Development of the endoplasmic reticulum in the rodlet cell of two teleost species.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 283A:239 –249 (2005) Development of the Endoplasmic Reticulum in the Rodlet Cell of Two Teleost Species EDITH BIELEK* Center of Anatomy and Cell Biology, Institute for Histology and Embryology, Medical University of Vienna, Vienna, Austria ABSTRACT The rodlet cell found in different tissues and the blood of teleosts is distinguished by a thick capsule and bipartite rodlets, each consisting of club-like sac and a dense protein core. The development of its rough endoplasmic reticulum (RER) was ultrastructurally investigated in gill and intestinal epithelium of trout (Salmo trutta L., Oncorhynchus kisutch). The RER showed signs of hypertrophy beginning already in immature cells. The typical vesicular appearance noted in the mature cell as well as the apical labyrinth, an accumulation of dislodged mitochondria and vesicles, derives from RER dilatations shedding their ribosomes and pervading the cytoplasm. These dilatations extend also into extracapsular protrusions formerly supposed to be nutritive. A possible role of RER in the formation of the rodlets is indicated by the close association of RER-derived vesicles with rodlet sacs. Conspicuous undulations of the membranes of the sacs as well as the occurrence of tubules (ø ⫽ 30 nm) in the dilated RER, rodlet sacs, and along the core support the proposed hypothesis of hypertrophy. Both features signal the storage of (membrane) protein during altered conditions ranging from slowed metabolism to viral infections. The differentiation of apical microvillar projections replaced later by cytoplasmic blebbing and disruption is compatible with surplus production resulting in discharge by pressure. Together with the occasional presence of junctional complexes, these observations argue for an aberrant cell differentiation due to an unknown cause, representing an alternative to the current interpretation of the rodlet cell as a migrating secretory or leucocytic cell with a defensive function. © 2005 Wiley-Liss, Inc. Key words: rodlet cell; endoplasmic reticulum; ultrastructure; trout; Salmo trutta L.; Oncorhynchus kisutch The rodlet cell is a bafﬂing cell type found in many teleostean families. Characterized by a thick ﬁbrous capsule, a basal nucleus, and conspicuous inclusions, the rodlets which are club-shaped and consist of a basal rodlet sac and a dense central core. Over 100 years ago, the rodlet cell was ﬁrst discovered and considered as a parasitic stage due to its superﬁcial resemblance to Sporozoa, but shortly afterward interpreted as an endogenous and secretory cell. This dispute has still not been resolved to date (Leino, 1974, 1979, 1996; Desser and Lester, 1975; Barber et al., 1979; Grünberg and Hager, 1979; Viehberger and Bielek, 1982; Bielek and Viehberger, 1983; Barber and Mills Westermann, 1986a, b; Smith et al., 1995; Dezfuli et al., 1998, 2000, 2003a, 2003b; Fishelson and Becker, 1999; Bielek, 2002; Manera and Dezfuli, © 2005 WILEY-LISS, INC. 2004). The rodlet cells are preferentially found in epithelia, including endothelia, but also in the blood, hemopoietic organs, and connective tissue. Their number and tissue speciﬁcity seem to be varying both among species and *Correspondence to: Edith Bielek, Center of Anatomy and Cell Biology, Institute for Histology and Embryology, Medical University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria. Fax: 0043-01-4277-9613. E-mail: email@example.com Received 29 July 2004; Accepted 5 November 2004 DOI 10.1002/ar.a.20162 Published online 26 January 2005 in Wiley InterScience (www.interscience.wiley.com). 240 BIELEK among individuals (Mattey et al., 1979; Sulimanović et al., 1996; Reite, 1997; Fishelson and Becker, 1999). Various interpretations of their possible function range from a sensory or endocrine cell (Wilson and Westerman, 1967) to a migrating regulatory element responsible for osmoregulation, pH, or some defensive secretion (Leino, 1974, 1979, 1996). As the only secretory cells known to migrate into the blood are leucocytes, the rodlet cell was consequently considered as blood cell, probably a granulocyte or an inﬂammatory cell derived from a circulating stem cell (Duthie, 1939; Catton, 1951; Smith et al., 1995; El-Habback et al., 1997; Dezfuli et al., 1998, 2000, 2003a, 2003b, 2004; Manera et al., 2001). The secretion of antibiotic substances was proposed by Leino (1974, 1996), although the number of rodlet cells is not decreasing