Library

Notes: Available evidence provides little support for a recent proposal that the term “trophoblast” be applied solely to eutherian mammals. Arguments for such a restricted usage are based on a dichotomous interpretation of therian reproduction that underestimates the developmental, structural, and functional diversity of trophoblastic tissues occurring within the infraclass Eutheria. The occurrence of developmental patterns that are phenotypically intermediate between those of commonly studied eutherians and metatherians suggests that blastocyst development is not fundamentally different in marsupials and eutherians.The trophoblast of marsupials accomplishes most or all of the major functions of the eutherian trophoblast, including maternal-fetal physiological exchange, implantation, contribution to placental membranes, steroid metabolism, and possibly, immunological protection of the conceptus. Furthermore, application of the term “trophoblast” to marsupials is consistent with present and past usage, as well as with the original definition and etymological derivation of the term. Therefore, we recommend that the term “trophoblast” continue to be applied in a functional-morphological sense to the appropriate extraembryonic tissues of marsupials. Such use of functional (rather than taxonomic) criteria for application of this term avoids biasing interpretations of mammalian reproductive evolution.

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: In rat uterine luminal epithelial cells, nuclear bodies occur in the euchromatin in varying numbers in relation to the nuclear concentration of the estrogen receptor (Clark et al., 1978; Padykula et al., 1981, 1982). This functional responsiveness indicates that nuclear bodies may be useful indicators of the degree of cellular estrogenization. Because these filamentous bodies vary in size (200-1200 nm), shape, and composition, quantitative analysis of frequency of their occurrence has been difficult. A fundamental division into 2 categories can be made by the following criteria: 1) simple nuclear bodies (200-500 nm) consisting of a protein mesh of microfilaments, and 2) complex nuclear bodies (200-1200 nm) composed of an outer filamentous protein capsule enclosing a lucent core that may contain granules. Previous quantitative analyses at the electron microscopic level has excluded “simple bodies” because they might actually be ultrathin sections through the filamentous capsule of complex bodies (Le Goascogne and Baulieu, 1977; Clark et al., 1978). To resolve this sampling problem, we have performed serial ultrathin section analysis of nuclear bodies in hyperestrogenized luminal epithelial cells. Ultrastructural evidence presented here demonstrates that simple and complex nuclear bodies are anatomically separate entities. Ultrathin sections through the capsule of complex nuclear bodies will be misidentified as profiles of simple bodies during quantitative analysis. This anatomic distinctness of simple and complex nuclear bodies correlates with their differing responses to estrogenic stimulation and withdrawal (Fitzgerald and Padykula, pp. 131-141, this volume). Thus the existence of these two major categories should be taken into consideration during quantitative analyses.

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: Preliminary evidence has indicated that the number of nuclear bodies in uterine luminal epithelial cells of the immature rat may be related to the duration of nuclear retention of the estrogen receptor complex (Clark et al., 1978). To test this hypothesis, an ultrastructural analysis of nuclear and cytoplasmic differentiation was performed at 4, 12, 24, 48, and 72 hr after a single injection of estradiol or nafoxidine (synthetic estrogen agonist/antagonist) into 21 day female rats. Variations in nuclear and cytoplasmic differentiation and in the frequency of occurrence of nuclear bodies (simple and complex) were determined and compared with established biochemical changes in the concentration of nuclear estrogen receptor and RNA polymerase activity (Clark et al., 1978). Following nafoxidine there is sustained elevation of the nuclear concentration of the estrogen receptor as well as RNA polymerase I and II activities over the entire 72-hr period. From 4 to 72 hr the height of the luminal epithelial cells as well as the frequency of nuclear bodies increase at linear rates. Through steady expansion of the cytoplasmic membrane systems (RER and Golgi) the relatively undifferentiated epithelial cells of the control uterus are converted progressively into ones equipped for protein secretion. At 72 hr the effects of an estradiol implant resemble closely those observed after a single injection of nafoxidine; these include sustained nuclear receptor occupancy, elevated RNA polymerase activity, epithelial hypertrophy, and high frequency of nuclear bodies. However, after a single injection of estradiol, the luminal epithelial cells become slightly but significantly taller than the control cells and remain close to this size from 24 to 72 hr; the frequency of nuclear bodies decreases linearly from 4 to 72 hr to fall below the control level. In addition, limited cytoplasmic autolysis is evident from 24 to 72 hr. A single injection of estradiol results in short-term nuclear receptor occupancy and elevated RNA polymerase activities which return to control levels by 24 hr. This collective evidence offers further support to the hypothesis that the duration of nuclear occupancy by the estrogen receptor is reflected in the size of the nuclear body populations in these epithelial target cells. Also during hyperestrogenization, epithelial hypertrophy is accompanied by steady formation of nuclear bodies.

Notes: A single injection of estradiol or the antiestrogen, nafoxidine, into immature female rats results in a striking divergence in the frequency of observation of nuclear bodies in uterine luminal epithelial cells (Padykula et al., 1981). During the 72 hour period after a single injection of nafoxidine (estrogen antagonist with agonist effects), progressive cellular hypertrophy occurs and is accompanied by a linear increase in the total number of nuclear body profiles (TNB). Conversely, the 72 hour period after a single injection of estradiol is marked by cellular catabolism and a linear decrease in TNB. Our recent demonstration that the population of nuclear bodies consists of two major structural types, simple nuclear bodies (SNB), and complex nuclear bodies (CNB) (Padykula and Pockwinse, pp. 119-130, this volume), permits separation and analysis of the original TNB data in terms of the functional responses of SNB or CNB.During the 72 hour period after injection of nafoxidine, the luminal epithelial cells remain in an anabolic state because the nuclear concentration of estrogen receptor and RNA polymerase activity are elevated. Separate quantitative analysis of the complex and simple nuclear bodies demonstrates two distinct functional responses: (1) a linear increases in the frequency of CNB, and 2) a relatively constant frequency of SNB. Since the complex bodies do not increase in diameter during this period, the increase in the frequency of TNB may arise from an increase in the actual number of CNB per nucleus during cellular hypertrophy.Conversely, a single injection of estradiol results in a relatively short elevation of nuclear estrogen receptor concentration and RNA polymerase activity which returns by 24 hour to the control level. This brief estrogenic stimulation is reflected by a linear decrease in the frequency of TNB and a linear decrease in the frequency of SNB. The frequency of CNB decreases suddenly between 4 and 12 hours and levels off thereafter. The steady decrease in the frequency of observation of nuclear bodies during catabolism may represent a decrease in the actual number of SNB and CNB per nucleus rather than a reduction in their size.This study provides quantitative evidence that simple and complex nuclear bodies are functionally separate entities. Also, some evidence suggests that simple nuclear bodies may be the precursors of complex nuclear bodies.

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.