THE AMERICAN JOURNAL OF ANATOMY 187:137-157 (1990)

Blastocyst Implantation in the Chinese Hamster (Cricetulus griseus) THOMAS N. BLANKENSHIP, RANDALL L. GIVEN, AND TERRY A. PARKENING Department of Anatomy and Newosciences, University of Texas Medical Branch, Galveston, Texas 77550

ABSTRACT Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete areas. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert’s membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert’s membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents. INTRODUCTION

Mammalian implantation consists of a series of complex biochemical, physiological, and morphological interactions between the blastocyst and endometrium. The morphological events of implantation have been described for several species, although most reports have utilized only light microscopy. To understand better the nature of the interrelationship between the embryo gnd uterus these events need to be examined at the ultrastructural level. The purpose of this investi0 1990 WILEY-LISS, INC.

gation was to examine with light and electron microscopy the process of implantation in the Chinese hamster (Cricetulus griseus) and compare these events with what is known about this process in the more common laboratory rodents. Only a few reports are available on the early embryonic development of the Chinese hamster, although it has been used for many years as a laboratory rodent in studies of cytogenetics (Kamiguchi and Mikamo, 19821, diabetes mellitus (Sirek and Sirek, 1967), and neural development (Donkelaar et al., 1979). Gamete morphology and fertilization were examined by Yanagimachi et al. (1983). The sequence of ovulation, fertilization, and early cleavage were described by Pickworth et al. (1968), while the fate of paracrystalline inclusions in the oocyte and early embryo has been described by Parkening e t al. (1985). Later embryonic development, from the first appearance of somites (day 10 of pregnancy) until birth (day 211, were outlined by Donkelaar et al. (1979). Fortuyn (1929) investigated the incidence of prenatal death in these animals. However, except for a brief comment on the suspected time of implantation (Pickworth e t al., 19681, no information is currently available regarding this process in the Chinese hamster. MATERIALS AND METHODS

Chinese hamsters used in this study were obtained from a resident colony housed at this institution. All animals were maintained on a LD14:lO hour light cycle (lights on at 0700 hr) in a n environmentally controlled room (21-23°C) with food (Formulab Chow #5008, Ralston Purina Company, with occasional oatmeal supplements) and water provided ad libitum. Adult females 4-8 months of age (weighing approximately 33 gm) were examined in the late afternoon (1600-1700 hr) to determine the stage of the estrous cycle. Those hamsters experiencing late proestrus possessed a swollen vaginal orifice which formed a prominent ridge when pressed lightly on each side (Yerganian, 1967). Each proestrus female was housed with two males and examined on the following morning for a copulatory sperm plug or presence of sperm in the vagina (designated day 1 of pregnancy). Pickworth et al. (1968) reported that on day 4 of pregnancy in the Chinese hamster the embryos were located in the oviduct and were about to enter the uterus, Previous studies indicated that blastocysts could be easily flushed from the uterus on day 5 of

Received May 30, 1989. Accepted September 5, 1989.

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Fig. 1 a-c

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gestation (Parkening, unpublished). Therefore, the present investigation began with embryos of six days gestation. During the afternoon (1200-1600 hr) on day 6 , 7 , or 8, the animal was deeply anesthetized with sodium pentobarbital and injected with 0.15 ml of 1%Evans blue in Hanks’ balanced salt solution via the femoral vein to localize the implantation sites. Approximately 10 min later the animal was fixed by vascular infusion through the left ventricle with 2% paraformaldehyde2.5% glutaraldehyde in 0.10M phosphate buffer (pH 7.3). The uterus was removed, sectioned transversely to isolate each implantation site, and fixed for a n additional 2-4 hr. Tissues were rinsed overnight in phosphate buffer, postfixed in 1%osmium tetroxide, dehydrated in ethanol, and embedded in Durcupan Araldite (Fluka Chemical Corp., New York). Individual sites were sectioned a t a thickness of 1-2 pm and stained with alkaline Azure B for evaluation by light microscopy (53 sites from 20 animals). From these sites, 22 were selected (representing 12 animals), thin sectioned, and stained with uranyl acetate and lead citrate before examination with a Philips 300 transmission electron microscope.

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of separated uterine epithelium that lines opposite sides of the lumen. These observations indicate that the uterine lumen is only just closing around the embryo at this time and that the integrity of this closure is tenuous. Blastocyst

The trophoblast is closely apposed to the uterine epithelium over most of its surface and there is no evidence of a zona pellucida. Trophoblast cell membranes in contact with the uterine epithelium exhibit scattered microvilli and a n uneven contour that usually follows the apical surface of the epithelium (Fig. 2). Each flattened trophoblast cell overlies several epithelial cells. Distinct intercellular junctions between trophoblast cells are evident. The lateral membranes of these cells frequently show elaborate interdigitations, although the surfaces lining the blastocyst cavity are smooth. Polyribosomes are abundant while granular endoplasmic reticulum is represented by only a few short cisternae (Fig. 2). Mitochondria are moderate in number and are distributed throughout the cytoplasm. Multivesicular bodies and inclusions of various sizes containing cellular debris are common. A layer of fine filaments is often seen beneath the membrane adjacent OBSERVATIONS to the uterine epithelium, while small isolated bundles of filaments are also found throughout the cytoplasm Day 6 (Attachment of Blastocyst) By day 6 the blastocyst is fixed in position near the (Fig. 2). Coated vesicles are occasionally seen along the antimesometrial end of a crypt formed within the uter- cell membrane next to the uterine epithelium. No cyine lumen (Fig. l a ) . At this stage of gestation, the blas- tological differences between polar and mural trophotocyst cannot be flushed from the uterus without dam- blast were noted. The polar trophoblast is closely apage. Upon gross examination, faint but definite plied to the inner cell mass. No evidence of a basal evidence of a n Evans blue reaction is noted at each of lamina associated with the trophoblast is seen at this the small swellings located in one or both uterine time. The inner cell mass is situated mesometrially. Cells horns. The uterine lumen may be open or closed a t different of the inner cell mass have a typical embryonic appearimplantation sites on day 6. In those sites with a closed ance and are filled with polyribosomes and moderate lumen, the blastocyst is sequestered within a n implan- numbers of mitochondria (Fig. 3). Granular endoplastation chamber. Of those sites with a n open lumen, mic reticulum is sparse but contains some dilated cissome may have been closed initially and subsequently ternae. Contiguous cell membranes are parallel over pulled open during tissue preparation. This is sug- long distances and are connected by rudimentary puncgested by observing matching complementary profiles tate junctions. We use the term “punctate junction” because these intercellular junctions are small, distinct, have some cytoplasmic density, and exhibit reduced intercellular distances. They do not possess the distinct substructure of a typical desmosome. Golgi complexes are small in size and few in number in all Fig. 1. Light micrographs of the blastocyst on days 6, 7, and 8 of cells of the blastocyst. Although presumptive endopregnancy. a: On day 6 (1330 hr), the blastocyst is attached to the derm cells along the blastocyst cavity are somewhat antimesometrial portion of the uterine epithelium. Some embryos flattened (Fig. 31, there is no migration of these cells a t have a compressed appearance a t the abembryonic pole. A few perthis time. iluminal stromal cells (S) are enlarged a t this time. The epithelium at Variable numbers of paracrystalline inclusions are the bottom of the implantation chamber has a stratified appearance (asterisk). BC, Blastocyst cavity. b The uterine lumen is closed on distributed within cells of both the inner cell mass and day 7 (1400 hr), and the blastocyst is sequestered within the implan- trophoblast (Figs. 2-4). These aggregates are smaller tation chamber. Visceral endoderm (VE) and parietal endoderm (PE) in size and fewer than those seen in the oocyte and cells are now present. Decidual cells (D) are evident. Note the blood preimplantation blastocyst (Parkening et al., 1985). vessels near the implantation site (arrows). The mural trophoblast has penetrated the luminal epithelium (arrowhead) and has advanced These inclusions are not surrounded by a membrane, to the basement membrane (see also Fig. 8). c: The embryo has de- but small remnants of these structures can be found in veloped to the egg cylinder stage and fills the yolk sac cavity on day lysosome-like granules. Another inclusion often seen 8 (1400 hrj. A visceral endoderm (VE) layer covers the epiblast (EPIj. in trophoblast cells consists of arrays of thin rods assoThe forming proamniotic cavity is evident (asterisk).A blood channel (C) lined by mural trophoblast (TI is seen, while free maternal blood ciated with flocculent electron-dense material (Fig. 5 ). cells (RBC) are distributed around the embryo. Giant trophoblast cells These pleomorphic structures are surrounded by a (GT) are present. Large decidual cells (D) are abundant around the embryo. No uterine epithelium remains near the embryo and tropho- membrane and often found near the paracrystalline inclusions. blast is in contact with decidua. All x 390.

