developmental stages embryo-like bodies in ...j. clin. path. (1957), 10, 31. developmental stages of...

J. clin. Path. (1957), 10, 31.

DEVELOPMENTAL STAGES OF EMBRYO-LIKE BODIESIN TERATOMA TESTIS

BY

R. WINSTON EVANSFromn the David Lewis Northern Hospital, Liverpool

(RECEIVED FOR PUBLICATION JULY 11, 1956)

Small bodies, comparable in their morphologywith that of the early developmental stages ofmammalian embryos, are observed uncommonlyin certain testicular teratomata (Melicow, 1940;Dixon and Moore, 1953; Evans, 1956); andalthough an enormous number of these embryo-like structures may be scattered haphazardlythroughout a teratomatous growth, it is onlyrarely that such bodies (Figs. 2, 5) mimic closelythe form of normal early human embryos (Fig. 1).

In the case to be described numerous embryoidbodies were present in many areas of a testicularteratoma and many of these bore a remarkableresemblance to normal early primate embryos.The mimicry was so extraordinary that it was

considered worth while studying a few of theseembryo-like structures in detail in serial sectionsso that their anatomy could be compared withthat of normal presomite mammalian embryos ofcorresponding morphological stages of develop-ment. An attempt was made also to trace thevarious ontogenetic phases in the hope of gather-ing some information relevant to the evolution andultimate fate of these structures.

Clinical Note and Details of SpecimenThe testicular teratoma was removed by Mr.

Philip Hawe from a patient aged 41 years; thegrowth had been present for six months. Fiveyears after operation the man was alive and well,exhibiting no clinical evidence of recurrence or

metastases.The tumour formed an ovoid mass measuring

6 cm. by 5 cm. by 5 cm. in its main axes. Its cutsurface presented a variegated appearance withmany small cystic spaces and glistening, blue,translucent foci of cartilage.

Histologically, apart from numerous embryo-like structures, there were areas of tumourexhibiting well-differentiated cartilage, smoothmuscle, nerve tissue, large and small epithelial

tubules, glandular components and cystic spaceslined by cuboidal and sometimes columnar epi-thelium. There were also pilo-sebaceous units,cornifying structures, and primordia of enamelorgans. In addition, undifferentiated embryoniccells were observed and these, for the most part,were arranged in sheets and cords. Foci of cyto-trophoblastic and syncytiotrophoblastic differen-tiation also were noted.No hormone determinations were carried out.

Anatomically Well-formed TriploblasticEmbryoid Bodies

In well-formed embryoid bodies structures canbe discerned easily which resemble, both in formand relation to one another, the amniotic cavity,embryonic disc, intra-embryonic mesoderm andyolk sac, and gut endoderm of a normal presomitehuman embryo (Fig. 1).Four such morphologically well-formed embryo-

like structures were followed in series, and con-secutive sections of each embryoid body werephotographed at a constant magnification. Nomarkers were inserted into the paraffin blocks, butarbitrary points and lines were selected on thephotomicrographs so that rough graphic represen-tations of the embryoid bodies could be con-structed. The sections were cut at 5 microns.The overall shape of the embryoid bodies varied

from approximately spheroidal to globular andpyriform; sometimes an embryoid body wascylindroid in outline. Such bodies varied also insize and their calculated main axes are given in theTable. Although in this table measurements in two

TABLE

Embryo Height (mnl.) Width (mnm.) Length (mm.)

A 0-7 0-35 0-30B 0-28 0-28 0-30C 0 41 0-18 0-21E 0-36 0-30 0-40

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R. WINSTON EVANS

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FIG. 1.-Presomite-human embryo, age approximately 17-18 days.Section is through Hensen's node. The embryonic plate, due toa fixation artefact, is dislocated from the gut endoderni. Theintra- and extra-embryonic mesoderm is in continuity andangioblastic tissue is present on the wall of the yolk sac.

