implantation and early postimplantation...

/ . Embryo!. exp. Morph. Vol. 35, 3, pp. 535-543, 1976 5 3 5

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Implantation and early postimplantationdevelopment of the bank vole Clethrionomys

glareolus, Schreber

By WACfcAW OZDZENSKI1 AND EWA T. MYSTKOWSKA2

From the Department of Embryology, University of Warsaw and theLaboratory of Experimental Embryology, Medical Academy, Warsaw

SUMMARYThe development of the bank vole Clethrionomys glareolus is described from implantation

to the formation of the foetal membranes. The embryonic development of this species com-bines features of primitive rodent species, for example Geomys bursarius and highly specializedones, for example Mus musculus. The egg-cylinder is formed by invagination into the blasto-coelic cavity of the inner cell mass and polar trophoblast overlying it; this resembles in manyrespects 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 similarto that in Muridae. It is concluded that in the bank vole and also in other rodents, theextra-embryonic ectoderm of the egg-cylinder is derived from the polar trophoblast ratherthan from the inner cell mass.

INTRODUCTION

The early stages of post-implantation development in rodents are charac-terised by the phenomenon of inversion of germ layers. Although this process iscommon to all rodents studied so far, it occurs in different ways in variousspecies cf. Mossman, 1937; Snell & Stevens, 1966. While in some species forma-tion of the foetal membranes shows only slight modification as compared to thetypical pattern in mammals, for example Citellustridecemlineatus, the courseof early post-implantation development has undergone profound changes inothers, for example Mus musculus.

So far only a small number of representatives of the order Rodentia have beenexamined as regards early stages of post-implantation development. Therefore,it was of interest to investigate this period of development in a species that hasnot been included hitherto in these studies. The bank vole Clethrionomys glare-olus, family Microtidae is of particular interest in view of its increasing use as alaboratory animal.

1 Author's address: Department of Embryology, University of Warsaw, KrakowskiePrzedmiescie 26/28, 00-325, Warszawa, Poland.

2 Author's address: Laboratory of Experimental Embryology, Medical Academy, Karowa2, 00-315, Warszawa, Poland.

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536 W. OZDZENSKI AND E. MYSTKOWSKA

MATERIAL AND METHODS

The material consisted of 50 embryos from 17 females dissected between4 p.m. of the 5th day and 11 a.m. of the 9th day of pregnancy. The females werechecked for plugs between 9 and 11 a.m. The day of the plug was designated thefirst day of pregnancy. The uteri of pregnant females were fixed in 15 parts of96 % 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 thesection was perpendicular to the long axis of the uterus. The sections werestained with haematoxylin and eosin.

The volume of the giant cells described in this paper was estimated on thebasis of linear measurements taken on the largest section. The formula for thevolume of a sphere was used, the diameter being calculated as the mean of bothaxes of the section - it was assumed that the 3rd axis is intermediate between thetwo axes measured on the largest section. This kind of computation is burdenedwith a considerable error, particularly in the case of greatly flattened cells in themyometrium, but the difficulty of measuring the 3rd axis of the cells made moreprecise calculations unfeasible.

RESULTS

In the afternoon of the 5th day of development the blastocyst of the bank voleis already elongated along the polar axis (Fig. 1). It settles in a fold of the uteruson 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. Theendometrium forms a crypt around the embryo in continuity with the uterinelumen by a narrow canal filled with detritus. In the implanted blastocyst theinner cell mass is oriented towards the mesometrium. The polar trophoblastlies 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: proximalentoderm. 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 proximalentoderm. a: ectoplacental cone cavity; b: extra-embryonic endoderm; c: em-bryonic endoderm. x 100.

Fig. 7. Ectoplacental cone of a 9-day-old embryo, a: cavity between the peripheralparts 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 form-ation, a: primitive streak, x 250.

Implantation and postimplantation development of bank vole 537

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whereas the mural trophoblast becomes attached to the walls of the crypt. Theendoderm 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 particu-larly intense along the long axis so that the blastocyst becomes elongated.Simultaneously with the enlargement and elongation of the blastocyst as awhole, the inner cell mass and the polar trophoblast covering it begin to growinto the blastocoele (Fig. 3). The polar trophoblast invaginates to form a cup-shaped depression, with sides continuous with the peripheral trophoblast andbase in contact with the inner cell mass (Fig. 4). Hence the egg-cylinder isdivided into an embryonic and extra-embryonic part from the outset, both ofwhich are covered with proximal endoderm.

The epamniotic cavity arises at the moment of formation of the extra-embryonic part of the cylinder, as a consequence of the invagination of thepolar trophoblast. This cavity opens into the lumen of the implantation crypt.The embryonic part of the cylinder is at first compact. The amniotic cavitybegins to form between the 5th and 6th day of development (Fig. 4). In thecourse of the 6th day the cavities unite, forming a continuous lumen in thecylinder 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 bygrowth of the upper border of the extraembryonic part of the egg-cylindertowards 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 theegg-cylinder (Fig. 6). At first the walls surrounding the ectoplacental cavityare thin. On the 7th day they become thicker. In the course of the 8th dayfurther changes occur in the appearance of the ectoplacental cone. Theoriginally compact cell mass becomes less dense and numerous fissures appear.Simultaneously the recently formed chorion is elevated and its central partadheres to the basal part of the ectoplacental cone, leaving a peripheral circularcavity (Fig. 7).