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Fig. 2. Early interaction between trophoblast (T) and uterine epithelium (UE) on day 6. The trophoblast cell contains paracrystalline inclusions (PC), multivesicular bodies (MVB), lysosome-like granules

(L), and abundant free polyribosomes. A layer of fine filaments (F) is seen along the outer surface of the trophoblast cell. BC, Blastocyst cavity. x 18,500.

Uterine epithelium

oli. Apical junctional complexes are well developed. In addition, desmosomes and gap junctions are present along the lateral membranes. Adjacent membranes show interdigitations which may be elaborate in the basal regions. Mitochondria are found throughout the cytoplasm (Fig. 6 ) . Golgi complexes are small, multiple, and not confined to the perinuclear region. Electron-lucent vesicles are found within the cytoplasm near the microvillous border. Granular endoplasmic

Epithelium lining the lumen consists of a single layer of tall columnar cells (Fig. la). The apical surfaces are arranged primarily as numerous short microvilli (Fig. 6 ) interspersed with occasional small, pleomorphic, knob-like protrusions. These surfaces are typically subtended by a terminal web of fine filaments. Nuclei are located basally, are simple in outline with diffuse chromatin, and display prominent nucle-

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Fig. 3. Developing visceral endnderm WE) is present in the inner cell mass adjacent to the blastocyst cavity (BC). Paracrystalline inclusions (PC) are cnmmnn in both the epiblast (EPI) and endoderm. Day 6. x 19,000.

reticulum is not extensive and is generally restricted to short strands. Glycogen is distributed diffusely throughout the cytoplasm, with only a few small aggregates present. Small lipid droplets are sometimes seen near the nucleus. The epithelium at the antimesometrial end of the crypt often appears stratified (Fig. l a ) , probably a result of somewhat convoluted topography in this region of the lumen. In some implantation sites, the epithelium is punctuated by densely staining cells. These cells have pyknotic nuclei and may represent stages of cellular degeneration. The dark cells, when present,

are not limited to the area adjacent to the blastocyst but may also reside in the more mesometrial portions of the uterine epithelium. Stroma

At this early stage of embryonic development, most of the stromal cells are fusiform in shape and surrounded by abundant extracellular matrix. These cells have relatively little cytoplasm while the nuclei are variable in contour with dispersed chromatin and prominent nucleoli. Granular endoplasmic reticulum consists of a few short strands with dilated cisternae

Fig. 4.Paracrystalline inclusions (PC) within a trophoblast cell. A periodicity in the structure of these filaments can be seen. Small isolated bundles of fine filaments (F) are also common. Day 6. x 48,600.

Fig. 6. Typical uterine epithelial cell found a t the onset of implantation. Abundant microvilli (MV) and mitochondria (M) are present. Lateral cell membranes often interdigitate with adjacent epithelial cells. Well-developed Golgi complexes (GI are common. Day 6. x 19,000.

Fig. 5. A characteristic inclusion containing thin rods and flocculent

electron-dense material is shown. These structures are often associated with paracrystalline inclusions. Day 6 . X 33,800.

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Fig. 7. Juxtaembryonic stromal cell on day 6 of pregnancy. Small amounts of glycogen (GLY) are present. Golgi complexes (GI are prominent, and dilated cisternae of granular endoplasmic reticulum (arrowheads) are seen. x 23,000. Flg. 8. Penetration of uterine epithelium (UE) by mural trophoblast (T) on day 7. A trophoblast process (arrowheads) has advanced through the epithelium. Decidual cells (D) are well developed, and blood vessels (BV) are present near the blastocyst. BC, Blastocyst cavity. x 975.

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Fig. 9. Area of penetration in an implantation site on day 7 similar to Figure 8. The trophoblast (T)is adjacent to the basal lamina (BL) of the uterine epithelium (UE). Note the folded basal lamina sometimes found in these areas. Portions of epithelial cells have been

phagocytized by the trophoblast and are present as deteriorating cellular debris (CD). Uterine epithelium in contact with the trophoblast shows little evidence of degeneration. BC, Blastocyst cavity; F, fine filaments; D, decidual cell. x 9,200.

containing flocculent material. Golgi complexes are not prominent. Lipid droplets are rare. These cells are in contact via thin cytoplasmic processes that sometimes form primitive punctate junctions. Stromal cells found near the blastocyst have begun the process of decidualization (Figs. la, 7). These cells are enlarged and appear epithelioid with greatly reduced intercellular spaces. Cells nearest to the embryo

are arranged with their long axes parallel to the basal lamina of the uterine epithelium. Cisternae of granular endoplasmic reticulum are somewhat elongated and filled with homogeneous dense material. These developing decidual cells contain greater numbers of lipid droplets, form more intercellular junctions, and possess more Golgi complexes than the undifferentiated stroma1 cells. Glycogen is seen to accumulate around lipid

Fig. 10. Uterine epithelial apical protrusions (P) increase in both size and number on day 7. These protrusions are surrounded by the trophoblast cell (TI. UE, Uterine epithelium. x 21,300. Fig. 11. Larger apical protrusions (PI may be attached to the uterine epithelium (UE) by a narrow stalk (asterisk). Profiles of these large apical protrusions are sometimes seen isolated within trophoblast cells. T, Trophoblast. Day 7. x 15,700.

