BIOLOGY OF REPRODUCTION 15, 593-603 (1976) Intercellular Junctions Between Decidual Cells in the Growing
Deciduoma of the Pseudopregnant Rat Uterus’
RUTH G. KLEINFELD, HENRY A. MORROW and VINCENT J. DeFEO
Department of Anatomy and Reproductive Biology, The John A Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822 Downloaded from https://academic.oup.com/biolreprod/article/15/5/593/2768190 by guest on 01 October 2021
ABSTRACT The appearance of gap junctions between decidual cells and the restructuring of the intercellular matrix of the endometrium was studied by electron microscopy in the developing primary deciduoma of the pseudopregnant rat uterus. Decidualization was induced by intraluminal injection of Hanks balanced salt solution at the time of peak sensitivity (Day 4). The progression of decidualization was followed through Day 9 of pseudopregnancy. At the time of sensitivity the periluminal stromal cells of the antimesometrial region are surrounded by an abundant collagenous matrix and there are relatively few contacts between processes of neighboring cells. When junctions exist they are of the maculae adherentes type. On the day following the deciduogenic stimulus (Day 5) contacts between cells are numerous and gap junctions are present. The presence of gap junctions between the early differentiating decidual cells suggests that cell to cell communication may be involved in the spread of decidualization. As decidualization progresses a rapid reduction in the amount of intercellular matrix occurs. With continued growth extensive infoldin and interdigitations of the plasma membranes develop between adjoining decidual cells forming a complex membranous labyrinth. Numerous gap junctions are present involving extensive areas of the cell surfaces. By Day 9 the antimesometrial region consists of large, polyploid, glycogen-rich decidual cells with extensive surface info!din that are joined by gap junctions.
INTRODUCTION h later. This is a transitory state lasting only a
Preparation of the rat uterus for ovum few hours. In normal pregnancy and pseudo- implantation involves specific interactions of pregnancy this occurs on Day 4 (DeFeo, 1967; the two ovarian hormones, progesterone and Psychoyos, 1973). At this time the uterus is estrogen (for reviews see: DeFeo, 1967; Psy- capable of responding to a variety of stimuli choyos, 1973; Glasser and Clark, 1975). The which elicit the decidual response. uterus of the rat and of many other mammals is Decidualization, a specific response of the normally not receptive to ovum implantation progestational endometrium, involves an exten- and must undergo specific hormone dependent sive reorganization of the endometrial cell changes before it develops a sensitive or recep- populations, analogous in many ways to em- tive state. Experiments with ovariectomized bryonic organogenesis. A period of rapid rats have established that uterine sensitivity growth transforms the mesenchymal-like stro- (the ability to decidualize in response to an mal cells into a densely confluent population of implanting blastocyst) depends on the comple- epithelioid cells. RNA and protein synthesis tion of a basic hormone sequence: daily expo- increase, DNA replication and mitosis are great- sure to progesterone for at least 48 h and the ly stimulated, cell migrations occur and the presence of minute amounts of estrogen at the stromal cells differentiate into large polyploid end of this period (Tachi et al., 1972; Psy- cells filled with glycogen and lipid. Within five choyos, 1973). When this sequence is com- days the uterine weight may increase more than pleted, a state of peak sensitivity occurs 20-24 10-fold (DeFeo, 1967; Finn, 1971). Decidualization involves an orderly coordi- nated progression of stromal cell differentiation Accepted July 29, 1976. starting with a population of periluminal stro- Received June11, 1976. ma! cells at the antimesometrial pole, then ‘This work was supported in part by National spreading toward the peripheral regions and the Institutes of Health grant HD 02399, a grant from the Ford Foundation 660-0202A and a research grant mesometrial pole as more stromal cells divide from the University of Hawaii. and differentiate. In each zone, cell division
593 594 KLEINFELD ET AL. precedes cell differentiation. When fully differ- tion on the day of estrus, Day 0 (DeFeo, 1%6). The entiated, the decidual cells stop dividing and decidual response was induced between 12 noon and 1 pm on Day 4 of pseudopregnancy by injection of eventually degenerate. The sequential pattern 0.1 ml of Hanks balanced salt solution (Cibco) into of cellular changes suggests that cell to cell the tubal end of the uterine lumen of each cornu interactions are involved. under ether anesthesia. The uterus of anesthetized rats A consistent feature of decidual tissue, was perfused via the dorsal aorta on Days 4, 5, 7 or 9 of pseudopregnancy with 3 percent glutaraldehyde in demonstrated by electron microscopy, is the 0.1 M phosphate buffer at pH 7.2. The uterine horns presence of junctional complexes between de- were removed and cross sectional slices (0.5-1 mm
