Electron microscopy of an early implantation stage, with a postulated mechanism of implantation

DEVELOPMENTAL

Electron with

BIOLOGY

10,

395-410

Microscopy a Postulated

(1964)

of an Early Mechanism

Implantation Stage, of Implantation’

ALLEN C. ENDERS Depcrrtment

of Anutomy,

Washington

University

S&no1

of

Medicine,

St. Louis, Missouri Accepted

August

17, 1964

INTRODUCTION

Implantation is a sequence of events starting with an unattached intrauterine blastocyst and resulting in an attached blastocyst with an altered relationship to the endometrium. The individual events in the sequence vary with the species and are, in the majority of instances, inadequately known. In species in which a hemochorial placenta is eventually established, penetration of the luminal epithelium is a relatively early and necessary event. However, the mechanisms by which the trophoblast comes to pass through the epithelium into the stroma of the endometrium have not been determined, although several hypotheses have been presented. It is the purpose of this study to report some unique observations concerning implantation in the armadillo, and to postulate a possible mechanism of implantation which has received little or no attention. Implantation in the armadillo is more readily compared to implantation in the primates than to implantation in members of other orders with hemochorial placentas. The single blastocyst of the armadillo implants in the fundic tip of the uterus simplex (Patterson, 1913). The trophoblast penetrates the epithelium and spreads out in the endometrium (Enders, 1960). It penetrates more deeply than does the trophoblast of the macaque, does not form a trophoblastic plaque, and shows no tendency to form a placenta on the abembryonic side. On the other hand, the embryonic cell mass is more superficial than in the human, ‘This study Foundation.

was

supported

by

grant 395

GB-1166

from

the

National

Science

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ALLES

C.

ENDERS

and no decidua capsularis is formed over the conceptus. In the postimplantation development, placental formation differs more markedly from the primates, in that the villi of the hemochorial placenta penetrate maternal blood spaces rather than forming intratrophoblastic lacunae, and an early yolk sac inversion occurs. A chronological description of the events in implantation in the armadillo has been published recently ( Enders, 1963). MATERIALS

AND

METHODS

In the course of eight years of studying reproduction in the armadillo, we have collected more than 200 blastocysts and over 50 early implantation sites (presomite stage). During the last two years, early implantation sites were sought specifically for examination with the electron microscope both by collections from the wild population and by inducing implantation (Buchanan et al., 1956; Enders and Buchanan, 1959). The earliest stages obtained other than the specimen reported here were two blastocysts that became detached during the processing; they showed intermediate features between unattached blastocysts and our earliest attached stages in which invasion of the endometrium had already occurred. The specimen that forms the subject of this paper appeared on examination with a dissecting microscope to be an unattached blastocyst, but did not float free of the endometrium on immersion of the endometrium in saline. The immediate area of endometrium surrounding the blastocyst was excised with the aid of a dissecting microscope and was placed together with the adhering blastocyst in 1.2% potassium permanganate in 0.9% sodium chloride. Following fixation, the material was dehydrated rapidly in ethyl alcohol and embedded in epoxy resin. Sections were taken from all parts of the specimen. As many sections as possible were obtained from the region of closest association. Since well over 100 sections from the most crucial region were examined, we are confident that all the endometrial cells in close proximity to’ the trophoblast were studied. DESCRIPTION

The blastocyst was found to be less than 0.3 mm across and to lie on the luminal epithelium at a slight angle. The embryonic cell mass was close to the epithelium, with a region of the trophoblast at the

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margin of the embryonic cell mass in particularly close association to the epithelium (Fig. 1). The rest of the blastocyst, including almost all the abembryonic trophoblast, was free in the uterine lumen (Fig. 2). On the side away from the uterine epithelium, the blastocyst was slightly indented. It otherwise appeared undistorted. Within the region of close association, the intimacy of the relationship between the trophoblast and the endometrial epithelium varied. Over much of the surface, the cells were closely apposed, but otherwise little altered

FIG. 1. Phase micrograph of the blastocyst at the edge of the region where it was attached to the endometrium. The trophoblast cells in close association with the uterine epithelium are at the margin of the embryonic cell mass, which almost completely fills the blastocyst at this end. Note that the section is somewhat tangential to the endometrium. Magnification: x 400.

(area of close apposition). Some of the trophoblast cells were very closely applied to the uterine epithelium (area of adhesion), and in one spot some of the uterine epithelial cells had ruptured (area of rupture). Where the blastocyst was in close proximity to the uterine epithelium but not directly apposed to it, the normal characteristics of the cells of the respective tissues could be seen (Figs. 3 and 5). The surfaces of the luminal epithelial cells were conical, with irregular projections and

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small microvilli (60-80 rnp in diameter). Some of the cells in this region were ciliated. The large size of the cilia makes their identification obvious, despite the fact that only a trace of the internal fibrillar pattern could be seen in this permanganate-fixed preparation. (Cilia are characteristic of osmium-fixed preparations of the armadillo uterus,) The trophoblast cells were elongated, with a convex outer surface following an even contour, except for the presence of numerous, rather uniform microvilli (85 rnp in diameter) (Figs. 4 and 5).