Fig. 12. A basal lamina (arrows) representing the early Reichert’s membrane appears between parietal endoderm (PE) and trophoblast (T). Apical protrusions (PI replace the microvilli on the uterine epithelium. Note the close proximity of the protrusions and trophoblast cell membrane. Day 7. x 24,500.

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Fig, 13. On day 7 the embryonic cell mass includes visceral endoderm (VE), parietal endoderm (PE), and epiblast (EPI)cells. Granular endoplasmic reticulum (ER) forms a reticular pattern typical of en-

doderm at this stage of development. Reichert’s membrane is evident (arrows). The uterine epithelium (UE) is somewhat compressed. BC, Blastocyst cavity; T, trophoblast. x 10,900.

droplets. There is very little intervening intercellular space separating the uterine epithelium from the closest stromal cells. The ground substance throughout the stroma has a mottled appearance, with collagen or other fibrils seen only rarely. A few mitotic figures are seen throughout the stroma. Occasionally, endothelial cell processes are seen in contact with the decidual cells (Fig. 23). Uterine glands are not numerous and some are filled with dense, homogeneous, basophilic material. Glandular epithelium is composed of pyramidal cells containing abundant granular endoplasmic reticulum, Golgi complexes, and apical granules. The structure of

these glands did not appear to change throughout the duration of this study. Day 7 (Penetration of Uterine Epithelium)

Embryonic development at this time proceeds rapidly. Therefore, it is during this period that embryos of the same apparent gestation age may appear most asynchronous. This disparity is further exacerbated by the focal nature of initial trophoblast penetration of the epithelium, the primary event occurring on this day. Gross examination of the uterus reveals implantation sites larger than those of the previous day and a more intense Evans blue reaction.

RLASTOCYST IMPLANTATION IN THE CHINESE HAMSTER

Fig. 14. Microvilli of the trophoblast (TI and uterine epithelium WE) are bent and flattened on day 7. This compression suggests active expansion of the blastocyst within the implantation chamber.

Despite this close contact, no junctions were found between the trophoblast and epithelium. x 23,000.

Fig. 15. Well-differentiated decidual cell from a day 7 implantation site. Large, dense glycogen (GLY) deposits are common. Note the abundance of granular endoplasmic reticulum and the Golgi complex (GI. x 12,600.

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Flg. 16. By day 8 the visceral endoderm WE) is well developed. A distinct basal lamina (BL) separates visceral endoderm and epiblast (EPI). Contrast the electron-lucent cytoplasm of the endoderm with the dense cytoplasm of the polyribosome-rich epiblast. Lysosome-like granules (L) are present in the endoderm, as are electron-dense apical vesicles (V). x 9,200.

Blastocyst

On day 7 the uterine lumen is closed with the embryo encased within the implantation chamber (Fig. lb). Mitotic figures may be seen in both the inner cell mass and trophoblast. Areas of uterine epithelial penetration are initially quite focal with advancing mural trophoblast spreading beneath adjacent epithelial cells (Figs. lb, 8).These foci are not restricted to a particular region of the mural surface of the blastocyst. Thereafter, increasing numbers of trophoblast cells come to lie adjacent to the basal lamina of the epithelium, but no penetration of this layer is noted (Fig. 9). Within the trophoblast are abundant inclusions of various sizes containing cellular debris that are most likely derived from phagocytized, deteriorating epithelial cells. Although no junc-

tions appear to develop between the uterine epithelium and trophoblast, areas of close, parallel membrane apposition between these cells do occur (Fig. 9). In addition, no areas of cytoplasmic continuity between trophoblast and epithelium are observed. Trophoblast cells sometimes are seen to insinuate between the base of a n epithelial cell and its basal lamina. The leading edge of the advancing trophoblast often has bundles of fine filaments just beneath its surface. The remaining trophoblast shows a n increased complement of lipid droplets and glycogen compared to day 6 embryos. Polyribosomes remain abundant, while granular endoplasmic reticulum is sparse. The polar trophoblast layer is composed of flattened cells that are sometimes slightly separated from the embryonic cell mass. The inner surface of the mural trophoblast lining

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Fig. 17. The visceral endoderm (VE) layer is close to the well-developed Reichert’s membrane (arrows), while parietal endoderm (PE) forms a discontinuous layer. Trophoblast cells (TImay be quite thin, as seen in this figure. Although no uterine epithelium remains by day

8, portions of the basal lamina and underlying connective tissue separate the trophoblast from decidual cells (D) in this area. Note the uneven contour of Reichert’s membrane where it is not in contact with a parietal endoderm cell. YS, Yolk sac cavity. Day 8. x 10,900.

the blastocyst cavity remains smooth but now has a thin basal lamina, thus forming the predecessor of Reichert’s membrane (Fig. 12). Microvillous interactions between trophoblast and epithelium similar to those found on day 6 remain, but these regions are greatly reduced and are replaced by apical protrusions from the epithelial cells (Figs. 10, 12). The protrusions present profiles of various shapes and sizes and are intimately surrounded by trophoblast. Some of the larger protrusions are attached to the epithelium by a short, narrow stalk (Fig. 11).These structures are filled with homogeneous cytoplasm containing fine filaments. Further, profiles of these large ectoplasmic protrusions are occasionally seen isolated in nearby trophoblast with two membranes separating the cytoplasm of the protrusion from that of the trophoblast. The embryonic cell mass has differentiated into a n epiblast and a layer of visceral endoderm (Fig. lb). These two cell types are usually separated by a basal lamina continuous with Reichert’s membrane (Fig. 13). A discontinuous layer of parietal endoderm is seen along the mural trophoblast. Microvilli festoon the apices of both visceral and parietal endoderm. Anastomosing granular endoplasmic reticulum with dilated cisternae typical of endoderm are present (Fig. 13), as are lipid droplets. Although endoderm has become distinct, the remaining epiblast retains the undifferentiated ap-

pearance of the attachment phase blastocyst. The paracrystalline inclusions are now decreased in both size and number. Uterine epithelium