after the end of an infection as expected (Leino, 1996; Koponen and Myers, 2000). Moreover, some inconsistencies remain due to the fact that rodlet cells are absent under pathological conditions (Fishelson and Becker, 1999) or, by contrast, present in apparently sound ﬁshes (Mattey et al., 1979; Smith et al., 1995). Some other open questions pertain for several morphologic features, which are difﬁcult to ﬁt into the general scheme of a blood cell with defensive function, e.g., the thick capsule that has been interpreted as protective structure (Iger and Abraham, 1997) or as contractile mechanism for the discharge of the rodlets (Leino, 1974, 1979; Barber et al., 1979), the latter opinion frequently taken over by other authors. Besides different opinions on the occurrence of cell junctions and the mode of discharge and possible solubility of the rodlets, additional open questions remain with respect to the secretory cycle. Most authors assume that the development of rodlets follows the classical secretory pathway, i.e., increase of RER and Golgi apparatus and the appearance of secretory vacuoles at the trans face of the Golgi stacks, which develop into the rodlets (Leino, 1974, 1979; Desser and Lester, 1975; Flood et al., 1975; Barber et al., 1979). A possible direct involvement of the RER in rodlet formation was only mentioned by Desser and Lester (1975), Flood et al. (1975), and, more recently, Kramer and Potter (2002). The last described the rodlets arising within the dilated cisternae of the endoplasmic reticulum (ER) without discussing this conﬂicting evidence. For this reason, further ultrastructural examination with the aim of clearing up some of these discrepancies was performed on rodlet cells in gill Fig. 1. Rodlet cell stages. Light microscopic survey of intestinal epithelium containing rodlet cells with typical inclusions and thick capsule. Part of the cells show basal vacuoles at the outside of the capsule. Narrow dense rodlet cells represent degenerating compressed forms (arrowheads), while migrating leucocytes (mostly lymphocytes) are seen at the base of the epithelium and in a damaged area (X). Semithin section (0.5 m). Toluidine blue staining. Fig. 2. Rodlet cell stages. Transmission electron microscopical (TEM) survey of intestinal epithelium with different rodlet cell stages: (A) immature cell without capsule, identiﬁed by its dilated endoplasmic reticulum; (b) immature cell with capsule; (C) rodlets apparently ejected into normal epithelial cells; and (D) dense rodlet cells probably degenerating. The cell to the left displays only a large condensing vacuole and an external vacuolar projection at its side, while the cell to the upper right appears compressed. Fig. 3. Rodlet cell stages. Immature rodlet cell from the base of the intestinal epithelium displaying smooth dilatations of rough endoplasmic reticulum, which extend through the developing capsular zone (boxed and intestinal epithelium of trout (Salmo trutta L. and Oncorhynchus kisutch). The study focused on the development of the endoplasmic reticulum and showed evidence for signs of hypertrophy at all stages supporting the concept of a transformed cell rather than a normal secretory cycle. MATERIALS AND METHODS In the course of diverse ultrastructural studies, adult specimen of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta L.) were obtained either from commercial suppliers or caught wild in different regions of lower Austria. The ﬁshes showed no pathological signs but minor infections (e.g., protozoan parasites could not entirely be ruled out). Three specimen of wild Salmo trutta L. (measuring 26, 29, and 50 cm) contained numerous rodlet cells in gill and intestine. In eight adult specimen of Oncorhynchus mykiss (30 –35 cm; three of them wild catches) and two immature cultured ones (8 and 11 cm), only one wild ﬁsh was rich in rodlet cells, while the probes of the others contained only few or none. Small blocs (1–2 mm3) from different organs including intestine and gills were dissected out and processed after standard electron microscopic methods, i.e., ﬁxation in 2% glutaraldehyde buffered in phosphate buffer or cacodylate buffer (0.1 M, pH 7.2–7.4, at 4°C) for 1–2 hr, postﬁxation in 1% OsO4 in the same buffer for 1 hr, followed by dehydration in a graded ethanol series and embedding in epoxy resin (Epon 812 or Glycidether). Semi- and ultrathin sections (0.5 m and ⬃ 70 nm, respectively) were cut on a Reichert ultramicrotome (OM U3, Ultracut S), stained with Toluidine blue or uranyl acetate/lead