FIGURES 9-14

Fig. 9. Formation of the amniotic fold (am). Three trophoblast giant cell arevisible, x 80.Fig. 10. Cylinder with formed foetal membranes: amnion (am), chorion (c) andallantois (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 ofdevelopment). x400.Fig. 13. 'Migrating' trophoblast giant cells in myometrium (9th day of develop-ment), x 150.Fig. 14. 'Migrating' trophoblast giant cell found on the 6th day of pregnancy(estimated volume -12-7 x 106 /*m3). x 30.


The proximal endoderm covering the egg-cylinder consists at first of cells ofsimilar appearance. However, as early as the 6th day of development differen-tiation into two zones takes place. In the embryonic part of the cylinder theendoderm cells become flattened, while in the extra-embryonic part they arehigh and vascularized (Fig. 6). There is a gradual transition between the twokinds of endoderm.

On the 7th day the primitive streak arises in the future posterior part of theembryo (Fig. 8). The mesodermal cells penetrating through it disperse in alldirections. The head process, which will constitute the axis of the future embryo,grows out towards the front. The mesoderm also spreads outwards and upwardsto the extra-embryonic part of the cylinder, where it will be involved in theformation of the foetal membranes.

The first step in the development of the amnion is folding of the ectodermlining the egg-cylinder cavity above the upper border of the embryonic part(Fig. 9). Mesoderm penetrates into the developing fold and divides into twoparts - one underlying the ectoderm of the forming amnion, the second con-stituting a component of the yolk-sac wall. The amniotic fold does not formsimultaneously around the cylinder circumference - it is formed first over theposterior part of the embryo. In the night between the 7th and 8th day ofdevelopment the edges of the amniotic fold join, separating the amniotic fromthe epamniotic cavity (Fig. 10). The lower part of the fold facing the embryoforms the amnion, and the upper part the chorion. The extra-embryonic cavityarises between the amnion and the chorion. On the 8th day of development aclub-shaped mesodermal process with a spongy structure - the allantois - startsto 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 mergeswith the chorion (Fig. 7). At a later stage the blood vessels of the embryo willpass through this junction to the chorio-allantoic placenta.

The trophoblastic giant cells appear in the bank vole between the 5th and 6thday of pregnancy (Fig. 11). They are derived initially from the peripheraltrophoblast, and later from the ectoplacental cone also. Between the 5th and6th day of pregnancy single giant cells may be observed; subsequently theyincrease in number to form a broad loose layer surrounding the embryo. Thetrophoblastic giant cells may be distinguished from the mucosa cells by theirlarger dimensions, deeply-staining chromatin and poorly delineated cell surface.These cells resemble trophoblastic giant cells of the mouse in both size andmorphology. Apart from the giant cells lying in the direct vicinity of the embryo,others of exceptionally large dimensions may be seen, mostly at some distancefrom the embryo, in the mucosa and even in the myometrium of the uterus(Brambell & Rowlands, 1936). These cells, characterized by an ellipsoid shapeand 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


Table 1. Volume of'migrating' trophoblast giant cells

Day of 6 7 8 9development

Volume ±S.D. 11-4 ±8-8* 30-3 ±14-3 262-7 ±197-8 918-9 ±593-3(103x/*m3)

* 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/tmfrom the embryo. The presence of cytoplasmic processes suggests that thesecells are capable of amoeboid movement. On the 9th day they are found inlarge numbers at the border of the mucosa and myometrium or only in thelatter. They then assume the shape of lenses arranged tangentially to the uterinesurface (Fig. 13). The volume of one such giant cell situated at a considerabledistance from the nearest embryo in the uterus on the 6th day of pregnancy wasestimated to be more than 12 x 106/tm3 (Fig. 14). Since this cell exceeded insize not only the giant cells found on the 6th, but even those occurring on laterdays of pregnancy, we believe that it must have been a residual cell from anearlier pregnancy.

DISCUSSION

In rodents the formation of foetal membranes is modified as compared toother mammals. The embryo sinks into the yolk-cavity, so that the region of theyolk-sac oriented with its endoderm to the outside becomes the external coveringof the developing embryo and forms an important organ of exchange betweenthe mother and the foetus (yolk sac placenta). This phenomenon, described asgerm layer inversion, occurs in different ways in different rodents.