The epithelium appears to change little from that seen on the previous day, except for cells adjacent t o areas of trophoblast penetration. In these restricted regions, some of the epithelium has been phagocytized by the trophoblast but the basal lamina remains intact (Fig. 9). The trophoblast flange extends along the basal lamina, giving a rounded profile to adjoining epithelial cells (Fig. 8). The remaining epithelium apposed to the blastocyst is compressed to approximately half its former height. There is some increase in the number of lysosome-like granules and glycogen in these cells. The epithelial microvilli which remain in contact with the blastocyst tend to be bent and flattened (Figs. 13,14). Most of the apical surfaces in this region either are comprised of large, blunted processes (Figs. 10, 11) or are smooth and lie parallel to the trophoblast membrane. The cytoplasm within and immediately underlying these apical protrusions is often devoid of organelles and contains a network of fine filaments. The appearance of these ectoplasmic protrusions and their interactions with the trophoblast has already been described. Epithelium located mesometrial to the embryo re-

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Fig. 18. On day 8 many maternal red blood cells are in contact with trophoblast cells. Some trophoblast cells (TIform blood channels (C) that contain blood cells. Decidual cells (D) adjacent to the embryo are epithelioid with little intercellular space. EPI, Epiblast; VE, visceral endoderm; arrow, Reichert’s membrane. x 975. Fig. 19. Electron micrograph of a trophoblast blood channel. Note the attenuated trophoblast cytoplasm (T)around the red blood cell (RBC) in the channel. The thin trophoblast layer separates the blood cell from Reichert’s membrane (arrow)and the yolk sac cavity (YS). V, Vesicle; VE, visceral endoderm. Day 8. x 32,000.

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Fig. 20. Giant trophoblast cell (GT) forming a portion of a blood channel. The trophoblast (TI and decidual cells (D)are in close proximity, with no intervening basal lamina. A decidual cell process (PI and collagen fibrils (C) a r e surrounded by the trophoblast cell. R, Reichert’s membrane; RBC, red blood cell; WBC, leucocyte. Day 8. x 15,700.

tains a brush border that tightly interdigitates with that of cells from the opposite side. The number of dark cells within the epithelium appears to increase, but this is not a consistent feature. Stroma

Decidual cells near the uterine epithelium continue to enlarge, especially those adjacent to the blastocyst (Figs. lb, 8). The area occupied by differentiating decidua in the antimesometrial region is greater than that seen in the mesometrial direction. Three general

areas of stroma are apparent, each comprising approximately one-third of the width of the stroma. The layer next to the uterine epithelium consists of well-differentiated decidual cells while the area nearest the myometrium contains undifferentiated fibroblasts with extensive extracellular matrix, The intervening stratum contains stellate cells that are intermediate in development. As decidual cells continue to enlarge the extracellular matrix separating them decreases; and there appears to be a modest increase in the number of collagen

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fibrils. Large, dense accumulations of glycogen are found in many of the cells (Fig. 15). Granular endoplasmic reticulum is much more abundant, and free polyribosomes remain prevalent. Multiple Golgi complexes are present in many cells. Punctate junctions are common and gap junctions are seen. Decidual cells may lie very close to the basal lamina of the uterine epithelium and occasionally extend a flange of cytoplasm toward that layer. However, none of these processes was seen to penetrate the basal lamina. Occasional leukocytes are seen in the extracellular matrix, both near the epithelium and elsewhere. Blood vessels, primarily sinusoids, are prevalent and are sometimes seen adjacent to the epithelial basal lamina (Fig. l b ) . Generally, however, these vessels are separated from the epithelium by two or more decidual cells. Day 8 (Invasion of Uterine Stroma)

By day 8, the implantation chamber is elongated and the embryo has developed to the egg cylinder stage (Fig. lc). Paracrystalline inclusions are not seen a t this stage of development. The blastocyst cavity is now a yolk sac which constitutes a bilaminar omphalopleure. In gross appearance, a prominent swelling indicates the position of each embryo along the uterus, as does the focally intense Evans blue staining reaction. Blastocyst

The embryonic cell mass has proliferated and forms a column of cells which extends into the yolk sac. The endoderm is clearly distinct from the epiblast and forms a continuous visceral layer confluent with a discontinuous parietal layer (Fig. lc). Visceral endoderm forms a single columnar layer and is separated from the epiblast by a prominent basal lamina (Fig. 16) which is continuous with Reichert’s membrane. These endoderm cells possess a comparatively lucent cytoplasm which includes diffusely distributed polyribosomes, moderate amounts of granular endoplasmic reticulum containing electron-dense material, and lysosome-like granules. These cells do not display the interconnected granular endoplasmic reticulum seen earlier. The mitochondria are moderate in number. Apical Golgi complexes are present which are associated with vesicles containing material of the same electron-density a s Reichert’s membrane. The nuclei compose a large portion of these cells and display dispersed chromatin with one or two nucleoli. These cells possess apical junctions, narrow intercellular spaces, and numerous microvilli. Inclusions containing cellular debris are common. Coated vesicles are seen forming a t the interface with the yolk sac cavity. Because of growth of the embryonic cell mass, many areas of the visceral layer are in contact with the parietal layer or Reichert’s membrane, thus largely obliterating the yolk sac cavity (Figs. lc, 17-19). Parietal endoderm is continuous with the visceral layer a t the polar region and extends as a discontinuous sheet along Reichert’s membrane adjacent to the mural trophoblast. These cells have numerous microvilli and cytological features similar to those of the visceral layer, but are squamous in shape. Where parietal endoderm is present, Reichert’s membrane is composed of a basal lamina next to the trophoblast and a thin layer of flocculent electron-dense material in

