A Freeze-Fracture Study of Exocytosis and Reflexive Gap Junctions in Human Ovarian Decidual Cells JOHN C. HERR AND PAUL M. HEIDGER, JR. Department ofdnatomy, Uniuersity of Iowa, College of Medicine, Iowa City,Iowa 52242
ABSTRACT
Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, t h e membrane-leaflets of which exhibited a particlepoor E face adjacent to t h e vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. I n fractures revealing t h e E face of the plasmalemma, cytoplasmic portals at t h e bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed t h a t such gap junctions share basic similarities with intercellular gap junctions. These similarities include particle size (8-10 nm) ; number of particles within clusters (20-40); and t h e presence of 5-15 nm particlefree aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and t h e cell soma, a s well a s between peduncles, suggests t h a t the original definition of reflexive gap junctions as those existing between processes of t h e same cell should be broadened to include any gap junctional specialization formed between portions of t h e plasma membrane of one cell.
In a previous study employing transmission electron microscopy of conventional thin sections (Herr e t al., '781, ovarian decidual cells were shown to secrete small osmiophilic granules, approximately 30-60 nm in (diameter, by the well known mechanism of exocytosis. Interestingly, the release of these granules occurred from i n t a c t peduncular processes which protruded through a n external lamina of fine filaments surrounding t,he decidual cells. Although the secretory product(s1 might be suspected to include a protein component from observation of secretory vesicles on t h e maturing face of t h e Golgi apparatus, the precise chemical moieties present are as yet unknown. Ovarian decidual cells have, as well, been shown by lanthanum treatment and thin sections to possess reflexive gap junctions, i.e., gap junctions between processes of t h e same AM. J . ANAT. (1978)152: 29-44.
cell (Herr, '761, a feature shared with human uterine decidual cells (Lawn et al., '71). Several investigators have demonstrated t h a t freeze-fracture produces a unique preferential split through t h e hydrophobic portion of t h e lipid bilayer, thus revealing internal membrane structure (Branton, '66; Pinto da Silva and Branton, '70; Tillack and Marchesi, '70). The fracture results in two complementary halves of the membrane: t h e cytoplasmic half conventionally labelled the P face, and the exterior half conventionally labelled t h e E face (Branton e t al., '75). The application of freezefracture to studies of secretion mechanisms and membrane fusion has received only recent attention (Burwin and Satir, '77; Pinto da Silva and Nogueira, '77; Satir e t al., '73; Orci Accepted January 23, '70
29
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JOHN C. HERR A N D PAUL M. HEIDGER, JR.
et al., '73; Tandler and Poulsen, '76)and no such studies have been conducted utilizing ovarian or uterine decidua. The freeze-fracture observations presented in this study provide information on the planar distribution of intramembranous particles in ovarian decidual cell membranes. Attention has been focused upon the plasmalemma extending as peduncular processes, upon the membrane of the secretory vesicle, and upon vesicular subunits and small particles which occur a t exocytotic sites. Of particular interest is the arrangement of intramembranous particles within reflexive gap junctions occurring on decidual cell processes, and their topology in comparison with intercellular gap junctions. This study presents 3-dimensional images which serve to clarify further the unique mode of merocrine secretion exhibited by decidual cells. MATERIALS A N D METHODS
Biopsy of the human ovarian cortex a t term provided foci of decidual cells as described previously (Herr et al., '78). Razor-blade dicing was used to isolate these foci, which were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4,2% sucrose, for 15 to 20 minutes, and then transferred to 30%aqueous glycerol for three to four hours. Small pieces of the glycerinated tissue were mounted in the mirror-image device, frozen rapidly in Freon cooled in liquid nitrogen and fractured a t a stage temperature of - 123°C in a Balzers freeze-fracture device, model BAE 121 (Santa Anna). Platinum-carbon shadowing of the fracture face was performed without etching. The platinum-carbon replicas were cleaned in Chlorox in distilled water, washed with distilled water and mounted on 300-meshuncoated copper grids and examined in a Philips-300 electron microscope. Electron micrographs of freeze-fracture replicas were printed as positive images, mounted with the shadow from bottom to top; all shadows are white. Measurements of particle size on freeze-fracture replicas are uncorrected for shadow angle and platinum deposition. The freeze-cleave nomenclature proposed by Branton et al. ('75) is employed to designate membrane fracture faces. RESULTS
Cell overview The general cell surface topography and ap-
pearance following freeze-fracture of ovarian decidual cytoplasmic organelles are demonstrated in figure 1. Profiles of the Golgi apparatus were recognized as regions of crescentic cisternae with associated vesicles of varying diameter. Freeze-fracture failed to reveal the luminal contents of Golgi-associated vesicles; thus, we were unable to confirm with freeze fracture previous TEM observations of secretory bodies originating in the Golgi region. The cytoplasm typically was filled with widely dispersed polymorphous vesicles, many undoubtedly corresponding to saccules of endoplasmic reticulum and mitochondria. The cytoplasm itself exhibited a coarse-grained texture and a darker grey tone when compared to either the membrane fracture-surfaces or the eutectic of the extracellular matrix. The scalloped nature of the cell cortex was a prominent feature in low-magnification micrographs, such as figure l. The continuation of triangulate bulges of cortical cytoplasm into the stalks of peduncular processes was seen, but most often, the distal peduncle tip was observed without its attachment to the cell. Sites of triangulate bulges and lateral notches appeared to be reliable indicators of regions of peduncular processes, even though the peduncular tip might not lie within the plane of fracture. Although fractures revealing collagen in the eutectic of the extracellular matrix were common, it was uncommon to observe collagen within the interpeduncular extracellular matrix. In this region, small particles with a mean diameter of 7 nm were observed; these were more highly concentrated in a band immediately peripheral to the cell (figs. 2, 9). This latter zone corresponded to the region of the lamina externa.
