Cell Tissue Res (t987) 247:359 366

Reissner's fiber, massa eaudalis and ampulla caudalis in the spinal cord of lamprey larvae (Geotria australis) Light-microscopic immunocytochemical and lectin-histochemical studies*

and T't ue R e s e a r c h �9 Springer-Verlag 1987

Sara Rodriguez 1, Pabio A. Rodriguez 1, Carlos Banse 1, Est6ban M. Rodriguez 1, and Andreas Oksche 2 Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile;

2 Department of Anatomy and Cytobiology, Justus Liebig University of Giessen, Giessen, Federal Republic of Germany

Summary. The subcommissural organ (SCO), Reissner's fiber (RF) and its massa caudalis of lamprey larvae (Geotria australis) were investigated immunocytochemically by use of an antiserum raised against bovine RF as primary anti- body. The affinities of RF and massa caudalis for Ricinus communis agglutinin I (RCA) with and without previous acid hydrolysis, concanavalin A (Con A), wheat-germ ag- glutinin (WGA), aldehyde fuchsin, and PAS reaction were also studied.

SCO and massa caudalis were strongly immunoreactive, whereas RF proper was distinctly negative. RF did not react with Con A and RCA. Only the periphery of RF was WGA-positive. RCA showed affinity for RF only after acid hydrolysis. RF was homogeneously stained by the al- dehyde-fuchsin and PAS-methods. At variance with RF proper, the periphery of the massa caudalis reacted with RCA without previous acid hydrolysis, but its core was WGA-positive and reacted with RCA only after hydrolysis. It is suggested that (i) RF has a coat of glycoproteins con- taining sialic acid as terminal residue, whereas the massa caudalis possesses a coat with galactose as terminal residue; (ii) in RF proper and the massa caudalis the spatial arrange- ment of glycoproteins might be different.

Routine transmission electron-microscopic observations indicate that in larvae of Geotria australis an open commu- nication exists between the ampulla caudalis and blood ca- pillaries via large cavities or lacunae.

Key words: Reissner's fiber Massa caudalis- Subcommis- sural organ Spinal cord Central canal - Immunocyto- chemistry Lectin histochemistry- Lamprey, Geotria aus- tralis

In mammals the central canal of the spinal cord extends along a typical terminal filum, whereas in lower vertebrates beyond the caudalmost border of the spinal cord proper

Send offprint request to. Prof. Estbban M. Rodriguez, Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile

* Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Direcci6n de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Cientifico y Tecnol6- gico, Chile

Acknowledgements. The authors wish to acknowledge the valuable help of Mrs. Elizabeth Santibfifiez and Mr. Genaro Alvial

the central canal is encompassed merely by an ependymal tube directly surrounded by meninx primitiva (Studni~ka 1895; Olsson 1955; Wislocki et al. 1956). At its distal end the central canal displays a dilatation know as terminal ventricle or ampulla caudalis (cf. Olsson 1955).

Reissner's fiber (RF) is a thread-like structure secreted by the subcommissural organ (SCO); it extends along the Sylvian aqueduct, fourth ventricle and central canal of the spinal cord. When reaching the terminal ventricle, RF ends as an irregular mass, the so-called massa caudalis (Olsson 1955, 1958; Hofer 1963). RF and massa caudalis can be stained as a complex by a number of dyes and histochemical methods, including the Gomori and PAS procedures (Ols- son 1955, 1958; Wislocki etal. 1956; F~ihrrnann 1963; Hofer 1963; Naumann 1968).

During recent years immunocytochemistry by use of antisera raised against RF-material (Sterba etal. 1982; Rodriguez et al. 1984a) and lectin histochemistry (Meiniel and Meiniel 1985; Rodriguez et al. 1986) have been used to study the secretory material of the SCO. The combined application of both methodological approaches supported new evidence for the processing of the secretory material at its site of synthesis (Rodriguez et al. 1986). This stimu- lated us to use the same methods for studying RF and its massa caudalis with the aim to clarify whether the RF- material undergoes some kind of modification when shifted along the central canal and especially when forming the massa caudalis.

