/ . Embryo!. exp. Morph. Vol. 35, 3, pp. 535-543, 1976
Printed in Great Britain
535
Implantation and early postimplantation
development of the bank vole Clethrionomys
glareolus, Schreber
By WACfcAW OZDZENSKI 1 AND EWA T. MYSTKOWSKA 2
From the Department of Embryology, University of Warsaw and the
Laboratory of Experimental Embryology, Medical Academy, Warsaw
SUMMARY
The development of the bank vole Clethrionomys glareolus is described from implantation
to the formation of the foetal membranes. The embryonic development of this species combines features of primitive rodent species, for example Geomys bursarius and highly specialized
ones, for example Mus musculus. The egg-cylinder is formed by invagination into the blastocoelic cavity of the inner cell mass and polar trophoblast overlying it; this resembles in many
respects the early stages of development of primitive species. The fully formed egg-cylinder,
however, resembles that of the mouse and the formation of foetal membranes is also similar
to that in Muridae. It is concluded that in the bank vole and also in other rodents, the
extra-embryonic ectoderm of the egg-cylinder is derived from the polar trophoblast rather
than from the inner cell mass.
INTRODUCTION
The early stages of post-implantation development in rodents are characterised by the phenomenon of inversion of germ layers. Although this process is
common to all rodents studied so far, it occurs in different ways in various
species cf. Mossman, 1937; Snell & Stevens, 1966. While in some species formation of the foetal membranes shows only slight modification as compared to the
typical pattern in mammals, for example Citellustridecemlineatus, the course
of early post-implantation development has undergone profound changes in
others, for example Mus musculus.
So far only a small number of representatives of the order Rodentia have been
examined as regards early stages of post-implantation development. Therefore,
it was of interest to investigate this period of development in a species that has
not been included hitherto in these studies. The bank vole Clethrionomys glareolus, family Microtidae is of particular interest in view of its increasing use as a
laboratory animal.
1
Author's address: Department of Embryology, University of Warsaw, Krakowskie
Przedmiescie 26/28, 00-325, Warszawa, Poland.
2
Author's address: Laboratory of Experimental Embryology, Medical Academy, Karowa
2, 00-315, Warszawa, Poland.
34-2
536
W. OZDZENSKI AND E. MYSTKOWSKA
MATERIAL AND METHODS
The material consisted of 50 embryos from 17 females dissected between
4 p.m. of the 5th day and 11 a.m. of the 9th day of pregnancy. The females were
checked for plugs between 9 and 11 a.m. The day of the plug was designated the
first day of pregnancy. The uteri of pregnant females were fixed in 15 parts of
96 % ethyl alcohol, 4 parts of 40 % formalin, 1 part of glacial acetic acid;
embedded in paraffin and cut into 6 jum sections. In most cases the plane of the
section was perpendicular to the long axis of the uterus. The sections were
stained with haematoxylin and eosin.
The volume of the giant cells described in this paper was estimated on the
basis of linear measurements taken on the largest section. The formula for the
volume of a sphere was used, the diameter being calculated as the mean of both
axes of the section - it was assumed that the 3rd axis is intermediate between the
two axes measured on the largest section. This kind of computation is burdened
with a considerable error, particularly in the case of greatly flattened cells in the
myometrium, but the difficulty of measuring the 3rd axis of the cells made more
precise calculations unfeasible.
RESULTS
In the afternoon of the 5th day of development the blastocyst of the bank vole
is already elongated along the polar axis (Fig. 1). It settles in a fold of the uterus
on the antimesometrial side and comes into close contact with the uterine wall.
The decidual reaction appears at the site of the attachment of the blastocyst. The
endometrium forms a crypt around the embryo in continuity with the uterine
lumen by a narrow canal filled with detritus. In the implanted blastocyst the
inner cell mass is oriented towards the mesometrium. The polar trophoblast
lies close to the canal connecting the implantation crypt with the uterine lumen,
FIGURES 1-8
Fig. 1. Blastocyst in the uterine lumen, x 500.
Fig. 2. Implanted blastocyst. Short arrow: polar trophoblast; long arrow: proximal
entoderm. x400.
Fig. 3. Beginning of egg-cylinder formation, a: polar trophoblast invaginated;
b: inner cell mass and arrow: proximal endoderm x 400.
Fig. 4. Formed egg-cylinder, a: epamniotic cavity; b: amniotic cavity. x400.
Fig. 5. Beginning of ectoplacental cone formation, arrow, x 250.
Fig. 6. Cylinder with formed ectoplacental cone and differentiated proximal
entoderm. a: ectoplacental cone cavity; b: extra-embryonic endoderm; c: embryonic endoderm. x 100.
Fig. 7. Ectoplacental cone of a 9-day-old embryo, a: cavity between the peripheral
parts of the cone and the chorion; b: distal part of the allantois. x 40.
