J. Anat. (1987), 150, pp. 247-263 247With 13 figuresPrinted in Great Britain

A morphological study of the development of thechorion of rat embryos

S. K. L. ELLINGTONPhysiological Laboratory, Downing Street, Cambridge CB2 3EG

(Accepted 21 March 1986)

INTRODUCTION

The murine chorion develops from the mesoderm and extra-embryonic ectodermof the amniotic folds. During the primitive streak stage of development, mesodermcells accumulate at the extra-embryonic pole of the primitive streak between theextra-embryonic ectoderm and endoderm. With further increase in the number ofmesoderm cells in this region, the extra-embryonic ectoderm and the containedmesoderm begin to bulge into the proamniotic cavity, forming the posterior amnioticfold. Small lacunae appear amongst the mesoderm cells and then coalesce to form acentral cavity in the mesodermal tissue. The amniotic fold and the central cavityboth enlarge, extending around the egg cylinder until a complete, hollow annulus isformed between the embryonic and the extra-embryonic regions of the egg cylinder.The amniotic folds continue to enlarge, constricting the proamniotic cavity until

all that remains of it is a thick duct, the proamniotic tube. The central cavity ofthe amniotic folds, which develops to form the exocoelom, increases in size and themesoderm cells become confined to a single layer of cells lining the ectoderm of theamniotic folds. The proamniotic tube is further constricted by the enlarging amnioticfolds until the tissue on the embryonic side of the amniotic folds forms an almostintact sheet, the amnion, which divides the amniotic cavity from the remainingregion of the egg cylinder. Similarly, the tissue on the extra-embryonic side of theamniotic folds forms the chorion which divides the exocoelom from the ecto-placental cavity (Fig. 1).The development of the chorion up to this stage, as observed by light microscopy,

has been described in the rat (Jolly & Ferester-Tadie, 1936) and in the mouse(Bonnevie, 1950).As the egg cylinder enlarges the chorion increases in diameter and gradually

comes to lie closer to the ectoplacental cone. The ectoplacental cavity progressivelydecreases in size forming a disc-shaped cavity which is then further reduced to asmall cavity encircling the peripheral regions of the developing chorio-allantoicplacenta and finally disappears altogether. The chorion fuses with the extra-embryonic ectoderm cells of the ectoplacental cone and then with the allantois. Thusit forms an integral part of the chorio-allantoic placenta which is essential for fetalnourishment and excretion.The gross structural changes occurring during these later stages of development of

the chorion have been described for both the mouse (Amoroso, 1952; Snell &Stevens, 1966; Theiler, 1972) and the rat (Steven & Morriss, 1975) but there are nodetailed studies.

In this paper the growth and differentiation of the chorion of the rat, as seen by9 ANA 150

248

Ectoplacentalcavity

S. K. L. ELLINGTON

Mesoderm of chorion

Proamniotictube

EndodermExocoelom

Mesoderm 1

Ecodrof amnion

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Fig. 1. Drawing of a section prepared from a Bouin-fixed, wax-embedded embryo from a 9-25days rat. The section was cut parallel to the longitudinal axis of the egg cylinder and passesthrough most of the central cavity of the proamniotic duct, the amniotic cavity and the ecto-placental cavity.

Abbreviations for all Figuresa, amniotic cavity; all, allantois; c, chorion; cj, cell junction; e, ectoderm; ec, ectoplacentalcavity; epc, ectoplacental cone; er, rough endoplasmic reticulum; ex, exocoelom; g, Golgicomplex; m, mesoderm; mi, mitochondria; n, nucleus; pt, proamniotic tube; v, intracellularvesicle; vy, visceral yolk sac.

Development ofthe rat choriondirect observation of freshly dissected embryos, conventional histological techniquesand scanning and transmission electron microscopy, are described in detail.

MATERIALS AND METHODS

AnimalsCFHB rats were used throughout this study. Females were caged with males

overnight and those females with sperm in the vagina the following morning wereregarded as half a day pregnant (0 5 day) at noon that day. At six hourly intervals,between 8-25 and 10-25 days, pregnant females were lightly anaesthetised with ethervapour and then killed by cervical dislocation. The embryos were dissected out asdescribed by New (1971) and the majority were immediately processed for histology;a few were maintained in tissue culture for two hours prior to fixation.

HistologyThis report was based on embryos from 40 rats killed specifically for this study and

on the numerous slides and micrographs of normal development in my collection.