Ultrastructural studies of the ampulla and massa cauda- lis are scarce. Two of these studies have been performed in the lamprey, one with adult specimens (Sterba and Nau- mann 1966), the other with larvae (ammocoetes) of Lampe- tra planeri (Hofer et al. 1984). The detailed and provoca- tive ultrastructural analysis of the ampulla caudalis, massa caudalis and neighboring structures of lamprey lar- vae provided by Hofer et al. (1984) initiated us to conduct the present investigation with larvae of the Southern-Hemi- sphere lamprey, Geotria australis, and to extend our meth- ods to immunocytochemical and lectin-histochemical pro- cedures. The conventional transmission electron-micro- scopic observations included in the present report are re- stricted to basic ultrastructural features; an ultrastructural immunocytochemical analysis will be presented in a subse- quent communication (Peruzzo et al. 1986).

Materials and methods

Thirty lamprey larvae, Geotria austratis (De Buen 1961), ranging in length from 80 to 150 mm were used. They were

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collected in November and March from the Huichahue River near Valdivia, Chile.

Light microscopy

Fixation and embedding. The living animals were immersed in Bouin's fixative for 30 min. Subsequently, from each ani- mal three different types of blocks were prepared under a dissecting microscope. One, about 2 cm long, included the head and a portion of the spinal cord. A parasagittal cut through the head served to expose the brain tissue to the fixative. The second group of blocks included several pieces of the trunk, ca. I cm long, containing various seg- ments of the spinal cord. Lastly, a block approximately 1 cm long contained the tip of the tail where the ampulla and massa caudalis are located. All blocks were immersed in fresh fixative for 48 h. All samples from the head and the trunk, and also some from the tail, were dehydrated in increasing concentrations of ethanol and embedded in Paraplast. Some blocks from the tail were dehydrated in increasing concentrations of ethanol and pure acetone and embedded in butyl-methyl methacrylate according to a pro- cedure previously described (Rodriguez et al. 1984b).

Sectioning. Serial sagittal sections, 6 pm thick, were ob- tained from all samples embedded in Paraplast. To localize the SCO and the central canal of the spinal cord, every tenth section of a series was mounted and stained with hematoxylin-eosin. All sections containing the SCO (or the central canal) were mounted separately, so that adjacent sections could be stained with different methods. The tail blocks embedded in methacrylate were cut in the sagittal, horizontal or frontal planes. To localize the ampulla cauda- lis, sections about 2 ~tm thick were obtained by use of an ultramicrotome and stained with toluidine blue. When the area of the ampulla caudalis was reached, serial 1-1am thick sections were obtained and mounted separately.

Staining procedures

Immunocytochemistry. The immunoperoxidase method of Sternberger et al. (1970) was applied using an antiserum raised against an extract of bovine Reissner's fiber as prima- ry antiserum. Since the medium for the extraction of RF contained urea, this antiserum was labeled AFRU (A antise- rum, FR fiber of Reissner, U urea). For details concerning production and characterization of antibodies, see Rodri- guez et al. (1984a). AFRU was used at a dilution of I : |000. Incubation time was 18 h. An anti-rabbit IgG raised in sheep in our laboratory was used as second antibody at a dilution of 1:30. PAP (Bioproducts, Brussels, Belgium) was used at a dilution of 1:75. All incubations took place at 22 ~ C. All antibodies were diluted in Tris-buffer, pH 7.8, containing 0.7% lambda carrageenan (Sigma) and 0.5% Triton X-100 (Sofroniew et al. 1979). In most cases the im- munoreaction performed in 1-1am thick methacrylate sec- tions was enhanced with silver methenamine (Rodriguez et al. 1984c).

Lectins. The following lectins (Sigma), labeled with peroxi- dase were used: concanavalin A (Con A), wheat-germ ag- glutinin (WGA) and Ricinus communis agglutinin I (RCA). All lectins were dissolved in Tris-buffer, pH 7.8, at a con- centration of 1 mg/ml. This stock solution was aliquoted

in 25 ~tl samples, which were kept frozen. Each time a fresh working solution was prepared using an aliquot of the stock solution. The final dilution for individual lectins was 25 gg/ ml for Con A, 5 lag/ml for WGA, and 20 rtg/ml for RCA. After removal of Paraplast or methacrylate by use of xy- lene, the sections were rehydrated and washed for 15 min with Tris-buffer, pH 7.8. Then the sections were covered with the labeled lectin and placed in a moist chamber at 22 ~ C for 45 rain. After incubation the sections were washed three times (5 min each) with Tris-buffer, pH 7.8. Then the sections were processed for the demonstration of peroxidase using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) and HzO 2 (Merck), as employed in the immunoper- oxidase procedure. In most cases the peroxidase reaction was enhanced with silver methenamine following the same procedure as used for the enhancement of the immunoper- oxidase staining (Rodriguez et al. 1984c). Silver methen- amine used as enhancer of the peroxidase reaction (5 min incubation without previous acid oxidation) per se did not stain any of the structures investigated.

