Cellular Mechanisms Involved in Cyclic Stromal Renewal of the Uterus I. THE OPOSSUM, DIDELPHIS VIRGINIANA HELEN A. PADYKULA

AND

' j 2

J. MARY TAYLOR

Laboratory of Electron Microscopy, Wellesley College, Wellesley, Massachusetts, 02181, U.S.A. und Department of Zoology, University of British Colzrmbiu, Vancower, British Columbia, V6T 1W5, Canada

ABSTRACT 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 a t 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. I t 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.

Considerable effort has been directed toward analysis of uterine cyclic activity, especially in relation to the progressive build-up and differentiation of the tissues that occurs in response to follicular maturation and formation of the corpus luteum. However, less attention has been directed toward the strategic problem which exists at the close of a cycle (whether or not pregnancy has occurred) at which time large amounts of uterine epithelial, stromal, and vascular tissue will be reduced to prepare for the next cycle. I n some primates (Old World monkeys and humans) this elimination is achieved by menstrual ANAT. Rec., 184: 5-26.

sloughing of the outer portion of the endometrium; however, in other mammals the loss of endometrial mass generally occurs without sloughing, especially after a nonpregnant cycle. Thus in most mammals, uterine regression is a more subtle event which is still largely undefined. In this and the companion (Padykula and Campbell, '76) study, uterine regression is histologically and cytologically analyzed in two strikingly different mamReceived Mar. 11, '75. Accepted Apr. 10, '75. 1 This study is dedicated to the memory of the late Dr. Helen Wendler Deane. zThis work was supported by U. S. Public Health Research Grant HD 01026 (awarded to Dr. Padykula).

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HELEN A. PADYIWLA AND J. MARY TAYLOR

mals, the North American opossum which is generally considered to be a primitive marsupial and the albino rat which is a specialized eutherian. Despite the remarkable differences in their reproductive physiology, simultaneous study of these two mammals has facilitated interpretation and possibly uncovered common cellular and tissue mechanisms for effecting cyclic stromal remodelling in the endometrium. It has long been appreciated that the endometrial macrophages (rat, rabbit, and mouse) play a role in the reduction of stromal volume. These phagocytes appear in the stroma during the early postpartum period (Lobe1 and Deane, '62; Banon et a]., '64; Brandes and Anton, '69; '69; Anton et al., '69), and apparently ingest the collagen fibrils (Schwarz and Giildner, '67; Parakkal, '69, '72). At this time a high level of collagenase activity arises in the extracellular compartment of the endometrium, and it initiates the breakdown of the collagen fibrils (review by Gross, '74). It is assumed that the partially degraded collagen fibrils are then engulfed by macrophages which then complete the degradation intracellularly through their elaborate phagolysosomal systems. The fate of these cyclic macrophages has been unknown, other than that they subsequently vanish for the most part. The present cytochemical-ultrastructural investigation of the uterus of the opossum (Didelphis virginiana) concentrates on stromal reorganization in the early postpartum period and during diestrus of the non-pregnant cycle. Evidence is offered in support of a hypothesis that macrophages, laden with engulfed extracellular material, emigrate from the stroma by passing through the glandular and luminal epithelia to gain entrance to the glandular and the uterine lumens. This hypothesis offers explanation for previous observations of the presence of cellular material of unknown identity within the lumens of the opossum uterine glands at diestrus (Hartman, '23). In the accompanying paper (Padykula and Campbell, '76) evidence is presented for comparable postpartum reorganization in the rat endometrium, which suggests that cyclic emigration of macrophages may occur widely among mammals.

MATERIALS AND METHODS

Adult female opossums purchased from Ray Singleton and Co. in Tampa, Florida were flown to Boston, Massachusetts during January and February. I n Florida the major breeding period occurs during these months, and hence these animals were either in a phase of the estrous cycle, pregnant, or lactating. Some pregnant opossums delivered their young in the Wellesley laboratory. During the past ten years we have studied 71 opossums, of which eight were pregnant and nine were lactating. In addition, nine animals were in a regressing luteal phase, after a non-pregnant cycle or a n interruption of pregnancy. The stage of the estrous cycle or pregnancy was tentatively established by gross examination of the pouch and reproductive tract. Final determination of reproductive condition depended on study of serial sections of ovaries, using the criteria of Hartman ('23) and MartinezEsteve ('42) or by comparing the embryos with stages described by McCrady ('38). Light microscopic preparations For each opossum, both ovaries were sectioned serially in paraffin, one was preserved in Serra's fixative for staining with eosin and methylene blue at pH 4.1, whereas the other was fixed in Rossman's fluid for the periodic acid-Schiff reaction. The same procedures were also used on samples of the uteri. Lipids were localized in uterine specimens that were fixed in 10% neutral buffered formalin, embedded in gelatin, sectioned with a freezing microtome. and stained with Sudan black B. The localization of acid phosphatase activity in the endometrium was performed with the Barka-Anderson ('63) medium using glyceraphosphate and tris maleate buffer on the following types of sections: (1) Unfixed cryostat sections (5-7 or 40 pm) were dried and incubated for 30-60 minutes. ( 2 ) Fixed endometrium (3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 1-2 hours) was sectioned at 225 pm with a Sorvall TC-2 tissue chopper, incubated for 60-90 minutes, rinsed in 0.1 M cacodylate buffer at pH 7.4, postfixed i n 2% unbuffered osmium tetroxide, dehydrated in ethanol, and embedded in Epon. In this latter procedure, alternate