cidual cells (Jollie and Bencosme, 1965; Enders thick) were immersed in the buffered glutaraldehyde Downloaded from https://academic.oup.com/biolreprod/article/15/5/593/2768190 by guest on 01 October 2021 and Schlafke, 1967; Finn and Lawn, 1967; solution for 24 h at 4#{176}C.The tissue samples were Lawn et al., 1971; Kleinfeld and DeFeo, 1971). washed several times in the same buffer and postfixed for 4-6 h in 1 percent 0s04 in 0.1 M phosphate Gap junctions between decidual cells (earlier buffer pH 7.2, dehydrated in a graded series of ethanol referred to as tight junctions) have been noted solutions and embedded in Epon 812. in the pregnant mouse uterus (Finn and Lawn, Lanthanum was used according to the procedure of 1967; Finn, 1971), in the pseudopregnant rat Revel and Karnovsky (1967) to outline the extracellu- lar space and delineate the specialized gap junctions. deciduoma (Kleinfeld and DeFeo, 1971), and The uterus was perfused with 3 percent glutaraldehyde between predecidual cells in the human endo- buffered with s-collidine containing a final concentra- metrium during the late luteal phase of the tion of 1 percent lanthanum nitrate. The tissue was menstrual cycle (Lawn et a!., 1971). Gap then processed in a manner similar to that of uterine junctions are regions of membrane specializa- tissue not treated with lanthanum except that 1 percent lanthanum nitrate was added to all solutions tion where adjacent cell membranes are closely up until dehydration. apposed (2-3 nm) and are believed to be the To select specific areas of the uterus semithin structural basis of a form of communication sections (1 Mm) were stained with toluidine blue (0.5 between cells allowing free passage of ions, percent toluidene blue in 2.5 percent sodium car- metabolites and other low molecular weight bonate) and blocks were trimmed accordingly. Thin sections (silver to gold) cut with a diamond knife were substances (Lowenstein, 1968; Bennett, 1973). mounted on 200 mesh grids and stained with 3 There is currently much interest in the role percent aqueous uranyl acetate followed by lead such communicating junctions play in facilitat- citrate (l eynolds, 1963). The sections prepared from ing synchronous responses to hormonal signals the lanthanum experiments were lightly stained with lead citrate (Venable and Coggeshall, 1965). Sections (Bergman, 1968; Merck et a!., 1972, 1973; were scanned in a Phillips EM 300 electron microscope Albertini and Anderson, 1974) or the coordina- at 60KV. tion of growth and differentiation (Gilula et al., 1972; Lowenstein, 1973). RESULTS The fine structure of decidual cells in the pseudopregnant rat was previously described by The cells of the antimesometrial region of Jollie and Bencosme (1965). This report de- the uterus are the first to respond to a scribes the fine structural features of intercellu- deciduogenic stimulus. There is a wave-like lar contacts that exist between stromal cells and progression of mitotic activity and differentia- decidual cells in the developing deciduoma of tion starting with the periluminal stromal cells the rat from Days 4 to 9 of pseudopregnancy. then spreading toward the peripheral region as Particular attention is focused on 1) the time of more cells become involved. The antimesome- appearance of gap junctions and 2) the exten- trial region reaches peak development and sive increase in cell surface area of decidual cells growth by Day 9, at which time cells in the in relation to the reduction of the intercellular inner region start to degenerate. Mesometrial matrix and free space. decidualization starts on Day 7 and reaches peak development by Days 11-13. MATERIALS AND METHODS The endometrial stromal cells resemble em- Three month old Holtzmann virgin female rats bryonic mesenchyme, consisting of irregular were made pseudopregnant by vaginal-cervical stimula- stellate cells with large ovoid nuclei and rela-
All electron micrographs are taken from the antimesometrial pole of the endometrium. FIGS. 1-5. Day 4 of pseudopregnancy prior to administration of the deciduogenic stimulus.
FIG. 1. Stromal cells from the periluminal area are larger, more rounded with lobate processes which extend out in all directions. X5,100. INTERCELLULAR JUNCTIONS BETWEEN DECIDUAL CELLS 595 Downloaded from https://academic.oup.com/biolreprod/article/15/5/593/2768190 by guest on 01 October 2021
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FIG. 2. A contact of the macula adherens type junction between two periluminal stromal cells. X 38,200. FIG. 3. An elongated area of contact between two periluminal stromal cells resembling the zonula adherens type junction. X40,200. FIG. 4. Stromal cells just below the periluminal area resemble active fibroblasts and are surrounded by an abundant collagenous matrix. X 5,300. FIG. 5. A portion of a stromal cell showing a newly polymerized collagen fibril at the surface of a small recess. X35,500. 596 KLEINFELD ET AL.