Acid hydrolysis. After hydration some sections were incu- bated in a 0.1 N HCL solution, at 80 ~ C, for I h. They were then washed in Tris-buffer for 15 min and subse- quently stained with RCA. Adjacent sections were stained with RCA without previous acid hydrolysis.

Other staining methods. Sections containing RF or massa caudalis were also stained with aldehyde fuchsin (Gabe 1953) or with the periodic acid-Schiff (PAS) method accord- ing to McManus (Pearse 1980).

Electron microscopy. The living animals were immersed in a threefold aldehyde mixture containing 4% paraformalde- hyde, 2% acrolein and 2.5% glutaraldehyde, buffered to pH 7.4 with 0.2 M monosodic-disodic phosphate (Rodrl- guez 1969). After 20 min the tip of the tail (about 3 mm) was cut off and immersed in fresh fixative to complete a period of 2 h of aldehyde treatment. The blocks were washed with 0.1 M phosphate buffer, pH 7.4, and then fixed for 2 h in 1% OsO4, buffered to pH 7.4 with phos- phate. After a few washes in distilled water the blocks were dehydrated in increasing concentrations of ethanol and pure acetone, and embedded in a mixture of Epon and Araldite. The blocks were oriented to obtain cross and sa- gittal sections of the ampulla caudalis. Ultrathin sections were stained with uranyl acetate and lead citrate. One-~tm thick sections, adjacent to the ultrathin sections, were stained with toluidine blue-borax.

Results

Light microscopy

Subcommissural organ (SCO). The ependymal cells of the SCO were strongly immunoreactive. In the thinnest region of the organ these cells displayed immunoreactive basal pro- cesses that, after traversing the posterior commissure, ended at the 'external limiting membrane' of the brain (Fig. 1). The thickest portion of the posterior commissure contained a few scattered immunoreactive hypendymal cells (Fig. 1).

Reissner'sfiber. In Paraplast sections, RF - regardless of the segment of the spinal cord either lacked immunoreac-

Fig. 1. Sagittal section of SCO immunostained with AFRU. Para- plast-embedded material. Small arrows hypendymal cells; large ar- row ependymal processes projecting to the leptomeninges, x 180

Fig. 2-4. Sagittal sections through Paraplast-embedded spinal cord immunostained with AFRU (Fig. 2) and stained with aldehyde fuchsin (Fig. 3) or PAS (Fig. 4). RF Reissner's fiber. Fig. 2 • 700; Fig. 3 x 900; Fig. 4 x 700

Figs. 5, 6. Sagittal sections through Paraplast-embedded spinal cord stained with WGA (Fig. 5) and RCA after acid hydrolysis (Fig. 6). Arrows lectin-binding material at the periphery of RF. Fig. 5 • 1150; Fig. 6 x 1400

tivity completely (Fig. 2) or displayed only a very weak reaction. It was, however, strongly aldehyde-fuchsin posi- tive (Fig. 3) and moderately PAS-positive (Fig. 4). The se- cretory material of RF did not exhibit Con-A binding sites. The fiber showed a strong WGA staining at its border but a very weak reaction in the remaining area (Fig. 5). When stained with aldehyde-fuchsin RF showed a diameter of

1 ~tm. However, when stained with WGA the fiber diame- ter was ~1.4 lain, and the non-reactive core measured

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0.85 gm. Regarding the thickness of RF (~ 1 lam) in rela- tion to that of the section (6 ~tm) RF is seen "in toto" in such microscopic preparations. Therefore, the WGA re- action pattern can be explained by the presence of a periph- eral ring of WGA-positive material. This may also account for the larger diameter of RF when stained with WGA in comparison to the calibers obtained with the use of alde- hyde fuchsin. Without previous acid hydrolysis RCA gave a negative reaction. After acid hydrolysis, however, the "pe- ripheral ring" of the fiber displayed affinity for RCA (Fig. 6).

M a s s a caudalis and ampul la caudalis. As established by early investigations (cf. Olsson 1955) the ventral and dorsal walls of the ampulla caudalis differ considerably in their struc- ture. Thus, whereas the ventral wall is formed by a rather compact layer of tall and ciliated ependymal cells, the dorsal wall appears as a loose and irregular layer of elongated cells, the nature of which could not be established with the methods used (Figs. 7-10). A transitional zone between the thick ventral wall and the thin dorsal wall could be clearly seen at the lateral aspect of the ampulla (Fig. 10). The massa caudalis was always found in close contact with the dorsal wall. From this site, RF-material penetrated via the dorsal wall into the adjacent structures (Figs. 7-10).

