J. Anat. (1976), 122, 2, pp. 211-230 211With 15 figuresPrinted in Great Britain

An ultrastructural study of implantation in the golden hamster

II. Trophoblastic invasion and removal of the uterine epithelium

TERRY A. PARKENING*Department of Biology, University of Oregon, Eugene, Oregon 97403

(Accepted 27 September 1975)

INTRODUCTION

During implantation in the rabbit and rodents the development of a haemochorialplacenta is accompanied by the degradation of the uterine epithelium. Cellular deathof the epithelium is thought to precede invasion by the trophoblast cells (Larsen,1961; Abraham et al. 1970; Finn & Bredl, 1973) and there are primarily two hypo-theses as to how the epithelium is eliminated. Tachi, Tachi & Lindner (1970) havesuggested that the epithelium is actively removed by trophoblastic invasion andphagocytosis, while Abraham et al. (1970) contend that the epithelium is decomposedthrough autolysis. The latter researchers have postulated that lysosomes accumulatein the epithelium and then their membranes break down, releasing enzymes whichautolyse the cells. Thus, they consider the function of the trophoblast in the removalof the uterine epithelium is a secondary and passive one.A previous study on implantation in the golden hamster described the loss of the

zona pellucida and the attachment of the blastocyst to the uterine epithelium(Parkening, 1976). This investigation is a continuation of that study and isconcerned with the penetration and removal of the uterine epithelium by thetrophoblast.

MATERIALS AND METHODS

The methods of housing and breeding golden hamsters (Mesocricetus auratus) andthe techniques employed for the examination of embryos during implantation werethe same as described earlier (Parkening, 1976). Eighteen females (3-5 months) wereinjected with Pontamine blue via the femoral vein for localization of implantationsites at 4, 44, 5 and 5- days of pregnancy (post-ovulation). After 15 minutes theanimals were perfused via the abdominal aorta with 2-5 % glutaraldehyde (phosphatebuffered, pH 7*4 at 4 °C). The blue areas were excised, fixed for an additional 2 hoursin the same solution of glutaraldehyde and then post-fixed for l 2 hours in 20osmium tetroxide. After dehydration and embedding in Epon-Araldite, thick sec-tions (1-3 ,tm) were cut to locate implanting embryos. Sixty six implantation siteswere photographed unstained using phase contrast microscopy and were then re-embedded for electron microscopy, using the technique of Schabtach & Parkening(1974). Thin sections cut from at least one thick section of every implantation site

* Present address: Department of Anatomy, The University of Texas Medical Branch, Galveston,Texas 77550.

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'' ~~~~~~~~~~~~~~~MFig. 1. A blastocyst from a hamster pregnant for 4 days (post-ovulation) in which the tropho-blast (Tr) is adhering tightly to the uterine epithelium (UE). Epithelial microvilli remain visiblealong the maternal-embryonic junction and degradation bodies (DB) are abundant throughoutthe blastocyst. x 9500. Inset: A thick section (0 5 ,tm) of the same blastocyst shown in theelectron micrograph. This blastocyst has not started to invade the uterine epithelium. x 325.

were stained with uranyl acetate and lead citrate and examined in a Philips 300 elec-tron microscope.

OBSERVATIONS

As described in other species (Reinius, 1967; Tachi, Tachi & Lindner, 1970),golden hamster blastocysts from the same female or from different females pregnantfor the same number of hours post-ovulation were not always at the same stage ofimplantation. Such differences in development were most apparent at 4 days ofpregnancy when some blastocysts were tightly enclosed by the uterine epithelium as

Fig. 2. Trophoblast cells (Tr) of a blastocyst at 4 days of pregnancy which are preparing tophagocytose the uterine epithelium (UE). A network of microfilaments has formed along theapical edge of the trophoblast (arrow). The microfilaments apparently help form the tropho-blastic processes which invade and engulf the uterine epithelium. Four epithelial cells adjoiningone another are in a state of early degeneration. At this implantation site other epithelial cellsadjoining the necrotic cells appear the same as those in Figure 1. x 10300.Fig. 3. A trophoblast cell of a blastocyst, at 4 days of pregnancy, which has started to phago-cytose the uterine epithelium. Some lysosomes (Ly) appear to be present within the epithelium.x 9500.