Light microscopyThe parietal yolk sacs were removed and the remaining embryonic and extra-

embryonic components of the conceptus were rinsed in 0 9 % NaCl and either fixedin alcoholic Bouin's solution for at least 48 hours or processed as though for electronmicroscopy. The Bouin-fixed conceptuses were dehydrated, cleared and embeddedin paraffin wax. Blocks were sectioned at a thickness of 5 #tm and sections werestained with haematoxylin and eosin for routine histology. Seventy two of theBouin-fixed conceptuses (eight from each of the stages of pregnancy examined) werestained by the periodic acid-Schiff technique (PAS) to demonstrate the presence ofPAS-positive carbohydrates. The measurements of the egg cylinders and the cavitieswere taken from longitudinal sections of Bouin-fixed, wax-embedded specimens.The egg cylinders were measured from the tip of the embryonic endoderm to apoint in the middle of the ectoplacental cone on the level at which the proximal anddistal extra-embryonic endoderm join. The length of the exocoelom and ectoplacentalcavity at their intersection with the same measuring line was recorded.To calculate the mitotic index of the cells in the chorions of embryos cultured in

media containing colchicine (see below), 5 ,um thick serial sections were cut parallelto the longitudinal axis of the egg cylinder and every fourth section was analysed.The number of arrested mitotic figures and the total number of nucleated cells in themesoderm layer and the ectoderm layer were counted and the mitotic index wascalculated as the number of arrested mitotic figures per 100 nucleated cells. Tensections were analysed from each embryo (except from some of the smallest in whichonly eight were analysed). The significance of the results was tested using Student'st-test. The index was not calculated for embryos which still had distinct amnioticfolds because of the very small number of cells and sections available for analysisand the difficulty of predicting which cells were destined to become chorion.

Sections 1 ,um thick were cut from the resin blocks prepared as for electronmicroscopy and were stained with 1 % toluidine blue in borax.

9-2

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S. K. L. ELLINGTON

Electron microscopyAdditional specimens were fixed in 3 % glutaraldehyde in phosphate buffer

(300 mosmol) for six hours, or overnight, at 4 'C. They were rinsed in buffer andspecimens for transmission electron microscopy were postfixed in 1% osmiumtetroxide in phosphate buffer for two hours at room temperature. The fixed materialwas dehydrated in ethanol and propylene oxide, and embedded in Araldite. Thinsections were stained in saturated uranyl acetate in 50 % ethanol and lead citrate(Reynolds, 1963).

Following glutaraldehyde fixation, specimens for scanning electron microscopywere thoroughly rinsed in buffer and either halved along their longitudinal axis or alarge window was made in the visceral yolk sac to expose the chorion. The specimenswere then postfixed in osmium tetroxide, dehydrated in ethanol, critical-point driedand gold coated prior to being viewed in a JEOL scanning electron microscope.

Conceptuses of less than 9 0 days are very difficult to dissect neatly because theyare so small. Twenty four embryos from each stage up to, and including, 9 0 dayswere dissected and the better dissections were preserved for scanning. A minimumof eight specimens from each stage (including the later stages) was examined byscanning electron microscopy.

CulturePreliminary experiments indicated that the development of the chorion in vitro

appeared similar in all respects to that occurring in vivo. In order to examine thedistribution of cell division in the chorion, embryo-culture techniques were used aspreviously described (Ellington, 1985) with the exception that in this present studythe culture medium contained 50 % Dulbecco's modification of Eagle's medium(Gibco) and 50 % serum, with 0-001 % colchicine. A total of 72 explants was culturedand eight embryos from each of the stages of pregnancy examined. All cultures wereterminated after two hours, the specimens were rinsed in saline, fixed in alcoholicBouin's and processed for light microscopy.

OBSERVATIONS

The development of the chorion was studied from its first appearance at theprimitive streak stage until its incorporation into the developing chorio-allantoicplacenta with the fusion of the allantois, chorion and extra-embryonic ectodermcells of the ectoplacental cone. During the earlier stages of its development, thechorion developed to form a diaphragm separating the exocoelom and the ecto-placental cavity. With further development the exocoelom enlarged, the chorioncame to lie closer to the ectoplacental cone and the ectoplacental cavity was initiallyreduced in size and finally disappeared altogether (Table 1). In this paper, thedevelopment of the chorion and the relevant changes of the exocoelom and theectoplacental cavity are divided into three stages: (i) formation of the chorion,exocoelom and ectoplacental cavity, (ii) enlargement of the chorion and exocoelom,(iii) incorporation of the chorion into the chorio-allantoic placenta. The developmentis summarised in Figure 2.