In Citellus tridecemlineatus (Mossman & Weisfeldt, 1939) the invagination ofthe embryo starts late, when it is already surrounded by the amnion formed byfolding of the somatopleure. This invagination includes only a small part of theyolk sac surface. In Geomys bursarius (Mossman & Hisaw, 1940) the mode offormation of amnion is similar to that in Citellus tridecemlineatus, but the pro-cess of sinking of the embryo into the yolk cavity begins relatively earlier so thatthe amniotic cavity is connected for some time with the epamniotic cavity. Theepamniotic cavity remains in this species open to the uterine lumen. In Musmusculus the proliferation of the inner cell mass into the blastocyst cavity pre-cedes not only the formation of foetal membranes, but also the formation of theembryo itself. The mass of cells growing into the blastocoele forms what iscalled the egg-cylinder, containing primary ectoderm covered on the outsidewith endoderm. The amniotic and epamniotic lumina are formed by the de-hiscence of the cells of the egg-cylinder and remain connected until the amnionis formed by folding of somatopleura. The epamniotic cavity has no connexion


with the uterine lumen since, even before it is formed, the polar trophoblastproduces the ectoplacental cone, the primordium of the embryonic part of thefuture chorio-allantoic placenta. Formation of the egg-cylinder and ectoplacen-tal cone is also observed in Cavia cobaya, but in this species the amniotic and theepamniotic cavities arise independently and have no connection at any stage ofdevelopment.

Developmental processes in C. glareolus resemble the development of lessspecialized (G. bursarius) as well as more specialized (M. musculus) forms.Formation of the egg-cylinder precedes that of the embryo and amnion. On theother hand, the epamniotic lumen appears simultaneously with egg-cylinderformation and does not occur as in the mouse or the guinea pig by dehiscence ofcells, but by invagination. An apamniotic cavity is initially in continuity with theuterine lumen, as in Microtus arvalis (Kupffer, 1882). The amniotic and epam-niotic cavities become separated by folding of the somatopleura. Amnion for-mation is preceded by the development of the ectoplacental cone. The final resultis 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 participationof two of the components of the blastocyst, i.e. the polar trophoblast and theinner cell mass, in the formation of the embryo in rodents. There is no doubtthat the inner cell mass is the precursor of the embryonic part of the egg-cylinderin the bank vole. But from which tissue does the extra-embryonic part of theegg-cylinder form? According to the present observations it would seem that itforms from the polar trophoblast.

Rodent species examined so far exhibit varying degrees of reversal of thegerm layers, of which Muridae and Cavidae are the most extreme. In view of thecomplete similarity of the fully formed egg-cylinder in the bank vole to that ofhigher forms, it would seem that in higher specialized forms the origin of theextra-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 extra-embryonic ectoderm of the egg-cylinder by Gardner & Papaioannou's (1975)observations on rat-mouse chimeras, made by injecting ICM from rats intomouse blastocysts. When the rat inner cell mass adhered to the mouse ICMafter transplantation, they formed a single egg-cylinder. In this cylinder rat cellswere present in the embryonic ectoderm and in the endoderm but were completelyabsent from the extra-embryonic ectoderm. When the rat ICM adhered to theinner surface of the trophoblast, an independent egg-cylinder was formed. Theembryonic part of the 'rat ' cylinder was built exclusively of rat cells but theextra-embryonic ectoderm was of pure mouse origin. These facts show that theextra-embryonic ectoderm in Muridae is formed by trophoblast rather than bythe 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 fromtrophoblast is a general rule for the development of all rodents.


The above description of the formation of the egg cylinder in the bank volebridges a gap in our knowledge of early post implantation development inrodents. In these animals one observes an increasing role of the yolk sac, both asan exchange organ (yolk-sac placenta) and as an outer coating for the develop-ing embryo. Beginning with Sciuridae, which are the most primitive in this re-spect, and ending with the most highly specialized Muridae and Cavidae, thisprocess progresses towards an increase in the proportion of the total surface ofthe yolk sac facing the uterine wall, as well as towards an earlier occurrence ofthis process. The bank vole resembles Muridae in both these respects but in theformation of the egg cylinder some processes resemble the less specialized forms.It would be interesting to extend the investigations on foetal membrane for-mation to other rodents, since information is lacking for many species and evenwhole families. A better knowledge of the evolution of so conservative a trait asthe formation of foetal membranes (Mossman, 1937) could make an importantcontribution to our knowledge of the taxonomic relationships within this order.

We are grateful to Professor Andrzej K. Tarkowski for his help and valuable criticismduring the course of the work.

REFERENCES

BRAMBELL, F. W. R. & ROWLANDS, I. W. (1936). Reproduction of the bank vole (Evotomysglareolus, 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 andinner 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 themouse. Tijdschr. ned. dierk. Vereen. 2, 124-198.

KUPFFER, C. (1882). Das Ei von Arvicola arvalis und die vermeintliche Umkehr der Kaim-blatter an demselben. Sber. Akad. Wiss. Wien. II Cl., 5, 621-637.

MOSSMAN, H. W. (1937). Comparative morphogenesis of the fetal membranes and accessoryuterine 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, illustrat-ing an intermediate type of rodent membrane formation. I. From the unfertilized tubal eggto 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, thethirteen-striped ground squirrel. Am. J. Anat. 64, 59-109.

SNELL, & STEVENS, (1966). Early embryology. In Biology of the Laboratory Mouse, 2ndedition, (ed. E. L. Green), pp. 205-245. New York: McGraw-Hill.

{Received 13 October 1975)

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