Haematoxylin and eosin x 180.

planes are listed as width and length the terms are

used in a descriptive sense and have no anatomicalsignificance, for no cephalo-caudal orientation ofthese embryoid bodies is possible; the valuesgiven for the main axes serve only to indicate thegeneral contours and the variations which occur

in the size and shape.A chorionic membrane with villi is not distin-

guishable as such, but adjacent to the " amnion"or "yolk sac," or both, of these embryoid bodiesa few syncytial giant cells are sometimes seen (Figs.7, 10). Frequently such a body terminates in astructure which may be interpreted as a type of"body stalk" (Figs. 10, 11); this is circular or

oval in cross-section and composed of concentric-ally disposed spindle cells often arranged looselyto impart a characteristic appearance. Within or

immediately adjacent to this structure cytotropho-blastic and syncytiotrophoblastic differentiationoccurs in small foci; also within this distinctivecorpuscle a tubule is frequently included.

The homologue, in embryoid bodies, of the yolksac and gut endoderm of normal early embryosvaries in size and shape. Commonly it extendsbeyond the limits of the " embryonic plate " andis often continued within the stalk-like terminalcorpuscle, sometimes as a narrow duct (Fig. 8) butnow and again as a dilated flask or sausage-shapeddiverticulum (Figs. 13, 15, 16). The narrow ducttype of prolongation may be considered as homo-logous with the primate type of allantois, but thedilated form of outpouching corresponds more tothe allantois of carnivores and ungulates.Sometimes the yolk-sacs of two neighbouring

embryoid bodies communicate with each otherthrough a small opening; at other times twoembryo-like structures share a common " yolksac" (Fig. 9).The yolk sac of embryoid bodies may be com-

posed completely or partially of flattened,cuboidal, or columnar cells, and these are disposedgenerally as a single layer. When, however, sucha yolk sac protrudes beyond the limits of theembryonic plate there is a tendency for the cellsto become vacuolated and arranged in two ormore layers; especially toward the blind end ofthe outpouching, representing the allantois, endo-dermal cells may hypertrophy and proliferate insolid masses (Fig. 15).Although the gut endoderm is usually sharply

demarcated from the embryonic plate and meso-derm (Figs. 2, 3, 4, 6), at various points it appearsto mingle imperceptibly with the latter (Figs. 5,12, 13). In one embryoid body (Fig. 6) a patch ofthickened endoderm forms a conspicuous featurein the roof of the yolk sac and may be consideredas homologous with the primordium of the pro-chordal plate of the normal embryo. However, nostructure corresponding to a head process couldbe identified at either extremity of this abnormalprochordal plate.An amorphous coagulum (Fig. 9) and, infre-

quently, a few isolated desquamated cells areformed within the yolk-sac. Angioblastic tissuewas not noted in association with the yolk sac.The amnion of embryoid bodies is dome-shaped

or bell-like (Figs. 2, 6, 7) and shows much variationin size. Whereas sometimes the transition isgradual, in most cases, as in the normal presomiteembryo, the amnion arises abruptly from themargins of the embryonic plate (cf. Figs. 1 and 2)and apparently consists of a single ectodermallayer, for any delicate external mesodermallayer is difficult to perceive and distinguish fromthe layers of mesoblastic cells which in concentricmanner invest closely the whole embryoid body.

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FIG. 4

FIG. 2.-Well-formed, reasonably symmetrical embryoid body show-ing a large amniotic cavity (Am.), an obliquely positionedembryonic plate (Em.P.), intra-embryonic mesoderm (Mes.),yolk sac (Y.S.), and gut endoderm (End.). The section is throughthe region of the " primitive streak." Haematoxylin and eosinx 104.

FIG. 3.-This section is 250 microns removed from the previous figure.A diverticuluni is developing in the roof of the yolk sac and gutendoderm in relation to an abnormally situated nodal prolifera-tion in the embryonic plate. In this section the amnion isobviously double layered. Haematoxylin and eosin x 104.

FIG. 4.-Detail of Fig. 2 x 250.

