Unusually Complex Basement Membranes in the Midgut of Two Decapod Crustaceans, the Stone Crab (Menippe Mercenaria) and the Lobster (Homarus Americanus)
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7/ THE ANATOMICAL RECORD 200:253-258 (1981) Unusually Complex Basement Membranes in the Midgut of Two Decapod Crustaceans, the Stone Crab (Menippe mercenaria) and the Lobster (Homarus americanus) JAN ROBERT FACTOR Smithsonian Institution, Ft. Pierce Bureau, Ft. Pierce, Florida 33450 ABSTRACT UltrastructuraJ studies of the stone crab (Menippe mercenaria) and the lobster [Homarus americanus) demonstrate that the basement membrane of the midgut (intestine) is unusually complex. In both species, the basement membrane is three-layered and has processes that form extensive networks pro- truding into the connective tissue. The possible functional significance of this complex structure is discussed. The basement membrane underlying epithe- clude cylinders or grid-like patterns, some- lial tissues is generally considered to be a con- times composed of hexagonal units. tinuous, electron-dense, extracellular sheet Ultrastructural studies of the connective tis- which ranges in thickness from 200 to 50,000 sue layer surrounding the digestive epithelium A and often appears fibrous or flocculent. in two decapod crustaceans, the stone crab When viewed at low magnifications, it has Menippe mercenaria (Brachyura: Xanthidae) been variously described in textbooks as and the lobster Homarus americanus "amorphous" (Threadgold, 76) or "homogene- (Macrura: Nephropidae), demonstrate that the ous" with poorly defined inner and outer limits basement membrane of the adult midgut (Fawcett, '66). Closer examination shows that (intestine) is unusually complex. This is the this layer may be a mat or meshwork of fine fil- first description of a complex basement aments embedded in an amorphous matrix membrane in this major group of (Bloom and Fawcett, 75). The basement mem- invertebrates. brane (also commonly referred to as the basal lamina or lamina densa) often appears to be MATERIALS AND METHODS separated from the epithelial cells by an inter- The stone crabs used in this study were vening transparent layer (the lamina rara). The trapped in the Indian River coastal lagoon of structure, function, and biochemistry of the Florida and the American lobsters were ob- basement membrane have recently been re- tained from commerical sources. The digestive viewed by Kefalides et al. (79). tissues of both species were excised from living Several elaborations of this basic plan have animals, fixed for light microscopy in seawater been reported. The basement membrane of ver- Bouin's fluid, and embedded in Paraplast. Sec- tebrate skin may be connected to the underly- tions were stained with Harris's hematoxylin ing connective tissue by anchoring fibrils em- and eosin, Mallory's triple connective tissue bedded in this tissue (Briggaman et al., 71). stain, 1% alcoholic aniline blue and picric acid- Additionally, Fox (76) has shown that larger acid fuchsin, and periodic acid-Schiff s reagent anchoring fibers may be composed of a varia- (Humason, '62). Tissues were fixed for electron ble number of fibrils in the skin of several microscopy in 3% glutaraldehydein 0.2M sodi- aquatic vertebrates. Among the invertebrates, um cacodylate buffer at pH 7.0. The buffer con- several authors have described unusual struc- tained 30 mg/ml NaCl and 20 /tg/ml CaCL Fix- tural patterns for the basement membrane of ation was carried out at 6°C for 3-6 hours and the midgut in a variety of insects (Terzakis, was followed by 5 washes in solutions of buffer '67; Richards and Richards, '68; Gouranton, with decreasing concentrations of salts. Speci- 70; Holter, 70; Reinhardt and Hecker, 73; Hess and Pinnock, 75; Bayon and Francois, 76). These substructural variations usually in- Received December 2. 1980; accepted December 19. 1980. 0003-276X/81/2003-0253S02.00 1981 ALAN R LISS. INC. 254 JAN ROBERT FACTOR mens were post-fixed in 1.0% OsO, in buffer for lated appearance and is coated on both sides 5 hours, treated with 2% uranyl acetate, dehy- by outer layers of fibrous material (Fig. 6). The drated in ethanol which was replaced by propy- outer layer facing the epithelium is a simple, lene oxide, and embedded in a mixture of Epon thin, electron-dense sheet approximately 0.06 812-Araldite 506. Thin sections were stained urn thick. The outer layer on the connective-tis- with uranyl acetate and lead citrate. sue side of the basement membrane is general- ly thicker (approximately 0.2 /*m) and relative- OBSERVATIONS ly less electron-dense. In addition, extensions The basement membrane of the midgut of of this layer protrude into the connective tis- the lobster is illustrated in Figures 1-3. When sue. These extensions are often T-shaped in viewed with the light microscope (Fig. 1) it can transverse section and the fibrous material is be seen to be a wavy structure which follows commonly drawn out into discrete, banded fi- the folding of the simple, columnar epithelium bers at the indistinct boundary between this of the midgut and separates it from the under- layer and the general matrix