contact with the endoderm. Where parietal endoderm is absent, Reichert’s membrane may be much thicker and variable in contour as a result of uneven deposition of the flocculent material (Fig. 17). Cells of the epiblast increase in number but otherwise appear similar to those seen in the previous stages (Fig. 16). The appearance of these cells remains typically embryonic. The cells are filled with polyribosomes, resulting in a denser cytoplasm than that seen in the endoderm. Mitochondria are sparse, granular endoplasmic reticulum is composed of a few short strands, and Golgi complexes are rare. The lysosomelike granules seen in the endoderm are not present. Primitive punctate junctions may be found along contiguous membranes. A proamniotic cavity is apparent on day 8 and appears to develop from expanding intercellular spaces originating between cells located in the center of the epiblast. Subsequently, these spaces coalesce to form a cavity surrounded by a primitive epithelium (Fig. lc). Uterine epithelium surrounding the embryo is lost, and mural trophoblast has achieved intimate contact with the surrounding stroma over most of its area (Figs. lc, 17, 18, 20). The epithelial basal lamina has largely disappeared, although remnants of this layer remain. In some areas where the basal lamina is present, i t is associated with increased amounts of collagen arranged roughly parallel to the basal lamina in the mesometrial-antimesometrial axis (Fig. 17). In other areas the basal lamina appears flocculent and of uneven thickness, as if deteriorating. It was not possible to determine whether the trophoblast or decidual cells were the first to penetrate the basal lamina. Giant trophoblast cells are found a t both the antimesometrial pole (primary giant cells) and the mesometrial pole (secondary giant cells). These large cells (approximately 100 pm in width) are in direct contact with the decidual cells and extravasated maternal blood cells (Fig. lc). Giant cells appear active with abundant euchromatin, polyribosomes, numerous strands of granular endoplasmic reticulum, and moderate numbers of diffusely distributed mitochondria (Fig. 20). In addition, many inclusions of phagocytized cell debris in various stages of deterioration are present. Lipid droplets are occasionally seen, and small Golgi complexes are scattered throughout the cytoplasm. Maternal erythrocytes and leukocytes are often in contact with the giant cells but do not appear to be degenerating. In some instances it is uncertain whether these blood cells have been phagocytized or are merely surrounded by the trophoblast. Few cells of the mural trophoblast reach the large size of the giant cells, but they do contain similar cytological features. In some areas the mural trophoblast appears to form blood channels containing tightly packed maternal erythrocytes, platelets, and leukocytes (Figs. 18-20). As a result, some blood cells are separated from Reichert’s membrane by a layer of trophoblast cytoplasm only 0.25 pm thick (Fig. 19).These blood cells do not appear deteriorated. In some areas alternating layers of trophoblast and decidual cells are seen, indicating regions of elaborate interdigitations between these cell types. Remnants of uterine epithelial basal lamina are sometimes present

Fig. 21. Decidual cells (D) contain short cisternae of dilated granular endoplasniic reticulum (ER). Glycogen accumulations iGLY) appear somewhat extracted as compared to day 7 (see Fig. 15). The endothelium (END) is thickened and without fenestrations. RBC, Red blood cell. Day 8. x 9,900. Fig. 22. A decidual cell process (asterisk) has penetrated the endothelial (END)basal lamina and is adjacent to the capillary lumen (L),

forming a portion of the capillary wall. Note the intercellular junctions between the decidual cell (D) process and the endotheliurn. GLY, Glycogen. Day 8. x 19,000. Fig. 23. A short process (arrow) from an endothelial cell (END)has penetrated the basal lamina and is in contact with a decidual cell (D). L, Capillary lumen. Day 6. x 21,100.

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among these layers in addition to varying amounts of extracellular matrix. Many junctions are found between trophoblast cells (Fig. 19). Some areas of lateral trophoblast membranes show profuse interdigitation, especially near Reichert’s membrane. Uterine epithelium

The uterine epithelium has completely disappeared in the vicinity of the embryo and is replaced by trophoblast. In some day 8 sites, the epithelium located mesometrial to the embryo is deteriorating with only the most distal cells appearing healthy. In other sites no connection is seen between the embryo and the residual lumen. Although it is possible that a connection remains between the embryo and the remaining epithelium located mesometrially, results from serial sectioning a single site do not support this contention. Thus the implantation chamber is isolated in the decidual tissue. The small amount of epithelium remaining appears normal except for its antimesometrial extreme, where the cells are deteriorating and in contact with extravasated blood. The lumen is otherwise tightly closed. Stroma

The area containing decidual cells continues to enlarge, extends to the myometrium in the antimesometrial region, and occupies approximately half the stroma1 area laterally. It appears that this region expands by recruitment of undifferentiated stromal cells in addition to mitotic activity of the decidual cells. Fewer cells undergo differentiation in the areas located a t the mesometrial end. The decidual cells are large but similar to those found on day 7. They contain numerous cisternae of dilated granular endoplasmic reticulum which is often filled with electron opaque material (Figs. 20, 21). Golgi complexes and vesicles are common. Large accumulations of glycogen are found, although this substance appears partially extracted from many cells. Isolated bundles of fine and intermediate filaments are seen in a few cells. Some of these cells display a n extensively folded surface, with many of the lamellae in close apposition, thus increasing surface area. Blood sinusoids and capillaries are plentiful near the embryo. In all the vessels examined, the endothelium was without fenestrations. Some of these vessels exhibit thickened endothelium (Fig. 21) with electronlucent cytoplasm and a discontinuous basal lamina. In these vessels, there are areas of the basal lamina which are flocculent, variable in width and focally absent. Processes of nearby decidual cells are in contact with the endothelium. In one instance a decidual cell process was seen to extend to the lumen of a sinusoid and form primitive intercellular junctions with the endothelium (Fig. 22). DISCUSSION

The results presented in this study establish the nature and time course of morphological interactions that occur between the blastocyst and endometrium during implantation in the Chinese hamster. Attachment of the blastocyst to the uterine epithelium occurs on day 6 of gestation, and penetration of the epithelium is achieved on day 7. By day 8 the trophoblast is in con-

tact with the uterine stroma. Pickworth et al. (1968) stated briefly that when the day of mating is designated day 0, implantation appeared to have begun on day 5 in the Chinese hamster, which corresponds to day 6 in this study. The developmental events described for the Chinese hamster on days 6 and 7 are similar to those that occur in the rat on the same days (Enders and Schlafke, 1967). Those investigators found that although blastocysts were in position within the uterus and a faint Pontamine blue reaction could be elicited on late day 5 , the blastocysts could be readily flushed from the lumen without apparent damage. Further, i t was not until day 6 that the rat embryos were adhered securely to the uterine epithelium. Reinius (1967) and Potts (1968) found that mouse blastocysts are tightly embraced by the uterine epithelium by day 5 of gestation, approximately 1day earlier than seen in the Chinese hamster. In contrast, implantation in the golden hamster begins approximately on the afternoon of day 3 (Young et al., 1968; Parkening, 1976a), substantially earlier than the mouse, rat, or Chinese hamster. By late day 3, the golden hamster blastocyst is completely enclosed within the uterine epithelium. Early interactions between the blastocyst and uterine epithelium on day 5 in the mouse and day 6 in the Chinese hamster and rat are quite similar. Initial microvillous interdigitation of trophoblast and epithelial cells are followed by the appearance of larger epithelial cytoplasmic protrusions. Subsequent diminution of apical membrane features are characteristic of these species. In contrast, Parkening (197613) did not describe this progression of changes for the golden hamster and stated that epithelial microvilli were present until these cells were phagocytized by trophoblast. Close membrane apposition between trophoblast and uterine epithelial cells occurred in the Chinese hamster, but no distinct intercellular junctions between these cell types were seen. Potts (1968) described desmosomes between trophoblast and epithelial cells in the mouse, while Smith and Wilson (1974) found mouse trophoblast cells attached to uterine epithelium by primitive junctions. In another study, however, no junctions between these cell types were observed in the mouse or rat (Parr et al., 1987). An important aspect of interstitial implantation is the removal of the uterine epithelium. Although the mechanism of this process is not completely understood, several reports have focused on this phenomenon. The apical cytoplasmic protrusions and their association with trophoblast cells observed in Chinese hamster epithelium have also been seen in other species during implantation (Nilsson et al., 1978). Parr et al. (1987) have found that these protrusions are involved in the process of uterine epithelium removal in the early implanting mouse and rat. Those workers documented the uptake of the protrusions by trophoblast cells and concluded that the epithelium disappears as a result of apoptosis rather than necrosis or autolysis. Nilsson (1974), in a study of mouse blastocysts induced to implant following a delay of implantation, also noted the presence of epithelial protrusions and observed that they appeared to be engulfed by trophoblast. El-Shershaby and Hinchliffe (1975) described degenerating epithelium in the implanting mouse. In that study, cells apposed to the blastocyst underwent