The E face plasmalemma In figure 2, a sheet of fractured E face plasmalemma is viewed, as from the cell interior. The small annular pits and elevations, ranging from 35-50nm in diameter and occurring consistently within the E face, were interpreted as apertures of endocytic vesicles (fig. 2);such diameters are similar to those of endocytic vesicles in endothelial cells described by Leak ('71).Ovoid depressions, approximately 80 nm in diameter, were seen to be continuous with the stalks of peduncular processes and appeared to represent the cytoplasmic portal a t the base of the peduncular
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS
stalk. Mature peduncles (as in fig. l ) ,which contained secretory bodies within their expanded tips, exhibited stalk widths of 95-120 nm. Allowing approximately 15 nm for two membrane thicknesses, the 80-nm diameter of the cytoplasmic portals in figure 2 was, therefore, not surprising. Secretory peduncles Both fracture faces of the plasma membrane of ovarian decidual cell secretory peduncles and both fracture faces of the secretory vesicle membrane were examined in some detail. Secretory peduncles which contained within their tips secretory vesicles surrounded by cytoplasm are shown in several planes of fracture in plate 3. In figure 3, the fracture revealed the concave E face of the peduncular stalk to be in continuity with the E face of the soma1 plasma membrane. The E face of the secretory vesicle (E') was designated as such in conformity with the nomenclature preferred by Branton et al. ('75) and represents the face of t h a t half membrane which is closest to the secretory vesicle lumen. We believe secretory vesicles con form to this terminology because of the TEM finding (Herr e t al., '78) that the secretory vesicles are derived from the Golgi apparatus and their lumen therefore represents exoplasmic space. A fracture plane which passed through the cytoplasm of the peduncular stalk and exposed much of a convex secretory vesicle E face is shown in figure 4.In contrast to the relatively smooth E face of the secretory vesicle, the concave P face of the secretory vesicle seen in figure 5 contained a population of randomly arranged particles 8-9 nm in diameter. In figure 6, a tangential fracture revealed a peduncular tip unattached to the cell soma, with the convex E face of the secretory vesicle rimmed by coarse-grained cytoplasm. Exocytosis
In plate 4, images interpreted as sites of exocytosis are presented. The fracture plane in figure 7 has exposed several E faces of the secretory vesicles within the dilated tips of peduncular processes; a t the left of this figure, a secretory vesicle has been fractured to expose its lumen. A more advanced stage of retraction of the peduncular lips from around the secretory product is presented in figure 8. Here, the secretory material appeared as a heterogeneous population of vesicles ranging from 20 nm to
31
130 nm in diameter. The most numerous and uniform of these vesicular profiles lay within a diameter range of 40-55 nm. Particles ranging from 8.5-11.5nm were also observed associated with regions of exocytosis (fig. 9). The concentration of these particles was much greater a t exocytotic sites than was the background of small particles in the extracellular matrix.
Particle arrangement in reflexive gap junctions I t is not possible to state from our freezefracture replicas alone that all gap junctions observed on decidual cell processes and on the cell surface were unambiguously reflexive gap junctions. The remote possibility exists that some represented gap junctions between adjacent cells, and that, due to plane of fracture, we were unable to detect the second adjacent cell or cell process. Our confidence that all gap junctions observed a t the periphery of decidual cells were reflexive rests on the following: (1) We have never observed any cell process, clearly seen to be from another cell, in the region of the gap junction or confluent with the junctional membrane; (2) Analysis of 600 or more micrographs of thin and serial sections has failed to show a single example of a n intercellular gap junction between either adjacent cells or decidual cells and another cell type (Herr, '76). In figure 10, a gap junction on an ovarian decidual cell process displayed both P face particles and E face pits. It is difficult to determine the overall geometry of this or other gap junctions on decidual cell processes, for although many macular junctions were seen, much of the junction was often obscured by shadow, as here. As in figure 13, E faces commonly revealed gap junctional membrane profiles a t peduncular tips, as the plane of fracture followed a concave peduncular depression. Whether certain junctions entirely encircled and encased a peduncle tip in a chalice-like fashion, as suggested by TEM (Herr, '761, was not resolved by freezefracture. The clusters of intramembranous particles in plate 5 were typical of all the reflexive gap junctions we observed. Within the clusters, t h e center-to-center spacing of particles ranged from 8-12nm and the particle diarneters were from 8-10 nm, with larger particles more typical a t the junction's periphery. Representative clusters contained from 20-40par-
32
JOHN C. HERR AND PAUL M. HEIDGER, JR
ticles separated by particle-free aisles, 5-15 nm wide. These particle-free aisles on the P face corresponded to slightly elevated bands observed between E face pits. In figures 11 and 12, a peduncular-shaped gap junction was observed on a broad sheet of t h e P leaflet and provided evidence for reflexive gap junction formation between a peduncle and the cell soma. DISCUSSION
One distinct difference between freeze-fracture and conventional TEM is t h a t tissue preparation does not include prolonged chemical fixation and dehydration. Freeze-fracture thus affords the opportunity to examine cellular fine structure following only brief fixation and freezing and permits comparison of the fine structure seen without dehydration with t h a t observed in tissue preserved with conventional techniques. I n this regard, we noted t h a t peduncular-shaped processes containing dense bodies were easily demonstrated in Epon-embedded tissue, but t h a t routine paraffin sections did not readily show this feature. Accordingly, we considered the possibility t h a t conventional TEM processing might induce distortion at the decidual cell periphery and secondary collapse of plasma membrane around secretory bodies, and t h a t exocytosis from peduncular processes thus might in some fashion be artifactual. However, t h e freeze-fracture data presented in this study not only clearly confirm earlier TEM observations of the pedunculate nature of ovarian decidual cell secretory processes, but demonstrate also the secretory vesicle membrane leaflets, t h e vesicle content, and sites of exocytosis. The secretory vesicle and its content By TEM, the 0.4-to 0.9-pm secretory bodies (termed secretory vesicles in freeze-fracture) contain a predominant population of electrondense granules, 30-60 nm in diameter (Herr e t al., '78). At exocytotic sites, however, TEM demonstrates both this major population and vesicles of larger diameter. The freeze-fracture images (plate 4) substantiate the presence at exocytotic sites of both a major population of vesicles 40-55 nm in diameter and a quantitatively smaller population of vesicles varying from 20-120 nm in diameter. The former population falls well within the 30- to 60-nm size range for the contents of t h e secretory body as observed by TEM. I t appears thus