Fig. 8. Cross section through an egg-cylinder at the moment of mesoderm formation, a: primitive streak, x 250.
Implantation and postimplantation development of bank vole 537
!
*\.M
F
^'*+!.\
'%$& '.•&**iL*1f
538
W. OZDZENSKI AND E. MYSTKOWSKA
14
Implantation and postimplantation development of bank vole 539
whereas the mural trophoblast becomes attached to the walls of the crypt. The
endoderm begins to form on the blastocoelic surface of the inner cell mass
(Fig. 2).
The blastocyst begins to increase in size after implantation; growth is particularly intense along the long axis so that the blastocyst becomes elongated.
Simultaneously with the enlargement and elongation of the blastocyst as a
whole, the inner cell mass and the polar trophoblast covering it begin to grow
into the blastocoele (Fig. 3). The polar trophoblast invaginates to form a cupshaped depression, with sides continuous with the peripheral trophoblast and
base in contact with the inner cell mass (Fig. 4). Hence the egg-cylinder is
divided into an embryonic and extra-embryonic part from the outset, both of
which are covered with proximal endoderm.
The epamniotic cavity arises at the moment of formation of the extraembryonic part of the cylinder, as a consequence of the invagination of the
polar trophoblast. This cavity opens into the lumen of the implantation crypt.
The embryonic part of the cylinder is at first compact. The amniotic cavity
begins to form between the 5th and 6th day of development (Fig. 4). In the
course of the 6th day the cavities unite, forming a continuous lumen in the
cylinder in the form of a narrow duct opening into the uterine crypt.
The ectoplacental cone begins to develop in the morning of the 6th day by
growth of the upper border of the extraembryonic part of the egg-cylinder
towards the mesometrium (Fig. 5). At first it is shaped like an open cylinder.
On the 6th day the upper edges of the cylinder draw near to one another,
forming a cone which covers the wide cavity connected with the lumen of the
egg-cylinder (Fig. 6). At first the walls surrounding the ectoplacental cavity
are thin. On the 7th day they become thicker. In the course of the 8th day
further changes occur in the appearance of the ectoplacental cone. The
originally compact cell mass becomes less dense and numerous fissures appear.
Simultaneously the recently formed chorion is elevated and its central part
adheres to the basal part of the ectoplacental cone, leaving a peripheral circular
cavity (Fig. 7).
FIGURES 9-14
Fig. 9. Formation of the amniotic fold (am). Three trophoblast giant cell are
visible, x 80.
Fig. 10. Cylinder with formed foetal membranes: amnion (am), chorion (c) and
allantois (al). x 50.
Fig. 11. Formation of trophoblast giant cells: arrows, x 150.
Fig. 12. 'Migrating' trophoblast giant cell with a cytoplasmic process (7th day of
development). x400.
Fig. 13. 'Migrating' trophoblast giant cells in myometrium (9th day of development), x 150.
Fig. 14. 'Migrating' trophoblast giant cell found on the 6th day of pregnancy
(estimated volume -12-7 x 106 /*m3). x 30.
540
W. OZDZENSKI AND E. MYSTKOWSKA
The proximal endoderm covering the egg-cylinder consists at first of cells of
similar appearance. However, as early as the 6th day of development differentiation into two zones takes place. In the embryonic part of the cylinder the
endoderm cells become flattened, while in the extra-embryonic part they are
high and vascularized (Fig. 6). There is a gradual transition between the two
kinds of endoderm.
On the 7th day the primitive streak arises in the future posterior part of the
embryo (Fig. 8). The mesodermal cells penetrating through it disperse in all
directions. The head process, which will constitute the axis of the future embryo,
grows out towards the front. The mesoderm also spreads outwards and upwards
to the extra-embryonic part of the cylinder, where it will be involved in the
formation of the foetal membranes.
The first step in the development of the amnion is folding of the ectoderm
lining the egg-cylinder cavity above the upper border of the embryonic part
(Fig. 9). Mesoderm penetrates into the developing fold and divides into two
parts - one underlying the ectoderm of the forming amnion, the second constituting a component of the yolk-sac wall. The amniotic fold does not form
simultaneously around the cylinder circumference - it is formed first over the
posterior part of the embryo. In the night between the 7th and 8th day of
development the edges of the amniotic fold join, separating the amniotic from
the epamniotic cavity (Fig. 10). The lower part of the fold facing the embryo
forms the amnion, and the upper part the chorion. The extra-embryonic cavity
arises between the amnion and the chorion. On the 8th day of development a
club-shaped mesodermal process with a spongy structure - the allantois - starts
to grow into the extra-embryonic cavity from the posterior end of the embryo.
