Structural interactions of trophoblast and uterus during hemochorial placenta formation.

It is argued that the successful placental arrangements are those that decrease the exposure of the trophoblast to the efferent side of the cellular immune response. Examples are taken from three different groups with hemochorial placentation, to show that their placental morphology places most of the trophoblast in contact with maternal blood, not maternal connective tissue. In addition, maternal cells of the junctional area are modified either before contact with trophoblast (rat) or just after trophoblast invasion (primate), or the region of contact is limited as in the armadillo, in which maternal blood sinuses are expanded to form intervillous spaces.

Chorioallantoic placenta formation in the rat. III. Granulated cells invade the uterine luminal epithelium at the time of epithelial cell death.

Rat gestation sites were microscopically examined on Days 7, 8, and 9 of pregnancy to determine the fate of epithelial cells lining the uterine lumen mesometrial to the implantation chamber. This region of the uterine lumen and the mesometrial region of the implantation chamber comprise the site of chorioallantoic placenta formation. The fate of the uterine luminal epithelium was closely associated with a subepithelial accumulation and epithelial invasion of granulated cells (granulated metrial gland cells). On Day 7, mature granulated cells were not detected in the subepithelial stroma, and epithelial cells appeared healthy. On Day 8, when granulated cells began populating the subepithelial stroma and a few crossed the basal lamina, some epithelial cells degenerated in situ. On Day 9, when large numbers of granulated cells invaded the epithelium, large numbers of epithelial cells were displaced from their basal lamina and were degenerating. Degenerating cells were of two morphological types. One type conformed to the standard description of apoptosis; that is, cells rounded up, detached from their neighbors, and their nuclei fragmented, although other organelles remained intact. The other type of cell degeneration resembled apoptosis in some respects, but the cells were very electron-dense, adjacent cells often remained attached, membranous organelles including mitochondria were swollen, and nuclei did not fragment. Granulated cells are related to natural killer cells, cells that induce target cell apoptosis, and we suggest that granulated cells may be involved in uterine luminal epithelial cell death. The conceptus remained in an antimesometrial region of the implantation chamber during the times studied and did not come into direct contact with these epithelial cells.

Uterine cell death during implantation and early placentation.

During blastocyst implantation and placentation in common laboratory rodents, trophoblast cells come into increasingly more intimate associations with the endometrium and, eventually, are in contact with maternal blood. Uterine cell death is one mechanism for removing uterine tissues, primarily epithelial cells, and decidual cells that intervene between trophoblast cells and maternal blood. Mechanisms of cell death and the signals that initiate and regulate it are not well understood. According to current theories, cell death is either gene-directed or the result of traumatic injury, and classification of cell death is based on ultrastructural and biochemical criteria that hypothetically reflect underlying molecular mechanisms. Although the term apoptosis is extensively used to describe all aspects of gene-directed cell death and the term necrosis to describe traumatic death, ultrastructural studies indicate that there are morphological variations of the established criteria, and these could reflect variations of underlying mechanisms. Recent light and electron microscopic work has shown that timing and ultrastructure of uterine cell death at the gestation site varies with region suggesting that initiation and control of cell death is complicated and that more than one mechanism of cell death may be operative. Current information indicates that uterine cell death is most likely part of an intrinsic response of the endometrium to the conceptus, and other than acting as a stimulus to elicit the uterine response, the conceptus probably plays only a minor role in regulating the death of endometrial cells in these species.

Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.

Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat.

Implantation sites from rats were studied on days 6, 7, and 8 of pregnancy to determine the sequence of events in the formation of blood spaces in the trophoblast that is part of the parietal wall of the yolk sac placenta and to determine how trophoblast gains access to maternal blood. The maternal blood flowing through these spaces is the source of nutrients that reach the embryo via the visceral endoderm. Tissues were prepared for light microscopy, scanning electron microscopy, and transmission electron microscopy. Trophoblast blood spaces are derived from the lateral intercellular spaces of trophoblast cells and are present in a collapsed condition until day 8, when maternal vessels are tapped by trophoblast. These spaces then contain circulating maternal blood, and trophoblast cells reflect adaptations for metabolic exchange including thinning of trophoblast covering Reichert's membrane and the appearance of numerous fenestrations, with and without diaphragms, in the areas where trophoblast is attenuated. Between days 6 and 7 decidual cells appear to form a barrier between the maternal circulation and trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelial basal lamina, and then the decidual cells that are juxtaposed to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain access to the maternal vessels by replacing decidual cells or by direct imposition of trophoblast cell processes between endothelial cells.

Penetration of the basal lamina of the uterine luminal epithelium during implantation in the rat.

During early stages of implantation in the rat, as in other species that form a hemochorial placenta, there is a progressive increase in intimacy between blastocyst and endometrium. After initial invasion of the uterine luminal epithelium by trophoblast cells and displacement of epithelial cells, the trophoblast comes to lie adjacent to the residual basal lamina of the displaced epithelium but does not penetrate it. After a pause at the basal lamina, this temporary barrier is breached. To study the interrelations of trophoblast, uterine epithelium, and decidual cells with the epithelial basal lamina during the time of penetration of the basal lamina, implantation sites collected on day 7 of pregnancy were oriented so that the implantation chamber could be sectioned either longitudinally or transversely. Neither trophoblast nor uterine epithelial cells have processes that extend through the basal lamina. However, flange-like processes from the decidual cells penetrate the basal lamina and underlie both trophoblast and, more rarely, epithelium. Smaller folds of the surface of decidual cells partially surround bundles of collagen fibrils oriented parallel to the long axis of the implantation chamber. Initially the area of penetration of basal lamina by decidual cell processes is quite restricted; as implantation proceeds the basal lamina becomes displaced and is sometimes not discernible, extracellular materials accumulate, and the relationships become more difficult to follow. It is concluded that the initial breaching of the basal lamina is an activity of the decidual cells, and that contact of basal lamina with trophoblast is not necessary to permit this penetration.

Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration.

Rat gestation sites were obtained on days 10 through 16 of normal pregnancy. Light and electron microscopic examination of day-10 sites revealed a consistent complex pattern of stromal cell morphologies. Six distinct regions were identified: an antimesometrial region of epithelioid decidual cells that form the gestation chamber containing the embryo and extraembryonic membranes; an abembryonic antimesometrial decidual region, the decidual crypt, where the cells are separated by large extracellular spaces; a mesometrial region with granule-containing cells and mesometrial decidual cells; a region of spiny cells that are lateral to the antimesometrial decidual cells and continuous with the mesometrial decidual cells; and a region of undifferentiated stromal cells adjacent to the myometrium. Between days 12 and 16, the antimesometrial decidua becomes thinner and is eventually sloughed into the newly formed uterine lumen. The role of the antimesometrial decidual cells is discussed with reference to trophoblast invasiveness, protein synthesis, and especially remodeling of the gestation chamber. Differences between decidua and deciduoma are considered.

Implantation in the rhesus monkey: Endometrial responses.

In this continuing series of studies of implantation in the rhesus monkey, eight specimens, ranging in gestation age from 9.5 days to 16.5 days after ovulation, were examined with a focus on localized modifications in the endometrium as a response to implantation. Additionally, evidence of continuing changes in early pregnancy was provided by three specimens at the end of the first month of gestation (days 24, 28, and 35). The responses of the endometrium to pregnancy start with a localized accumulation of stromal eosinophils, which is rapidly followed by epithelial plaque formation in the basal cells of the luminal epithelium and gland necks. Plaque cells hypertrophy, develop marginal dense granules, and accumulate glycogen. They form a pad underlying the margins but not the central zone of the implantation site. However, some degenerating plaque cells are found as early as day 15; and little more than a region of leukocytic infiltration remains of the plaque by day 35. Shortly after the plaque response is initiated there is a striking subepithelial edema surrounding the plaque, and the venular capillaries enlarge by engorgement and by endothelial hyperplasia. The endothelial cells subsequently hypertrophy, resulting in a largely columnar endothelium. There is a localized decidual cell response, consisting of an increase in rough endoplasmic reticulum and in filaments, but only a moderate amount of hypertrophy of these cells. Endometrial granular cells become more conspicuous in the area as they accumulate glycogen. Patches of large pale cells appear in the lumen and walls of arterioles subjacent to the implantation site, but the cytology of these cells provided insufficient clues to their origin (cytotrophoblast?). Although the endometrial responses described are impressive and diverse, their advantages to the organism are not obvious. The hypertrophy of the anastomotic capillary bed that accompanies plaque formation may well provide an extensive vascular network available to the developing trophoblastic lacunae. The role of endometrial granular cells, decidual cells, and even plaque cells may be more related to their largely unexplored secretory activity than to their physical contribution to the formation of the basal plate.

Occlusion and reformation of the rat uterine lumen during pregnancy.

Implantation sites were obtained from rats at various stages of pregnancy and were studied by light microscopy and scanning electron microscopy. Early in pregnancy the uterine luminal epithelium and the decidual cells in the implantation site formed an implantation chamber containing the conceptus. The epithelial cells lining the chamber and the mouth of the chamber degenerated, and the uterine lumen that was mesometrial to the conceptus was obliterated such that the uterine lumen became discontinuous, and the luminal epithelia of intersite areas were isolated. As the conceptus continued to grow, the decidua-conceptus unit bulged into the intersite areas and was partially covered by an epithelium that eventually became discontinuous and degenerated. Once this had occurred, the luminal epithelium of the intersite areas reestablished contact antimesometrial to the decidua-conceptus unit, and the uterine lumen was again continuous. However, the epithelium lining the lumen was not complete in the mesometrial region because of the vascular connections between the uterine stroma and the placenta. Factors influencing the restructuring of the uterine luminal epithelium were discussed.

Trophoblastic and uterine luminal epithelial surfaces at the time of blastocyst adhesion in the rat.

Fixed uteri from rats on the afternoon of day 6 of pregnancy were split to expose the implantation chambers, their enclosed blastocysts, and the imprints of the blastocyst on the adjacent epithelium of the chamber. Some of the implantation chambers were prepared for scanning electron microscopy; other chambers were treated with colloidal iron hydroxide, with cationized ferritin, or with the tannic acid method, and subsequently were prepared for transmission electron microscopy. In this manner, the disposition of the surface-coat markers on the surface of the blastocysts, surface of the uterus within the chamber, and the surface of the uterus that had been apposed to a blastocyst were compared. Despite the pronounced morphological differences between the microvilli of the uterine luminal epithelium in the imprint and those in the rest of the chamber, the binding of the markers was remarkably similar. No evidence of removal of surface coat could be found in that area of the uterus in contact with the blastocyst. In addition, in two instances in the cationized ferritin-treated material, and in another instance in tannic acid-stained material, regions of the apparently adhering trophoblastic cell membranes and uterine cell membranes had abundant coat materials and, possibly, even secretory materials interposed. When blastocyst-sized glass beads were introduced into uteri from animals made pseudopregnant or unilaterally pregnant, the beads failed to elicit a decidual response and made an imprint that did not resemble the imprint of a blastocyst in an implantation chamber. It was concluded that, at least in the initial stages of adhesion, the blastocyst does not bring about a physical removal of the demonstrable aspects of the surface coat of the uterus. It was concluded further, that glass beads are not a suitable object for mimicking a blastocyst in the rat uterus.