that the 30- to 60-nm electron-opaque, granular subunits of TEM are, in fact, vesicular as revealed by freeze-fracture. Also revealed by freeze-fracture at degranulation sites are small 8.5-11.5 nm particles; such a population of small particles has been difficult to demonstrate by TEM. The location of these particles within the secretory peduncles is unclear. I t should not be overlooked, however, t h a t they may provide a n approximate molecular diameter of t h e decidual cell secretory product. Our TEM and serial-section observations t h a t degranulation occurs from peduncles which are in cytoplasmic continuity with the soma during the exocytotic event a r e also corroborated (cf. fig. 9 ) by these freeze-fracture studies. Reflexive gap junctions Freeze-fracture studies have shown t h a t not all intercellular gap junctions are morphologically identical. Among the differences noted are variations in size of subunit particles, their density per unit area and their arrangement (Staehelin, '72; Larsen, '77). For example, some intercellular gap junctions are diminutive and macular (Friend and Gilula, '72; McNutt and Weinstein, '73; Satir and Gilula, '73); others exhibit a diversity of contours as in the outer plexiform layer of the retina (Raviola and Gilula, '731,while gap junctions of granulosa cells may range to as large as 6 p m in diameter (Albertini et al., '75). Considerable diversity also has been reported in particle diameters and in center-to-center spacing within the particle clusters. I n t h e study of Raviola and Gilula ('73), for example, gap junctional particles demonstrated between photoreceptor cells of monkeys, rabbits and turtles had diameters of from 8-14 nm and center-to-center spacing t h a t varied from 8-18 nm. The spatial arrangement of the particle clusters of reflexive gap junctions demonstrated in this study is similar to t h a t of intercellular gap junctions described in ovarian granulosa cells (Albertini e t al., '751, in t h e ciliary epithelium (Kogon and Pappas, '75) and in normal and transformed chick embryo fibroblasts (Pinto da Silva and Gilula, '72). The features shared in common include size of intramembranous particles, their center-tocenter spacing, and most notably the grouping of intramembranous particles into clusters of 20 to 40 particles separated by particle-free aisles. Thus, a fundamental parity between re-
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS
flexive gap junctions and intercellular gap junctions from a variety of tissues seems clearly established from this and previous studies (Herr, '76; Larsen, '77). The term, reflexive gap junction, was originally introduced to describe gap junctions between adjacent processes from the same cell (Herr, '76). In this study, we present evidence interpreted as gap junction formation between a peduncular process and the cell soma and i t seems appropriate t h a t this type of gap junction be termed reflexive, as well. In regard to the functional significance of decidual cell gap junctions, intercellular gap junctions have been examined in the uterus of the pregnant mouse (Finn and Lawn, '67) and in the developing deciduoma of rats, where possible functions in cell-to-cell communication and subsequent involvement in t h e spread of decidualization have been hypothesized (Kleinfeld et al., '76). It is yet uncertain whether the association of reflexive gap junctions with secretory peduncles implies a cytophysiological relationship. I t is worthy of note that many of the cells in which reflexive gap junctions occur are actively secretory (Larsen, '77). We have observed, in thin and serial sections of routine and lanthanumtreated tissue (Herr, '76; Herr e t al., '781, reflexive gap junctions both between processes which exhibit secretory bodies and between processes undergoing exocytosis. However, we have demonstrated frequent instances of both early-stage and exocytotic peduncles on which gap junctional membrane is not observed. It is important to recognize in this regard that the surface area occupied by peduncles in an actively secreting ovarian decidual cell is extensive, and when junctional membrane does occur on these peduncles i t may be a randon association and not necessarily related to the secretory event. The recent suggestion that gap junctional particles may be hormone binding sites (Albertini et al., '75; Larsen, '77) should not be overlooked in assessing the possible role of gap junctions in decidual tissue. ACKNOWLEDGMENTS
The authors thank Doctor William J. Larsen for preparing the freeze-fracture replicas, for aid in interpretation, and for helpful discussions. We also gratefully acknowledge Paul J. Reimann and Mary J o Henington for photographic assistance, and Doctor Richard
33
D. Sjolund for critical reading of t h e manuscript. These studies were supported by grants from the National Institutes of Health (GM00148-191, GRS funds from the Univ. of Iowa (to W. J. Larsen) and The American Cancer Society (PDT-84, to W. J. Larsen and P. M. Heidger). LITERATURE CITED Albertini, D. F., D. W. Fawcett and P. J. Olds 1975 Morphological variations in gap junctions of ovarian granulosa cells. Tissue and Cell, 7: 389-405. Branton, D. 1966 Fracture faces of frozen membranes. Proc. Natl. Acad. Sci. ( U S A . ) , 55: 1048-1056. Branton, D., S. Bullivant, N. Gilula, M. Karnovsky, H. Moor, K. Muhlethaler, D. Northcote, L. Packer, B. Satir, P. Satir, V. Speth, I,. Staehelin, R. Steere and R. Weinstein 1975 Freeze-etching nomenclature. Science (Washington, D.C.), 190: 54-56. Burwen, S. J . , and B. H. Satir 1977 A freeze-fracture study of early membrane events during mast cell secretion. J. Cell Biol., 73: 660-671. Finn, C. A,, and A. M. Lawn 1967 Specialized junctions between decidual cells in the uterus of the pregnant mouse. J. Ultrast. Res., 20: 321-327. Friend, D. S., and N. B. Gilula 1972 A distinctive cell contact in the rat adrenal cortex. J. Cell Biol., 53: 148-163. Herr, J. C. 1976 Reflexive gap junctions. Gap junctions between processes arising from the same ovarian decidual cell. J. Cell Biol., 69: 495-501. Herr, J. C., P. M. Heidger, J. R. Scott, J. W. Anderson, L. B. Curet and H. W. Mossman 1978 Decidual cells in the human ovary a t term. 1. Incidence, gross anatomy and ultrastructural features of merocrine secretion. Am. J. Anat., 152: 7-28. Kleinfeld, R. G., H. A. Morrow and V. J. DeFeo 1976 Intercellular junctions between decidual cells in the growing deciduoma of the pseudopregnant rat uterus. Biol. Reprod., 15: 593-603. Kogon, M., andG. D. Pappas 1975 Atypical gap junctions in the ciliary epithelium of the albino rabbit eye. J. Cell Biol., 60: 671-676. Larsen, W. J. 1977 Structural diversity of gap junctions. A review. Tissue and Cell, 9: 373-394. Lawn, A. M., E. W. Wilson and C. A. Finn 1971 The ultrastructure of human decidual and predecidual cells. J. Reprod. Fert., 26: 85-90. McNutt, N. S., and R. S. Weinstein 1969 Carcinoma of the cervix: deficiency of nexus intercellular junctions. Science, 165: 597-599. Orci, L., M. Amherdt, F. Malaisse-Largae, C. Rouiller and A. E. Renold 1973 Insulin release by emiocytosis: Demonstration with freeze-etching. Science (Washington, D. C.), 179: 82-83. Pinto da Silva, P., and D. Branton 1970 Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J. Cell Biol.. 45: 598-605. Pinto da Silva, P., and N. B. Gilula 1972 Gap junctions in normal and transformed fibroblasts in culture. Exp. Cell Res., 71: 393-401. Pinto da Silva, P., and M. L. Nogueira 1977 Membrane fusion during secretion. A hypothesis based on electron microscopic observation of Phytophthora palmivora zoospores during encystment. J. Cell Biol., 73: 161-181.