On the evening of the 8th day the widened distal end of the allantois merges
with the chorion (Fig. 7). At a later stage the blood vessels of the embryo will
pass through this junction to the chorio-allantoic placenta.
The trophoblastic giant cells appear in the bank vole between the 5th and 6th
day of pregnancy (Fig. 11). They are derived initially from the peripheral
trophoblast, and later from the ectoplacental cone also. Between the 5th and
6th day of pregnancy single giant cells may be observed; subsequently they
increase in number to form a broad loose layer surrounding the embryo. The
trophoblastic giant cells may be distinguished from the mucosa cells by their
larger dimensions, deeply-staining chromatin and poorly delineated cell surface.
These cells resemble trophoblastic giant cells of the mouse in both size and
morphology. Apart from the giant cells lying in the direct vicinity of the embryo,
others of exceptionally large dimensions may be seen, mostly at some distance
from the embryo, in the mucosa and even in the myometrium of the uterus
(Brambell & Rowlands, 1936). These cells, characterized by an ellipsoid shape
and distinctly demarcated cytoplasmic border, will be referred to as 'migrating'
giant cells. They may be seen first in the evening of the 5th day of pregnancy,
and thereafter both their number and dimensions increase with the embryo's
Implantation and postimplantation development of bank vole 541
Table 1. Volume of'migrating' trophoblast giant cells
Day of
development
6
7
8
Volume ±S.D.
(103x/*m3)
11-4 ±8-8*
30-3 ±14-3
262-7 ±197-8
9
918-9 ±593-3
* With exception of one 'gigantic' cell (see text for explanation).
age (Table 1, Fig. 12). These cells can be found up to a distance of 1700/tm
from the embryo. The presence of cytoplasmic processes suggests that these
cells are capable of amoeboid movement. On the 9th day they are found in
large numbers at the border of the mucosa and myometrium or only in the
latter. They then assume the shape of lenses arranged tangentially to the uterine
surface (Fig. 13). The volume of one such giant cell situated at a considerable
distance from the nearest embryo in the uterus on the 6th day of pregnancy was
estimated to be more than 12 x 106/tm3 (Fig. 14). Since this cell exceeded in
size not only the giant cells found on the 6th, but even those occurring on later
days of pregnancy, we believe that it must have been a residual cell from an
earlier pregnancy.
DISCUSSION
In rodents the formation of foetal membranes is modified as compared to
other mammals. The embryo sinks into the yolk-cavity, so that the region of the
yolk-sac oriented with its endoderm to the outside becomes the external covering
of the developing embryo and forms an important organ of exchange between
the mother and the foetus (yolk sac placenta). This phenomenon, described as
germ layer inversion, occurs in different ways in different rodents.
In Citellus tridecemlineatus (Mossman & Weisfeldt, 1939) the invagination of
the embryo starts late, when it is already surrounded by the amnion formed by
folding of the somatopleure. This invagination includes only a small part of the
yolk sac surface. In Geomys bursarius (Mossman & Hisaw, 1940) the mode of
formation of amnion is similar to that in Citellus tridecemlineatus, but the process of sinking of the embryo into the yolk cavity begins relatively earlier so that
the amniotic cavity is connected for some time with the epamniotic cavity. The
epamniotic cavity remains in this species open to the uterine lumen. In Mus
musculus the proliferation of the inner cell mass into the blastocyst cavity precedes not only the formation of foetal membranes, but also the formation of the
embryo itself. The mass of cells growing into the blastocoele forms what is
called the egg-cylinder, containing primary ectoderm covered on the outside
with endoderm. The amniotic and epamniotic lumina are formed by the dehiscence of the cells of the egg-cylinder and remain connected until the amnion
is formed by folding of somatopleura. The epamniotic cavity has no connexion
542
W. OZDZENSKI AND E. MYSTKOWSKA
with the uterine lumen since, even before it is formed, the polar trophoblast
produces the ectoplacental cone, the primordium of the embryonic part of the
future chorio-allantoic placenta. Formation of the egg-cylinder and ectoplacental cone is also observed in Cavia cobaya, but in this species the amniotic and the
epamniotic cavities arise independently and have no connection at any stage of
development.
Developmental processes in C. glareolus resemble the development of less
specialized (G. bursarius) as well as more specialized (M. musculus) forms.
Formation of the egg-cylinder precedes that of the embryo and amnion. On the
other hand, the epamniotic lumen appears simultaneously with egg-cylinder
formation and does not occur as in the mouse or the guinea pig by dehiscence of
cells, but by invagination. An apamniotic cavity is initially in continuity with the
uterine lumen, as in Microtus arvalis (Kupffer, 1882). The amniotic and epamniotic cavities become separated by folding of the somatopleura. Amnion formation is preceded by the development of the ectoplacental cone. The final result
is the formation of an egg-cylinder similar to that in Muridae and Cavidae.