212

Implantation"in the golden hamster 213

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Fig. 4. A trophoblast cell (Tr) from a blastocyst in the process of invading and engulfing theuterine epithelium (UE) at 4 days of pregnancy. x 10050. Inset: A thick section (0 5 jtm) of thesame blastocyst shown in the electron micrograph. The pulling away of the trophoblast fromthe lateral uterine epithelium is an artifact. x 325.

in 31 day implantation sites (Parkening, 1976), while others had reached a moreintimate relationship with the epithelium (adhesive phase) and still others had begunto invade the uterine epithelium (invasive phase).

AttachmentMaternal-blastocyst adherenceDuring this phase of implantation the trophoblast came into intimate contact

with the uterine epithelium (Fig. 1). The plasma membrane of the trophoblast becameirregular as it closely contacted the microvilli and cytoplasm of the uterine epi-thelium. The two membranes were separated by approximately 20 nm, except incertain regions where deposits of dense material appeared between them. Microvilliof the uterine epithelium retained their integrity throughout this close association.At some implantation sites a fine network of filaments appeared intracellularly alongthe trophoblast interface adjacent to the epithelium (Fig. 2). These filaments re-sembled those of the contractile system of non-muscle cells (Wessels et al. 1971) andwere probably concerned with the imminent formation of trophoblastic processesfor invasion of the uterine epithelium. At two implantation sites three or four of the

214

Implantation in the golden hamster

Fig. 5. A trophoblast cell (Tr) from a blastocyst at 4 days of pregnancy which is phagocytosingthe uterine epithelium (UE). Trophoblastic processes are beginning to surround some portionsof the epithelium, while other portions already appear to have been ingested (arrows). x 18 500.

epithelial cells showed signs of necrosis (Fig. 2), as judged by the swollen and emptyappearance of their mitochondria. Cells adjacent to these seemed normal, but theinterface of the trophoblast was more irregular in these blastocysts where it madecontact with the microvilli and cytoplasm of the epithelium. Microfilaments werepresent along the marginal membrane of the trophoblast cells in both these blasto-cysts. Lysosomes were not abundant in any of these epithelial cells, although morewere present than at 32 days of pregnancy.

Trophoblastic invasion of the uterine epitheliumIn a few blastocysts at 4 days of pregnancy the trophoblast had begun to invade

and phagocytose the uterine epithelium (Figs. 3, 4). Initial invasion occurred alongthe lateral-central portion of the maternal-embryonic junction and proceeded anti-mesometrially. Trophoblastic processes extended into the epithelium, surroundingand engulfing portions of it (Fig. 5).During the invasion of the uterine epithelium the trophoblast cells contained

numerous polyribosomes, much granular endoplasmic reticulum and many cylin-drical mitochondria containing expanded lamelliform cristae with prominent intra-cristal spaces. Also present were lipid bodies, some quite large as compared with the

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Implantation in the golden hamsterlipid droplets found at earlier stages, as well as various electron-dense inclusions,many of which contained membranous material (Fig. 4). Some degradation bodieswere difficult to distinguish from these inclusions. Cytoplasmic plaques were com-pletely absent but there were numerous vesicles, some apparently associated withGolgi complexes.

Cells of the inner cell mass were also devoid of plaques at the time of epithelialinvasion. Numerous autophagic vacuoles were present in most of the cells, withdegradation bodies undergoing changes as they became larger, assuming variousshapes. Mitochondria were morphologically similar to those of the trophoblast cellsexcept they exhibited fewer cristae. Endodermal cells were characterized by moreabundant and dilated granular endoplasmic reticulum containing an amorphousmaterial.The uterine epithelium at the time of trophoblastic invasion was generally in a

state of deterioration. Lysosomes were present in all the epithelial cells. At this timecollagen fibres appeared for the first time between the epithelium and the developingdecidual cells.At 41 days of pregnancy some portion of the epithelium had been eliminated at

