ectoplacental region and the
extraembryonic ectodern. The latter is found to be, in both the mouse and the
guinea pig, a highly cellular tissue which contains numerous mitoses and shows
no evidence of giant-cell formation (Table 3B). If the in vitro-denved guinea-pig
egg cylinder is dissected into its presumptive extraembryonic ectodermal,
embryonic ectodermal, and ectoplacental components, isolated extraembryonic
ectodern becomes giant, embryonic ectoderm remains diploid (2-4 c), and the
ectoplacental region continues to endoreduplicate (Fig. 6).
DISCUSSION
In contrast to previous proposals (Blandau, 1971), the results of the present
study suggest that the growth of guinea-pig trophectoderm in vitro is under the
control of the inner cell mass. Thus, if guinea-pig trophectoderm is isolated from
the ICM, it becomes giant. However, in the presence of the ICM, trophectodermal growth appears to continue thereby forming structures similar to the
extraembryonic ectodermal and ectoplacental giant-cell regions of the 10-5-day
27
EMB 60
414
E. B. ILGREN
Table 3. Development of isolated mural trophectoderm and polar
trophectoderm-ICMfragments in vitro
Table 3 A
0 days in vitro
Mural
trophectoderm
Polar
trophectoderm
Cell
number
Nuclear DNA
content
Guinea pig
23 (a)
100% (2-4 5)(<?)
(4)
ND
Mouse
Guinea pig
18 + 4(6)
10 (a)
95%(2-4c)(/)
100%(2-4c)(e)
2% (b)
Mitotic
index
ND
(7)
Mouse
16 ±3 (6)
95%(2-4c)(/)
12% (b)
Table 3 B
5 days in vitro
A
Cell
number
Mural
trophectoderm
Guinea pig
Mouse
Extra-embryonic
ectoderm
Guinea pig
Mouse
Ectoplacental
region (h)
Guinea pig
Mouse
15± 10
(9)
13±7
(14)
331±37(c)
(6)
355±32 (d)
(2)
41 +12 (c)
(2)
984±72(d)
(2)
Nuclear DNA
content
Mitotic
index
83-8% (> 4 c)
0%
(9) (*)
(3) U)
791 % ( > 4c)
0%
(14 (k)
(3)(7)
NGC (g)
2-8% (c)
(2)
(6)(m)
NGC(s)
5-8 + 2(rf)
(2)
(2)(0
94-3% (> 4c)
0%(c)
(6) (k)
(5)
Cone -> NGC
4-5 ±0-9%
Periphery -M5-8•%
0%
(>4c)
(4) (A:)
(a) From one serially-sectioned blastocyst but probably maximal estimates since (7) whole
blastocysts had 38 ± 16 cells; most embryos at attachment cone stage; (6) data from Copp
(1979), consistent with Surani & Barton (1977); (c) Data presented taken from one seriallysectioned egg cylinder (416 cells) that was comparable in size to (12) others used for histological controls, DNA analyses, and tissue layer separations; also includes cell-number
counts performed on presumptive extraembryonic ectoderm that was isolated from in vitroderived egg cylinders and grown for an additional 72 h in vitro - assumes little or no increase
in cell number has occurred as seen in isolated 10-5-day extraembryonic ectoderm grown for
72 h in culture (see Table 1); (d) counts made on serially-sectioned, in v/7ro-derived, egg
cylinders kindly provided by Dr A. J. Copp; (e) and (/), individual regions not studied;
data extrapolated from measurements made on dissociated whole blastocysts; for mouse, from
Barlow, Sherman & Graham (1972); (g) DNA measurements not performed; visual estimates
made; (li) in 10-5-day guinea-pig embryo, ectoplacental cone is not present-only rim of
giant cells (see Figs 1,2); (/) polar trophectoderm presumably forms extraembryonic ectoderm
and ectoplacental giant cells in guinea pig (Sansom & Hill, 1931); for mouse, see Gardner &
Papaioannou (1975); ND, not determined. (;) see also Fig. 6. Mitotic indices determined on
(k) non-colcemid treated tissue explants dissociated for cytophotometry, (/) alternate histological sections of non-colcemid treated, in vitro egg cylinders, or (m) both of the former.
Trophoblastic growth in the guinea pig
40
80
415
120
160
200
Arbitrary DNA-index units
Fig. 5.Nuclear DNA contents of (a) 5-5-day guinea-pig and (b) 3 -5-day mouse mural
trophectodermal cells after 5 days in vitro.