(i) Formation of the chorion, exocoelom and ectoplacental cavityDetailed accounts of the changes occurring during this phase in rat embryonic

development, as observed by light microscopy, have been given by Jolly &

250

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Table 1. The lengths ofegg cylinders, exocoeloms and ectoplacental cavitiesfrom 8.5 to 100 days

All measurements were taken from Bouin-fixed specimens sectioned parallel to the longitudinal axis of theegg cylinder.

Length of egg Length of Length of ecto-Age of embryos cylinder (jam) exocoelom (,um) placental cavity

(days) Number ofembryos mean ± S.E. mean± S.E. (im) mean± S.E.

8-50 6 529±24 -8 75 8 683±20 -9 00 5 853±23 -9-25 8 924±23 223±21 247±24950 6 1112±34 449+33 151±409 75 8 1277±42 609+76 45±1910 00 6 1965±118 719±32 2±2

Ferester-Tadie (1936). The present account will therefore concentrate primarilyon ultra-structural details.The youngest egg cylinders examined by transmission electron microscopy were

from 8 5 days rats at which stage the posterior and lateral amniotic folds projectedinto the proamniotic cavity causing a slight constriction in the cavity between theembryonic and extra-embryonic regions of the egg cylinder. The majority of themesoderm cells in the amniotic folds were irregular in shape with short, broadcytoplasmic processes extending into the extracellular space and sometimes ontoneighbouring cells. By contrast, the cells in the centre of the posterior amniotic foldhad longer and thinner cytoplasmic processes, some of which enclosed smalllacunae. The lacunae appeared to coalesce to form a single cavity, the rudiments ofthe exocoelom. No specialised intercellular junctions were observed betweenmesoderm cells at this stage of development. The cytoplasm of the mesoderm cellscontained numerous free ribosomes, a few small mitochondria with lamellatecristae, and short strands of rough endoplasmic reticulum. Light microscopy showedthe presence of a slight PAS-positive response in the cytoplasm of the mesodermcells at this stage of development.The mesoderm was separated from the proamniotic cavity by ectodermal cells

which were rounded or elongated cells forming a layer two or three cells deeparound the amniotic folds. The cells contained spherical vesicles about 3 ,um indiameter which, when viewed by light microscopy following staining by the PAStechnique, were shown to be intensely PAS-positive. The cell cytoplasm contained

Fig. 3. Transmission electron micrograph ofa mesodermal cell from the chorion of an 8-75 daysegg cylinder to show numerous free ribosomes, mitochondria and rough endoplasmic reticulum.Scale bar: 0-5 jim.Fig. 4. Transmission electron micrograph of regions of two adjoining ectoderm cells from thechorion of an 8-75 days egg cylinder to show numerous free ribosomes, small mitochondria andrough endoplasmic reticulum. Scale bar: 0 5 jm.Fig. 5. Light micrograph of a transverse section of a 9 0 days egg cylinder to show the develop-ing exocoelom and proamniotic tube. The mesoderm cells around the exocoelom are flattened,those in other parts of the amniotic folds are still rounded or irregular in shape. The innersurfaces of the amniotic folds fomi the proamniotic tube. Scale bar: 25 jim.

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numerous free ribosomes, small mitochondria, short strands of rough endoplasmicreticulum and membrane-bound vesicles, about 3 ,um in diameter, containing agranular, electron-dense material.By 8-75 days the exocoelom had enlarged in diameter and was also beginning to

lengthen, extending into the lateral amniotic folds. The mesodermal cells lining theexocoelom were slightly flattened especially on the surface bordering the exocoelom.The remaining mesodermal cells in the amniotic folds were widely dispersed andintercellular contact was uncommon. There appeared to be a slight increase in thenumber of organelles in the mesodermal cells compared to that observed in the8-5 days egg cylinders. The cytoplasm contained numerous free ribosomes, smallmitochondria, rough endoplasmic reticulum and, occasionally, small Golgi complexesin the perinuclear regions (Fig. 3). Some of the cells also contained electron-densevesicles about 1 ,um in diameter.The ectodermal cells appeared to be similar to those at 8-5 days with very