most implantation sites (Fig. 6), allowing the trophoblast to make contact with thebasement membrane of the uterine epithelium. Other trophoblast cells showed pro-cesses surrounding and apparently phagocytosing the remaining areas of the epithe-lium. Frequently, portions of the epithelium were almost or completely enclosed withinthe trophoblast. In other regions, remnants of cells still existed along the epithelial-decidual junction as the trophoblast continued to engulf this material (Fig. 8). Alayer of collagen fibres encompassed and separated the embryo from the decidualcells, except for a small region where the cells were forming the ectoplacental cone.This collagen layer was well formed by the time the trophoblast had eliminated theepithelium. Occasionally leucocytes were visible in this region (Fig. 7). In some areas,the uterine epithelium, including the basement membrane of the epithelial cells, hadcompletely disappeared. Trophoblastic processes were still present in these regionsas they extended toward the decidual cells. Although collagen fibres generallyseparated the trophoblastic processes from the decidual cells, in a few places theprocesses looked as if they were trying to invade the decidua (Fig. 9).

Inclusions of various shapes, sizes and electron densities were present throughoutthe cytoplasm of the trophoblast cells (Figs. 6, 7). Much of this material came fromthe ingested epithelium. Trophoblastic nuclei at the abembryonic pole of theembryo enlarged during phagocytosis of the uterine epithelium, while mitochondriaremained cylindrical with numerous cristae. Large lipid droplets accumulated atrandom in the trophoblast, the amount of granular endoplasmic reticulum increasedand ribosomes remained abundant. Micropinocytic vesicles, some bristle-coated,appeared along the invading interface of the trophoblast (Figs. 7, 9). Vesicles of thelatter type have been associated with protein uptake (Fawcett, 1965). Fine filaments,resembling microfilaments (Wessels et al. 1971), were visible in select regions of thetrophoblast away from the invading interface. These were probably contractile,being similar to those in the trophoblastic processes.

Within the blastocyst a single layer of cuboidal cells, the visceral (proximal) endo-derm, encompassed the egg cylinder, while the parietal (distal) endoderm, originating

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Implantation in the golden hamsterat the embryonic pole, was spreading along the trophoblast's interior surface. Theinner ectodermal cells of the egg cylinder were characterized by numerous poly-ribosomes, scattered mitochondria with well-defined cristae, small amounts ofgranular endoplasmic reticulum and few inclusions (Fig. 10). Ultrastructurally, thevisceral endodermal cells resembled those of the ectoderm, except they containedgreater amounts of granular endoplasmic reticulum with an amorphous materialfilling the cisternae (Fig. 11). The parietal endodermal cells were generally similar tothe visceral endoderm, but they were flatter as they made close contact with thetrophoblast.

DevelopmentElimination of the uterine epitheliumBy day 5 the uterine epithelium adjacent to most embryos had been completely

eliminated (Fig. 12). During this time the trophoblast cells expanded, becomingthinner at their sites of cellular contact, and filling the space left by the engulfed epi-thelial cells. The layer of collagen fibres between the embryo and the decidual cellsbegan to lose its integrity (Figs. 13, 14), becoming a hazy mass of fine filaments.Blood lacunae became enlarged around the implantation site, with certain regionsexisting where red blood corpuscles came into direct contact with the trophoblast.Polymorphonuclear leucocytes were occasionally visible in these lacunae.

Large inclusions of various densities remained in many trophoblast cells as intra-cellular epithelial digestion continued. Other organelles within the trophoblast cellsremained similar to those seen 12 hours earlier. One ultrastructural difference wasthe presence of Reichert's membrane, a faint thin non-cellular amorphous coating,which began to form along the inner surface of the trophoblast. Glycogen granulesalso became numerous in some trophoblast cells, primarily those at the mesometrialside of the uterus.Although there was some enlargement of trophoblast cells at the abembryonic

pole of the blastocyst just before and during the early invasion and phagocytosis ofthe uterine epithelium, only at day 5 did trophoblast cells with nuclei approximately3-4 times larger than normal nuclei appear. Trophoblast cells which enlargedthroughout implantation were classified as primary giant trophoblast cells (Ward-Orsini, 1948; Dickson, 1963). Ultrastructurally these cells were similar to othertrophoblast cells except for the nuclear chromatin. The nucleoli possessed granularand fibrillar zones resembling those of cells which underwent mitosis in the pre-implanted hamster blastocyst.