The present observations throw a new light on the problem of the participation
of two of the components of the blastocyst, i.e. the polar trophoblast and the
inner cell mass, in the formation of the embryo in rodents. There is no doubt
that the inner cell mass is the precursor of the embryonic part of the egg-cylinder
in the bank vole. But from which tissue does the extra-embryonic part of the
egg-cylinder form? According to the present observations it would seem that it
forms from the polar trophoblast.
Rodent species examined so far exhibit varying degrees of reversal of the
germ layers, of which Muridae and Cavidae are the most extreme. In view of the
complete similarity of the fully formed egg-cylinder in the bank vole to that of
higher forms, it would seem that in higher specialized forms the origin of the
extra-embryonic part of the cylinder should be the same as in the vole.
Recently a new light was thrown on the problem of the origin of the extraembryonic ectoderm of the egg-cylinder by Gardner & Papaioannou's (1975)
observations on rat-mouse chimeras, made by injecting ICM from rats into
mouse blastocysts. When the rat inner cell mass adhered to the mouse ICM
after transplantation, they formed a single egg-cylinder. In this cylinder rat cells
were present in the embryonic ectoderm and in the endoderm but were completely
absent from the extra-embryonic ectoderm. When the rat ICM adhered to the
inner surface of the trophoblast, an independent egg-cylinder was formed. The
embryonic part of the 'rat' cylinder was built exclusively of rat cells but the
extra-embryonic ectoderm was of pure mouse origin. These facts show that the
extra-embryonic ectoderm in Muridae is formed by trophoblast rather than by
the inner cell mass, as suggested by Jenkinson (1902).
The results of our observations and Gardner & Papaioannou's data (1975)
enable us to suppose that the origin of the extra-embryonic ectoderm from
trophoblast is a general rule for the development of all rodents.
Implantation and postimplantation development of bank vole 543
The above description of the formation of the egg cylinder in the bank vole
bridges a gap in our knowledge of early post implantation development in
rodents. In these animals one observes an increasing role of the yolk sac, both as
an exchange organ (yolk-sac placenta) and as an outer coating for the developing embryo. Beginning with Sciuridae, which are the most primitive in this respect, and ending with the most highly specialized Muridae and Cavidae, this
process progresses towards an increase in the proportion of the total surface of
the yolk sac facing the uterine wall, as well as towards an earlier occurrence of
this process. The bank vole resembles Muridae in both these respects but in the
formation of the egg cylinder some processes resemble the less specialized forms.
It would be interesting to extend the investigations on foetal membrane formation to other rodents, since information is lacking for many species and even
whole families. A better knowledge of the evolution of so conservative a trait as
the formation of foetal membranes (Mossman, 1937) could make an important
contribution to our knowledge of the taxonomic relationships within this order.
We are grateful to Professor Andrzej K. Tarkowski for his help and valuable criticism
during the course of the work.
REFERENCES
BRAMBELL, F. W. R. & ROWLANDS, I. W. (1936). Reproduction of the bank vole (Evotomys
glareolus, Schreber). I. The oestrus cycle of the female. Phil. Trans. R. Soc. Ser. B, 226,
71-101.
GARDNER, R. L. & PAPAIOANNOU, V. E. (1975). Differentiation in the trophectoderm and
inner cell mass. In The Second Symposium of the British Society for Developmental Biology
(The Early Development of Mammals) (ed. M. Balls & A. W. Wild) pp. 107-132.
Cambridge University Press.
JENKINSON, J. W. (1902). Observations on the histology and physiology of the placenta of the
mouse. Tijdschr. ned. dierk. Vereen. 2, 124-198.
KUPFFER, C. (1882). Das Ei von Arvicola arvalis und die vermeintliche Umkehr der Kaimblatter an demselben. Sber. Akad. Wiss. Wien. II Cl., 5, 621-637.
MOSSMAN, H. W. (1937). Comparative morphogenesis of the fetal membranes and accessory
uterine structures. (Carnegie Instit. Wash. Pub. no. 479). Contr. Embryol. 158, 129-246.
MOSSMAN, H. W. & HISAW, F. L. (1940). The fetal membranes of the pocket gopher, illustrating an intermediate type of rodent membrane formation. I. From the unfertilized tubal egg
to the beginning of the allantois. Am. J. Anat. 66, 367-391.
MOSSMAN, H. W. & WEISFELDT, L. A. (1939). The fetal membranes of a primitive rodent, the
thirteen-striped ground squirrel. Am. J. Anat. 64, 59-109.
SNELL, & STEVENS, (1966). Early embryology. In Biology of the Laboratory Mouse, 2nd
edition, (ed. E. L. Green), pp. 205-245. New York: McGraw-Hill.
{Received 13 October 1975)