tively small amounts of cytoplasm with cell involving long stretches of apposing membranes processes extending randomly from their sur- are characteristically seen within this mem- faces. Few contacts exist between neighboring branous labyrinth (Fig. 16). It is difficult to cells. The cells are surrounded by an abundant determine if the gap junctions are always ground substance with collagenous fibers dis- between membranes of adjoining cells or also persed throughout. At the time of sensitivity form between membranes of the same cell. The (Day 4) the so called predecidual cells of the entire antimesometrial region consists of large, antimesometrial pole adjacent to the Iuminal polyploid, glycogen-rich decidual cells (Fig. 11) whose surfaces are extensively intertwined
epithelial cells are enlarged, more rounded and Downloaded from https://academic.oup.com/biolreprod/article/15/5/593/2768190 by guest on 01 October 2021 have numerous lobate processes (Fig. 1). Junc- (Figs. 15, 16). A kind of functional syncytial tions between adjoining cells are of the macula mass is thus produced, characterized by an adherens type (Figs. 2, 3). Stromal cells in the exaggerated increase in total surface area of more peripheral region are fibroblast-like, have dimensions rarely achieved in other tissues. At fewer processes and contacts between cells are the peripheral region, toward the myometrium, rare. Large amounts of intercellular matrix rich newly differentiated stromal cells feed into the in collagen surround the cells (Figs. 4, 5). decidual mass. Gap junctions are also present Junctions between cells when present are also between these cells (Fig. 17). of the macula adherens type. Localized cell death of decidual cells occurs On the day following the deciduogenic at Day 9 in the central region of the anti- stimulus, i.e., Day 5, the periluminal stromal mesometrial pole. In the pregnant animal the cells enlarge and nucleolar structure indicates trophoblast of the developing embryo would increased activity which is reflected in the have invaded and phagocytosized much of this increased concentrations of ribosomes in the cell population. This phenomonon of pro- cytoplasm (Figs. 7, 8). Junctions between cells grammed cell death remains unexplained. There are numerous and now gap junctions as well as are lysosomes present but few autophagic bod- the adherentes type junctions are present (Figs. ies exist during this early phase of degeneration. 6, 8, 9, 10). The many cup shaped and annular Autophagy is more widespread during the forms of gap junctions present suggest that the initial transition period of cell differentiation. extending processes interact to form gap junc- One of the earliest cellular changes noted is the tions upon contact with neighboring cells (Figs. condensation of the chromatin. The pattern of 8, 9, 10, 14). Lysosomes and autophagic cell death usually involves islands of adjoining vesicles are especially numerous during this cells, suggesting some coordinated sequence or early phase of decidualization. intercellular transfer of signals. Figure 18 shows As decidualization progresses, i.e. Days 7-9, portions of such a group of degenerating cells. the cells enlarge further and nuclear shapes As the process of decidualization expands become irregular and lobated (Fig. 11). A rapid and more peripheral stromal cells differentiate, reduction in the amount of intercellular matrix a basal zone can be delineated at the endome- occurs during the rapid growth of the decidual trial-myometrial interface. Within this zone the cells and gap junctions between apposing sur- glandular epithelial buds long since discon- faces are extensive (Figs. 12, 13, 14). With nected from the luminal epithelium come to lie. continued growth, elaborate interdigitations of The formation of a massive deciduoma in the the plasma membrane develop within the same rat involves the entire length of the uterine cell and between processes of adjoining cells horn and thus differs from the localized de- forming a complex labyrinth of plasma mem- cidua of the implantation site of pregnancy. branes (Figs. 15, 16). The free intercellular The entire luminal epithelium usually degener- space is reduced and the surface areas are ates, the straight portions of the glands lose enormously expanded. Gap junctions often their connection to the lumen as the stromal
FIGS. 6-10. Day 5 of pseudopregnancy, 24 h after administration of the deciduogenic stimulus.
FIG. 6. Portions of two decidual cells. Nuclei and nucleoli are enlarged and cytoplasmic volume is increased. An abutment gap junction joins the two cells (circle: inset). X 5,800; inset, X40,600. FIG. 7. A portion of a nucleolus from a differentiating decidual cell shown at high magnification. The open fibrillar component and the prominent granular component suggest increased ribosomal RNA synthesis. X 34,000. INTERCELLULAR JUNCTIONS BETWEEN DECIDUAL CELLS 597
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FIG. 8. Portions of two decidual cells with many points of contact between processes. A cup shaped gap junction between two processes is shown (circle: inset). Note the high concentration of free ribosomes in the cytoplasm. X8,300; inset, X62,500. FIG. 9. A process from one decidual cell enveloped within an invagination of an apposing cell form a cup shaped gap junction seen in longitudinal view. X 51,200. FIG. 10. The so-called annular gap junction probably represents an invading process enveloped by a gap junction and sectioned transversely. )< 50,000. 598 KLEINFELD ET AL.