FIG. 2. Phase micrograph of the ahembryonic trophoblast. This end of the blastocyst was free in the uterine lumen. Note that the blastocyst is slightI> collapsed and that the huninal epithelium is sectioned in a more normal plane than in the previous micrograph. Magnification: x 400.

None of the trophoblast cells were ciliated. A line drawn along the distal ends of the projecting microvilli averaged 410-600 rnp from the basal ends of these structures. In the area of apposition, the endometrial cells tended to follow the contour of the trophoblast cells (Fig. 6). Since the trophoblast cells are 4-5 times the width of the epithelial cells, the concave cups formed by the epithelial cells were 4-5 cells wide. In the areas of FIG. 3. Luminal epithelium of the uterus peripheral to the blastocyst. Note the irregular surface of the cells. The large projecting structures in the upper right are cilia. The internal structure of cilia is more readily apparent in osmium-fixed material. Magnification: X 11,000.

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closer association (area of adhesion), the distance between the two surfaces was only 200400 m p. This distance was less than the average distance (410-600 mp) of the distal tips of the microvilli from the convex general surface of the trophoblast in unassociated areas. As might be expected in such close association, the free space between the two cell surfaces was largely obliterated by the commingled micro-

FIG. 4. A trophoblast cell away from the site of implantation. of the microvilli along its free surface. Magnification: x 8000.

Note the height

villi (Fig. 7). Where the epithelial cells were ciliated, the space between the two surfaces was approximately l?h times the width of the cilia. In other words, the cilia were almost completely flattened between the two surfaces. At one spot in the region of close apposition, several of the epithelial cells in contact with two trophoblast cells had disrupted apical ends, with granular material that was apparently cytoplasm extending beFIG. 5. epithelium. the uterine trophoblast Magnification:

Margin of the area of attachment of the blastocyst to the uterine Note that the apical ends of the luminal epithelial cells projecting into lumen at the right are conical. In this and in subsequent figures, the is at the top, and the uterine epithelium at the bottom of the picture. X 6200.

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FIG. 6. Parts of two trophoblast cells are seen apposed to the uterine epithelium. Note the smooth contour of the trophoblast cells against the uterine epithelium and that the apical ends of the epithelial cells conform to this contour. To the left in the cleft between the epithelia are a few cilia from the uterine epithelial cells (arrow). Magnification: x 10,200.

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FIG. 7. In this micrograph, the trophoblast is especially closely apposed to the uterine epithelial cells. The commingled microvilli occupy most of the interposed space. Magnification: x 12,300.

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FIG. 8. This is a section of the area in which the apical ends of the uterine epithelial cells have been ruptured. The trophoblast is intact and appears to be unaltered, as is the rest of the epithelial cell cytoplasm. Note that substance from the cells extends into the interposed space (arrow), Magnification: x 7,920.

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tween the two surfaces (Fig. 8). In this area of rupture the discontinuities in the individual epithelial cells were not simple membrane discontinuities, but involved displacement of portions of the apical cytoplasm. The proximal and perinuclear regions of the endometrial cells appeared unaltered, as did the overlying trophoblast cells. INTERPRETATION

OF OBSERVATIONS

The armadillo has a uterus simplex, implantation occurring at the fundic tip. The blastocyst does not expand to the extent that it could be pushing against folds of endometrium opposite the abembryonic trophoblast. The association of the trophoblast with the epithelium must, therefore, be an active one rather than a passive one. In addition, the distance between the apposed surfaces of the trophoblast and uterine epithelial cells is closer than would be anticipated in a simple contact situation. Furthermore, in some places at the margins of the implantation site, the uterine epithelial cells appeared stretched toward the trophoblast cells by their adhering apical ends (Fig. 5). It is particularly interesting that it is the endometrial cells that take on the contour of the blastocyst cells, and not the reverse. This could be due in part to the turgor (reestablished?) of the blastocyst, although the blastocyst is slightly indented, or to a relatively greater rigidity of the trophoblast cells, as opposed to the endometrial cells. The latter seems more likely, since the trophoblast cells show little indication of individual indentations from the endometrial cells. The rupture of the endometrial cells occurred not at the margin, but toward the center, of the region of close association. The endometrial cells were broken prior to fixation, in that the cytoplasm streams toward the trophoblast; that is, it was fixed in this position. The observed rupture, therefore is not a simple membrane discontinuity, which is a frequent fixation artifact. A few membrane discontinuities were observed elsewhere in the preparation, but only at this site was there any evidence of displacement of protoplasm or loss of apical ends of the cells. The position of the break in cell membranes and of the streaming just mentioned indicates that this disruption was probably not an artifact of handling the site. However, even should the break have occurred in handling, it would nevertheless serve to illustrate that the trophoblast cells are resistant to mechanical disruption, whereas the endometrial cells in association with them were disrupted. The possibility is suggested, therefore, that implantation in this species can be