2-4 c
175 —
2-4 c
g 140-
-150
-125
4c
o 105 —
I
-100
-75
70-
-50
35-
16 c
c 16c
32c
128 c - 2 5
0
i
r \ r
15 30 45 60 0
I
15
30
0 15
Arbitrary DNA-index units
30
60 70
180
200
Fig. 6. Nuclear DNA contents of presumptive guinea-pig (a) embryonic ectoderm
(b) extraembryonic ectoderm and (c) ectoplacental tissues after isolation from the
in v/Vro-derived egg cylinder and an additional 72 h in culture.
guinea-pig embryo. If this in v/Vra-derived extraembryonic ectodermal tissue is
subsequently isolated and grown in culture for an additional 72 h, it too becomes
giant, a finding consistent with its proposed trophoblastic origin. Although
these observations suggest that guinea-pig trophectodermal growth is under
ICM control, it is still not possible to exclude an ICM contribution to presumptive guinea-pig trophoblast without performing reconstitution experiments.
Nevertheless, the cytoarchitecture, nuclear DNA contents, mitotic indices, and
cell numbers of the embryonic and extraembryonic ectodermal tissues of the
guinea pig frequently resemble those of the mouse not only in vivo but also after
isolation and growth in vitro. These observations therefore suggest that guineapig ICM cells do not make a substantial contribution to presumptive trophectodermal derivatives. In addition, they also suggest that the results of the present
study are not artifacts of the tissue-culture environment.
27-2
416
E. B. ILGREN
The attachment cone of the guinea-pig blastocyst is probably not due to an
ICM-independent proliferation of trophectoderm but is most likely derived from
a transient accumulation of abembryonic trophectodermal cells. The fact that
this is a temporary rather than a persistent trophectodermal growth is further
supported by the observation that in vivo the attachment cone degenerates shortly
after implantation (Sansom & Hill, 1931). Alternative explanations for the origin
of this abembryonic thickening are possible but less likely. For example, the
attachment cone could be, either partially or totally, composed of nontrophectodermal cells. Blandau & Rumery (1957) actually describe 'macrophage-like'
cells in the abembryonic trophectoderm of the guinea pig. However, such presumptive mesodermal derivatives are generally confined to the exocoelomic
cavity of the postimplantation, guinea-pig embryo (Ptyler & Strasser, 1925;
Kaufman & Davidoff, 1977) or to the stroma of trophoblastic villi (Amoroso,
1952). Since primitive endoderm does not cover the abembryonic portion of the
guinea-pig blastocoele cavity (Mossman, 1937; Amoroso, 1952), it would also
not be expected to participate in the formation of the attachment cone. Therefore, the attachment cone is most likely to be totally trophectodermal in origin
and, being located directly opposite the inner cell mass, probably destined to
cease cell division (Copp, 1978) and become giant (Dickson, 1963, 1966) in a
manner similar to mouse mural trophectoderm. However, definitive proof that
guinea-pig trophectodermal growth depends on the ICM will only be obtained
after blastocyst reconstitution experiments are performed.
I am very greatly indebted to Professor R. L. Gardner for his supervision of this work.
I should also like to thank Dr A. J. Copp, Dr F. A. L. Clowes, Dr E. P. Evans, Dr C. F.
Graham, Dr Anne McLaren, Dr P. W. Barlow, Professor A. C. Braun, and my laboratory
colleagues for extremely helpful criticism, and Miss R. E. Woolston, Mr J. Haywood and
Mr P. L. Small for excellent technical assistance. I am grateful to Professors F. R. Whatley
(Botany School, Oxford) and H. Harris (Pathology School, Oxford) for providing the facilities
to complete this work and to Professor J. W. Pringle and the Department of Zoology, Oxford,
where this project was started. This work has been supported by fellowships to EB1 from the
International Agency for Research on Cancer, World Health Organization (IACR/R.882),
the American Cancer Society (SPF 14), and the National Institutes of Health (1F32-H D0559201). This study would not have been possible without the generous assistance of Mr H. Elvidge
(Nuffield Institute for Medical Research, Oxford) who provided timed guinea-pig matings.
Mr T. Smy (Zoology) and Mr C. Dear (Biochemistry, Oxford) have also kindly provided
assistance with animals.
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(Received 5 April 1980, revised 20 June 1980)