ENDOMETRIAL REGRESSION IN THE OPOSSUM

semi-thin sections (1-2 p m ) were reacted with ammonium sulfide to visualize the reaction product or they were stained with toluidine blue. Controls for this enzymic localization were incubated in a medium that lacked substrate.. Electron microscopic preparations Our best material resulted from double fixation using 5-696 glutaraldehyde in 0.1 M cacodylate buffer followed by 2% Os04 buffered in veronal acetate. E n bloc staining with 0.5% uranyl acetate i n veronal acetate buffer at pH 5.5 (Farquhar and Palade, ’65) was routinely used. Ultra-thin sections were usually doubly stained with both lead hydroxide and uranyl acetate and examined in a Siemens IA electron microscope. RESULTS

The opossum endometrium during pregnancy is a highly glandular mucosa with a rich thin-walled superficial vasculature and a n empty-appearing stroma concentrated beneath the luminal epithelium (Selenka, 1887; Hartman, ’ 2 3 ) . This characteristic empty appearance led early investigators to describe this extracellular stromal material as ‘‘lymph.’’ However, our histochemical and electron microscopic observations indicate that the so-called “lymph” is more appropriately described as a n abundant ground substance. The histological appearance of the luteal endometrium just before birth (twelfth day of pregnancy) is illustrated in figure I, which also shows the apposition of the simple columnar luminal epithelium with the bilaminar or non-vascularized portion of the yolk sac placenta. Venules and large diameter capillaries occur in the stroma subjacent to the luminal epithelium. Deeper i n the stroma, the glands and blood vessels seem to be floating, an appearance caused by the remarkable paucity of stromal cells and fibers. The stromal cells at this time are almost entirely fibroblasts, which are widely separated. Near term occasional macrophages occur as well (fig. 3 ) . Figure 5 shows the ultrastructural appearance of the extracellular stromal material; collagen fibrils are scarce and the ground substance contains a flocculent precipitate which most likely reflects the presence of carbohy-

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drate-protein polymers which are generally known to be extracellular stromal components. It should be noted that the endometrium of the luteal phase during a nonpregnant cycle has a similar appearance, and that this fact led Hartman ( ’ 2 3 ) and other early investigators of the marsupial uterus to refer to this state as “pseudopregnancy.” This designation is being abandoned in marsupial physiology, since the term “pseudopregnancy” has a n entirely different meaning in eutherian physiology ( S h a m a n , ’70). During the first two days postpartum, the endometrium undergoes striking temporary alterations. A conspicuous change is the appearance of numerous free cells throughout the storma which tend, moreover, to aggregate beneath the glandular and luminal epithelia (Compare fig. 1, 3 with fig. 2 , 4). Another impressive modification is the conversion of the largely secretory glandular epithelium of pregnancy (compare figs. 3 and 4) into one that is composed mostly of ciliated cells (figs. 4 , 12, 1 4 ) . It seems likely that the secretory cells of the luteal glands are shed during the first postpartum day; however, we have no direct evidence concerning their mode of disappearance. Mitotic figures are frequent in the postpartum glandular epithelium, and this presumably reflects the proliferation of a new population composed of ciliated cells. Thus, during the first day postpartum, the glands temporarily relinquish their secretory function and acquire a ciliated lining for a new function. The postpartum strornal population is a varied one consisting of macrophages, monocytes, lymphocytes, and plasma cells along with the resident population of fibroblasts. The remainder of this analysis will concentrate on the various locations of the macrophages, and attempt to interpret the significance of their positions in the endometrium. It is well established that macrophages appear in the stroma of regressing endometria in many species, where they function i n removal of extracellular material. In the postpartum opossum endometrium they are numerous during the first three days and are made conspicuous by their elaborate phago-lysosomal systems (figs. 6, 10) and lipid droplets (figs. 9,

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HELEN A. PADYKULA AND J. MARY TAYLOR

10). At the ultrastructural level their highly active cell surfaces (figs. 5, 9, 10, 1 1 ) serve a s further markers. Another cytochemical marker for these macrophages is derived from the fact that they engulf ground substance (fig. 5) which is strongly periodic acid-Schiff positive (PAS), particularly in the deeper stromal regions. Thus the phagosomes contain a PAS-positive material (fig. 7) which is composed of a carbohydrate polymer that resists digestion with a-amylase. At the light microscopic level, macrophages are observed i n the following locations in the regressing endometrium: ( 1 ) free in the stroma (figs. 4, 6 ) ; ( 2 ) as protrusions on the basal surfaces of the glands (figs. 4, 7, 8, 9 ) ; ( 3 ) within the glandular and luminal epithelia (figs. 4, 1 2 ) ; ( 4 ) in the lumens of the glands (figs. 12, 14), and (5) in the uterine cavity (figs. 15-17). Figure 4 illustrates their various positions in relation to a gland; they occur in the stroma, as a bulge on the basal surface of the gland, and in a n intraepithelial position. Their intraluminal location in the glands is more easily secn in figures 12 and 14, while figures 15-17 show their presence in the uterine cavity. In addition, histochemical preparations that demonstrate the contents of macrophages (figs. 7, 8 ) allow clear visualization of their position as basal protrusions on the gland. Because of the high acid phosphatase activity of the macrophages associated with the basal surface of the glands they are readily distinguished from the glandular epithelium which has low activity (fig. 8 ) . The basement membrane of the glandular epithelium which is PAS-positive appears to diverge from the contour of the gland to encircle the macrophagic protuberances which are also PASpositiye (fig. 7). The intraepithelial position of such macrophages is confirmed by electron microscopy (fig. 9 ) ; the basal lamina is typically closely adherent to the epithelium, except in regions where a macrophage has been incorporated. The basal lamina in the vicinity of a n incorporated macrophage loses its association with the epithelium. Here it is slack, diverges outward toward the stromal compartment and encloses the macrophage. Thus, macrophagic surface is placed in direct contact