numerous free ribosomes, small mitochondria, a few short strands of rough endo-plasmic reticulum and intracellular vesicles (Fig. 4) but, in contrast to the observa-tions on 8 5 days old embryos, desmosome junctions were observed between theectodermal cells at 8 75 days.The amniotic folds and the contained exocoelom gradually enlarged, extending

laterally around the egg cylinder (Fig. 5) until a hollow annulus completely encircledit. The mesodermal cells lining the exocoelom became increasingly flattened but noultrastructural changes were observed. The ectodermal cells were similar to thoseobserved at earlier stages.As the exocoelom enlarged, the amniotic folds bulged further into the proamniotic

cavity until the proamniotic cavity became almost dumb-bell in shape with a narrowproamniotic tube connecting the embryonic and the extra-embryonic regions of thecavity.

(ii) Enlargement of the chorion and exocoelomBy 9 25 days almost all the embryos had distinct amnions and chorions dividing

the amniotic cavity, exocoelom and ectoplacental cavity (Table 1; Fig. 2). As theexocoelom enlarged the proamniotic tube became progressively thinner until itfinally ruptured (Fig. 6); in the majority of the 9-25 days embryos examined the pro-amniotic tube was still patent as a duct stretching between the amnion and thechorion. The mesoderm of the chorion formed a single layer of flattened cells, oftenno thicker than 5,/m, adjacent to the exocoelom (Fig. 7). (This layer was oftendifficult to see using light microscopy but could always be seen by use of electronmicroscopy.)At the ultrastructural level, the mesodermal cell contents were seen to resemble that

Fig. 6(a-c). Light micrograph of transverse sections of the proamniotic tube to show variousstages of its development. (a) Section from a 9 0 days egg cylinder in which the proamniotic tubeforms a thick duct connecting the amniotic and ectoplacental cavities, (b) and (c) are from9 25 and 9 5 days egg cylinders respectively; at 9-25 days the tube is much thinner but stillforms a patent duct, by 9 5 days it has begun to degenerate. Scale bar: 25 #sm.Fig. 7. Scanning electron micrograph of a 9-25 days egg cylinder dissected to show the pro-amniotic tube and the developing exocoelom. The layer of flattened mesoderm cells lining theexocoelom is conspicuous. Scale bar: 100 jtm.

254 S. K. L. ELLINGTON

Development of the rat chorion

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Table 2. The mitotic index of cells of the 'chorion' during its early developmentEmbryos were cultured for two hours in medium containing 0-001 % colchicine. The mitoticindex is calculated as the mean number of mitotic figures for 100 cells.

Mitotic index (mean ± S.E.)Age of embryos at start of A

culture (days) Number of embryos Ectoderm Mesoderm

9 25 8 17-7±1-9 8-6+2-2950 8 16-7+0-8 90±1419 75 7 30 9+2-0** 9-6±2-01000 7 19-0±1-7 8-1±1-510-25 6 12-0+1-3* 1-9±0-4t

* Significantly different from the mitotic indices of ectoderm at 9 5 and 10 0 days. P < 0 01.** Significantly different from the mitotic indices of ectoderm at all other stages examined. P < 0-001.t Significantly different from the mitotic indices of mesoderm at 9 5, 9-75 and 10-0 days. P < 0 01.

of earlier stage mesoderm except for an increased number of electron-dense intra-cellular vesicles (Fig. 8).The ectoderm cells still formed a layer two or three cells thick which was closely

applied to the mesodermal cells. The ectodermal cells were beginning to showpolarity with the nuclei tending to be nearer the mesoderm side of the cell and aslight accumulation of PAS-positive vesicles in the cytoplasm near the ectoplacen-tal cavity. The cells looked more active than at earlier stages with an increase inboth the size and the number of mitochondria and the number of electron-densevesicles (Fig. 9).The mitotic index of the ectoderm was about double that of the mesoderm