Fig. 7. An electron microscope montage of an implanting embryo at 4j days of pregnancy,illustrating how the trophoblast cells (Tr) surround and engulf remaining portions of the uterineepithelium (UE). Inclusions (In) within the trophoblast represent the breakdown of previouslyingested epithelium. A leucocyte (Le) appears at the site of phagocytosis. L, lipid; C, collagen.x 6800. Upper inset: An enlargement of the fibres, presumed to be collagen, which formbetween the trophoblast and decidua (D). x 41 000. Lower inset: A thick section (055,m) ofthe same embryo from which the electron micrograph was taken. x 325.

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.7Fig. 8. All that remains of the uterine epithelium (UE) in one region of an implantation site at41 days of pregnancy. Note the abundance of collagen fibres just below the epithelium. Tr,trophoblast. x 12600.

Differentiation of the inner cell massThe inner cell mass continued to differentiate, forming a proamniotic cavity,

embryonic ectoderm (to become the embryo proper) and extraembryonic ectoderm,in addition to parietal and visceral endoderm. At times dark cells were noted withinthe newly formed regions of the ectoderm. These appeared necrotic and probablyrepresented spontaneous degeneration of certain embryonic cells, as described forthe mouse (Potts, 1969; Wilson & Smith, 1970; El-Shershaby & Hinchliffe, 1974).The fine structure of the majority of interior cells of the egg cylinder was similar tothat described at 44 days. Endodermal cells remained much as before except thatsome cells within the visceral endoderm exhibited moderately dense elongatedstructures. These have been referred to as filiform or dictyosome-like tubules in thevisceral endoderm of the mouse (Bartel, 1972), but, as yet, no function has beenattributed to them. Cells along the mesometrial side of the uterine lumen began toform the ectoplacental cone. Several layers of flattened cells adhered closely to oneanother in this region, with most cells containing abundant deposits of glycogen.Most implantation sites at 54 days of pregnancy showed trophoblast cells com-

pletely surrounded by blood lacunae except for the ectoplacental cone. Reichert'smembrane was well formed at this stage of pregnancy (Fig. 15). Trophoblast cells

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Fig. 9. A trophoblast cell of an embryo after 4- days of pregnancy. A trophoblastic processextends itself (arrow) in an apparent attempt to invade the decidua. Collagen fibres (C) appearbetween the invading trophoblast and the decidual cells (D). x 16400.

were somewhat flattened as they extended thin processes to join adjacent cells. In-clusions, so prevalent at 41 and 5 days, were absent in the trophoblast and the cellsappeared similar to those of the parietal endoderm described at 44 days.The number of cells within the embryonic and extraembryonic ectoderm was

considerably greater than 12 hours earlier, but the appearance and distribution of theorganelles remained much the same.

DISCUSSION

During implantation in the golden hamster the trophoblast clearly invades,phagocytoses and digests the uterine epithelium. Death of the uterine epitheliumprobably precedes invasion by the trophoblast, but the phagocytic action of thetrophoblast is primarily responsible for its removal. Ultrastructural studies examiningimplantation in mice and rats (Enders & Schlafke, 1967; Finn & Lawn, 1968; Smith& Wilson, 1974; Schlafke & Enders, 1975) indicate that some epithelial cells becomedissociated from their underlying basal lamina before trophoblastic penetration andthese are phagocytosed by the trophoblast. Later, during displacement of theepithelium, the trophoblast extends processes between the epithelial cells and engulfsentire cells. The hamster differs from these species in that trophoblastic processes