with the basal surface of the glandular epithelial cells, and the macrophages are therefore in a n intraepithelial position. Macrophages were also often observed i n close association with lymphocytes within the epithelium. The next observations relate to a possible mechanism for transfer of stromal macrophages into the intraepithelial position. Slender cytoplasmic processes of a fibroblast-like cell are aligned in general conformity with the contours of the macrophagic protrusion (fig. 9 ) . This alignment is more clearly evident in figure 10 which shows three macrophages located in a stromal region below the luminal epithelium. These macrophages are almost entirely enveloped by a loose fitting extracellular coat. The significant feature here is the association of another stromal cell with long sheet-like cytoplasmic processes that follow the contours of the coated macrophages. In addition, there is a n interruption in the cellular investment over the uncoated portion of one of the macrophages in this figure. This occurrence of partially coated macrophages is a regular feature located in the stroma subjacent to epithelia (fig. 11). In other words, a few uncoated macrophagic processes remain freely exposed to the ground substance while most of the macrophagic surface is isolated by the extracellular coat and enveloping cellular processes. These images suggest that macrophages destined for union with a n epithelium acquire a n extracellular coat that resembles a basal lamina in its appearance. Furthermore another type of stromal cell which resembles a fibroblast may be involved in the process of coating the macrophage. It is not known how the macrophage achieves a n intraepithelial position. Some evidence in support of transepithelial migration of macrophages is illustrated in figure 12 which is a longitudinal section through a gland. The glandular epithelium is composed largely of ciliated cells with a few non-ciliated cells interspersed (fig. 12). There is, however, a third category of cells which have heterogeneous inclusions. These are most likely macrophages which occur as insertions in the epithelium. Cytoplasmic extensions from the basal surface suggest that actively motile macrophages

ENDOMETRIAL REGRESSION IN THE OPOSSUM

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tween cycles the secretory epithelium is converted to a highly ciliated one that facilitates the transportation of stromal cells. This funlction is manifested after either a non-pregnant or a pregnant cycle. The cellular transport function of the uterine glands was not perceived in earlier studies, although the occurrence of intraluminal cells at diestrus was reported by Hartman (’23). He described the glands at diestrus as follows: “. . . the lumen is filled with cellular material that is often made up of normal or slightly degenerating cells” and asked, “What is the origin of these cells?” (pp. 375-376). An answer to Hartman’s 50-year old question is provided in the present study: The intraluminal cells are primarily macrophages and lymphocytes which have most likely emigrated from the stroma through the glandular epithelium. On the basis of Corner‘s (’21) description of the sow‘s uterus, it seems likely that cellular transport is a function of these uterine glands at diestrus. Immediately after birth, a rapid dramatic reconstruction of the glandular epitheDISCUSSION lium occurs in the opossum; the secretory Dual function of the uterine cells of the luteal phase vanish, mitosis glands in the o p o s s u m commences, and within 48 hours postThe secretory function of the endome- partum ciliated cells predominate (figs. 12, trial glands during the luteal phase in vari- 14). Among these abundant ciliated cells, ous species is well-recognized, although a relatively small population of non-ciliwe are only now beginning to gain infor- ated cells exists (fig. 12); these cells are mation concerning the chemical composi- most likely the precursors for the next tion of the eutherian uterine fluid (e.g., crop of secretory cells that will multiply Breed et al., ’72; Murray et al., ’72; Squire and differentiate during the succeeding folet al., ’72; Chen et al., ’73). As seen in licular phase. figure 3, secretory cells predominate in Whether or not pregnancy occurs in the the opossum glands during the luteal opossum, cellular and tissue differentiation phase; from their ultrastructural appear- during luteal expansion of the endomeance, these cells are most likely synthesiz- trium, as well as its subsequent regression, ers of macromolecular components of the are similar. This correlates with the recent uterine fluid. Only a small population of demonstration by Shorey and Hughes (’73) ciliated cells is scattered among the secre- that blood levels of progesterone are comtory cells, and the current created by these parable in the pregnant and non-pregnant cells may assist in the movement of secre- brush-tailed possum, Trichosurus vulpection toward the glandular orifice. ula. Another biologic fact relevant to interA new function of uterine glands, pretation is that in Didelphis virginiana namely of cellular transportation, has the gestation period occurs within the been revealed through these observations luteal phase of the cycle. The occurrence o f intraluminal cells in on endometrial regression in the opossum. The glands temporarily become avenues of the regressing endometrial glands of maregress for certain stromd cells. During the supials has been mentioned in several pubendometrial reorganization that occurs be- lications. Sharman (’55) reported that

