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Notes: [Auszug] WE present here a view of marsupial-eutherian origins and evolutionary trends, which constitutes an alternative to the view of Lillegraven1'2 recently discussed by Cox in a Nature News and Views article3. The trophoblast constitutes the outer foetal boundary of the eutherian ohorioallantoic ...

Notes: Summary Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (∼0.25%) considerably less concentrated with respect to OsO4 than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry.

Notes: The postovulatory period in the primate endometrium of the menstrual cycle is characterized by rapid growth of the coiled arterioles. A great variety of developing microvascular components occurs among a well-differentiated microvasculature of coiled arterioles, capillaries, and venules. Endometrial biopsies were obtained by hysterotomy during progesterone dominance at 5, 6, 7, 10, 12, and 14 days following the peak of the estrogen surge as determined by serum radioimmunoassay. Arteriolar ultrastructural differentiation is remarkably similar on each of these days.Ultrastructural evidence of elastogenesis in the extracellular matrix adjacent to certain endothelial tubes provides the initial sign of coiled arteriolar formation. The cellular primordia of the tunica intima and media are identified by spatial location and glycogen storage in smooth muscle cells. Endothelial projections span the incipient internal elastic membrane to make contact with the surfaces of the innermost vascular smooth muscle cells.Subsequent arteriolar differentiation centers on formation of a muscular media composed of 1 or 2 muscle layers separated by a spiraling lamellar elastic matrix that appears initially between the endothelial tube and the first muscle layer. Vascular smooth muscle cells are highly branched and linked across the elastic matrix by surface contacts. Definitive coiled arterioles consist of interlinked endothelial and smooth muscle cells within a thick, spiraling elastic matrix that provides flexibility for rapid changes in shape. Progressive differentiation of coiled arterioles continues up to the premenstrual stage. This abundant angiogenesis may reflect preparation and maintenance of a suitable uterine environment for the possibility of implantation and pregnancy during each menstrual cycle.

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: 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: The ultrastructural organization of 40 soleus neuromuscular junctions from ten normal young adult male and female Sprague-Dawley (SD)-derived rats (Charles River Breeders, CD-Crl:COBS (SD)BR) has been studied. A smaller sample of motor endplates from the gastrocnemius, diaphragm, and extensor digitorum longus muscles of these rats as well as from the soleus muscles of two adult Wistar (W) rats (Crl:COBS(WI)BR) was included. Widespread ultrastructural reorganization was evident at the soleus neuromuscular junction during the growth period from three to five months of age. A major characteristic of reorganization is the presence of junctional folds not associated with axonal terminals; such sites occur within a single endplate adjacent to areas with typical intact synaptic associations. Additional features possibly related to remodelling are: (1) spatial separation of axonal terminals from the myofiber, (2) intervention of Schwann cell cytoplasm between an axon terminal and myofiber, (3) aggregates of satellite cells, and (4) folded or multilayered basal lamina. These features are most pronounced in the soleus muscle but occur to varying degrees in the neuromuscular junctions of other muscles of SD-derived rats.Distinctive characteristics of the rat soleus postjunctional sarcoplasm include the widespread occurrence of myofibrillar components, abundant free and membrane-associated polysomes, and triads oriented in various planes. Away from such discrete sites, myofibers possess the usual highly oriented organization of myofibrils, T tubules, sarcoplasmic reticulum, and mitochondria.The soleus muscle is a postural muscle that responds directly to rising workload imposed by continuous body growth during young adulthood by steady myofiber hypertrophy and conversion of motor units (Kugelberg, '76). This changing structural-functional relationship may be reflected also by ultrastructural remodelling of the neuromuscular junctions reported here.

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: 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.