JOHN C. HERR AND PAUL M. HEIDGER, J R Raviola, E., and N. B. Gilula 1973 Gap junctions between photoreceptor cells in the vertebrate retina. Proc. Natl. Acad. Sci. (U.S.A.), 70: 1677-1681. Satir, B., C. Schooley and P. Satir 1973 Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J. Cell Biol., 56: 153.176. Satir, P., and N. B. Gilula 1973 The fine structure of membranes and intercellular communication in insects. Ann. Rev. Entomology, 28: 143-166. Staehelin, L. A. 1972 Three types of gap junctions in
interconnecting intestinal epithelial cells visualized by freeze-etching. Proc. Natl. Acad. Sci. (USA.), 69: 1318-1321. Tandler, B., and J. J. Poulsen 1976 Fusion of the envelope of mucous droplets with the luminal plasma membrane in acinar cells of the cat submandibular gland. J. Cell Biol., 68: 775-781. Tillack, T. W., and V. T. Marchesi 1970 Demonstration of the outer surface of freeze-etched red blood cell membranes. J. Cell Biol., 45: 649-653.
PLATE 1 EXPLANATION OF FIGURE
1 Low-magnification view of human decidual cell displaying surface pedunculation. The peduncles of this cell range from 0.8-1.2 p m in length; several peduncles are continuous with the cell soma a t arrows, and secretory vesicles are evident in certain expanded peduncle tips (large arrowheads). Collagen cross-fractures are seen in the lower right portion of the micrograph and cross-fractures of fine filaments believed to be components of the external lamina are seen in close proximity to the cell soma a s small particles (see also figs. 2, 8, 9 at higher magnification). Note the absence of collagen from the interpeduncular region immediately surrounding the cell. Cytoplasmic features a r e displayed in cross-fracture; the cytoplasm is more coarsely textured and of greyer tone than are the membrane surfaces and eutectic. Prominent among cell organelles are Golgi regions (arrow). X 20,825.
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M Heidger, Jr
PLATE 1
35
PLATE 2 EXPLANATION OF FIGURE
2 Cross-fracture through cytoplasm (bottom left) contiguous with a broad sheet of the E half-membrane. The annuli of endocytic vesicles are evident in this membrane leaflet a t arrowheads. At the arrows, ovoid depressions ca. 80 nm in diameter are interpreted to represent the cytoplasmic portal a t the base of the peduncular stalk. A band of small particles of ca. 7 nm exists a t the cell periphery in the ice table of the
extracellular matrix. E, E face.
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X
40,625.
FREEZE FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C Herr and Paul M. Heidger. Jr.
PLATE 2
37
A bbreuiations P, P face C, Cytoplasm
E , E face of secretory vesicle membrane
PLATE 3 EXPLANATION OF FIGURES
3 Fracture through peduncular process. The E face of the plasmalemma is seen to extend as a thin stalk to the base of the dilated portion of the peduncle, where the fracture passes across the cytoplasm (arrowhead) of the peduncle to the E face of the secretory vesicle (E7. The particle-rich P face of the plasmalemma of the dilated portion of the peduncle obscures most of the secretory vesicle. X 41,200. 4
Fracture through tip of a peduncular process. Much of the E face of the secretory vesicle (E') is evident here. The fracture also passes through the cytoplasm of the peduncular stalk (C) which is seen to be continuous with the cytoplasm of the soma. X 58,900.
5 Fracture through secretory peduncles. Concave or P faces of secretory vesicles are here surrounded by cytoplasm (C) outside of which is the eutectic of the extracellular matrix. X 76,800. 6 Tip of a peduncular process containing a secretory vesicle is depicted toward the top of figure. Here the fracture has passed tangentially through the tip of a peduncular process. The P face of the peduncular plasmalemma, E face of the secretory vesicle (E') and the homogeneous cytoplasm surrounding the secretory vesicle are seen. Other fracture profiles, perhaps of the same process, exhibit E face and P face leaflets. X 53,100.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M. Heidger. Jr.
PLATE 3
39
PLATE 4 EXPLANATION OF FIGURES
7 Decidual cell processes tangentially fractured to expose not only the E faces of the secretory vesicles (E'), but also the contents of a secretory vesicle lumen. The vesicle lumen is continuous a t arrow with the extracellular matrix and the vesicle's content presents both particulate and vesicular profiles. Note the thin folium of E face secretory vesicle membrane a t the arrowhead. X 34,350. 8 Higher-magnification view of a site of exocytosis demonstrating heterogeneity of released material. Vesicular profiles with both concave and convex surfaces are present. The smallest are ca. 20 nm diameter, and the largest are ca. 130 nm diameter. The most numerous and uniform profiles lie within a diameter range of 40-55 nm. X 46,300. 9 An attached peduncular process cross-fractured through the lumen of the secretory vesicle is shown here with exocytotic material a t its margin. Other adjacent sites of exocytosis a r e visible. In addition to the vesicular profiles noted in figure 8, numerous small (ca. 8.5-11.5 nm) particles are dispersed among the vesicular components. X 34,350.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C Herr a n d Paul M Heidger, Jr.
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
10 Gap junction on ovarian decidual cell process from the region of the cell periphery shown in the inset; arrowhead indicates this process in inset. Intramembranous P face particles are arranged in linear arrays two to four particles in width, and of variable length. The rows of 8-10nm particles are separated by particle-free aisles ca. 5-15 nm in width. Such aisles correspond to slight elevations (arrows) seen between E face pits. X 106,250; inset, X 13,400. 11, 12 A broad sheet of particle-rich P face is continuous over a peduncular process (bottom) and exhibits a club-shaped gap junctional profile (upper right). Unlike the gap junction in figure 10, this junction represents gap junction formation between peduncle and soma. The particle size and spacing are similar in both junctions, as seen at higher magnification in figure 12. Figure 11 X 56,250; figure 12 X 168,950. 13 A peduncular process exhibits a n extensive E face containing gap junctional pits. The E face of the peduncular membrane is continuous through the peduncular stalk with t h a t of the soma1 plasmalemma. X 113,480.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M. Heidger, Jr.