penetrate the epithelium. Other examples of intraepithelial macrophages are visible in figures 4 and 7. Macrophages are similarly associated with the luminal epithelium, though not illustrated here. Thus, transepithelial emigration of macrophages may occur in both epithelia. During these first postpartum days the glandular lumens are crowded with free cells which are mostly macrophages but also inlclude lymphocytes (figs. 12, 14). The acid phosphatase activity of the luminaI contents is very high (fig. 13), an observation consistent with the large population of lysosome-rich rnacrophages. It is likely that the intraluminal cells are swept toward the uterine cavity by the ciliary action of the transformed postpartum glands, since macrophages are present at the orifices of the glands (fig. 2 ) and within the uterine cavity (fig. 15). Within the uterine lumen, the macrophages retain their high level of acid phosphatase activity (fig. 16) as well as their ultrastructural integrity and conspicuous surface activity (fig. 17).

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HELEN A. PADYKULA AND J. MARY TAYLOR

some uterine glands in the wallaby, Setonix brachyurus, are invaded by polymorphonuclear leucocytes which aggregate in the lumens. We have observed, furthermore, that heterophil emigration from the opossum (fig. 15) and rat endometria precedes that of macrophages; it is most conspicuous during the first 24 hours postpartum (see Padykula and Campbell, '76 for illustrations). In the brush-tailed possum, Trichosurus vulpecula, Pilton and Sharman ('62) report that "degenerated cells collect in the cavities of some glands" (p. 132) during regression. Recently Shorey and Hughes ('73) described glandular regeneration in the brush-tailed possum during the early follicular phase and arrived at a considerably different interpretation from that presented here for the North American opossum concerning the possible significance of invasion of the glands by stromal cells. Shorey and Huges suggested that the stromal cells which surround the glands have a regenerative function, and that this may involve conversion of a stromal cell into glandular cell. Thus, in their interpretation, the glandular epithelium is the destination of the stromal cells, whereas our study on the North American opossum suggests that stromal cells are merely passing through the epithelia en route to the uterine cavity. The occurrence of ciliated cells in endometrial epithelia as well as their cyclic differentiation is only now beginning to be described with some precision because of improved cytological preparations available through the techniques of electron microscopy. In the human uterus, ciliated cells occur in both luminal and glandular epithelia where they are most frequent in the late proliferative and early secretory phase (Schueller, '68, '73). Morgan ('46) recognized the presence of ciliated cells in both epithelia of the opossum, and noted their dependence on hormonal stimuli. The present study demonstrates that ciliated cells predominate in the opossum's glands during postpartum or diestrous reorganization. A relatively complete ciliary carpet is thereby created which might provide widespread waves of force to propel the stromal cells toward the glandular orifice. After several days of endometrial reorganization, the intraluminal cells are eliminated from

the glands into the uterine cavity, and the epithelial and stromal differentiations of proestrus are again initiated. Interim reorganization of the endometrium between cycles Although the appropriate measurements have not been made on the hormonal levels of the North American opossum, it may be assumed that endometrial reorganization occurs during a hormonal changeover state in which progesterone blood levels have declined and estrogen levels are rising again. The histological appearance of the endometrium reflects simultaneously occurring regenerative and regressive events. Mitoses in the epithelia reflect rising blood estrogen levels. This regenerative event is, however, occurring simultaneously with the transepithelial emigration of macroyhages, which is a regressive event. The hormonal control of this remarkable transient stromal differentiation is unknown, although it seems likely from the data on the brush-tailed possum (Shorey and Hughes, '73) that i t occurs during a period of very low levels of circulating progesterone. Cellular and tissue mechanisms involved in stromal regression of the endometrium Current cytological evidence indicates that endometrial phagocytes function in the removal of collagen fibrils in the rat endometrium (Schwarz and Guldner, '67; Parakkal, '69, '72). The present study on the opossum reveals, for the first time, that during endometrial regression macrophages emigrate from the stroma by passage through luminal and glandular epithelia. The abundance of uterine glands in the opossum has facilitated recognition of this remarkable phenomenon. Intraluminal aggregations of macrophages and lymphocytes are conspicuous in the lumens of the glands (fig. 12). Endometrial macrophages have been studied most often in the rat uterus (Lobe1 and Deane, '62; Schwarz and Guldner, '67; Anton et al., '69; Parakkal, '69, '72) which has relatively less epithelial tissue, and is especially lacking in glands. In both species, macrophages penetrate the luminal and glandular epithelia