At variance with the lack of immunoreactivity of RF proper along the central canal, the massa caudalis and the closest distal portion of RF were strongly immunoreactive (Fig. 9). Adjacent l-gm thick sections showed that the massa caudalis and the secretory material that had escaped from the ampulla caudalis were both aldehyde-fuchsin-posi- tive (Fig. 7) and immunoreactive (Fig. 9). Although the massa caudalis was PAS-positive, we were not able to deter- mine to what extent the extraventricular immunoreactive material continues to be PAS-positive. We feel that this important point requires further investigation.

The massa caudalis was Con A-negative. RCA without previous hydrolysis showed a strong reaction in the periph- ery of the massa caudalis and a very weak reaction in the main mass (Figs. 11, 13). After acid hydrolysis, the periph- ery of the massa caudalis continued to be RCA-positive and the main mass became strongly reactive to RCA (Figs. 12, 14).

Although unevenly distributed, the periphery of the dis- tal portion of RF was RCA-positive (Fig. 11). This RCA affinity became stronger after acid hydrolysis (Fig. 12). RCA showed an affinity for nuclear material of all cells, a reactivity that disappeared after acid hydrolysis (Figs. 11, 12). Noteworthy after acid hydrolysis was a distinct RCA reactivity of the glycocalyx of the ependymal lining in the ventral wall of the central canal and ampulla caudalis (Fig. 12).

WGA stained the massa caudalis but in a rather non- uniform pattern; throughout the massa areas of high affini- ty alternated with others displaying a lower affinity (Fig. 15).

Electron microscopy. At this point only a brief account on some of the basic results obtained from conventional ultra- structural studies will be presented (for further details, see Peruzzo et al. 1986).

The ependymal cells lining the lateral and ventral walls of the ampulla caudalis were tall and attached by junctional complexes, which appeared to represent zonulae adhaer- entes (Figs. 16, 19). Intercellular spaces in the ependymal

362

Figs. 7, 8. One-gm thick sagittal (Fig. 7) and frontal (Fig. 8) sections through the ampulla caudalis (star). Methacrylate- embedded material. Aldehyde- fuchsin stain. Ventral (v) and dorsal (d) walls of terminal ventricle; mc massa caudalis; small arrows secretory material outside the ampulla caudalis; sc spinal cord; n notochord. Fig. 7 • Fig. 8 x850

Fig. 9. Adjacent section to that in Fig. 7, immunostained with AFRU. Open star ampulla caudalis ; f u l l star cilia of ventral wall; cc central canal; r f Reissner's fiber; small arrows immunoreactive material outside the ampulla caudalis x 720

Fig. 10. Frontal methacrylate section through the ampulla caudalis (star) stained with PAS. n Notochord; v ventral wall; small arrow PAS-positive material outside the ampulla; s skin. x 770

Figs. 11, 12. One-~tm thick methacrylate sections stained with RCA without (Fig. I 1) and after (Fig. 12) acid hydrolysis. Dorsal (d) and ventral (v) walt of central canal; r f Reissner's fiber; n notochord ; star ampulla caudalis; small arrow massa caudalis; double arrows (Fig. 12) layer of posthydrolytic RCA-positive material on surface of ventral wall. Fig. 11 x680; Fig. 12 x630

Fig. 13, 14. Higher magnification of the massa caudalis shown in Figs. 11 and 12, respectively. Fig. 13: RCA without acid hydrolysis. Strong reaction at the periphery (small arrows), weak reaction in the core (open star) of massa caudalis. Full star reaction in nuclei. Fig. 14: RCA after acid hydrolysis. Strong reaction in the periphery (arrows) and core (full star) of massa caudalis. No reaction in the nuclei (open star). x 3400

Fig. 15. Frontal methacrylate section through the ampulla caudalis stained with WGA. Positive reaction in massa caudalis (arrow) and skin (s). x 1300

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Fig. 16. Cross section through the ampulla caudalis (ac). Dorsal (d) and ventral (v) aspects of the tail. N notochord; r f Reissner's fiber; mc massa caudalis; c capillaries; open star fibrillar material resembling massa caudalis; rectangle lateral opening shown at higher magnification in insert; S skin. • 2200. Insert: Fibrillar material in the ampulla caudalis (ac), lumen of latero-dorsal opening (star) and extra-ampullar space

