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Notes: In most marsupials, placentation involves only the yolk sac; however, in the bandicoot family, Peramelidae, a functional chorioallantoic placentation develops in addition (Hill, 1895, 1897, 1900; Flynn, ′22, ′23). This duality is viewed as having evolutionary significance because most eutheria have both placentae. Furthermore, the bandicoot trophoblast was reported to vanish from the chorioallantoic site in late gestation (Hill, 1897; Flynn, ′23); whereas, the eutherian trophoblast is identifiable throughout later pregnancy and may act as an immunological barrier between maternal and fetal genotypes (Kirby, ′68). Thus we have re-examined this singular chorioallantoic placenta of the bandicoot in plastic sections with light and electron microscopy.A distinctive feature of bandicoot placentation is the transformation of the uterine simple columnar luminal epithelium into a highly vascular lining composed almost entirely of discrete syncytial masses (homokaryons). Endometrial blood vessels penetrate among the homokaryons to create a rich network of large diameter capillaries at extremely superficial locations near the maternal surface.In the chorioallantoic placenta (7 mm to 10-11 mm crown-rump embryos) the microvillous surface of the maternal homokaryons interdigitates with the microvillous border of the fetal trophoblast with desmosomal interaction. This trophoblast consists of a single layer of tall columnar undifferentiated cells rich in ribosomes-polysomes, poor in cytoplasmic membranes, and with large nuclei that have distinct clumps of heterochromatin and conspicuous nucleoli. It is thus remarkable that these undifferentiated cells disappear as a recognizable layer later in gestation (12 mm crown-rump embryos).Flynn's hypothesis that the trophoblastic cells disapppear by fusing with maternal syncytia gains support from the existence of two populations of nuclei in the syncytial masses only at the chorioallantoic site. One population is comparable to that occurring in the homokaryons of the yolk sac placenta, i.e., pale staining nuclei with little heterochromatin and small peripheral nucleoli. However, the other nuclei resemble those of the trophoblast cells. Since the trophoblastic cells before their disappearance as a layer possess properties associated with potential for further differentiation, the possibility of fusion between the maternal homokaryons and the fetal trophoblastic cells to form heterokaryons composed of two genotypes merits further consideration.The disappearance of the trophoblastic layer and the superficial positioning of the maternal capillaries bring the maternal and fetal bloodstreams into closest proximity near term (12 mm crown-rump embryo). The thinnest parts of the barrier consist of delicate cytoplasmic extensions from the syncytial masses (that may be maternal in origin or jointly maternal and fetal) and a layer of maternal stroma intervening between the maternal and fetal endothelia. Thus the chorioallantoic placental barrier of the marsupial bandicoot is unlike any thus far described for eutherian mammals.

Notes: Rapid collagen breakdown in the postpartum rat uterus is accompanied by rising collagenase activity (Jeffrey and Gross, '70) and a transient infiltration of the stroma by heterophils, eosinophils, monocyte-macro-phages, lymphocytes, and plasma cells (Padykula and Campbell, '76), cells usually associated with inflammatory response. This uterine catabolism is initiated soon after birth while blood estrogen and progesterone levels are low. To investigate the hormonal factors involved in regulation of this postpartum stromal differentiation, we analyzed the cytological effects of experimentally elevating progesterone and estradiol levels in the peripartum period by following the protocol of biochemical experiments that have demonstrated inhibition of collagenase activity by progesterone (Koob and Jeffrey, '74) and estradiol (Ryan and Woessner, '74).Prolonged gestation (progesterone, 10 mg/day starting on day 19 gestation) was used as a condition to prevent the prenatal drop in blood progesterone; this treatment was the most effective in blocking postpartum stromal differentiation. It preserved the state of prepartum uterine differentiation and most importantly it prevented monocytic-macrophagic conversion. Progesterone (40 mg/day) given at birth delayed but did not block stromal differentiation during the first 48 hours; by 72 hours collagen loss was extensive in both control and progesterone-treated rats and numerous macrophages were present. Estradiol (100 μg/day) given at birth caused a greater delay in stromal differentiation than progesterone given at birth; for approximately 48 hours the number of eo-sinophils, heterophils, and macrophages was less than normal. By day 3 the number and distribution of the macrophages resembled that of the day 1 control uteri. Overall these experiments indicate that the low estrogen and progesterone levels at birth are essential for normal stromal regression. Since these transient cells originate from the blood, the temporal pattern of their emigration into the uterus may be under hormonal control. Experimental disturbance of this pattern influences the course of collagen resorption.

Notes: The principal cell types associated with the humoral immune response (monocyte-macrophages, lymphocytes, and plasma cells) are numerous in the endometrial stroma of the uterus during the first four postpartum days in two types of mammals, the marsupial North American opossum and the eutherian albino rat. This transient cellular differentiation coincides with the physiologic period of rapid uterine regression which includes massive reduction in the amount of extracellular stromal material. In addition, heterophils and eosinophils, cell types also known to be associated with phagocytic and immunologic activity, appear in the stroma during the first two postpartum days; their presence may, however, be associated more directly with the postpartum estrus that occurs on day 1 postpartum than with endometrial regression. Thus, the five cell types, which are known in pathologic conditions to be components of an inflammatory response to a foreign antigen, are conspicuously present in the normal regressing endometrium. Furthermore, there is ample ultrastructural evidence of frequent macrophagic-lymphocytic interaction, transformation of lymphocytes, and active secretion by plasma cells during this early postpartum period.An hypothesis has been derived by uniting this new description of endometrial stromal cell differentiation with the existing literature on uterine collagenase activity, an important feature of postpartum regression (reviews by Gross, 1974; Harris and Krane, 1974). It is based on the assumption that during regression the extracellular action of neutral collagenase (and possibly other extracellular proteases) release new antigenic sites in proteins located in the ground substance. In the case of collagenase, these transient antigenic sites would arise at the locus of enzymic cleavage as well as from the subsequent denaturation of the fragments of the collagen molecule. This endogenous antigenic stimulus would be strong and temporary, and would lead to the cellular manifestations of the transient humoral immunologic response which are evident in the regressing stroma of these two mammals. This humoral immune reaction may be one of the regulatory mechanisms involved in the cyclic renewal of the extracellular compartment of the uterine stroma.