ENDOMETRIAL REGRESSION IN THE OPOSSUM

(also Padykula and Campbell, '76), but this event is less noticeable in the rat because of the paucity of endometrial glands. The origin of stromal macrophages in the uterus remains to be determined experimentally. However, the appearance of monocytes in the early postpartum stroma suggests that some of the macrophages may originate from blood monocytes. This possibility is consistent with the concept introduced by Volkmann and Gowans ('65A,B) that, under experimental pathological conditions, blood monocytes enter the stroma and convert into tissue macrophages. However, the regressing uterus is not a pathologic but a normal condition. Under pathologic conditions, exogenous antigens create the milieu that attracts blood monocytes into the stroma. In the normal cyclic invasion of the endometrium, the stimuli are now unidentified but would presumably be endogenous rather than exogenous. The endometrial stroma during the luteal phase of the opossum is rich in a gelatinous ground substance but poor in collagen fibrils and cells (figs. 1, 3, 5). The rapid shrinkage of this highly expanded endometrium during the early postpartum period suggests that withdrawal of extracellular water is a n early event in regression. Although the biochemistry of this particular ground substance has not been studied, investigation of the rabbit uterus indicates that this organ, as a whole, contains glycosaminoglycans, hyaluronic acid, chondroitin sulfate, and heparin sulfate (Endo and Yosizawa, '73). Thus, the principal macromolecular substances to be removed by the stromal macrophages are most likely protein-polysaccharide complexes. In appropriate histologic material the ground substance is periodic acidSchiff positive (Padykula, unpublished observation). Figure 5 illustrates the macrophagic engulfment of ground substance with the formation of PAS-positive phagosomes (fig. 7). This cytochemical and ultrastructural study demonstrates that, during regression, laden macrophages occur in the following locations: (1) entirely free in the stroma (fig. 6 ) , (2) free in the stroma, but partially enclosed in the stroma by an extracellular coat as well as a nearly all-encom-

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passing cellular vesture (figs. 10, l l ) , ( 3 ) as protuberances within the basal laminae of the glands (figs. 7, 8, 9), ( 4 ) within the glandular epithelium (figs. 9, 12), (5) in the lumens of the glands (figs. 12, 1 4 ) , and (6) within the uterine cavity (figs. 16, 17). These various positions suggest a pathway of emigration from the stroma to the uterine cavity that involves transepithelial passage. This hypothesized pathway is illustrated in text figure 1. Direct evidence for such migratory activity would depend on the use of cell labelling techniques. Additional evidence in support of this hypothesis exists in the accompanying studies (Padykula and Campbell, '76; Padykula, '76). An intriguing step in this hypothesized migration is the isolation of laden niacrophages with an extracellular coat which resembles the basal lamina of the nearby epithelium (figs. 10, 11; also text figure 1, stage 4). This enclosure may prepare effete macrophages and lymphocytes for transport and elimination. The origin of this special coating requires further investigation. However, circumstantial evidence indicates participation of the associated fibroblast-like investing cell in its synthesis. It is possible, of course, that the macrophage itself may be involved i n the synthesis of this loose fitting surface coat; other specialized cells, such as smooth muscle cells, may secrete a "basement membrane-like material" in culture (Ross, '71). Acquisition of the macrophagic coat seems to occur in the vicinity of the epithelia. As shown in figure 11,the macrophagic coating is incomplete in regions where pseudopodia extend free and active in the stroma. Such images are frequent and suggest that a temporary immobilization of the macrophage is effected by this enclosure. The ultrastructural similarity between the macrophagic coat and the basal laminae suggests that fusion of these extracellular sheets may facilitate transfer of the macrophage to an intraepithelial position. As may be seen in figure 9, the basal lamina of the glands tends to be slack and convoluted during postpartum reorganization; this may be a result of a rapid shedding of secretory cells from the epithelial population. Once the macrophage

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HELEN A. PADYKULA AND J. MARY TAYLOR

3

Figure I

ENDOMETRIAL REGRESSION IN THE OPOSSUM

is enclosed within the basal lamina, it has an active surface suggesting that penetration through the intercellular space of the epithelium is accomplished by active movement of the macrophage. This passage, which requires separation of intercellular junctions, needs further analysis. Morphological and cytochemical evidence suggests that the macrophages remain viable as they are swept through the uterine glands and that self-destruction in the uterine cavity eliminates them. ACKNOWLEDGMENTS

Ann G. Campbell prepared the biologic material for cytochemical and ultrastructural study, Richard V. T. Stearns produced the photomicrographs and the h a 1 electron micrographic plates. Dr. Susan SmithStiles made the original drawing for text figure 1 which was later modified and extended by Irene Schwartz (Dental Research Text fig, 1 Hypothetical representation of t h e pathway of macrophngic and lymphocytic d i f f e r entiation and migration during regression in t h e early postpartum endometrium of t h e opossum (Didelphis virginiana). (1) Blood monocytes enter the stroma after parturition. ( 2 ) As the monocytes ingest various extracellular stromal macromolecules (or the products of their enzymic degradation), they transform into macrophages with conspicuous phagolysosomal systems. The surfaces of macrophages and lymphocytes become closely apposed, a relationship suggesting functional interaction. ( 3 ) Mature plasma cells appear in the stroma, a cellular signal that antibody synthesis is occurring. ( 4 ) Effete macrqhages with (or without) accompanying lymphocytes are packaged in the stroma within an inner extracellular coat (black line) that usually appears discontinuous. Macrophagic cytoplasmic processes, sometimes resembling pseudopodia, extend through these discontinuities. A cell resembling a fibroblast (hatched lines) encircles this coated cellular association with sheet-like cytoplasmic projections that form an incomplete investment. (4, 5 ) Fusion of the macrophagic extracellular coat with the epithelial basal lamina places the macrophagic-lymphocytic association into a n intraepithelial position. (6, 7 ) Macrophages and lymphocytes actively penetrate through the intercellular compartment of the epithelium to gain entrance to the glandular lumen. (8, 9 ) Currents created by the newly ciliated glandular epithelium sweep the emigrating macrophages and lymphocytes toward the glandular orifice and uterine lumen. The glands acquire a temporary function in cellular transportation during involution; after regression is complete, the non-ciliated precursor cells proliferate and differentiate to create a new population of secretory cells.