(Table 2).During the 12 hours following 9-25 days the length and diameter of the egg

cylinder increased, the exocoelom enlarged, the proamniotic tube became constrictedto a fine duct and finally broke (Figs. 2d, 6) and the ectoplacental cavity becamemuch smaller and occasionally was reduced to a small ring around the peripheralregion of the ectoplacental cone (Table 1). Since there was considerable variation inthe rate of these changes (Table 1) they will be described in chronological order butnot by reference to the age of the embryo.As the exocoelom enlarged and the chorion came closer to the ectoplacental cone,

the mesodermal cell layer in the central region of the chorion became less closelyattached to the ectoderm. Some of the mesodermal cells became rounded so that smallintercellular spaces were formed. The cytoplasm of the rounded mesodermal cellsappeared much more active than at the earlier stages, with an increase in the numbersof mitochondria, Golgi complexes and strands of rough endoplasmic reticulum.Many such cells, when observed by light microscopy, were seen to contain PAS-positive vesicles. In the peripheral regions of the chorion the mesodermal cells

Fig. 8. Transmission electron micrograph of a mesodermal cell from the chorion of a 9 25 daysegg cylinder to show the numerous ribosomes, small mitochondria, rough endoplasmic reti-culum and electron-dense vesicles. The cells form a very thin layer between the ectoderm andthe exocoelom. Scale bar: 0-5 ,Am.Fig. 9. Transmission electron micrograph of ectoderm cells from the chorion of a 9 25 daysegg cylinder to show mitochondria, rough endoplasmic reticulum, and intracellular vesicles.The cells are joined by desmosome junctions (shown at higher magnification in the inset).Scale bar: I gm (inset: 0 25 ,tm).

256

257Development ofthe rat chorion

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S. K. L. ELLINGTONremained flattened, closely packed and appeared less active. The mesodermal cellscontinued to divide and the mitotic index was similar to that of the 9-25 days oldembryos.The ectodermal cell layer initially became thinner until only a single layer of cells

remained, then, shortly before the chorion made contact with the ectoplacental cone,

there was a sudden increase in the mitotic index (as observed in the embryos ex-planted at 9*75 days - Table 2). With the increase in cell numbers the ectodermallayer of the chorion once again became more than one cell thick and also formedinvaginations extending into the ectoplacental cavity and towards the ectoplacentalcone. With further increase in the number of ectodermal cells in the chorion, theectoplacental cavity was reduced in size until the central region of the chorion cameto lie against the ectoplacental cone. No ultrastructural changes were observed inthe ectodermal cells themselves.

(iii) Incorporation of the chorion into the chorio-allantoic placentaBy 10-0 days the ectoplacental cavity was usually reduced to a ring extending

around the peripheral regions of the developing chorio-allantoic placenta. Themesodermal cells in the central region of the chorion continued to change. As thecells began to round up, small extracellular spaces appeared. These extracellularspaces increased in size as the cells became more rounded until, in the central regionof the chorion, quite large areas of the chorion appeared to be devoid of theirmesodermal layer (Fig. 10). Thus the ectodermal cells, with their PAS-positiveextracellular coat, were exposed to the exocoelom and the tip of the allantois. Thecytoplasm of the rounded mesodermal cells contained numerous mitochondria,Golgi complexes and short strands of dilated rough endoplasmic reticulum; somecells contained intracellular vesicles of about 1 gm in diameter. The cell cytoplasmand the intracellular vesicles stained intensely with PAS. The flattened mesodermalcells in the peripheral regions of the chorion contained many fewer organelles andPAS-positive vesicles. The mitotic index was similar to that of the earlier stages(Table 2).

After the initial fusion of the central region of the chorion and the ectoplacentalcone, the area of contact extended laterally towards the peripheral parts of thechorion. The ectodermal cells of the chorion became columnar and the cell surfaceadjacent to the mesodermal cell layer became covered by a thin layer of PAS-positive extracellular material. Desmosome junctions became restricted to the cellmargins abutting the exocoelom and long convoluted extracellular spaces wereformed between the remaining parts of the cells (Fig. 11). There was a reduction inthe mitotic index of the chorionic ectodermal cells (Table 2).

Following the fusion of the ectodermal cells of the chorion with those of theectoplacental cone, the mesodermal surface of the chorion fused with the allantoiswhich, in the rat, is also mesodermal in origin (Amoroso, 1952; Ellington, 1985).By 10-25 days the allantois was in contact with the chorion in all the embryos

examined. In most embryos the initial contact between the allantois and the chorionwas in the central region of the chorion. In a few embryos (about 20 %) the allantoisfirst came into contact with a peripheral region of the chorion then continued togrow parallel to the surface of the chorion until it made contact and fused with thecentral region. The allantois was never seen to fuse with a region of chorion coveredby flattened mesodermal cells.Once the allantois had come into contact with the central region of the chorion it

258

Development ofthe rat chorion

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Fig. 10. Scanning electron micrographs of the surface of the chorion from a 9 5 days embryo.The inset is a micrograph taken at low magnification to show the flattened mesodermal cells atthe periphery of the chorion and the gradual change in cell morphology nearer the centre of thechorion. The main micrograph is of the central region of the chorion showing the roundedmesodermal cells and the exposed ectodermal cells. Scale bar: 0 5 8sm (inset: 10 ,sm.)