PLATE 5
43
ABSTRACT
Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, t h e membrane-leaflets of which exhibited a particlepoor E face adjacent to t h e vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. I n fractures revealing t h e E face of the plasmalemma, cytoplasmic portals at t h e bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed t h a t such gap junctions share basic similarities with intercellular gap junctions. These similarities include particle size (8-10 nm) ; number of particles within clusters (20-40); and t h e presence of 5-15 nm particlefree aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and t h e cell soma, a s well a s between peduncles, suggests t h a t the original definition of reflexive gap junctions as those existing between processes of t h e same cell should be broadened to include any gap junctional specialization formed between portions of t h e plasma membrane of one cell.
In a previous study employing transmission electron microscopy of conventional thin sections (Herr e t al., '781, ovarian decidual cells were shown to secrete small osmiophilic granules, approximately 30-60 nm in (diameter, by the well known mechanism of exocytosis. Interestingly, the release of these granules occurred from i n t a c t peduncular processes which protruded through a n external lamina of fine filaments surrounding t,he decidual cells. Although the secretory product(s1 might be suspected to include a protein component from observation of secretory vesicles on t h e maturing face of t h e Golgi apparatus, the precise chemical moieties present are as yet unknown. Ovarian decidual cells have, as well, been shown by lanthanum treatment and thin sections to possess reflexive gap junctions, i.e., gap junctions between processes of t h e same AM. J . ANAT. (1978)152: 29-44.
cell (Herr, '761, a feature shared with human uterine decidual cells (Lawn et al., '71). Several investigators have demonstrated t h a t freeze-fracture produces a unique preferential split through t h e hydrophobic portion of t h e lipid bilayer, thus revealing internal membrane structure (Branton, '66; Pinto da Silva and Branton, '70; Tillack and Marchesi, '70). The fracture results in two complementary halves of the membrane: t h e cytoplasmic half conventionally labelled the P face, and the exterior half conventionally labelled t h e E face (Branton e t al., '75). The application of freezefracture to studies of secretion mechanisms and membrane fusion has received only recent attention (Burwin and Satir, '77; Pinto da Silva and Nogueira, '77; Satir e t al., '73; Orci Accepted January 23, '70
29
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JOHN C. HERR A N D PAUL M. HEIDGER, JR.
et al., '73; Tandler and Poulsen, '76)and no such studies have been conducted utilizing ovarian or uterine decidua. The freeze-fracture observations presented in this study provide information on the planar distribution of intramembranous particles in ovarian decidual cell membranes. Attention has been focused upon the plasmalemma extending as peduncular processes, upon the membrane of the secretory vesicle, and upon vesicular subunits and small particles which occur a t exocytotic sites. Of particular interest is the arrangement of intramembranous particles within reflexive gap junctions occurring on decidual cell processes, and their topology in comparison with intercellular gap junctions. This study presents 3-dimensional images which serve to clarify further the unique mode of merocrine secretion exhibited by decidual cells. MATERIALS A N D METHODS
Biopsy of the human ovarian cortex a t term provided foci of decidual cells as described previously (Herr et al., '78). Razor-blade dicing was used to isolate these foci, which were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4,2% sucrose, for 15 to 20 minutes, and then transferred to 30%aqueous glycerol for three to four hours. Small pieces of the glycerinated tissue were mounted in the mirror-image device, frozen rapidly in Freon cooled in liquid nitrogen and fractured a t a stage temperature of - 123°C in a Balzers freeze-fracture device, model BAE 121 (Santa Anna). Platinum-carbon shadowing of the fracture face was performed without etching. The platinum-carbon replicas were cleaned in Chlorox in distilled water, washed with distilled water and mounted on 300-meshuncoated copper grids and examined in a Philips-300 electron microscope. Electron micrographs of freeze-fracture replicas were printed as positive images, mounted with the shadow from bottom to top; all shadows are white. Measurements of particle size on freeze-fracture replicas are uncorrected for shadow angle and platinum deposition. The freeze-cleave nomenclature proposed by Branton et al. ('75) is employed to designate membrane fracture faces. RESULTS
Cell overview The general cell surface topography and ap-
pearance following freeze-fracture of ovarian decidual cytoplasmic organelles are demonstrated in figure 1. Profiles of the Golgi apparatus were recognized as regions of crescentic cisternae with associated vesicles of varying diameter. Freeze-fracture failed to reveal the luminal contents of Golgi-associated vesicles; thus, we were unable to confirm with freeze fracture previous TEM observations of secretory bodies originating in the Golgi region. The cytoplasm typically was filled with widely dispersed polymorphous vesicles, many undoubtedly corresponding to saccules of endoplasmic reticulum and mitochondria. The cytoplasm itself exhibited a coarse-grained texture and a darker grey tone when compared to either the membrane fracture-surfaces or the eutectic of the extracellular matrix. The scalloped nature of the cell cortex was a prominent feature in low-magnification micrographs, such as figure l. The continuation of triangulate bulges of cortical cytoplasm into the stalks of peduncular processes was seen, but most often, the distal peduncle tip was observed without its attachment to the cell. Sites of triangulate bulges and lateral notches appeared to be reliable indicators of regions of peduncular processes, even though the peduncular tip might not lie within the plane of fracture. Although fractures revealing collagen in the eutectic of the extracellular matrix were common, it was uncommon to observe collagen within the interpeduncular extracellular matrix. In this region, small particles with a mean diameter of 7 nm were observed; these were more highly concentrated in a band immediately peripheral to the cell (figs. 2, 9). This latter zone corresponded to the region of the lamina externa.