Figs. 17-19. Detailed magnification of a capillary (Fig. 17), massa caudalis (Fig. 18), and lateral wall of ampulla caudalis (Fig.19); compare with Fig. 16., Fig. 17: Fibrillar material inside and outside a capillary (asterisks). The area framed in the rectangle, but from an adjacent section, is shown in the inserL • 6700. lnsert: Large opening (arrow) in endothelial wall. Fig. 18: R F Reissner's fiber; M C massa caudalis. • 12000. Fig. 19: Lateral wall of ampulla. Small arrows basal lamina; thick arrow beyond this point basal lamina is missing; arrowheads junctional complexes; D dorsal wall. • 6700

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layer became wider in the ventro-dorsal direction. The epen- dymal cells of the ventral wall were provided with cilia and microvilli (Fig. 16).

Dorsally the ampulla caudalis was lined by thin and elongated cells (Figs. 16, 18), which were not attached by junctional complexes. This thin portion of the wall dis- played small lateral gaps (Fig. 16) and a large central open- ing, which communicated with large cavities or lacunae. The basal lamina that covered the latero-ventral walls was missing in the dorsal wall of the ampulla (Figs. 18, 19). Certain cells forming part of the dorsal wall or located in the ampulla caudalis, free or embedded in the massa caudalis, were characterized by large lamellar bodies, nu- merous ribosomes and bundles of filaments ~ 7.5 nm thick.

At low magnification the massa caudalis appeared ho- mogeneous and of low electron density (Fig. 16). At higher resolution it resembled a loose meshwork consisting of short filaments (Fig. 18). RF proper was formed by a simi- lar, however more densely packed material. The massa cau- dalis was in close apposition to the dorsal wall and detached from the ventral wall of the ampulla.

Material with ultrastructural characteristics similar to those of massa caudalis was seen in the openings of the dorsal wall (Fig. 16), in the extraventricular spaces (Figs. 16, 17), and also in the lumen of the adjacent blood capillaries (Figs. 16, 17). These capillaries were character- ized by a very thin endothelium, large openings in the endo- thelial wall, and by the lack of a basal lamina (Figs. 16, 17).

Discussion

Our results obtained with larvae of the lamprey, Geotria australis, are in principal agreement with the concept pre- sented by Hofer et al. (1984), based on their pioneer work with ammocoetes of Lampetra planeri. The introduction of immunocytochemical and lectin-histochemical methods, however, provides more precise tools for the identification and tracing of secretory material (see the results of the pres- ent study).

With the use of antibodies against aqueous extracts of RF, Sterba et al. (1982) reported that in non-mammalian species the SCO is strongly immunoreactive, whereas the corresponding RF is not. However, Vullings and Diederen (1983), who used the same antibodies, showed that the RF of the frog, Rana temporaria, is definitely immunoreactive. Rodriguez et al. (1984a), after complete dissolution of bo- vine RF, were able to obtain antibodies that reacted with RF of mammalian as well as amphibian and reptilian spe- cies. These antibodies, however, did not stain RF of chick embryos (Schoebitz et al. 1985) and, as has been shown in the present report, they did not react with the RF proper of lamprey larvae. By comparing the immunoreactivity of RF in different vertebrate species (our collection of Bouin- fixed and paraffin-embedded material) it becomes evident that there is a gradual decrease in the intensity of the immu- nostaining of RF in a reverse phylogenetic sequence, i.e., from mammals to cyclostomes. In all the species investi- gated, however, the massa caudalis is strongly immunoreac- tive. This holds true even for lamprey larvae, the RF of which is completely negative (present report). If, as in lam- preys, the secretory material is strongly immunoreactive at the site of synthesis (SCO) and at the distal site of accumu- lation (massa caudalis), the lack of immunoreactivity in

the "intermediate" location of the material (RF proper) might be regarded as a transitory stage, indicating that some factor prevents the accessibility of the antibodies to the immunoreactive sites or interferes in some unknown way with the reaction mechanism. Which, in fact, are the events leading to a masking of the immunoreactive sites in RF? Although the evidence is still scarce, we offer two alternative explanations. In species possessing an immuno- reactive RF, the latter is not evenly immunostained. Thus, in bovine, RF is strongly reactive at its periphery, while the core is only weakly stained. Within this core, longitudin- ally oriented, threadlike structures are strongly immunos- tained (S. Rodriguez etal. 1985). The areas displaying strong immunoreaction (peripheral and central, threadlike structures) appear to correspond to the loosely arranged 5-10-nm filaments (Rodriguez et al. 1986). This may sug- gest that the degree of packaging or condensation of RF material and the extent of immunoreactivity might be inter- related phenomena.