Uncommonly, as in Fig. 3, a double layer isobvious and the external mesodermal membraneis well marked. The "amnion" is composedusually of flattened cells, but sometimes cuboidaland columnar cells, frequently of clear aspectR aredeveloped also, often in a patchy manner (Figs.12, 13, 14).The embryonic disc varies in its configuration in

different embryoid bodies; in some it is,,reason-ably symmetrical and convex about and inrflie twoupright planes, whereas in other cases no sym-metrical pattern is maintained and this asymmetryin the position of the embryonic plate is reflectedby a corresponding asymmetry of the yolk sac andgut endoderm (Fig. 3).

EMBRYOID BODY AX

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FIG. 2

FIG. 3

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EMBRYO B

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EMBRYOID BODY C

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EMBRYOID BODY D

4^'~~~~~~~~>2FIG. 9 FiG. II

FIG. 5.-Section through region of " primitive streak." The endo-derm is well separated from the ectoderm except at the right-hand side ofthe figure. The mesoblastic cells possess cytoplasmicprolongation. Haematoxylin and eosin x 230.

FIG. 6.-Section 50 microns from Fig. 5 shows a thickening in theroof of the yolk sac. ? homologue of prochordal plate (X).Haematoxylin and eosin x 230.

FIos. 7 and 8.-Small embryoid body near " body stalk" endshowing syncytial giant cells in association with arnnion andyolk sac. Haemnatoxylin and eosin x 126.

FIG. 9.-Embryoid body containing two embryonic plates and acomnlon yolk sac. Haematoxylin and eosin x 130.

FIG. 10.-" Body stalk " of embryoid body showing extension intoit of yolk sac. Haematoxylin and eosin x 130.

FIG. 11.-Vacuolated syncytial giant cells in " body stalk." Haema-toxylin and eosin x 130.

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EMBRYOID BODY E

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E-MBRYOID BODY G

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EMBR YOID BODY F

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DEVELOPMENT OF EMBRYOID BODIES IN TERATOMA TESTIS

EMBRYOID BODY H

FIG. 16

FIG. 12.-Embryoid body cut through region of " primitive node andstreak." Haematoxylin and eosin x 175.

FIG. 13.-Extension of yolk sac of same embryoid body as in previousfigure as a large " allantois " of the carnivore type. This largeendodermal outpouching is not included in the measurements ofthe main axis of this embryoid body. Haematoxylin and eosinx 120.

FIG. 14.-Embryoid body showing continuity of intra- and extra-embryonic mesoderm. Haematoxylin and eosin x 250.

FIG. 15.-Embryoid body showing extension of yolk sac into " bodystalk " as a large type of allantois. At X there is a homologueof the cloacal membrane. Haematoxylin and eosin x 250.

FIG. 16.-Embryoid body showing extension of the endoderm to linea large blastocystic cavity. Haematoxylin and eosin x 120.

FIG. 17.-Embryonic plate showing typical primitive streak tissue.Haematoxylin and eosin x 180.

The shield consists of cuboidal and columnarcells staining darkly with their oval or elongatednuclei generally arranged in a stratified fashion togive the impression of two or more rows of cells(Figs. 12, 17). For the most part the cells are setat right angles to the free surface of the shield,but in many planes and instances their long cells

EMBRYOID BODY I

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are arranged obliquely. The oblique dispositionof cells and their nuclei is particularly evident inlocations where the shield thickens to form anorganization centre composed of tissue homo-logous with that of Hensen's node and primitivestreak (Figs. 12, 14, 17).

In one embryoid body, which appears to be cutalmost in a longitudinal plane, the endoderm of theallantois comes into continuity with the ectodermof the amnion immediately at one end of theembryonic plate, and this connexion can be regardedas a homologue of the cloacal membrane. Such atransition from primitive streak to " cloacal mem-brane" is shown in Fig. 15.

Intra-embryonic mesoderm from one to four ormore cells thick is present and obvious in mostwell-formed embryoid bodies. These mesodermalcells are packed closely together or connected toone another by branching processes (Figs. 4, 5).At the peripheral margins of the shield they maybe seen in some specimens to extend in continuitywith the mesodermal cells surrounding theembryoid body (Fig. 14). No doubt, as in normalhuman early embryos, some of the intra-embryonic

37



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R. WINSTON EVANS

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FIG. 18.-Embryoid body at innercell mass stage showing earlyformnation of amnion. The delanlinated endoderm extends toIline the blastocystic caevity. Haematoxylin and eosin x 250.