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Center, The University of North Carolina). Their fine skills are acknowledged here with respect and gratitude. LITERATURE CITED Anton, E., D. Brandes and S. Barnard 1969 Lysosomes in uterine involution: Distribution of acid hydrolases in luminal epithelium. Anat. Rec., 164: 231-252. Banon, P., D. Brandes and J. K. Frost 1964 Lysosomal enzymes i n the rat ovary and endometrium during the estrous cycle. Acta Cytol., 8: 416-425. Barka, T., and P. J. Anderson 1963 Histochemistry. Theory, Practice and Bibliography. Hoeber Medical Division, Harper & Row, New York. Brandes, D., and E. Anton 1969 Lysosomes in uterine involution: Intracytoplasmic degradation of myofilaments and collagen. J. Gerontol., 24: 55-69. 1969 A n electron microscopic cytcchemical study of macrophages during uterine involution. J. Cell Biol., 41: 450-461. Breed, W. G., P. V. Peplow, P. Eckstein and S. A. Barker 1972 The chemical composition of flushings from rat uteri with and without intrauterine devices. J. Endocrinol., 52: 575-584. Chen, T. T., F. W. Bazer, J. J. Cetorelli, W. E. Pollard and R. M. Roberts 1973 Purification and properties of a progesterone-induced basic glycoprotein from the uterine fluid of pigs. J. Biol. Chem., 248: 8560-8566. Comer, G . W. 1921 Cyclic changes in the ovaries and uterus o f the sow and their relations to the mechanism of implantation. Carnegie Inst., Washington. Contrib. Embryol., 1 3 : 117-146. Endo, M., and 2. Yosizawa 1973 Hormonal effect on glycoproteins and glyco-saminoglycans in rabbit uteri. Arch. Biochem. Biophys., 156: 397-403. Farquhar, M. G., and G . E. Palade 1965 Cell junctions in amphibian skin. J. Cell Biol., 26. 263-292. Gross, J. 1974 Collagen biology: Structure, degradation, and disease. In: The Harvey Lectures, 1972-73. Academic Press, New York, pp. 351-432. Hartman, C. 1923 The oestrous cycle in the opossum, Didelphis wirginiana. Am. J. Anat., 32: 353-421. Lobel, B., and H. W. Deane 1962 Enzymic activity associated with postpartum involution of the uterus and with its reeression after hormone withdrawal in the rat. Endocrinology, 70: 567-578. Martinez-Estkve, P. 1942 Observations on the histology of the opossum ovary. Carnegie Inst. Washington. Contrib. Embryol., 30: 17-26. McCrady, F., Jr. 1938 The embryology of the opossum. Am. Anat. Mem. No., 16: 1-233. Morgan, C. F. 1946 Sexual rhythms in the reproductive tract of the adult female opossum and effects of hormonal treatments. Am. J. Anat., 78: 411-463.

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HELEN A. PADYKULA AND J. MARY TAYLOR

Selenka, E. 1887 Studien uber EntwickelungsMurray, F. A., Jr., F. W. Bazer, H. D. Wallace geschichten viertes heft das opossum. C. W. and A. C. Warnick 1972 Quantitative and Kreidel, Verlag, Wiesbaden, Germany. qualitative variation in the secretion of protein by the porcine uterus during the estrous cycle. Sharman, G. B. 1955 Studies on marsupial reBiol. Reprod., 7: 314-320 production. 11. The estrous cycle of Setonix Padykula, H. A., and A. G. Campbell 1976 brachyurus. Aust. J. Zool., 3. 44-55. Cellular mechanisms involved in cyclic stromal 1970 Reproductive physiology of marrenewal of the uterus. 11. The albino rat. Anat. supials. Science, 167: 1221. Rec., 184: 2 7 4 8 . Shorey, C. D., and R. L. Hughes 1972 Uterine Parakkal, P. F. 1969 Involvement of macroglandular regeneration during the follicular phages in collagen resorption. J. Cell Biol., 41; phase in the marsupial Trichosurus vulpecula. 345-354. Aust. J. ZOO^., 20: 235-247. 1972 Macrophages: The time course 1973 Cyclictal changes in the uterine and sequence of their distribution in the postendometrium and peripheral plasma concenpartum uterus. J. Ultrastr. Res., 40: 284-291. trations of progesterone in the marsupial Pilton, P. E., and G. B. Sharman 1962 ReproTrichosurus vulpecula. Aust. J. ZOQ~., 21: 1-19. duction i n the marsupial Trichosurus vulpecula. Squire, G . D., F. W. Bazer and F. A. Murray, Jr. J. Endocrinol., 25: 119-136. 1972 Electrophoretic patterns of porcine uterRoss, R. 1971 The smooth muscle cell. 11. ine protein secretions during the estrous cycle. Growth of smooth muscle in culture and formaBiol. Reprod., 7: 321-325. tion of elastic fibers. J. Cell Biol., 50: 172-186. Thorburn, G. D., R. I. Cox and C. D. Shorey Srhueller, E. F. 1968 Ciliated epithelia of the 1971 Ovarian steroid secretion rates in the human uterine mucosa. Obst. Gynec., 31: 215marsupial Trichosurus vulpecula. J. Reprod. 223. Fert., 24: 139. 1973 Ultrastructure of ciliated cells in the human endometrium. Obst. Gynec., 41: Volkmann, A., and J. L. Gowans 1965A The production of macrophages in the rat. Brit. J. I 88-1 94. Exp. Path., 46: 50-61. Schwarz, W., and R. H. Giildner 1967 Elektronenmikroskopische Undersuchingen der Kol1965B The origin of macrophages from lagenabbaus im Uterus der Ratte nach der bone marrow in the rat. Brit. J. Exp. Path., Schwangerschaft. Z. Zellforsch., 83: 416-426. 46: 62-70.