BLASTOCYST IMPLANTATION I N T H E CHINESE HAMSTER

autolysis without a n increase in lysosomes, while those located antimesometrially accumulated degradation bodies prior to cell death. In addition, apparently viable cells showed a n increase in dense bodies characterized as lysosomes, which were morphologically similar to the granules seen to increase in Chinese hamster uterine epithelium. These workers concluded that the cells located antimesometrially underwent autonomous degeneration, and not as a result of trophoblast contact. Smith and Wilson (1974) noted isolated dark epithelial cells in the mouse endometrium both prior to and during implantation. They found that these cells were deteriorating and that some were directly associated with invading trophoblast. In the present study a few dark cells were seen throughout the luminal epithelium, but no specific correlation with trophoblast was noted. These results support the possibility that a t least some uterine epithelial cells deteriorate spontaneously and perhaps facilitate trophoblast penetration. Cytological features of the uterine epithelium in the mouse, rat, and golden hamster are similar to those found in the Chinese hamster. However, the abundance of lipid inclusions seen in the mouse epithelium is not present in the Chinese hamster. That the uterine epithelium of these and other species is active in protein synthesis is evidenced by the plethora of mitochondria, granular endoplasmic reticulum and Golgi complexes. A review of the uterine epithelium during implantation is provided by Given and Enders (1989). Invasion of the epithelium begins along the mural trophoblast in each of these species. By day 7 in the Chinese hamster and rat, by day 6 in the mouse, and by late day 4 in the golden hamster, the trophoblast has advanced to but remains separated from the stroma by the basal lamina of the uterine epithelium. In each case this event occurs approximately 24 h r after blastocyst attachment has begun. The layer of fine filaments often seen along the membrane of the advancing trophoblast in the present study may be associated with cell motility. By day 8 in the Chinese hamster, the basal lamina of the uterine epithelium is discontinuous and has largely disappeared, leaving trophoblast cells in direct contact with decidua. In other areas, the basal lamina was flocculent and appeared to be undergoing dissolution. It was not possible to determine which cell type first penetrated the basal lamina. Tachi et al. (1970) found that the epithelial basal lamina in the rat began to disappear approximately 48 h r after the onset of implantation, leaving trophoblast in progressively intimate contact with stromal cells. In a study of implantation in the rat, decidual cells were seen to first breach the basal lamina barrier on day 7 (Schlafke et al., 1985). This report is at odds with the prevailing assumption that the trophoblast is responsible for the penetration of this temporary barrier. It remains to be seen whether this interesting finding is unique to the rat or if it occurs in other species. In the golden hamster, maternal blood is in contact with the trophoblast approximately 2 days following the onset of blastocyst adhesion (Parkening, 1976b), indicating that the epithelial basal lamina is no longer intact. The biochemical mechanisms by which the uterine epithelial basal lamina is penetrated in vivo are not known. However, several reports indicate that trophoblast cells are capable of degrading basal laminae in

155

vitro. Fisher et al. (1985) found that human cytotrophoblast cells are able to degrade basal lamina components in vitro, but only in areas immediately adjacent to the cells. In another report, degradation of smoothmuscle-derived extracellular matrix by cultured mouse blastocysts was limited to areas of contact (Glass et al., 1983). These findings suggest that the proteolytic enzymes are located on trophoblast cell membranes. Yagel et al. (1988) found that human trophoblast cells and specific tumor cells were comparable in their ability to invade human amnion basement membrane. The invasiveness of those cell types was blocked when the cells were challenged with proteinase inhibitors. These results indicate that trophoblast cells are capable of focal destruction of basal laminae and that these mechanisms may be similar to those of metastatic tumor cells known to cross basal laminae. Further study of interactions between trophoblast, basal laminae, and decidual cells in situ are needed to clarify the processes by which basal laminae are degraded during implantation. The trophoblast formed a sometimes thin but continuous layer in these embryos. The elaborate interfolding of contiguous trophoblast cell membranes seen in the Chinese hamster was also reported in the mouse (Potts, 1968) and rat (Welsh and Enders, 1987). These folds contain intercellular junctions and may serve to limit the intercellular passage of materials into or out of the blastocyst cavity. Channels containing maternal blood cells were seen in the mural trophoblast of the day 8 Chinese hamster (Fig. 18).Welsh and Enders (1987) described the presence of similar blood spaces in the day 8 rat embryo, although these channels included fenestrations in the trophoblast which were not seen in the Chinese hamster. These spaces appear at the time of blood vessel penetration by the trophoblast, thus establishing blood circulation in the yolk sac placenta. Intracellular inclusions of phagocytized degenerating cellular debris similar to those found in the Chinese hamster were also noted in mouse (Potts, 1968; Smith and Wilson, 1974; El-Shershaby and Hinchliffe, 1975; Parr et al., 19871,rat (Enders and Schlafke, 1967; Tachi et al., 1970; Parr et al., 19871, and golden hamster trophoblast (Parkening, 197613). It is likely that most of this debris is derived from deteriorating epithelial cells. Paracrystalline inclusions characteristic of the preimplantation Chinese hamster blastocyst (Parkening et al., 1985) were also present on days 6 and 7 in the present study. Although the periodic structure of these elements appeared unchanged from the previous description, their numbers and sizes were progressively diminished. These structures were not seen in the day 8 embryo. Similar, but not identical inclusions are seen in the mouse, rat, and hamster (Nilsson, 1980). The function and fate of these inclusions remains unclear, although various workers have concluded that they were composed of protein (Enders, 1971; Parkening et al., 1985). Remnants of these inclusions were seen in lysosome-like granules both in the present study and in the golden hamster (Enders, 1971). In the rat, similar inclusions were also present in the implanting blastocyst but greatly diminished during subsequent days, becoming absent by day 7 (Enders and Schlafke, 1967).