citrate, respectively, and examined on a Jeol 1200 EXII electron microscope (for LM survey: Leitz DMRB). Samples of gill and intestine from ﬁshes especially rich in rodlet cells (e.g., in focal accumulations) were selected for the ultrastructural analysis. No essential differences between the rodlet cells of rainbow or brown trout were observed. RESULTS In the comparatively high intestinal epithelium, the different developmental stages could be easily observed (Figs. 1 and 2). Immature ovoid stages without or with only an indistinct capsule were identiﬁed by their dilated cisternae of rough endoplasmic reticulum (RER). Mature area). The apically located rodlet sacs (s) show varying condensation and the central dense core. Note prominent nucleolus in the uncondensed nucleus and desmosomes (arrowhead). bm, basal membrane; f, capsular zone; L, migratory lymphocyte; s, rodlet sac. a: Detail. The cisternae of the rough endoplasmic reticulum are irregularly distended and project into neighboring cisternae, mitochondria (arrowhead), and through the developing basal capsule (f), thereby losing their ribosomes. At the right border contact with a lymphocyte (L) and a desmosome-like junction with an adjacent cell process. Fig. 4. Rodlet cell stages. Apex (to the left) of an immature rodlet cell slightly more developed than that in Figure 3, exhibiting numerous cores, dilated ER partly invaginating the aggregated mitochondria (arrowheads), and three desmosomal junctions with neighboring cells. The ﬁrst traces of the capsule appear as marginal zone free of organelles (f). a: Similar stage with RER dilatations shedding their ribosomes (asterisk) and invaginating into mitochondria (m; arrowhead). f, capsular zone; s, rodlet sac. ENDOPLASMIC RETICULUM IN RODLET CELL Figures 1– 4. 241 242 BIELEK elongated cells with the conspicuous capsule were packed with rodlets and situated near or in contact with the epithelial surface (Fig. 1). Smaller or narrower dense cells represented probably degenerating stages and often had a compressed appearance (compare Figs. 1, 2, and 19). Vesicular protrusions at the cell base were large enough to be discernible by light microscopy (Fig. 1). Ejected intact rodlets (Figs. 14, 16, gill, and 17) or whole rodlet cells (not shown) were found near the surface, although occasionally rodlet sacs protruded from mature cells situated in the middle zone of the epithelium into the neighboring intestinal cells (Fig. 2). In the gill, similar stages occurred but were more scattered. Their axis was usually oriented not perpendicularly but obliquely due to the lower height of the squamous epithelium. Immature Stage The most immature rodlet cells were located near the basal membrane, showed a basal euchromatic nucleus with great nucleolus, and were identiﬁed by an increase and dilatation of RER. Simultaneously, the ﬁrst traces of the ﬁbrous capsule or at least a marginal cytoplasmic zone free of organelles were discernible (Fig. 3). Centrioles, Golgi complex, and associated vacuoles with ﬂocculent content sometimes already displaying the typical dense core were found in a supranuclear position. Already, at this early stage, the RER cisternae often showed vesicular dilatations losing their ribosomes and penetrating the forming capsule, usually at the base of the cell but occasionally also at the lateral sides, thereby causing vesicular protrusions (Fig. 3). The enlarging and apparent fusion of these vesicles led to the characteristic basal or lateral processes found in more mature stages (Fig. 8 and Figs. 18 and 19, respectively) mentioned above and visible by LM (Fig. 1). Smaller vesicular dilations invaginated the mitochondria, especially in the more apical cell area (Fig. 4), thus deforming them into crescent shapes. Rarely even the nuclear envelope was seen bulging into mitochondria. Desmosomal junctions with neighboring cells were found in varying numbers (Figs. 3 and 4). In the intestinal epithelium, intraepithelial migrating leucocytes (mostly lymphocytes) were often seen in the vicinity or in contact with rodlet cells (Fig. 3; see also Fig. 1). Advanced Immature Stage With progressive maturation, dilatations of the RER cisternae and the Golgi stacks grew more prominent. With respect to the development of the rodlets, small evaginations of the RER without ribosomes were observed, apparently giving rise to small vesicles showing increasing size and density and even forming rows. Their content was comparable to the structure of the adjacent rodlets and cores (Fig. 5). Dilated