The E face plasmalemma In figure 2, a sheet of fractured E face plasmalemma is viewed, as from the cell interior. The small annular pits and elevations, ranging from 35-50nm in diameter and occurring consistently within the E face, were interpreted as apertures of endocytic vesicles (fig. 2);such diameters are similar to those of endocytic vesicles in endothelial cells described by Leak ('71).Ovoid depressions, approximately 80 nm in diameter, were seen to be continuous with the stalks of peduncular processes and appeared to represent the cytoplasmic portal a t the base of the peduncular
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS
stalk. Mature peduncles (as in fig. l ) ,which contained secretory bodies within their expanded tips, exhibited stalk widths of 95-120 nm. Allowing approximately 15 nm for two membrane thicknesses, the 80-nm diameter of the cytoplasmic portals in figure 2 was, therefore, not surprising. Secretory peduncles Both fracture faces of the plasma membrane of ovarian decidual cell secretory peduncles and both fracture faces of the secretory vesicle membrane were examined in some detail. Secretory peduncles which contained within their tips secretory vesicles surrounded by cytoplasm are shown in several planes of fracture in plate 3. In figure 3, the fracture revealed the concave E face of the peduncular stalk to be in continuity with the E face of the soma1 plasma membrane. The E face of the secretory vesicle (E') was designated as such in conformity with the nomenclature preferred by Branton et al. ('75) and represents the face of t h a t half membrane which is closest to the secretory vesicle lumen. We believe secretory vesicles con form to this terminology because of the TEM finding (Herr e t al., '78) that the secretory vesicles are derived from the Golgi apparatus and their lumen therefore represents exoplasmic space. A fracture plane which passed through the cytoplasm of the peduncular stalk and exposed much of a convex secretory vesicle E face is shown in figure 4.In contrast to the relatively smooth E face of the secretory vesicle, the concave P face of the secretory vesicle seen in figure 5 contained a population of randomly arranged particles 8-9 nm in diameter. In figure 6, a tangential fracture revealed a peduncular tip unattached to the cell soma, with the convex E face of the secretory vesicle rimmed by coarse-grained cytoplasm. Exocytosis
In plate 4, images interpreted as sites of exocytosis are presented. The fracture plane in figure 7 has exposed several E faces of the secretory vesicles within the dilated tips of peduncular processes; a t the left of this figure, a secretory vesicle has been fractured to expose its lumen. A more advanced stage of retraction of the peduncular lips from around the secretory product is presented in figure 8. Here, the secretory material appeared as a heterogeneous population of vesicles ranging from 20 nm to
31
130 nm in diameter. The most numerous and uniform of these vesicular profiles lay within a diameter range of 40-55 nm. Particles ranging from 8.5-11.5nm were also observed associated with regions of exocytosis (fig. 9). The concentration of these particles was much greater a t exocytotic sites than was the background of small particles in the extracellular matrix.
Particle arrangement in reflexive gap junctions I t is not possible to state from our freezefracture replicas alone that all gap junctions observed on decidual cell processes and on the cell surface were unambiguously reflexive gap junctions. The remote possibility exists that some represented gap junctions between adjacent cells, and that, due to plane of fracture, we were unable to detect the second adjacent cell or cell process. Our confidence that all gap junctions observed a t the periphery of decidual cells were reflexive rests on the following: (1) We have never observed any cell process, clearly seen to be from another cell, in the region of the gap junction or confluent with the junctional membrane; (2) Analysis of 600 or more micrographs of thin and serial sections has failed to show a single example of a n intercellular gap junction between either adjacent cells or decidual cells and another cell type (Herr, '76). In figure 10, a gap junction on an ovarian decidual cell process displayed both P face particles and E face pits. It is difficult to determine the overall geometry of this or other gap junctions on decidual cell processes, for although many macular junctions were seen, much of the junction was often obscured by shadow, as here. As in figure 13, E faces commonly revealed gap junctional membrane profiles a t peduncular tips, as the plane of fracture followed a concave peduncular depression. Whether certain junctions entirely encircled and encased a peduncle tip in a chalice-like fashion, as suggested by TEM (Herr, '761, was not resolved by freezefracture. The clusters of intramembranous particles in plate 5 were typical of all the reflexive gap junctions we observed. Within the clusters, t h e center-to-center spacing of particles ranged from 8-12nm and the particle diarneters were from 8-10 nm, with larger particles more typical a t the junction's periphery. Representative clusters contained from 20-40par-
32
JOHN C. HERR AND PAUL M. HEIDGER, JR
ticles separated by particle-free aisles, 5-15 nm wide. These particle-free aisles on the P face corresponded to slightly elevated bands observed between E face pits. In figures 11 and 12, a peduncular-shaped gap junction was observed on a broad sheet of t h e P leaflet and provided evidence for reflexive gap junction formation between a peduncle and the cell soma. DISCUSSION
One distinct difference between freeze-fracture and conventional TEM is t h a t tissue preparation does not include prolonged chemical fixation and dehydration. Freeze-fracture thus affords the opportunity to examine cellular fine structure following only brief fixation and freezing and permits comparison of the fine structure seen without dehydration with t h a t observed in tissue preserved with conventional techniques. I n this regard, we noted t h a t peduncular-shaped processes containing dense bodies were easily demonstrated in Epon-embedded tissue, but t h a t routine paraffin sections did not readily show this feature. Accordingly, we considered the possibility t h a t conventional TEM processing might induce distortion at the decidual cell periphery and secondary collapse of plasma membrane around secretory bodies, and t h a t exocytosis from peduncular processes thus might in some fashion be artifactual. However, t h e freeze-fracture data presented in this study not only clearly confirm earlier TEM observations of the pedunculate nature of ovarian decidual cell secretory processes, but demonstrate also the secretory vesicle membrane leaflets, t h e vesicle content, and sites of exocytosis. The secretory vesicle and its content By TEM, the 0.4-to 0.9-pm secretory bodies (termed secretory vesicles in freeze-fracture) contain a predominant population of electrondense granules, 30-60 nm in diameter (Herr e t al., '78). At exocytotic sites, however, TEM demonstrates both this major population and vesicles of larger diameter. The freeze-fracture images (plate 4) substantiate the presence at exocytotic sites of both a major population of vesicles 40-55 nm in diameter and a quantitatively smaller population of vesicles varying from 20-120 nm in diameter. The former population falls well within the 30- to 60-nm size range for the contents of t h e secretory body as observed by TEM. I t appears thus