259

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260 S. K. L. ELLINGTON

Development ofthe rat chorion 261became impossible to distinguish between the rounded mesodermal cells from thetip of the allantois and those of chorionic origin (Fig. 12). Both contained mito-chondria, Golgi complexes, short strands of rough endoplasmic reticulum and smallelectron-dense vesicles (Fig. 13). The cytoplasm and intracellular vesicles werePAS-positive.The layer of flattened mesodermal cells in the peripheral regions of the chorion

became continuous with the layer of flattened cells encasing all but the tip of theallantois (Fig. 12). After the fusion of the chorion and allantois there was a significantreduction in the mitotic index of the chorionic mesodermal cells (Table 2). Fewmitotic figures were seen in the region of contact between the two structures.The ectodermal cells of the chorion appeared to be less active than at earlier stages

after the allantois had fused with the chorion. There was a decrease in the size andnumber of mitochondria and few other organelles were seen. The cells were joinedby desmosome junctions on the margins abutting the exocoelom but specialisedjunctions were not observed elsewhere. There was extensive extracellular space. Themitotic index was significantly lower than at 10 0 days (Table 2).

DISCUSSION

The above account describes in detail the morphological development of the ratchorion from its formation from the extra-embryonic region of the amniotic foldsuntil its fusion with the ectoplacental cone and allantois prior to the development ofthe chorio-allantoic placenta.

There is very rapid growth of both embryonic and extra-embryonic structuresduring the early post-implantation period. Since the chorion extends across the eggcylinder, increases in the diameter of the cylinder necessitate concurrent increasesin the size of the chorion. Growth of the chorion is probably a result solely of celldivision and cell growth within the chorion; there is no evidence of cell migrationinto the chorion from surrounding tissues.During its development the chorion moves relative to the other extra-embryonic

structures, gradually coming to lie against the ectoplacental cone. Following the de-velopment of the chorion as an intact diaphragm across the egg cylinder and theclosure of the proamniotic tube, the chorion becomes bowed, projecting into theectoplacental cavity and towards the ectoplacental cone. With increasing develop-ment, the chorion becomes more and more concave with respect to the exocoelomuntil, shortly before it fuses with the ectoplacental cone, it comes to lie against theextra-embryonic ectoderm lining the walls of the ectoplacental cavity. The con-figuration of the chorion coupled with the absence of any inherent rigidity in the

Fig. 11. Transmission electron micrograph of ectodermal cells from the chorion of a 10O25 daysconceptus to show the columnar cells with long, convoluted, extracellular spaces. The cellcytoplasm contains small mitochondria but few other organelles. Scale bar: 05,um.Fig. 12. Light micrograph to show the fusion of the allantois and chorion at 10-25 days. Themesodermal cells of the chorion are flattened in the peripheral regions but more rounded in thecentre of the chorion. The allantois has fused with the central part of the chorion. Scale bar:100 ,sm.Fig. 13. Transmission electron micrograph of part of a mesodermal cell from the area betweenthe allantois and the chorion to show the abundance of Golgi complexes, mitochondria andrough endoplasmic reticulum. Scale bar: 05 t%m.

chorion (such as extracellular fibres) is indicative of a relatively high hydrostaticpressure in the exocoelom compared to that in the ectoplacental cavity. The highincidence of desmosomes between the ectoderm cells of the chorion, prior to itsfusionwith the ectoplacental cone, would afford mechanical strength to maintain theintegrity of the ectoderm cell layer.The changes occurring in the mesodermal layer of the chorion are initiated before

the chorion fuses with either the ectoplacental cone or the allantois; it would there-fore appear not to be caused by the direct interaction of either of these tissues withthe chorion. The mitotic index of the mesodermal cells is considerably lower thanthat of the ectodermal cells (Table 2) and the mesodermal cells become increasinglythin as the chorion enlarges. The disruption of the mesodermal cell layer may be inresponse to mechanical forces as the layer becomes thinner.As the mesodermal cells round up, the surface of the ectoderm is exposed to the

approaching allantois. The allantois was only seen to fuse with areas of chorion inwhich the ectodermal cells, and their extracellular carbohydrate layer, were exposed.It is suggested that the glycocalyx on both the allantois (Ellington, 1985), and theectodermal cells of the chorion may be important in the fusion of the two tissuesperhaps by facilitating their initial adhesion.