of connective tis- lying connective tissue. The basement mem- sue (Fig. 6). Extensions of the fibrous layer are brane stains positively with PAS and aniline especially numerous and prominent where the blue and is slightly eosinophilic. It varies in basement membrane folds toward the lumen of thickness from 0.2 to 0.4 /im and can be seen the gut to follow the longitudinal infoldings of electron-microscopically to be pierced by the midgut epithelium. In these areas they branching channels (Figs. 2 and 3). The central form a network that extends approximately layer consists of a finely granular, extremely 1-2 ^m into the connective tissue (Fig. 5). Fi- uniform material and is coated on both sides broblasts are usually associated with this fi- with thin, fibrous, relatively electron-dense brous outer layer and its extensions (Fig. 6). outer layers (Fig. 3). In addition, structures The basement membranes in the midgut of which range from roughly circular to elongate both lobsters and stone crabs were often separ- in section lie adjacent to the basement mem- ated from the epithelium (Fig. 4), presumably brane proper and can be seen to consist of the as a result of mechanical damage during prep- same finely granular material coated with a aration, but were not separated from the con- similar electron-dense outer layer. Serial sec- nective tissue. tions indicate that the larger of these exten- sions, at least, are processes that extend from the basement membrane. The fibrous outer Abbreviations layer coating the basement membrane and its BM, Basement membrane processes is drawn out into strands which bridge the channels in the membrane and BS, Blood sinus which also interconnect its processes (Fig. 3). CL, Central layer of basement membrane The electron-dense strands, together with the processes of the basement membrane, form an CT, Connective tissue extensive network (approximately 0.4 pm EP, Epithelium thick) along the connective tissue surface of F, Fibroblast the basement membrane (Fig. 2). Processes of fibroblasts are commonly associated with this G, Granulocyte network (Fig. 3). A similar network is only in- L, Lumen of midgut termittently present on the surface of the base- ment membrane which faces the epithelium. OL, Outer layer of basement membrane The basement membrane of the midgut of the stone crab is illustrated in Figures 4-6. It Fig. 1. Basement membrane underlying the midgut epi- thelium of the lobster. Transverse section stained with 1% is considerably thicker than that of the lobster aniline blue (ale), counterstained with picric acid-acid fuch- (approximately 1.5 /tin) and appears to have sin. x 270, two layers when viewed with the light micro- scope (Fig. 4). Its staining characteristics are Fig. 2. Basement membrane (lobster) showing channel (arrowhead) and network of processes on connective tissue similar to the basement membrane in the mid- surface. X 9400. gut of the lobster — it is strongly PAS-positive, strongly aniline blue-positive, and slightly eo- Fig. 3. Portion of the basement membrane (lobster) sinophilic. When observed with the electron showing a branching channel, the central layer and fibrous outer layers, processes of the basement membrane (large ar- microscope, the basement membrane is three- rowheads), strands which interconnect processes and bridge layered and does not appear to have channels channels (small arrowheads), processes of fibroblasts, pro- (Fig. 5). The central layer has a coarse, reticu- cesses of epithelial cells, and connective tissue, x 46,000. COMPLEX BASEMENT MEMBRANES 255 G --'--.• 2 0 256 JAN ROBERT FACTOR COMPLEX BASEMENT MEMBRANES 257 DISCUSSION the three-layered glomerular capillary base- The use of the term "basement membrane" to ment membrane, in which the inner and outer describe the structures in the midgut of the layers are continuous with the surface coats of lobster and stone crab may require some justi- the surrounding cells (Latta and Johnston, fication, particularly in light of the complex na- '76). Although the basement membrane is of- ture of these structures. "Basement mem- ten described as "amorphous," substructure in brane" has been used in a variety of ways. Ke- the form of oriented fibrils (Jollie, '68; Clark et falides et al. (79) point out that the term was al., '75) or hexagonal patterns (Jakus, '64) has coined in 1857 by Robert Todd and William been reported, in addition to the reports of Bowman to describe the extracellular subepi- grid-like patterns in insect basement mem- thelial layer seen with the light microscope in a branes cited above. variety of tissues. Ultrastructural observa- Biochemically, basement membranes con- tions have shown that in some instances the sist of a collagenous component, shown in sev- basement membrane described from light mi- eral instances to be a unique type of collagen croscopy is actually a complex of structures molecule (type IV), and a non-collagenous com- that can be distinguished with the electron mi- ponent, possibly including glycoproteins and croscope, and includes the reticular layer, glycosaminoglycans (reviewed by Kefalides et which is now considered to be distinct from the al., '79).