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C. ENDERS

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FIG. 9. A diagrammatic presentation of the postulated mechanism of inplantation. (a) The blastocvst is free in the uterine lumen. (b) The blastocpst is attached near the embryonic cell mass, with the trophoblast cc& indenting the luminal epithelial cells of the endometrium. The partial collapse figured may not be a necessary part of attachment. (c) The rupture of the uterine epithelium by the overlying trophoblnst, as described in this paper, is indicated by the dots extending between the two cell types. It is suggested that such a mechanical of the disruption of the endometrium could provide an avenue of penetration through the epithelium by the trophoblast. (d) Tl le extension of the trophoblast luminal epithelimll and increase in size of the implantation region to include more of the trophoblast in the area of the embrvonic cell mass.

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brought about by mechanical disruption of the endometrial epithelium affixed to an overlying closely adhering trophoblast (Fig. 9). (It could be argued that the disruption of the endometrium was produced by lytic agents, rather than mechanically, but the absence of any difference in the cytological features of the trophoblast overlying the break is a point against such an argument. ) The observations, while giving ample evidence that the association between the trophoblast and the endometrium is an active one, do little to illustrate the mechanisms by which such an attachment is accomplished. The important question of how cells aggregate is further complicated here by the fact that genetically dissimilar cells are associating. DISCUSSION

Some of the difficulties in establishing the validity of a theory of implantation and applying this theory to diverse species have been reviewed recently by Biiving (1963). The difficulties have been amply illustrated by the recent work on the rabbit, in which the subject of implantation has been investigated by electron microscopy (Larsen, 1961), organ culture (Glenister, 1961, 1963), chemical and manipulative methods ( B6ving, 1962)) and biochemical methods ( LutwakMann, 1959, 1963). The first three of these authors have suggested three different methods by which implantation could be accomplished (fusion of syncytial and symplasmic masses, active invasion, and epithelial dissociation, respectively), and the last author has refrained from postulating a theory. Yet, even were there a consensus of opinion concerning implantation in the rabbit, it would be exceedingly difficult to determine which of the mechanisms involved in the rather unusual implantation in this species were applicable to implantation in species from different orders. Nevertheless, only by comparing implantation in different forms can we determine what, if any, are the salient features common to the majority. Some of the suggested mechanisms of penetration of the uterine epithelium in implantation in different animals have obviously limited application, Duval’s (1891) suggestion that the expanded decidua so affects the vasculature of the luminal epithelium that the latter undergoes necrosis would necessarily be limited to animals with a precocious decidual reaction, such as the myomorph rodents. The suggestions of dissociation of uterine epithelium by high pH (Biiving, 1959)) lysis of the epithelium by trophoblast (Wislocki and Streeter, 1938), or active invasion by the trophoblast of an altered or unaltered epithelium would seem to have more general possibilities.

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If mechanical disruption of the uterine epithelium by an attached blastocyst is a mechanism of penetration of the epithelium in the armadillo (a fact that cannot be considered fully established by this one observation), could it be a mechanism of implantation in some of the better known animals which also have hemochorial placentation? In order to assess the possibility that such a mechanism is widespread, it is necessary to consider the method by which disruption could occur. Once a distinct attachment has been formed between the trophoblast and the uterine epithelium, anything that tends to move the blastocyst in relationship to the epithelium would have a disrupting effect. If the trophoblast cells are more resistant than the epithelial cells, the latter would be disrupted. Uterine motility alone might be sufficient to tear the union, but in this instance the disruption would tend to be at the margin of the region of association. Such generalized motility would also tend to prevent the establishment of the initial attachment. Thus, there would have to be either a quiescent phase, then a more active phase of uterine motility, or a flaccid phase of the blastocyst. Another way in which force might be exerted on the union would be by swelling of the blastocyst without growth. Observation of primate blastocysts (Hertig et al., 1956) and on armadillo blastocysts (Enders, 1962) indicate that the blastocyst may collapse near the time of implantation. Such a collapse would permit establishment of fairly broad surface contacts with a subsequent tendency to disrupt the contact with reexpansion. It is interesting to note that periodic shrinkage and swelling of blastocysts of the rabbit and mouse have been observed in tissue culture ( Borghese and Cassini, 1963), If the expansion were merely a passive response to fluid uptake, the resultant enlargement would probably tend to disrupt the union of the trophoblast and endometrium at its margins. Active growth of the cells of the blastocyst, however, with concomitant linear expansion of the trophoblast, could exert a disruptive force at any position or over the entire area of attachment. This would be true whether unmodified trophoblast cells were growing or if an implantation cone first established contact, then in growing displaced the apical ends of the epithelial cells. Interpreted in this light, the mechanical disruption of the epithelium could be accomplished by unaltered trophoblast, trophoblastic knobs or other specialized areas, and could well be a widespread phenomenon, It remains to be seen by repeated observation whether such a mechanism is actually involved in implantation, but it appears to be worth considering.