that the 30- to 60-nm electron-opaque, granular subunits of TEM are, in fact, vesicular as revealed by freeze-fracture. Also revealed by freeze-fracture at degranulation sites are small 8.5-11.5 nm particles; such a population of small particles has been difficult to demonstrate by TEM. The location of these particles within the secretory peduncles is unclear. I t should not be overlooked, however, t h a t they may provide a n approximate molecular diameter of t h e decidual cell secretory product. Our TEM and serial-section observations t h a t degranulation occurs from peduncles which are in cytoplasmic continuity with the soma during the exocytotic event a r e also corroborated (cf. fig. 9 ) by these freeze-fracture studies. Reflexive gap junctions Freeze-fracture studies have shown t h a t not all intercellular gap junctions are morphologically identical. Among the differences noted are variations in size of subunit particles, their density per unit area and their arrangement (Staehelin, '72; Larsen, '77). For example, some intercellular gap junctions are diminutive and macular (Friend and Gilula, '72; McNutt and Weinstein, '73; Satir and Gilula, '73); others exhibit a diversity of contours as in the outer plexiform layer of the retina (Raviola and Gilula, '731,while gap junctions of granulosa cells may range to as large as 6 p m in diameter (Albertini et al., '75). Considerable diversity also has been reported in particle diameters and in center-to-center spacing within the particle clusters. I n t h e study of Raviola and Gilula ('73), for example, gap junctional particles demonstrated between photoreceptor cells of monkeys, rabbits and turtles had diameters of from 8-14 nm and center-to-center spacing t h a t varied from 8-18 nm. The spatial arrangement of the particle clusters of reflexive gap junctions demonstrated in this study is similar to t h a t of intercellular gap junctions described in ovarian granulosa cells (Albertini e t al., '751, in t h e ciliary epithelium (Kogon and Pappas, '75) and in normal and transformed chick embryo fibroblasts (Pinto da Silva and Gilula, '72). The features shared in common include size of intramembranous particles, their center-tocenter spacing, and most notably the grouping of intramembranous particles into clusters of 20 to 40 particles separated by particle-free aisles. Thus, a fundamental parity between re-
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS
flexive gap junctions and intercellular gap junctions from a variety of tissues seems clearly established from this and previous studies (Herr, '76; Larsen, '77). The term, reflexive gap junction, was originally introduced to describe gap junctions between adjacent processes from the same cell (Herr, '76). In this study, we present evidence interpreted as gap junction formation between a peduncular process and the cell soma and i t seems appropriate t h a t this type of gap junction be termed reflexive, as well. In regard to the functional significance of decidual cell gap junctions, intercellular gap junctions have been examined in the uterus of the pregnant mouse (Finn and Lawn, '67) and in the developing deciduoma of rats, where possible functions in cell-to-cell communication and subsequent involvement in t h e spread of decidualization have been hypothesized (Kleinfeld et al., '76). It is yet uncertain whether the association of reflexive gap junctions with secretory peduncles implies a cytophysiological relationship. I t is worthy of note that many of the cells in which reflexive gap junctions occur are actively secretory (Larsen, '77). We have observed, in thin and serial sections of routine and lanthanumtreated tissue (Herr, '76; Herr e t al., '781, reflexive gap junctions both between processes which exhibit secretory bodies and between processes undergoing exocytosis. However, we have demonstrated frequent instances of both early-stage and exocytotic peduncles on which gap junctional membrane is not observed. It is important to recognize in this regard that the surface area occupied by peduncles in an actively secreting ovarian decidual cell is extensive, and when junctional membrane does occur on these peduncles i t may be a randon association and not necessarily related to the secretory event. The recent suggestion that gap junctional particles may be hormone binding sites (Albertini et al., '75; Larsen, '77) should not be overlooked in assessing the possible role of gap junctions in decidual tissue. ACKNOWLEDGMENTS
The authors thank Doctor William J. Larsen for preparing the freeze-fracture replicas, for aid in interpretation, and for helpful discussions. We also gratefully acknowledge Paul J. Reimann and Mary J o Henington for photographic assistance, and Doctor Richard
33
D. Sjolund for critical reading of t h e manuscript. These studies were supported by grants from the National Institutes of Health (GM00148-191, GRS funds from the Univ. of Iowa (to W. J. Larsen) and The American Cancer Society (PDT-84, to W. J. Larsen and P. M. Heidger). LITERATURE CITED Albertini, D. F., D. W. Fawcett and P. J. Olds 1975 Morphological variations in gap junctions of ovarian granulosa cells. Tissue and Cell, 7: 389-405. Branton, D. 1966 Fracture faces of frozen membranes. Proc. Natl. Acad. Sci. ( U S A . ) , 55: 1048-1056. Branton, D., S. Bullivant, N. Gilula, M. Karnovsky, H. Moor, K. Muhlethaler, D. Northcote, L. Packer, B. Satir, P. Satir, V. Speth, I,. Staehelin, R. Steere and R. Weinstein 1975 Freeze-etching nomenclature. Science (Washington, D.C.), 190: 54-56. Burwen, S. J . , and B. H. Satir 1977 A freeze-fracture study of early membrane events during mast cell secretion. J. Cell Biol., 73: 660-671. Finn, C. A,, and A. M. Lawn 1967 Specialized junctions between decidual cells in the uterus of the pregnant mouse. J. Ultrast. Res., 20: 321-327. Friend, D. S., and N. B. Gilula 1972 A distinctive cell contact in the rat adrenal cortex. J. Cell Biol., 53: 148-163. Herr, J. C. 1976 Reflexive gap junctions. Gap junctions between processes arising from the same ovarian decidual cell. J. Cell Biol., 69: 495-501. Herr, J. C., P. M. Heidger, J. R. Scott, J. W. Anderson, L. B. Curet and H. W. Mossman 1978 Decidual cells in the human ovary a t term. 1. Incidence, gross anatomy and ultrastructural features of merocrine secretion. Am. J. Anat., 152: 7-28. Kleinfeld, R. G., H. A. Morrow and V. J. DeFeo 1976 Intercellular junctions between decidual cells in the growing deciduoma of the pseudopregnant rat uterus. Biol. Reprod., 15: 593-603. Kogon, M., andG. D. Pappas 1975 Atypical gap junctions in the ciliary epithelium of the albino rabbit eye. J. Cell Biol., 60: 671-676. Larsen, W. J. 1977 Structural diversity of gap junctions. A review. Tissue and Cell, 9: 373-394. Lawn, A. M., E. W. Wilson and C. A. Finn 1971 The ultrastructure of human decidual and predecidual cells. J. Reprod. Fert., 26: 85-90. McNutt, N. S., and R. S. Weinstein 1969 Carcinoma of the cervix: deficiency of nexus intercellular junctions. Science, 165: 597-599. Orci, L., M. Amherdt, F. Malaisse-Largae, C. Rouiller and A. E. Renold 1973 Insulin release by emiocytosis: Demonstration with freeze-etching. Science (Washington, D. C.), 179: 82-83. Pinto da Silva, P., and D. Branton 1970 Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J. Cell Biol.. 45: 598-605. Pinto da Silva, P., and N. B. Gilula 1972 Gap junctions in normal and transformed fibroblasts in culture. Exp. Cell Res., 71: 393-401. Pinto da Silva, P., and M. L. Nogueira 1977 Membrane fusion during secretion. A hypothesis based on electron microscopic observation of Phytophthora palmivora zoospores during encystment. J. Cell Biol., 73: 161-181.