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Fig. 10. Cells of the inner cell mass of an embryo at 41 days of pregnancy. Granular endoplasmicreticulum commonly appears in the visceral endodermal cells (VE), while it is seldom found inthe embryonic ectoderm (EE). The latter cells become the ectoderm ofthe embryo proper. x 9100.

penetrate directly into epithelial cells, phagocytosing portions of the cells. It appears,therefore, that the trophoblast of the hamster is more invasive than that of themouse and rat. Whether this invasiveness is considered active or passive is dependenton how these terms are defined. Any trophoblast which aids degeneration of theuterine epithelium can be considered active, whereas a trophoblast which invadesepithelial cells which are self-destroying may be considered passive. However,trophoblastic participation in the removal of dead epithelial cells may also be con-sidered as active or passive; in this respect, the hamster trophoblast can be consideredactive, but whether it influences autolysis of the uterine epithelial cells is not known.Another ultrastructural feature in the hamster is the retention of epithelial micro-

villi. During the adhesive phase of implantation in the rat (Tachi et al. 1970) andmouse (Potts, 1966, 1968) the epithelial surface becomes flattened and microvilli areno longer visible when electron-dense deposits begin to appear at the maternal-embryonic junction. This results in a wavy interface between the two adjoiningplasma membranes. During implantation in the hamster, however, epithelial micro-villi never completely disappear but remain until phagocytosed by the trophoblast.At no time is cytoplasmic fusion of the trophoblast and uterine epithelium detectableas reported by McLennan (1974).The formation of microfilaments along the apical surface of the trophoblast is

222

Implantation in the golden hamster

Fig.' 11. Visceral endodermal cells from an embryo at 4± days of pregnancy. These cells arecharacterized by granular endoplasmic reticulum with an amorphous material filling the cis-ternae. x 9800.

another feature not previously described in rodents. Only recently have such filamentsbeen illustrated in electron micrographs of the rabbit trophoblast (Schlafke & Enders,1975). In the hamster, these filaments apparently help to form the trophoblasticprocesses which extend, invade and engulf the epithelium.

Little evidence of epithelial autolysis was detectable before the cells' eliminationat implantation sites in the rat, bat, rabbit, guinea-pig, armadillo and ferret (Enders& Schlafke, 1969). In general, this is also true for hamster epithelial tissue, except intwo blastocysts where three or four adjoining cells appeared necrotic just beforeinvasion by the trophoblast. These cells may have been at the initial site of penetra-tion, or they may simply have been random degenerating epithelial cells as describedin other ultrastructural studies on the rat and mouse (Finn & Lawn, 1968; Tachi et al.1970). The majority of epithelial cells appear relatively normal until invasion by thetrophoblast, when cellular death is evident from organelle disruption and the generalappearance of the cytoplasm. Lysosomes present in the epithelium at this time maywell release enzymes which assist in the removal of the cells by the trophoblast.Ultrastructural cytochemistry could be employed to help determine the role oflysosomes in the degeneration of hamster epithelial cells. Proteolytic activity hasbeen described in the trophoblast of the guinea-pig (Owers & Blandau, 1971) and

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Fig. 12. A light microscope montage of an implanted embryo at 5 days of pregnancy. The innercell mass has differentiated into the extraembryonic ectoderm (EXE), embryonic ectoderm (EE),and visceral endoderm (yE). The parietal endoderm has also formed, but is not readily visiblein this section. At this stage of development blood lacunae (BL) are beginning to encompass theembryo. PC, proamniotic cavity; D, decidua. x 660.

Implantation in the golden hamsterrabbit (Denker, 1972), using gelatin substrates. In the latter species, similar butslightly modified techniques revealed that proteolytic enzymes are restricted to theblastocyst coverings (zona pellucida, mucoprotein layer and any attached uterinesecretion) rather than the trophoblast (Kirchner, 1972). Experiments to showthe presence of proteolytic enzymes in the rat and mouse trophoblast have failed(Blandau, 1949; Owers & Blandau, 1971; Bergstrom, 1970). The difference in pro-teinase activity in the guinea-pig and rat trophoblast has been related to differencesin trophoblastic invasiveness. Similar enzyme studies on the hamster are neededin view of the obvious invasive nature of its trophoblast shown in the present study.