cisternae of RER were apposed to the membranes of the rodlet sacs, shedding their ribosomes (Figs. 5–7) and often indenting the sacs (Fig. 6). Openings between the sacs occurred, as well as indications of occasional fusion between the membranes of the sacs and indenting vesicular elements possibly derived from the RER (Fig. 6). In rare cases, the regular scalloping of the membranes of the developing rodlet sacs seemed also to give rise to tubular elements found in the sacs (Fig. 7). The rodlets showed varying condensation of their ﬂocculent contents, with one or rarely several (Fig. 6) dense cores in the center. Apically, the membrane-bounded cores extended between the dislocated mitochondria that aggregated in the cell apex together with the mentioned vesicular ER elements indenting them. The basal endings of the cores were found in the Golgi area (Fig. 11) but were also stretched either to the nuclear membrane (Fig. 8) or to marginal RER cisternae under the capsule (Fig. 8). With the increasing development of the rodlet sacs, the Golgi apparatus became often dislocated from its supranuclear position (Fig. 9) to the cell base, while the nucleus assumed a bipartite or irregular shape (Figs. 8 and 10). At this stage, accumulations of free ribosomes and glycogen, as well as vacuoles similar to dense primary or heterogeneous secondary lysosomes, myelinand multivesicular bodies, could be detected in this region. In the trans and lateral zone of the Golgi apparatus, the expected variety of vesicles and vacuoles occurred (Figs. 8 and 11), including vacuoles with ﬂocculent contents representing developing rodlet sacs (Fig. 9). In some cases, only a single large condensing vacuole was found. These vacuoles fused with small vesicles derived from the trans Golgi face but were also in close association with vesicles and RER proﬁles from the intermediate compartment, i.e., the cis region of the Golgi stack. The RER elements were often dilated; the RER cisternae formed large evaginations around the whole area (Figs. 9 –11). The condensing vacuole sometimes contained tubular elements associated again with the membrane (Fig. 11) or the core. The tubules displayed a diameter about 30 nm, some of them with a distinct dense membrane, and represented possibly different elements. At times, similar straight tubules were also seen in extremely hypertrophying RER (Fig. 17). Mature Stage The fully developed and usually elongated cells near or in contact with the epithelial surface were packed with rodlets converging toward the apex, with microtubules running between them to the centrioles sometimes visible in the neck region of the cell. The terminal stage of the dilatation of the ER resulted in a vesicular appearance of the cytoplasm. In some cases, especially in the gill epithelium, extended ER lacunae surrounded the basally or apically dislodged organelles (Figs. 17). The nucleus of these rodlet cells showed occasionally dense accumulations (not shown). With respect to the extrusion of rodlets, the rodlet cells reaching the epithelial surface showed the known thinning and later loss of the capsule in the apical area. Junctional complexes with the adjacent epithelial cells were found often but not always at the lateral sides of the neck region and revealed varying differentiation, ranging from well-developed tight or desmosomal junctions to reduced desmosome-like connections (Figs. 12–15). In the high intestinal epithelium with its typical microvillar border, the apex of the rodlet cells displayed varying cytoplasmic formations. A smooth contour was found combined with intact organelles, i.e., mitochondria (Fig. 15), while cytoplasm ﬁlled with rodlets and apparently degenerating organelles displayed either a microvillar tuft (Fig. 12), a swollen cytoplasmic protrusion (a bleb: Fig. 13), or a disrupted zone with the organelles spilled into the intestinal lumen (Fig. 14). A few dense projections (Fig. 19) or a single thicker microvillar process (not shown) were visible in dense, probably degenerating or apoptotic cells. The discharged rodlet cores were found not only in the intestinal lumen but also near or between the microvilli, some- 243 ENDOPLASMIC RETICULUM IN RODLET CELL Fig. 5. Development of rodlet sacs. Immature rodlet cell in gill epithelium showing slightly condensed nucleus with large nucleolus, dilated ER cisternae, several rodlet sacs, and a well-developed capsule. bm, basal membrane. a: Detail showing series of small vesicles with ﬂocculent contents similar to the adjoining rodlet