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ENDOMETRIAL REGRESSION IN THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 1

EXPLANATION OF FIGURES

1

Endometrium, twelfth day of pregnancy. Toluidine blue. x 160. The bilaminar or non-vascular yolk sac placenta ( Y S ) is closely apposed to the simple columnar lurninal epithelium (LE). Blood vessels ( V ) of the maternal placenta are aggregated immediately beneath the luminal epithelium. Note the empty appearance of the stroma which is caused by a n abundance of ground substance and a paucity of fibers and cells. Compare this luteal appearance with the second day postpartum i n figure 2. G , glands.

2

Endometrium day 2 postpartum. Toluidine blue. x 160. A striking change occurs in the stroma i n the early postpartum period, as free cells become numerous in the stroma, especially beneath the luminal epithelium (LE) and near the glands ( G ) . The Iuminal epithelium is less regular i n appearance than during the late luteal phase. A glandular orifice ( A ) is filled with numerous free cells. Compare with figure 1.

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ENDOMETRIAL REGRESSION IN THE OPOSSUM Helen A. Padykula and J. Mary Taylor

EXPLANATION OF FIGURES

3

Gland and stroma, twelfth day of pregnancy. Toluidine blue. x 680. Secretory cells ( S ) predominate over ciliated cells (C) in the simple columnar glandular epithelium of the luteal phase. The stroma appears empty except for one cell, probably a macrophage ( M ) . Compare with figure 4.

4

Gland and stroma. Day 2 postpartum. Toluidine blue. X 720. The glandular epithelium has a less regular appearance than during pregnancy (fig. 3 ) . Ciliated cells ( C ) occur among non-ciliated cells. Macrophages ( M ) occur in the stroma, in apposition to the basal glandular surface, and in an intraepithelial position. Cells occur in the glandular lumen. Mitotic activity appears i n the epithelia during the early postpartum. Stromal cells are numerous and varied; they consist of fibroblasts, monocytes, lymphocytes, and plasma cells in addition to the macrophages.

5

Macrophage. Day 2 postpartum. Electron micrograph. x 12,000. The active phagocytic surface of a n endometrial macrophage is shown. The principal material being enclosed in phagosomes ( P ) is the ground substance ( G S ) which here retains a protein precipitate. Since the ground substance contains a macromolecular carbohydrateprotein material, these phagosomes can be identified at the light microscopic level in a periodic acid-Schiff preparation (fig. 7 ) . Note the paucity of collagen fibrils (cf).

6

Macrophage. Day 2 postpartum. Electron micrograph. x 9,600. The macrophage contains conspicuous large residual bodies (RE ) with heterogeneous content. Smaller homogeneous dense bodies may be lysosomes. The conspicuous inclusions most likely represent components of the phagolysosomal system. The clear vacuoles represent lipid droplets ( L ) . Note also the active cell surface, Golgi apparatus, and the widespread occurrence of rough endoplasmic reticulum and ribosomes.

PLATE 2

ENDOMETRIAL REGRESSION IN THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 3

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ENDOMETRIAL REGRESSION I N THE OPOSSUM Helen A. Padykula and J. Mary Taylor

EXPLANATION O F FIGURES

18

7

Glands. Day 2 postpartum. Periodic acid-Schiff preparation. x 450. The phagosomal derivatives of macrophages are Pas-positive, and stand out sharply here. At the lower left, there are some stromal macrophages ( M ) . Note that macrophages appear as rounded protrusions within the basement membrane of the basal glandular surface ( A ) . Some also occur as insertions i n the epithelium (arrow). PAS-positive material occurs i n the glandular lumens. Compare with figures 8 and 9.

8

Glands. Day 1 postpartum. Acid phosphatase, semithin plastic section. X 600. High acid phosphatase activity occurs in the glandular lumens and i n macrophages ( M ) that form basal protrusions. Compare with figures 9 and 10.