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SUMMARY

An early attachment stage of implantation in the armadillo has been found and studied with the electron microscope. The trophoblast cells were seen to indent the uterine epithelium. The trophoblast cells at the region of attachment were cytologically similar to the other trophoblast cells free of association with the uterine epithelium. The microvilli of the two types of cells were intermingled, and in some places the distance between the two cell types was less than the average length of microvilli of the trophoblast in free areas. At one place the epithelial cells in contact with two of the trophoblast cells were disrupted at their apical ends. The presence of granular cellular contents from the uterine cells in the intercellular space was taken as an indication that the cell rupture might not be an induced artifact of fixation. It is suggested that once attachment has taken place, mechanical disruption of the uterine epithelium, especially by growth of the trophoblast, is a possible mechanism of initial penetration of the endometrium in this species, and that such a mechanism might be widespread. REFERENCES E., and CASSINI, A. (1963). Cleavage of mouse egg. In “Cinemicrography in Cell Biology” (G. G. Rose, ed. ), pp. 263-278. Academic Press, New York. B~VING, B. G. (1959). Implantation. Ann. N.Y. Acad. Sci. 75, 700-725. B~VING, B. G. ( 1962). Anatomical analysis of rabbit trophoblast invasion. Contrib. Embryol. 37, 3355. BBVING, B. G. ( 1963). Implantation mechanisms. In “Mechanisms Concerned with Conception” (C. G. Hartman, ed. ), pp. 321-396. Macmillan, New York. BUCHANAN, G. D., ENDERS, A. C., and TALMAGE, R. V. (1956). Implantation in armadillos ovariectomized during the period of delayed implantation. J. Endocrinol. 14, 121-128. DUVAL, M. ( 1891). Le placenta des rongeurs. III. Le placenta de la souris et du rat. J. An&. (Paris) 27, 24-73. ENDERS, A. C. ( 1960). Development and structure of the villous haemochorial placenta of the nine-banded armadillo (Dasypus novemcinctus). J. Amt. 94, BORGHESE,

34-45. ENDERS,

A. C. (1962).

The

structure

of the armadillo

blastocyst.

J. Amt.

96,

39-48. A. C. ( 1963). Fine structural studies of implantation in the armadillo. In “Delayed Implantation” (A. E n d ers, ed.), pp. 281-292. Univ. of Chicago Press, Chicago, Illinois.

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A. C., and BUCHANAN, G. D. (1959). Some effects of ovariectomy and of ovarian hormones in the armadillo. J. Endocrinol. 19, 444-452. GLENISTER, T. \V. (1961). Organ culture as a new method for studying the implantation of mammalian blastocysts. Proc. Roy. Sot. B154, 428-431. GLENISTER, T. VV. ( 196s ). Observations on mammalian blastocysts implanting in organ culture. In “Delayed Implantation” (A. Enders, ed.), pp. 171-182. Univ. of Chicago Press, Chicago, Illinois. HERTIG, A. T., ROCK, J., and ADAMS, E. C. ( 1956). A description of 34 human ova within the first 17 days of development. Am. J. Anut. 98, 435. LARSEN, J. F. ( 1961). Electron microscopy- of the implantation site in the rabbit. Anut. Record 109, 319-334. LUTWAK-MANN, C. ( 1959). Biochemical approach to the study of ovum implantation in the rabbit. In “Implantation of Ova” (P. Eckstein, ed. ), pp. 35-49. Cambridge Univ. Press, London and New York. LUTWAK-MANN, C. ( 1963). Uterine-blastocyst relationships at the time of implantation: biochemical aspects. In “Delayed Implantation” ( A. Enders, ed. ), pp. 293-304. Univ. of Chicago Press, Chicago, Illinois. PATTERSON, J. T. ( 1913 ). Polyembryonic development in Z’rrttruicl noccmcinctus. J. Morphol. 24, 559-684. WISLOCKI, G. B., and STREETEH, G. L. ( 1958). On the placentation of the macaque (iZlacaca m&&u), from the time of implantation until the formation of the definitive placenta. Contrih. Embryol. 27, l-66. ENDEHS,

injection