sac (s) with a projection from the dilated RER, which has shed its ribosomes between them (arrowhead). Fig. 6. Development of rodlet sacs. Developing rodlet sacs with apposed membranes of dilated RER cisternae, which focally indent the sacs (arrows). Rarely, openings between the sacs (arrowheads) or a membrane-bounded cluster of several cores are seen. f, ﬁbrillar capsule. Fig. 7. Development of rodlet sacs. Dilated ER (ER) apposed to rodlet sacs containing tubular elements (arrowheads) probably derived from the strongly undulating membranes. times showing a strongly undulating membrane apparently giving rise to tubular elements (Fig. 16). Occasionally, rodlets were seen discharged into neighboring cells (compare Fig. 2) or penetrating through the lateral side of the capsule (Fig. 8). In the comparatively low gill epithelium with only irregular microridges, the apical openings of the rodlet cells showed similar disruption but sparse microvillar projections (Fig. 17). A regular scalloping of the outer membrane of the rodlet cells indicating possible contraction was lacking. cases, the cells appeared narrow and compressed (Fig. 19; compare also Figs. 1 and 2). Degenerative Stages In scattered cells belonging to different developmental stages, an increase in cytoplasmic density indicated degenerative changes (Figs. 18 and 19). The secretory organelles (ER cisternae and Golgi stack) were reduced (Fig. 18), although a large condensing vacuole was occasionally seen (Fig. 2). The number of rodlet sacs differed, and their contents appeared often clear and swollen, revealing again the mentioned tubular elements associated with ﬂocculent aggregations or the core (Fig. 18a). In other DISCUSSION As mentioned above, the controversy concerning the nature and function of the rodlet cell has been amply discussed (Manera and Dezfuli, 2004). The thick ﬁbrous capsule and the peculiar club-like inclusions with their dense core are reminiscent of a parasitic stage, whereas the electron microscopic investigation of the organelle development is compatible with secretory activity of an epithelial or a migrating blood cell. The most immature rodlet cells can be identiﬁed by dilated proﬁles of RER typical of the onset of protein synthesis, with the ﬁrst traces of the ﬁbrillar capsule appearing simultaneously or shortly thereafter (Leino, 1974; Desser and Lester, 1975; Flood et al., 1975; Barber et al., 1979). In trout, the present data show already at this early stage that part of the cells display vesicular protrusions of the RER shedding their ribosomes, a process usually indicating degeneration. Some of these dilatations extending through the forming capsule correspond to vesicular cytoplasmic exten- 244 BIELEK Fig. 8. Mature rodlet cell not yet reaching the surface of the intestinal microvillar border. The cell apex is ﬁlled with the mitochondrial labyrinth and the tips of the rodlets, with one core piercing the capsule laterally (arrowhead) and another ending near it (arrow). The basal part of the cell contains the irregular nucleus, a large Golgi area, and vacuoles extending through the capsule. One core is seen contacting the nucleus (boxed area). a: Detail with basally dislocated Golgi area displaying various vesicles at its trans side and dilated intermediate elements at the cis side. The ER cisternae (ER) and the nuclear envelope are partly dilated, and the core of a rodlet sac (s) touches the nuclear envelope. G, glycogen. sions presumed to have nutritive function (Kimura, 1973; Barber et al., 1979), a proposal often taken up but appearing now rather unlikely in view of its origin. Similarly, dilated protrusions of ER and rarely of the nuclear membrane bulge also into mitochondria, thereby deforming them into crescent-shaped forms. As these evaginations shed their ribosomes, an artifact due to ﬁxation and/or contraction of ﬁbrils seems improbable. The intermingling vesicular elements and degenerating mitochondria get apically displaced by the developing rodlet sacs, thereby forming a complex known in literature as whorled structures of the membranous labyrinth. The continuous formation of RER dilatations shedding their ribosomes proves the early assumption (Leino, 1974, 1979; Barber et al., 1979; Kramer and Potter, 2002) that the typical vesiculation in mature rodlet cells originates from the disappearing RER. As a consequence, accumulations of free ribosomes are found together with glycogen in the basal region of mature rodlet cells. The ﬁnal stage of the continued ER hypertrophy is