Prior to the fusion of the chorion and the allantois, the allantois appears to befilled with fluid, perhaps accumulating embryonic metabolic waste. Following thefusion there is a rapid decrease in the diameter of the allantois and a reduction inthe extra-embryonic spaces within the allantois (Ellington, 1985). The structure of theectodermal cell layer of the chorion also changes. The desmosomes become restrictedto the apical surfaces, there is a widening of the extracellular spaces and a decreasein the number of mitochondria. The structure of this cell layer resembles that ofepithelia involved in the flow of water from one compartment to another (as seen, forexample in the collecting duct of the kidney) and suggests that, at this stage ofdevelopment, the chorion may be of importance in the transport of water from theembryo to the maternal vascular system.

SUMMARY

The morphology of the developing chorion of the rat has been studied from itsinitial formation from the extra-embryonic region of the amniotic fold until itsfusion with the ectoplacental cone and allantois prior to the development of thechorio-allantoic placenta (that is from 8 25 to 10-25 days of gestation).The gross structural changes, the mitotic indices, the ultrastructure and the

distribution of carbohydrate in the mesodermal and ectodermal cells of the chorionhave been studied throughout this period of development.The chorion developed to form a diaphragm across the egg cylinder, separating

the exocoelom from the ectoplacental cavity. With further development, the ecto-placental cavity became smaller until the chorion was lying against the ectoplacentalcone, to which it fused.The mesodermal cells of the chorion formed a single layer of cells covering the

ectodermal cells. Shortly before the fusion of the chorion with the ectoplacental coneor the allantois, the integrity of the mesodermal cell layer in the central region of thechorion was disrupted. Intercellular contact was lost and the mesodermal cellsrounded up exposing the PAS-positive extracellular coat of the ectodermal cells.The allantois was only seen to fuse with the chorion in regions in which the ecto-

262 S. K. L. ELLINGTON

Development of the rat chorion 263dermal cells were exposed. It was suggested that the glycocalyx of the ectoderm cellsmay be of importance in the fusion of the allantois and chorion.

I thank Dr D. A. T. New and Professor M. H. Kaufman for their encouragementand advice. I am grateful to the Medical Research Council for financial support.

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AMOROSO, E. (1952). Placentation. In Marshall's Physiology of Reproduction (ed. A. S. Parkes), vol. 2,pp. 127-311. London: Longmans, Green & Co.

BONNEVIE, K. (1950). New facts on mesoderm formation and proamnion derivatives in the normal mouseembryo. Journal ofMorphology 86, 495-545.

ELLINGTON, S. K. L. (1985). A morphological study of the development of the allantois of rat embryosin vivo. Journal ofAnatomy 142, 1-1 1.

JOLLY, J. & FiRESTER-TADIP, M. (1936). Recherches sur l'oeufdu rat et de la souris. Archives d'anatomiemicroscopique 32, 323-390.

NEW, D. A. T. (1971). Methods for the culture of post-implantation rodents. In Methods in MammalianEmbryology (ed. J. C. Daniel), pp. 305-319. San Francisco: W. H. Freeman & Co.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron micro-scopy. Journal ofCellBiology 17, 208-211.

SNELL, G. D. & STEVENS, L. C. (1966). Early embryology. In The Biology of the Laboratory Mouse, 2nded. (ed. E. L. Green), pp. 205-245. New York, Toronto, Sydney, London: McGraw-Hill.

STEVEN, D. & MoRRiss, G. (1975). Development of the foetal membranes. In Comparative Placentation.Essays in Structure and Function (ed. D. H. Steven), pp. 58-86. London, New York, San Francisco:Academic Press.

THEILER, K. (1972). The House Mouse. Development and Normal Stagesfrom Fertilization to 4 weeks ofAge. Berlin, Heidelberg, New York: Springer-Verlag.