cells divide and differentiate, and the glandular between processes of adjacent cells. A similar buds are displaced to the basal zone by the sequence involving cell differentiation in the expanding deciduoma (Velardo et al., 1953; evolution of gap junctions has been reported Kleinfe!d, unpublished observations). The during folliculogenesis in the ovary of the rat glands in the basal zone are surrounded by (Merk et a!., 1973) and rabbit (Albertini and fibroblasts which remain refractory to the de- Anderson, 1974). ciduogenic stimulus. A portion of a gland and The functional role of gap junctions between neighboring fibroblasts from this area are decidual cells is not known. Correlative mor- shown in Fig. 19. Cell contacts between fibro- phological and physiological studies in various Downloaded from https://academic.oup.com/biolreprod/article/15/5/593/2768190 by guest on 01 October 2021 blasts when present are of the macula adherens other tissues have implicated gap junctions as type (Fig. 19, inset). No gap junctions exist be- the site of intercellular communication (Gilula tween these cells. et a!., 1972; Bennett, 1973; McNutt and Wein- stein, 1973). The putative role of such junc- DISCUSSION tions in facilitating the spread of decidualiza- This report has been primarily concerned tion becomes apparent if various aspects of with the appearance of gap junctions between endometrial priming and stromal cell differenti- decidual cells and the restructuring of the ation are analyzed. intercellular free space in the developing de- It was pointed out previously that DNA ciduoma of the pseudopregnant rat uterus. replication and cell division precede stromal cell Prior to decidualization at the time of peak differentiation. Various experimental findings sensitivity, few contacts are seen between adja- have suggested an essential role for mitosis in cent stromal cells. When junctions exist they the process of differentiation (Stockdale and are of the macula adherens type. The earliest Topper, 1966; Bishoff and Holtzer, 1970). recognizable decidual cell transformation is Very few stromal cell mitoses occur during the associated with the presence of gap junctions estrous cycle. At the time the endometrium is
FIGS. 11-14. Days 7-9 of pseudopregnancy.
FIG. 11. Portion of a mature decidual cell with a lobated polyploid nucleus (N) and containing dense aggregates of glycogen (G) in the cytoplasm. X 5,100. FIG. 12. A high magnification view through two junctional areas showing the pentalaminar structure of the junctional membranes. Xl 50,000. FIG. 13. An extensive junctional area between two decidual cells infiltrated with lanthanum, an electron opaque tracer, and shown from different angles. In regions where the junction is cut almost normally, a dense intermediate line can be seen as lanthanum penetrates between the junctional membranes. In regions cut tangentially the lanthanum outlines the quasi-hexagonal ordering of subunits characterizing the gap junction. X115,000. FIG. 14. Two gap junctions from an area of extensive interdigitations between decidual cells. The annulate form at the top probably represents a cross section through a junction-girdled cell process whereas the lower form is a longitudinal view of a cup shaded unction. Less dense areas underlying the plasma membrane are particularly conspicuous at the concave surface of the junctional complexes. X 54,000.
FIGS. 15-17. Day 9 of pseudopregnancy.
FIG. 15. A well differentiated group of decidual cells showing elaborate membrane interdigitations and the disappearance of the collagenous intercellular matrix. X13,000. FIG. 16. A higher magnification of the membrane labyrinth showing many gap junctions (circle: inset) and intercellular membrane whorls (arrows). X 14,200; inset, X 55,000. FIG. 17. Decidual cells from the peripheral region at the endometrial-basal zone region. Cells from this area may decidualize and feed into the growing deciduoma, or may degenerate and facilitate the detachment of the terminal deciduoma. Note the presence of gap junctions between the cells (circle: inset). Lysosomes and phagosomes are numerous. X6,700; inset, X 55,000.
FIGS. 18-19. Day 9 of pseudopregnancy.
FIG. 18. An area of degenerating decidual cells. The chromatin is considerably condensed and degenerative changes are indicated in the cytoplasm of all the conjoining cells. Numerous lysosomes are present. X4, 500. FIG. 19. An area from the basal zone showing glandular epithelial cells (GE) and adjacent fibroblasts. The few cell contacts present between the fibroblasts are of the macula adherens type (inset). X7,100; inset, X 28,500. INTERCELLULAR JUNCTIONS BETWEEN DECIDUAL CELLS 599 developing peak sensitivity, many stromal cells sponsive cells is thus produced (Shelesnyak and divide (Tachi et a!., 1972) and it is postulated Marcus, 1971). Morphologically larger more that a subpopulation of differentially re- densely aggregated predecidual cells can be
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