represented by cells displaying enormously distended ER proﬁles surrounding the decreasing secretory organelles, which are dislocated and packaged together against nucleus or apical capsule. This corresponds to uncommented micrographs in studies of ﬁshes in polluted water (Iger et al., 1994; Iger and Abraham, 1997; Pawert et al., 1998). Whether these extreme ER dilatations are typical for Salmonids or are a common reaction to adverse inﬂuences has to be further investigated. However, the varying extent or termination of this process of vesiculation at different stages would explain the described variations of maturing and degenerating rodlet cells in literature, as ENDOPLASMIC RETICULUM IN RODLET CELL 245 Fig. 9. Association of RER and Golgi apparatus (from gill region). Rodlet cell with immature uncondensed nucleus, large nucleolus, and indistinct capsule (f). The Golgi apparatus is well developed with a large condensing vacuole (cv) at its trans face and a row of vesicles and rodlets in varying stages of condensation laterally localized. The RER cisternae are dilated and conﬂuent to wide lacunae (ER) at the cell apex and show indentations toward the developing rodlets (arrows). Fig. 10. Association of RER and Golgi apparatus (from gill region). More mature rodlet cell than in Figure 9, displaying an irregular and slightly condensed nucleus. The developing rodlets (s) have dislocated the mitochondria to the apex and the Golgi stack with a condensing vacuole (cv) to the base. Dilated RER lacunae (ER) surround the latter. Fig. 11. Association of RER and Golgi apparatus (from gill region). Detail from a Golgi stack demonstrating grossly dilated RER lacunae (ER) bordering the Golgi area. The cis face displays dilatations (asterisk), the trans face a multitude of vesicles, while a large rodlet sac (s) contains tubular elements (diameter ⬃ 30 nm; arrows) running in various directions and possibly stemming from the membrane (arrowheads). The core appears to originate from the lateral side of the Golgi apparatus. well as possible species-speciﬁc differences. As an example, the rodlet cells of trout are characterized by an irregular nucleus and large vesicular distensions, while in Cyprinids and some other species a round nucleus and smaller cytoplasmic vesicles are typical [compare, e.g., Bielek and Viehberger (1983) and Dezfuli et al. (2004)]. As a possible consequence, the apically dislocated mitochondria in Cyprinidae often appear not deformed but dense and elongated between clear vesicles [“dense tubular bodies,” after Iger and Abraham (1997)]. With respect to the differentiation of the rodlets, many authors assume them to originate in the classical way from Golgi vesicles at the trans face. Corresponding morphological and cytochemical evidence indicating glycoprotein content in inner Golgi lamellae, associated vesicles, and developing rodlet sacs has been reported using routine phosphotungstic acid (PTA) or silver staining (Leino, 1979, 1982; Mattey et al., 1979; Bielek and Viehberger; 1983). The origin of the proteinaceous core seems less clear. Although its condensation in the center of the sac has been assumed, small vesicles of similar density can be found scattered in the cytoplasm distant from either developing sacs, condensing vesicles, or Golgi stack. Their exact ori- 246 BIELEK Figures 12–19. ENDOPLASMIC RETICULUM IN RODLET CELL gin and nature have to be decided cytochemically, but in the present study, small dense vesicles with contents similar to condensing core material were seen budding from dilated parts of RER cisternae losing their ribosomes, indicating the addition of a separate, probably proteinaceous component by direct participation of ER-derived elements. The close association of rodlet sacs and RER cisternae is very conspicuous also in more mature stages, and a direct participation of ER elements was repeatedly considered in literature. The fusion of the irregular marginal zones was proposed by Barber et al. (1979), and Desser and Lester (1975) assumed the complete investment of the rodlet sacs only at a later stage. Kramer and Potter (2002) described the development of the rodlets in the ER without referring to the role of the Golgi apparatus or the relevance of these unusual results. The direct sequestration of proteins from the RER is known from peroxisomes, a possibility discussed by Iger and Abraham (1997) based on a positive peroxidase reaction in the sacs. It occurs also in certain examples of intense protein production or various pathological conditions (Ghadially, 1997; Kopito and Sitia, 2000), but without speciﬁc markers the involvement of RER-derived elements must remain hypothetical. However, as an additional possibility, the participation of vesicles of the ER Golgi intermediate compartment (ERGIC) is implied by the very close association of ER cisternae, lateral side of the Golgi stack, and condensing vacuole, an observation leading Leino (1979) in an early study to consider the extra-Golgi pathway as an alternative to the classical secretion mode. An interesting parallel could be the hijacking of vesicles from the retrograde pathway from the Golgi trans face to the RER found in certain viral infections (Mackenzie et al., 1999). Another aspect of this problem is the observation of cores ending not only in the Golgi region but also near the marginal RER cisternae under the capsule and frequently Fig. 12. Release of rodlets and degenerating forms. Apical part of a rodlet cell showing microvillar border similar to the adjacent intestinal cells, a cell junctional complex in the neck region at the end of the capsule, and an accumulation of vesicles and deformed mitochondria. Fig. 13. Release of rodlets and degenerating forms. Similar region as in Figure 12, again with cell junctions. The apical cytoplasm is bulging into the lumen, revealing distended vesicles, cores, and the mitochondrial labyrinth. Fig. 14. Release of rodlets and degenerating forms. Apical region with apparently disrupted border, ejecting vesicles, and rodlets. Fig. 15. Release of rodlets and degenerating forms. Apical region with cell junctions, dense mitochondria, and smooth border. Fig. 16. Release of rodlets and degenerating forms. Released tip of rodlet caught in microvillar border with its limiting membrane displaying undulations forming tubular elements. Fig. 17. Release of rodlets and degenerating forms. Rodlet cell in gill epithelium showing apical disruption and expelled intact rodlet near the gill surface. In the cell, rodlets and organelles are again dislocated by RER lacunae (ER) containing tubules (arrowheads). Fig. 18. Release of rodlets and degenerating forms. Rodlet cell in the intestinal epithelium showing dense cytoplasm, swollen rodlets, and lateral vesicular protrusions. a and b: Details (boxed areas) showing sac and core associated with tubules (arrowheads) and a possible RER dilatation containing a core (arrowhead). Fig. 19. Release of rodlets and degenerating forms. Rodlet cell reaching the microvillar border but appearing compressed and displaying dense capsule, vacuolar cytoplasm, and vesicular extensions at the outer side of the capsule (arrows). 247 in contact with the nuclear envelope. Only Barber et al. (1979) mentioned “the stretching of the cores to the proximal side of the nucleus.” This observation as well as core projections from the base of mature sacs (Dezfuli et al., 2003b) could be explained by continuous growth of the core, but the ﬁnding of small cores identiﬁed by their crystalloid content in the immediate vicinity of the nucleus (Bielek, 2002) indicates again the derivation from ER (or nuclear envelope). The assumption of ER hypertrophy indicating storage of overproduction ﬁts with several other peculiar features, e.g., the crystalloid or at least striated structure of the cores reported in several studies (Leino, 1974; Desser and Lester, 1975; Della Salda et al., 1998; Fishelson and Becker, 1999; Bielek, 2002). Other features are the occasional strong undulation of apposed ER and limiting membranes of rodlet sacs as well as the occurrence of tubular elements partly derived from them (Bielek, 2002) but found also in dilated ER and condensing vacuoles. Straight or curved tubuli in the rodlet sacs were described only by Leino (1974, 1979), who interpreted them as a species-speciﬁc differentiation. As comparable tubules as well as strongly undulating ER membranes are associated with overproduction of a single or a few membrane proteins, forming sometimes a crystalloid or microtubular reticular complex (TRS) (Ghadially, 1997), they might also be taken as support for a pathological transformation or some physiological disturbance. These typical aggregates have been found in a variety of cells, either signaling storage of membranes due to slowing metabolism, genetic disorders, viral infections, or, more recently, the elevation, e.g., of interferon (Ghadially, 1997), an interesting link to the proposed defensive effect of rodlet cells in infections. With respect to the discharge of the rodlets, the observed increase of