In the mouse it appears to be well documented (Smith & Wilson, 1974; El-Shershaby & Hinchliffe, 1974; Poelmann, 1975) that autolysis of the uterine epi-thelium precedes the invasion of the trophoblast; but what initiates the death of theepithelial cells is not known. Various suggestions have been made, including eleva-tion in alkalinity (Boving, 1963), labilization of lysosomal membranes by steroidscausing the release of proteolytic enzymes (Abraham et al. 1970) and progressiveanoxia resulting from decidualization of the stroma (Enders & Schlafke, 1967;Smith & Wilson, 1974). Finn & Bredl (1973) hypothesized that the breakdown ofuterine epithelium in the mouse may be dependent upon DNA-directed RNAsynthesis. In their experiments, they administered actinomycin D and found itinhibited degeneration of the uterine epithelium, but was unable to halt invasion bythe trophoblast. They surmised that actinomycin D did not affect the trophoblastbecause it was unable to penetrate a hypothetical barrier between the embryo andmother.The anatomical barrier proposed by Kirby and his associates (1964, 1965) is not

seen during implantation in the hamster apart from the layer of collagen fibrespresent during trophoblastic invasion of the uterine epithelium. This region stainswith PAS and is diastase-resistant (Parkening & Soderwall, 1974). Unless the dis-appearance of the fibres aids in the formation of a chemical barrier, it is doubtful ifthis layer of fibres serves as an immunological barrier, since it disappears shortlyafter the uterine epithelium has been eliminated. Perhaps the fibres serve in some wayas a temporary barrier preventing further maternal phagocytosis by the trophoblastuntil the decidua can develop more fully. The possibility of decidual cells serving as aprotective barrier against the invading trophoblast has been discussed by Finn (1971).In the rat, close apposition (10-20 nm) of the trophoblast and decidual cells has beendescribed (Tachi et al. 1970), but at no time in the hamster are these cells in such closecontact except in a few areas where trophoblastic processes appear to be attemptinginvasion of the decidua. The embryo is separated from the decidua by collagenfibres until day 5, when the fibres begin to disappear and blood lacunae surroundthe implantation site (except for the ectoplacental cone); therefore, any immuno-logical barrier is most likely to be chemical in nature. Since the appearance anddisappearance of the collagen fibres is so closely related to the autolysis and removalof the uterine epithelium, the hypothesis (Smith & Wilson, 1974) that the fibres maycontribute to the isolation and hypoxia of the epithelial cells is a logical explanationfor their existence.

It is suggested that the degradation bodies, abundant in the trophoblast at thetime of uterine invasion, are lysosomes. Once the uterine epithelium has been

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Fig. 15. At 51 days of pregnancy red blood corpuscles (RBC) are free to make direct contact withtrophoblast cells as blood lacunae almost completely surround the embryo. Reichert's membrane(RM) is present between the parietal endoderm (PE) and the trophoblast (Tr). VE, visceralendoderm. x 7750. Inset: A thick section (0-5 ,ctm) of the embryo in the electron micrograph.x210.

phagocytosed they combine with the engulfed epithelium releasing enzymes for thedigestion of this material, thus forming various types of inclusions. The degradationbodies which accumulate in the trophoblast of the hamster probably correspond tothe acid phosphatase-rich material classified as enzyme-rich vesicles or lysosomes inhistochemical studies of implantation in mice (Smith & Wilson, 1971) and rabbits(Christie, 1967; Abraham et al. 1970). In the former study, light microscopic cryostatsections of implanting mouse blastocysts revealed a border of lysosomes in thetrophoblast during early phagocytosis of the uterine epithelium. A recent ultra-structural study (El-Shershaby & Hinchliffe, 1975) did not find such a border,

Fig. 13. At 5 days of pregnancy the uterine epithelium has been eliminated in most regions of theimplantation site. Glycogen (Gy) becomes abundant in many trophoblast cells near the em-bryonic pole and collagen fibres between the trophoblast and decidual cells (D) begin to dis-appear. x 10600. Inset: A thick section (0-5 ,um) of the same embryo shown in the electronmicrograph. x 325.Fig. 14. An enlargement of the deteriorating collagen fibres between the trophoblast (Tr) anddecidual cells (D) at 5 days of pregnancy. At this stage trophoblast cells are located further apartwith only thin extensions maintaining contact between them. x 24450.