JOHN C. HERR AND PAUL M. HEIDGER, J R Raviola, E., and N. B. Gilula 1973 Gap junctions between photoreceptor cells in the vertebrate retina. Proc. Natl. Acad. Sci. (U.S.A.), 70: 1677-1681. Satir, B., C. Schooley and P. Satir 1973 Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J. Cell Biol., 56: 153.176. Satir, P., and N. B. Gilula 1973 The fine structure of membranes and intercellular communication in insects. Ann. Rev. Entomology, 28: 143-166. Staehelin, L. A. 1972 Three types of gap junctions in
interconnecting intestinal epithelial cells visualized by freeze-etching. Proc. Natl. Acad. Sci. (USA.), 69: 1318-1321. Tandler, B., and J. J. Poulsen 1976 Fusion of the envelope of mucous droplets with the luminal plasma membrane in acinar cells of the cat submandibular gland. J. Cell Biol., 68: 775-781. Tillack, T. W., and V. T. Marchesi 1970 Demonstration of the outer surface of freeze-etched red blood cell membranes. J. Cell Biol., 45: 649-653.
PLATE 1 EXPLANATION OF FIGURE
1 Low-magnification view of human decidual cell displaying surface pedunculation. The peduncles of this cell range from 0.8-1.2 p m in length; several peduncles are continuous with the cell soma a t arrows, and secretory vesicles are evident in certain expanded peduncle tips (large arrowheads). Collagen cross-fractures are seen in the lower right portion of the micrograph and cross-fractures of fine filaments believed to be components of the external lamina are seen in close proximity to the cell soma a s small particles (see also figs. 2, 8, 9 at higher magnification). Note the absence of collagen from the interpeduncular region immediately surrounding the cell. Cytoplasmic features a r e displayed in cross-fracture; the cytoplasm is more coarsely textured and of greyer tone than are the membrane surfaces and eutectic. Prominent among cell organelles are Golgi regions (arrow). X 20,825.
FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M Heidger, Jr
PLATE 1
35
PLATE 2 EXPLANATION OF FIGURE
2 Cross-fracture through cytoplasm (bottom left) contiguous with a broad sheet of the E half-membrane. The annuli of endocytic vesicles are evident in this membrane leaflet a t arrowheads. At the arrows, ovoid depressions ca. 80 nm in diameter are interpreted to represent the cytoplasmic portal a t the base of the peduncular stalk. A band of small particles of ca. 7 nm exists a t the cell periphery in the ice table of the
extracellular matrix. E, E face.
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X
40,625.
FREEZE FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C Herr and Paul M. Heidger. Jr.
PLATE 2
37
A bbreuiations P, P face C, Cytoplasm
E , E face of secretory vesicle membrane
PLATE 3 EXPLANATION OF FIGURES
3 Fracture through peduncular process. The E face of the plasmalemma is seen to extend as a thin stalk to the base of the dilated portion of the peduncle, where the fracture passes across the cytoplasm (arrowhead) of the peduncle to the E face of the secretory vesicle (E7. The particle-rich P face of the plasmalemma of the dilated portion of the peduncle obscures most of the secretory vesicle. X 41,200. 4
Fracture through tip of a peduncular process. Much of the E face of the secretory vesicle (E') is evident here. The fracture also passes through the cytoplasm of the peduncular stalk (C) which is seen to be continuous with the cytoplasm of the soma. X 58,900.
5 Fracture through secretory peduncles. Concave or P faces of secretory vesicles are here surrounded by cytoplasm (C) outside of which is the eutectic of the extracellular matrix. X 76,800. 6 Tip of a peduncular process containing a secretory vesicle is depicted toward the top of figure. Here the fracture has passed tangentially through the tip of a peduncular process. The P face of the peduncular plasmalemma, E face of the secretory vesicle (E') and the homogeneous cytoplasm surrounding the secretory vesicle are seen. Other fracture profiles, perhaps of the same process, exhibit E face and P face leaflets. X 53,100.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M. Heidger. Jr.
PLATE 3
39
PLATE 4 EXPLANATION OF FIGURES
7 Decidual cell processes tangentially fractured to expose not only the E faces of the secretory vesicles (E'), but also the contents of a secretory vesicle lumen. The vesicle lumen is continuous a t arrow with the extracellular matrix and the vesicle's content presents both particulate and vesicular profiles. Note the thin folium of E face secretory vesicle membrane a t the arrowhead. X 34,350. 8 Higher-magnification view of a site of exocytosis demonstrating heterogeneity of released material. Vesicular profiles with both concave and convex surfaces are present. The smallest are ca. 20 nm diameter, and the largest are ca. 130 nm diameter. The most numerous and uniform profiles lie within a diameter range of 40-55 nm. X 46,300. 9 An attached peduncular process cross-fractured through the lumen of the secretory vesicle is shown here with exocytotic material a t its margin. Other adjacent sites of exocytosis a r e visible. In addition to the vesicular profiles noted in figure 8, numerous small (ca. 8.5-11.5 nm) particles are dispersed among the vesicular components. X 34,350.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C Herr a n d Paul M Heidger, Jr.
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
10 Gap junction on ovarian decidual cell process from the region of the cell periphery shown in the inset; arrowhead indicates this process in inset. Intramembranous P face particles are arranged in linear arrays two to four particles in width, and of variable length. The rows of 8-10nm particles are separated by particle-free aisles ca. 5-15 nm in width. Such aisles correspond to slight elevations (arrows) seen between E face pits. X 106,250; inset, X 13,400. 11, 12 A broad sheet of particle-rich P face is continuous over a peduncular process (bottom) and exhibits a club-shaped gap junctional profile (upper right). Unlike the gap junction in figure 10, this junction represents gap junction formation between peduncle and soma. The particle size and spacing are similar in both junctions, as seen at higher magnification in figure 12. Figure 11 X 56,250; figure 12 X 168,950. 13 A peduncular process exhibits a n extensive E face containing gap junctional pits. The E face of the peduncular membrane is continuous through the peduncular stalk with t h a t of the soma1 plasmalemma. X 113,480.
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FREEZE-FRACTURE OF HUMAN OVARIAN DECIDUAL CELLS John C. Herr and Paul M. Heidger, Jr.
PLATE 5
43