156

T.N. BLANKENSHIP ET AL

Paracrystalline inclusions in the golden hamster disappear by day 4, also only 1 day after implantation begins (Parkening, 1976a). Another inclusion found in the present investigation consists of patches of electron-dense flocculent material associated with thin rods. These membrane bounded structures were seen most often in the day 7 embryos and were typically associated with the paracrystalline inclusions. While the function of this inclusion remains unknown, it may be a type of degradation vacuole. In the Chinese hamster, endoderm begins to form a t the time of adhesion (day 6) and its distribution is restricted to the inner cell mass. By day 7 visceral endoderm covers the epiblast, and parietal endoderm is located along the mural trophoblast. Migration of parietal endoderm occurs along the trophoblast in the day 5 mouse (Potts, 1968; Enders et al., 1978) and day 6 rat (Enders and Schlafke, 1967; Enders et al., 1978). Parkening (1976a) reports that endoderm was visible in the day 4 golden hamster, soon after the onset of implantation. As the visceral endoderm develops in each of these species, it forms a complete epithelium that separates the epiblast from the blastocyst cavity, especially apparent during the egg cylinder stage. The coated vesicles seen in the day 8 Chinese hamster visceral endoderm are typical of vesicles involved in pinocytotic uptake. This process has been documented in these cells in implanting rat embryos exposed to horseradish peroxidase (Enders et al., 1978). Those workers suggest that this phenomenon may be important for embryo nourishment prior to the formation of the hemotrichorial chorioallantoic placenta. Reichert’s membrane was first seen as a rudimentary basal lamina along the trophoblast on day 7 in this study and in the rat (Enders and Schlafke, 1967), about 1day following adherence of the blastocyst to the uterine epithelium. A basal lamina lining the blastocyst cavity side of the mural trophoblast, representing the precursor of Reichert’s membrane, was seen in the day 4 golden hamster (Parkening, 197613). Potts (1968) reported that Reichert’s membrane had begun development in the day 5 mouse embryo, a t the beginning of implantation and relatively earlier than that seen in the Chinese hamster, rat, and golden hamster. As development continued in the Chinese hamster, electrondense material was seen to accumulate along the trophoblast basal lamina, thus forming the definitive Reichert’s membrane. This deposition was of uneven thickness, except where the trophoblast was juxtaposed to a parietal endoderm cell. Visceral endoderm cells of the day 8 Chinese hamster contained granules with electron-density similar to Reichert’s membrane. This observation provides circumstantial evidence for a contribution by these cells to the formation of Reichert’s membrane. The disposition of the uterine stroma in the Chinese hamster and its transformation to decidua is similar t o that in the mouse (Finn and Lawn, 1967; Potts, 1968; Abrahamsohn, 19831, rat (Enders and Schlafke, 1967; O’Shea et al., 1983), and golden hamster (Ward, 1948; Parkening, 1 9 7 6 ~ )Although . the structure and development of the decidua has been well investigated, the function of this tissue remains uncertain. Proposed functions have included modulation of the invasive properties of the trophoblast, immunologic isolation of

the embryo and mother, and nutrition of the early embryo, A few stromal cells near the antimesometrial end of the lumen are enlarged a t the time of blastocyst attachment. As decidualization proceeds, the number of cells undergoing hypertrophy increases, beginning with cells nearest the antimesometrial uterine epithelium and progressing outward. The compact layer of epithelioid decidual cells near the lumen corresponds to the rat primary decidual zone described by Parr et al. (1986). In addition, the numbers of intercellular junctions increase, including both punctate and gap junctions. Only small amounts of intracellular filaments were seen in the decidual cells of the Chinese hamster. In contrast, large accumulations of intermediate filaments were seen in the day 5 golden hamster (Parkening, 1976c),day 6 mouse (Abrahamsohn, 19831, and day 7 rat (Enders and Schlafke, 1967; Tachi et al., 1970) decidual cells. Glycogen content of the decidual cells increased dramatically in the Chinese hamster by day 7, with these accumulations often forming near lipid droplets. This increase is also seen in the mouse, rat, and golden hamster shortly after decidualization has begun. The apparent increase in collagen fibrils beneath the basal lamina seen on day 8 in the Chinese hamster is similar to that described for the mouse (Smith and Wilson, 19741, and golden hamster (Parkening, 1976b). In the present study a n increase in collagen subjacent to the epithelial basal lamina and its orientation roughly parallel to the long axis of the implantation chamber was similar to that found in the rat (SchlaRe et al., 1985). In addition, a small increase in the collagen content of the remaining stroma was seen in the Chinese hamster. A progressive decrease in collagen content of the extracellular matrix in preimplantation mice was followed by a n increase in collagen shortly after implantation (Zorn et al., 1986). However, the postimplantation collagen consisted of bundles of variable width, with thicker bundles associated with differentiated decidual cells. In day 8 Chinese hamsters, a few sinusoids displayed endothelial basal laminae that were disrupted, with a n appearance similar to that of the deteriorating uterine epithelial basal lamina. Although a few small cytoplasmic processes of decidual cells were in contact with the endothelium on days 6 and 7, these interactions were much more extensive in both size and distribution on day 8. Occasionally, a small flange of endothelium extended a short distance through its basal lamina and achieved contact with a decidual cell (Fig. 23). In one sinusoid of a day 8 animal, a decidual cell process was inserted between epithelial cells and adjacent to the lumen (Fig. 22). A similar finding in the rat was reported by Tachi et al. (1970) and Welsh and Enders (1987). The extensive contact between decidual cells and endothelium observed in the Chinese hamster was also reported in the pseudopregnant (O’Shea et al., 1983) and normally implanting r a t uterus (Parr et al., 1986; Welsh and Enders, 1987). Decidual cell processes were also the first to penetrate the uterine epithelial basal lamina in the day 7 rat implantation site (Schlafke et al., 1985). These results indicate that decidual cells have the ability to modulate the integrity of basal laminae of both uterine epithelium and endothelium during implantation.