ER supports a release by pressure (Bannister, 1966; Richards et al., 1994; Smith et al., 1995), an opinion contrary to the widespread theory of an expulsion by active and triggered contraction of the capsule in response, e.g., to a parasitic infection (Leino, 1974, 1979; Desser and Lester, 1975; Barber et al., 1979; Mattey et al., 1979; Kramer and Potter, 2002). The apical cytoplasmic corona-like structure observed in numerous studies was assumed to have a function during expulsion (Grünberg and Hager, 1978) or in the movement of the cells (Smith et al., 1995; Fishelson and Becker, 1999). Manera et al. (2001) described bleb-like and corona-like processes, the former increasing strongly during toxic conditions. A single thick microvillus was interpreted as anchoring structure between endothelial cells (Smith et al., 1995) or a chemoreceptor (Leino, 1996). The present observations show a sequence from more (intestinal epithelium) or less (gill) developed microvillar projections to varying stages of cytoplasmic blebbing, leading apparently to the rupture of the cell and the spilling of rodlets and organelles, which can be observed occasionally also in the middle of the comparatively high intestinal epithelium or at the lateral side as shown here and previously (Dezfuli et al., 2000; Bielek, 2002; Kramer and Potter, 2002). So while all the described features are difﬁcult to ﬁt into the pattern of a normal secretory cell, either epithelial or blood-borne, a possible malfunction of ER and/or transport mechanisms is compatible with the observed dilatations of the ER cisternae and the intermediate compartment at the cis side of the Golgi apparatus. The existence of immature rodlet cells at the base of the diverse epithelia, the 248 BIELEK occurrence of cell junctions, and a microvillar tuft after reaching the epithelial surface speak for an endogenous, probably transformed epithelial cell, but leaves open the cause for the proposed transformation and the function of capsule and presumed migration. The reason for the long controversy concerning the rodlet cell is the problem that each of the hypotheses (parasite, secretory cell, blood cell) is only covered by part of the observations and contradicted by others. The thesis of a defensive function has been supported by recent statistical studies showing an increase of rodlet cells under the inﬂuence of quite different stressors, ranging from parasites, toxins, neoplasm, viral infections, general tissue damage, to adverse environmental inﬂuences (Manera and Dezfuli, 2004) and even different depth of water (Winckler and Portela-Gomez, 1962). Therefore, most of these studies interpret the rodlet cell as regular element of the defense system, appearing in direct association with stressors (Iger and Abraham, 1997) and comparable to inﬂammatory cells (Dezfuli et al., 1998, 2000, 2003a, 2003b, 2004; Manera et al., 2001; Manera and Dezfuli, 2004). Nevertheless, there seem to exist convincing exceptions to this rule (Fishelson and Becker, 1999). As the rodlet cells are deﬁnitely found scattered in completely differing tissue compartments, including the blood stream, this distribution is usually only compatible with a leucocyte with defensive function. On the other side, while the question of junctions is just debatable for parasites, junctional complexes are without precedent in blood cells. Moreover, protection against aggressive granule components, a function for the capsule proposed by Iger and Abraham (1997), is managed by enzymatic masking (Rowley et al., 1988; Zucker-Franklin et al., 1988; Imagawa et al., 1998). The early suggestion of a modiﬁed epithelial secretory cell comparable, e.g., to goblet cells (Al-Hussaini, 1949), which also occur and increase at sites of epithelial infection, is not consistent with capsule or migration into connective tissue or blood. Searching again for a parallel under pathological conditions, an example might be found in neoplasm or virally infected cells, which are known to round up, change adhesion, reduce number and types of junctions, and migrate depending on the surrounding tissue (Gadhially, 1997). Some inconsistencies in literature with respect to number and type or complete absence of cell junctions could be due to the resulting variations. Considering the numerous open questions, the proposition of some submicroscopic infection (Fishelson and Becker, 1999) causing possibly an opportunistic reaction under stress conditions seems worth following up. 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