Notes: During the first four days postpartum, heterophils (polymorphonuclear leucocytes) and macrophages occur in the intercellular compartment of the luminal epithelium of the uterine endometrium. Cytochemical and ultrastructural evidence indicates that transepithelial emigration of these stromal cells to the uterine cavity is occurring. This event takes place while the luminal epithelium is proliferating in response to the estrogenic stimulus of the postpartum estrus.Heterophil emigration precedes that of the macrophages and is most conspicuous during days 1 and 2. Although it has been established that collagen fibrils occur in uterine phagocytes (Schwarz and Güldner, 1967) assumed to be macrophages (Parakkal, 1969, 1972), their precise role in collagen degradation remains undefined. It seems likely that the emigrating macrophages, heavily laden with phagolysosomal derivatives and lipid droplets, are hauling the remnants of the intercellular substance out of the endometrium during days 2-4 postpartum. Ultrastructural evidence indicates that the emigrating macrophage punctures the basal lamina and passes through the intercellular compartment of the luminal epithelium by active penetration.Another mode of macrophagic egress operates in the deep stroma of the endometrium and myometrium where lymphatic drainage occurs. Macrophages accumulate in the perilymphatic stroma as well as within lymphatic vessels.Thus macrophagic emigration through the luminal epithelium and lymphatic vessels may provide a cellular mechanism for elimination of the intercellular stromal substance in the regressing uterus. Transepithelial emigration is a mechanism which operates also in the marsupial uterus (Padykula and Taylor, 1976), and thus may be a fundamental mechanism among subprimate mammals that fulfills in part the function that menstruation effects in primates.

Notes: At the close of a uterine cycle, the remodelling of the endometrial stroma of the North American opossum involves removal of extracellular material by macrophages. This study provides cytochemical and ultrastructural evidence which indicates that the laden macrophages are eliminated from the endometrium through emigration across the glandular and luminal epithelia. During diestrus or the early postpartum period, the abundant uterine glands relinquish their secretory function to acquire a transient function in the transportation of emigrating stromal cells.During the first three postpartum days endometrial regression in the stroma is marked by sudden appearance of monocytes, macrophages, lymphocytes, and plasma cells. Ultrastructural and cytochemical evidence indicates that the macrophages engulf the extracellular macromolecular material which, in the opossum, consists primarily of ground substance. Macrophages filled with ingested extracellular material aggregate beneath the glandular and luminal epithelia, where they acquire an extracellular coat that resembles the material of the basal lamina elsewhere. A fibroblast-like cell closely invests the macrophage at the time the extracellular material appears. Simultaneously, the secretory glandular epithelium is being converted to a highly ciliated one. Macrophages, often accompanied by lymphocytes, acquire intraepithelial positions in the glands. From here these stromal cells gain entrance to the glandular lumens. At this time the luminal contents are rich in acid phosphatase activity which most likely reflects the high lysosomal content of the emigrating macrophages. Evidence suggests that these intraluminal macrophages and lymphocytes are swept, by the recently differentiated ciliary lining, toward the glandular orifices and into the uterine cavity. It is hypothesized that this cyclic appearance and transepithelial elimination of macrophages is a cellular mechanism for removing large amounts of extracellular material without disruption of the endometrium.

Notes: The steady decline in plasma progesterone level that occurs during the last week of pregnancy in the normal rat (Wiest, '70) provides good opportunity to study the effect of withdrawal of progesterone on uterine differentiation. Evidence is presented that tissue monocytes, heterophils, and eosinophils are regular components of the normal late gestational uterus and that their number increases as term approaches. Uterine monocytes and heterophils are located in the endometrial and myometrial stroma as well as within the basal intercellular compartment of the luminal epithelium. Stromal monocytes are distributed throughout the attenuated endometrium of late gestation, but are more common immediately beneath the luminal epithelium. In the myometrium, monocytes and heterophils occur, often as perivascular clusters, in the connective tissue septum that separates the two layers of smooth muscle. Eosinophils are present especially in the deep endometrial and myometrial stroma, and increase in number as plasma estrogen rises immediately before parturition. A small population of lymphocytes is regularly present.An important feature of the prepartum uterine stroma is the sparseness of macrophages. Near term, however, the beginnings of monocytic-macrophagic transformation are noticeable as the cell surface becomes more irregular and organelles associated with endocytic activity arise. The prepartum monocytes are positioned in the same histological sites that during the postpartum period of regression will be occupied by macrophages (Padykula and Campbell, '76). Since it is generally accepted that monocytes are precursors of macrophages, this spatial correlation raises the possibility that cellular preparations for regression commence before birth. The possible significance of prepartum monocytic infiltration is discussed in relation to the effect of changing plasma and uterine concentrations of progesterone on uterine collagenase activity. The steady increase in uterine tissue leucocytes which occurs concomitantly with decreasing uterine binding capacity for progesterone supports the hypothesis by Siiteri et al. ('77) that progesterone in high local concentrations has an antiinflammatory effect.