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although the distribution of acid phosphatase within the mouse trophoblast awaitsfurther study with the electron microscope. In an ultrastructural study of acidphosphatase activity during early implantation in the rabbit (Abraham et al. 1970),enzyme activity in the blastocyst was chiefly confined to bodies considered to belysosomes, but occasionally the enzyme was present within cisternae of the Golgicomplex and microvilli. Differences in the reaction produced between day 6 andday 8 of pregnancy were not detectable. During this same period, the uterus exhibitedan increased number of lysosomes, autophagic vacuoles and myeloid bodies with acorresponding increase in enzyme intensity. Abraham et al. (1970) concluded thatthese changes represented an enhanced autolysis of the uterine epithelium, and thatthis was the primary cause of the disintegration of these cells.Although acid phosphatase activity was not examined in the present study, auto-

lysis of the hamster uterine epithelium precedes or occurs simultaneously with theinvasion of the trophoblast. Whether the trophoblast initiates or aids in the auto-lysis of the uterine epithelium remains to be determined. However, the phagocyticactivity of the hamster trophoblast appears to be greater than that described thus farfor the mouse, rat and rabbit, and may reflect a species difference in the degree towhich the trophoblast participates in the removal of the uterine epithelium.

SUMMARY

Sixty six implantation sites from 18 golden hamsters were examined with light andelectron microscopy between 4 and 52 days of pregnancy (post-ovulation). At 4 dayssome blastocysts began to invade the uterine epithelium, with trophoblastic pro-cesses penetrating and engulfing portions of the uterine epithelium. The majority ofepithelial cells appeared normal before invasion, although at two implantation sitesthree or four adjoining epithelial cells were necrotic before penetration by thetrophoblast. In general the epithelial cells were degenerating at the time the tropho-blast invaded the epithelium. Inclusions, representing portions of the engulfedepithelium, and varying in size and electron density, were present throughout theinvading trophoblast cells at 42 and 5 days of pregnancy. At 51 days the uterineepithelium had disappeared and the embryo was now almost completely surroundedby blood lacunae.

The author would like to thank Dr A. L. Soderwall for his interest and supportduring this investigation. Appreciation is also extended to Dr A. C. Enders andS. Schlafke, Department of Anatomy, Washington University School of Medicine,and to Dr C. Lubicz-Nawrocki, Worcester Foundation for Experimental Biology,for critically evaluating the manuscript.

This study was supported in part by USPHS Grant No. HD 04234-03 and byNIH Physiology Training Grant No. 5 TOI GM00336.

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Implantation in the golden hamster 229

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BARTEL, H. (1972). Electron microscopic observations of the inner cell mass of a mouse embryo. Actaanatomica 83, 289-301.

BERGSTROM, S. (1970). Estimation of proteolytic activity at mouse implantation sites by the gelatindigestion method. Journal of Reproduction and Fertility 23, 481-485.

BLANDAU, R. J. (1949). Embryo-endometrial interrelationships in the rat and guinea pig. AnatomicalRecord 104, 331-360.

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CHRISTIE, G. A. (1967). Histochemistry of implantation in the rabbit. Histochemie 9, 13-29.DENKER, H. W. (1972). Blastocyst protease and implantation: effect of ovariectomy and progesterone

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EL-SHERSHABY, A. M. & HINCHLIFFE, J. R. (1975). Epithelial autolysis during implantation of the mouseblastocyst: an ultrastructural study. Journal of Embryology and Experimental Morphology 33,1067-1080.

ENDERS, A. C. & SCHLAFKE, S. (1967). A morphological analysis of the early implantation stages in therat. American Journal ofAnatomy 120, 185-226.

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