9

Intraepithelial macrophage i n the glands. Day 2 postpartum. Electron micrograph. X 4,300. Ciliated cells ( C ) predominate, and their cilia fill the glandular lumen ( L u ) . Along the basal epithelial surface, a macrophage ( M ) with residual bodies ( R E ) , lipid droplets (L), and a n active surface is included within the basal lamina (arrows), thus forming a basal protrusion. Another cell ( X ) which may be a lymphocytic derivative is also located within the basal lamina. Trace the basal lamina, starting at the right, to see that here it is closely apposed to the epithelial surface, then it is slack and loosened ( * ) from the epithelium, from there it follows the contour of the macrophage, and again i s slack o n the left near the other cell ( X ) . Thus, the macrophage and its companion cell occur in a n intraepithelial position. Note also the cytoplasmic processes ( p ) of another stromal cell are oriented in relation to the macrophage, and its investing basal lamina. A dividing macrophage occurs at the lower left. Study figures 7 and 8 for light microscopic evidence of this phenomenon.

PLATE 4

ENDOMETRIAL REGRESSION I N THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 5

19

ENDOMETRIAL REGRESSION I N THE OPOSSUM Helen A. Padykula and J. Mary Taylor

EXPLANATION OF FIGURE

10

20

Coated macrophages. Day 2 postpartum. Electron micrograph. x 4,200. Three heavily laden macrophages ( M ) occur in a stromal region just below the luminal epithelium. The nucleus ( N ) is included in only one macrophage. Their cytoplasm is filled with the products of phagocytic activity, residual bodies ( R B ) and lipid droplets ( L ) . These macrophages are enclosed almost entirely by a thin cellular coat (arrows); however, a portion of one macrophage ( * ) is freely exposed to the ground substance. Moreover, long, highly attenuated processes ( p ) , which extend from a fibroblast-like cell ( X ) , follow the contours of these coated macrophages and enclose them almost completely. Ly, lymphocyte; G S , ground substance.

PLATE 6

ENDOMETRIAL REGRESSION IN THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 7

EXPLANATION OF FIGURE

11

Coated stromal macrophage. Day 2 postpartum. Electron micrograph.

x 16,800.

This ultrastructural image suggests that a n extracellular coat is deposited along the surface of macrophages destined for extrusion from the endometrium. The extracellular coat (arrows) does not cover two cytoplasmic extensions ( * ) that have highly active surfaces. The cytoplasm of the macrophage contains many lipid droplets ( L ) and a well-developed rough endoplasmic reticulum. GS, ground substance; cf, collagen fibril.

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ENDOMETRIAL REGRESSION IN THE OPOSSUM Helen A. Padykula and J. Mary Taylor

EXPLANATION O F FIGURES

12

Intraluminal cells of the glands. Day 2 postpartum. Toluidine blue. X 1,008. The glands during postpartum regression are converted from a secretory epithelium (fig. 3 ) to the predominantly ciliated one shown here. Note that a few non-ciliated cells occur among the ciliated cells. Another characteristic feature is the presence of macrophages and lymphocytes in the lumen (GL). Evidence for possible transepithelial migration of a macrophage is indicated at the asterisk, which marks the location of a cell with heterogeneous inclusions that has a n extension (arrow) projecting into the stroma. In the lower right and left fields, processes ( A ) occur that most likely belong to macrophages which are largely out of the plane of section. Compare with figure 13.

13 Glands. Day 2 postpartum. Forty pm cryostat section, acid phosphatase

activity. x 220. The high acid phosphatase activity of the luminal content of the glands is evident in this thick section. It may originate in part from the lysosomal derivatives of the luminal macrophages shown i n figures 12 and 14.

PLATE 8

ENDOMETRIAL REGRESSION I N THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 9

EXPLANATION OF FIGURE

14

Intraluminal macrophage and lymphocyte i n a gland. Electron micrograph. x 4,000. A large macrophage ( M ) laden with residual material and lipid occurs in the glandular lumen ( G L ) ; its surface appears active. A lymphocyte (Ly) occurs in close association with the macrophage. The highly ciliated surface of the glandular epithelium is evident; slender microvilli (mv) occur among the cilia. Compare with figures 12 and 13.

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PLATE 10 EXPLANATION OH FIGURES

15

Uterine lumen and endometrial surface. Day 1 postpartum. Toluidine blue. x 640. Various cells occur in the uterine lumen ( U L ) during the first two days postpartum. During day 1 heterophil emigration through the luminal epithelium is conspicuous; heterophils ( A ) occur in the stroma, within the luminal epithelium (LE), and i n the uterine lumen. In addition, macrophages ( M ) and shed epithelial cells occur in the lumen.

16

Smear of uterine fluid. Day 1 postpartum. Acid phosphatase activity, 45 minutes incubation. x 680. The cells of the uterine fluid vary in acid phosphatase activity. Certain cells that are intensely active also contain lipid droplets (as shown better in the figure 16 inset, day 2 postpartum, 30 minutes incubation. x 1,100), and appear to be macrophages (M).

Electron micro17 Macrophage in the uterine lumen. Day. 1 postpartum. _ _ graph.- x 18,500. The intraluminal macrophages have active cell surfaces, are forming phagosomes ( P ) , and contain heterogeneous digestive vacuoles ( D V ) as well as lipid droplets ( L ) . N, nucleus; UL, uterine lumen.

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ENDOMETRIAL REGRESSION I N THE OPOSSUM Helen A. Padykula and J. Mary Taylor

PLATE 10

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