BLASTOCYST IMPLANTATION IN THE CHINESE HAMSTER

Cellular changes in the stroma at the time of implantation indicate that this tissue is undergoing substantial reorganization. Expansion of the decidua apparently involves recruitment of undifferentiated stromal cells. In addition, a few mitotic figures are seen throughout the stroma, a n indication that hyperplasia as well as hypertrophy are factors in decidual transformation. Interactions between the embryo and endometrium during implantation in the Chinese hamster, mouse, rat, and golden hamster have many common features. However, they are not identical. There are differences which are appreciated only by using ultrastructural techniques. Indeed, implantation has been comprehensively studied by electron microscopy in comparatively few mammalian species. Continued investigation into the fine structure of embryonic-maternal interactions is expected to reveal similarities and differences illustrative of reproductive strategies and phylogenetic relationships. Because interspecies differences do occur, one may safely assume that these differences reflect useful adaptations for successful reproduction. ACKNOWLEDGMENTS

This study was supported by grant S07-RR-07205 from the National Institutes of Health and funds from the state of Texas. The efforts of Ms. Emily Preslar and Ms. Helen Bug0 in preparing this manuscript are gratefully acknowledged. LITERATURE CITED Abrahamsohn, P.A. 1983 Ultrastructural study of the mouse antimesometrial decidua. Anat. Embryol. (Berl.), 166:263-274. Donkelaar, H.J. ten 1979 Stages in the prenatal development of the Chinese hamster (Cricetulus griseus). Anat. Embryol. (Bed.), 156: 1-28. El-Shershaby, A.M., and J.R. Hinchliffe 1975 Epithelial autolysis during implantation of the mouse blastocyst: An ultrastructural study. J. Embryol. Exp. Morphol., 33:1067-1080. Enders, A.C. 1971 Fine structure of the blastocyst. In: The Biology of the Blastocyst. R.J. Blandau, ed. University of Chicago Press, Chicago, pp. 71-94. Enders, A.C., and S. Schlafke 1967 A morphological analysis of the early implantation stages in the rat. Am. J. Anat., 120:185-226. Enders, A.C., R.L. Given, and S. Schlafke 1978 Differentiation and migration of endoderm in the rat and mouse at implantation. Anat. Rec., 190335-78. Finn, C.A., and A.M. Lawn 1967 Specialized junctions between decidual cells in the uterus of the pregnant mouse. J. Ultrastruct. Res., 20t321-327. Fisher, S.J., M.S. Leitch, M.S. Kantor, C.B. Basbaum, and R.H. Kramer 1985 Degradation of extracellular matrix by the trophoblastic cells of first-trimesterhuman placentas. J. Cell. Biochem., 27:31-41. Fortuyn, A.B. Droogleever 1929 Prenatal death in the striped hamster. Arch. Biol., 39r583-606. Given, R.L., and A.C. Enders 1989 The endometrium of delayed and early implantation. In: Biology of the Uterus, 2nd ed. R.M. Wynn and W.P. Jollie, eds. Plenum Publ. Corp., New York, pp. 175-231. Glass, R.H., J. Aggeler, A. Spindle, R.A. Pedersen, and Z. Werb 1983 Degradation of extracellular matrix by mouse trophoblast outgrowths: A model for implantation. J . Cell Biol., 96t1108-1116.

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Kamiguchi, Y., and K. Mikamo 1982 Dose-response relationship for induction of structural chromosome aberrations in Chinese hamster oocytes after X-irradiation. Mutat. Res., 103:33-37. Nilsson, B.O. 1974 The morphology of blastocyst implantation. J . Reprod. Fertil., 39:187-194. Nilsson, B.O. 1980 Comparative ultrastructure of the yolk material in preimplantation stages of the hamster, mouse, and rat embryos. Gamete Res., 3:369-377. Nilsson, B.O., S. Bergstrom, S. Hakansson, I. Lindqvist, I. Ljungkvist, 0. Lundkvist, and G. Naeslund 1978 Ultrastructure of implantation. Ups. J. Med. Sci. lSuppl.1, 22:27-38. O’Shea, J.D., R.G. Kleinfeld, and H.A. Morrow 1983 Ultrastructure of decidualization in the pseudopregnant rat. Am. J. Anat., 166: 271-298. Parkening, T.A. 1976a An ultrastructural study of implantation in the golden hamster. I. Loss of the zona pellucida and initial attachment to the uterine epithelium. J. Anat., 121:161-184. Parkening, T.A. 1976b An ultrastructural study of implantation in the golden hamster. 11. Trophoblastic invasion and removal of the uterine epithelium. J. Anat., 122:211-230. Parkening, T.A. 1976c An ultrastructural study of implantation in the golden hamster. 111. Initial formation and differentiation of decidual cells. J. Anat., 122:485-498. Parkening, T.A., A.F. Payer, and R.L. Given 1985 Characterization of paracrystalline inclusions in Chinese hamster oocytes and early embryos. Gamete Res., 12:373-384. Parr, M.B., H.N. Tung, and E.L. Parr 1986 The ultrastructure of the rat primary decidual zone. Am. J . Anat., I76:423-436. Parr, E.L., H.N. Tung, and M.B. Parr 1987 Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats. Biol. Reprod., 36t211-225. Pickworth, S., G. Yerganian, and M.C. Chang 1968 Fertilization and early development in the Chinese hamster (Cricetulus griseus). Anat. Rec., 162t197-208. Potts, D.M. 1968 The ultrastructure of implantation in the mouse. J. Anat., 103177-90, Reinius, S. 1967 Ultrastructure of blastocyst attachment in the mouse. Z. Zellforsch., 77:257-266. Schlafke, S., A.O. Welsh, and A.C. Enders 1985 Penetration of the basal lamina of the uterine luminal epithelium during implantation in the rat. Anat. Rec., 212:47-56. Sirek, O.V., and A. Sirek 1967 The colony of Chinese hamsters of the C.H. Best Institute. Diabetologia, 3:65-73. Smith, A,, and I.B. Wilson 1974 Cell interaction at the maternalembryonic interface during implantation in the mouse. Cell Tissue Res., 152:525-542. Tachi, S.,C. Tachi, and H.R. Lindner 1970 Ultrastructural features of blastocyst attachment and trophoblastic invasion in the rat. J. Reprod. Fertil., 21.37-56. Ward, M.C. 1948 The early development and implantation of the golden hamster, Cricetus aurutus, and the associated endometrial changes. Am. J. Anat., 82:231-276. Welsh, A.O., and A.C. Enders 1987 Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat. Anat. Rec., 21 7r203-219. Yagel, S., R.S. Parhar, J.J. Jeffrey, and P.K. Lala 1988 Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells. J. Cell. Physiol., 136:455-462. Yanagimachi, R., Y. Kamiguchi, S. Sugawara, and K. Mikamo 1983 Gametes and fertilization in the Chinese hamster. Gamete Res., 8t97-117. Yerganian, G. 1967 The Chinese hamster. In: UFAW Handbook on the Care and Management of Laboratory Animals. UFAW Staff, eds. E.&.S. Livingston, Ltd., Edinburgh, 21r340-351. Young, M.P., J.T. Whicher, and D.M. Potts 1968 The ultrastructure of implantation in the golden hamster (Cricetus uuratus). J. Embryol. Exp. Morphol., 19t341-345. Zorn, T.M.T., E.M.A.F. Bevilacqua, and P.A. Abrahamsohn 1986 Collagen remodeling during decidualization in the mouse. Cell Tissue Res., 244:443-448.