FIG. 19.Sections showing early development of amnion andexocoeloniic membrane. Haematoxylin and eosin x 126.

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mesoderm is formed by the spreading inwards ofextra-embryonic mesoderm.The elements of the middle layer sometimes fill

completely the interval between the embryonicshield and the gut endoderm (Fig. 4), and in manyembryoid bodies the mesodermal cells appear toissue forth from the deep surface of the embryonicdisc. In some embryoid bodies an intimate rela-tion exists. between the mesoderm and yolk sacendoderm, indicating that the latter is also furnish-ing mesodermal elements.

Diploblastic Embryoid BodiesNot infrequently structures were discovered

which resembled the blastocystic and inner-cellmass stage of normal primate embryos. Such earlyembryoid bodies are depicted in Figs. 18 and 19;Fig. 18 in particular shows the early formationof an amniotic cavity and the delamination of theendoderm. The delaminated endoderm spreadsquickly to line the blastocvstic cavity, which is arelatively large sac.

Carcature Triploblasc Embryoid BodiesApart from well-formed, embryo-like structures

there are bodies which, because of atypicalmorphology, appear to be caricatures of embryoidbodies, and as such their nature is not readilyrecognized (Figs. 20, 21). Despite distortions inmorphology the occurrence of two vesicular struc-tures in association with one another and evokinga mesodermal reaction permits their identificationas homologues of early embryos. Sometimes theembryonic plate overflows down and along thewall of the yolk sac forming an additional amni-otic cavity in the immediate vicinity of the latter.Further secondary differentiations, evocations, and

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FIG. 20.-Caricature embryoid bodies; there are four in this field.Haematoxylin and eosin x 108.

inductions follow the spread outside the embryoidbody not only of the ectodermal plate but also ofthe yolk sac and gut endoderm including the allan-tois. On other occasions ectodermal plate cellsand mesodermal cells proliferate haphazardlywithout the formation of a shield and amnioticcavity despite the presence of a well-formed yolksac and gut endoderm.

The Development and Fate of EmbryoidBodies

That embryoid bodies in the testes developthrough stages comparable with those of normalprimate embryos is obvious from a considerationof the different ontogenetic stages found in thisspecimen of testicular teratoma and are shown insequence in Figs. 18, 19, 20. Stages earlier thanthe inner-cell mass phase are perhaps also presentin this tumour, but because of difficulty in inter-pretation none has been identified.The minimum age of the tumour is six months,

yet the oldest well-formed embryoid body corre-

sponds, from a morphological standpoint, with a

normal human embryo of not more than 18 daysof age (Fig. 1). Later developmental phases, as

complete entities, were not found in the teratoma,

FIG. 21.-Embryoid body with two amniotic cavities. Haematoxylinand eosin x 150.

and it is reasonable therefore to assume that whilenew embryos are being formed others disappearby losing their identity. The older embryoid bodiesare obliterated by the ingrowth of mesoderm, bythe over-development of a part, notably the yolksac, gut endoderm, and allantois, and by the un-coordinated elaboration of ectodermal, endo-dermal, and mesodermal structures anddifferentiations. Such a fate may be due to lethalpoint mutations or to chromosomal aberrationsas well as to the abnormal mode of origin andenvironment. Some light may be shed on environ-mental factors by transplantations of such tumourtissue into male and female rats and noting iffurther developmental stages of embryoid bodies,as complete entities, can be obtained.

SummaryThe morphology and fate of embryo-like

structures have been described in a teratoma testisand the various ontogenetic phases compared withthose of normal presomite embryos.

REFERENCESDixon, F. J., and Moore, R. A. (1953). Cancer, 6, 427.Evans, R. Winston (1956). Histological Appearances of Tunours,

p. 694. Livingstone, Edinburgh.Melicow, M. M. (1940). J. Urol. (Baltimore), 44, 333.



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