A more attractive possibility to explain the lack of im- munoreactivity of RF, at least in the case of lamprey larvae (Geotria australis), refers to the pattern of distribution of its sugar moeities. WGA has an affinity for N-acetyl-glucos- amine and sialic acid. In all probability the WGA-positive reaction in the SCO and RF is due to the presence of sialic acid (Rodriguez et al. 1986). Furthermore, Sterba and Wolf (1969), who used histochemical methods, have shown the presence of sialic acid in the bovine RF. This has recently been confirmed in our laboratory by use of chemical meth- ods (Hein et al., unpublished results). The fact that only the periphery of the lamprey RF reacts with WGA may suggest that sialic acid residues are mostly or exclusively located at the surface of RF. This possibility is supported by the fact that the periphery of the lamprey RF also dis- plays affinity for RCA, a lectin binding to galactose, how- ever only after acid hydrolysis. Rodriguez et al. (1986) pro- vided evidence indicating that the secretory products of the SCO are N-linked glycoproteins with a high mannose-type core, and with sialic acid and galactose as the terminal and subterminal sugar residues, respectively. This has been largely confirmed by the use in our laboratory of specific glycosidases (Herrera et al., unpublished results). Since acid hydrolysis is known to remove sialic acid residues (Quintar- elli 1961), the RCA-staining of the periphery of RF after acid hydrolysis can only be explained by the assumption (i) that galactose is the subterminal sugar moeity, and (ii) that after removal of sialic acid it becomes the terminal sugar residue capable of binding to RCA. It is tempting to correlate the presence of this " coa t " of sialic acid in the lamprey RF with the lack of its immunoreactivity (nega- tive charges? steric effects?). The following two observa- tions may support this attempt of correlation. The massa caudalis of the lamprey is immunoreactive and has a "coa t " that is RCA-positive without previous acid hydrolysis sug- gesting that this coat lacks sialic acid residues. In material processed for ultrastructural immunocytochemistry (alde- hyde fixation and ultrathin sections) RF of Geotria australis is immunoreactive (Peruzzo et al. 1986). In this case of sec- tioned RF, the core of the fiber may become accessible to the antibodies and this may lead to the positive immuno- reaction. However, the aldehyde fixation could, per se, be a prerequisite of the enhanced immunoreactivity of the RF.

Regardless of the nature of the mechanism(s) leading to the masking of the immunoreactive sites in the lamprey

RF, it appears that they operate with different degrees of effectivity along the vertebrate phylum, as can be judged from the pat tern o f immunoreact ivi ty of R F in different species.

In contrast to R F proper, the core of the massa caudalis is WGA-pos i t ive and RCA-posi t ive after acid hydrolysis and its " c o a t " is RCA-posi t ive without previous hydroly- sis. Two impor tan t conclusions can be drawn from these results :

(1) The glycoproteins located at the periphery of the massa caudalis have lost their sialic acid residues and pos- sess galactose as the terminal residue, which implicates an impor tant chemical al teration. Experimental removal of sialic acid from circulating glycoproteins by sialidase leads to a dramat ic enhancement in their rate of clearance from the circulatory system; moreover, the sialoglycoproteins are rapidly taken up and catabolized by the liver. This uptake depends on recognit ion by the liver cells of galactose resi- dues exposed by removal of sialic acid (see Sharon and Lis 1982). Similarly, cleavage of sialic acid from surface glycoproteins of erythrocytes delivers the signal for the re- moval of old erythrocytes from the circulation and their phagocytosis by macrophages (see Sharon and Lis 1982).

(2) In the massa caudalis the glycoproteins, at least those containing sialic acid and galactose, are arranged in a man- ner different from that in the R F proper.

The changes observed in the massa caudalis appear to apply also to the terminal segment of RF.

With regard to the type of fixation and to the l imitat ion of the optical resolution, in the present investigation it was not possible to trace precisely the secretory material after escaping from the ampulla caudalis. Ul t ras t ructura l lectin histochemistry (cf. Meiniel and Meiniel 1985; Rodriguez et al. 1986) appears to be the appropr ia te tool for investiga- tion of probable further chemical modif icat ions of the secre- tory glycoproteins beyond the level of the ampul la caudalis.

Convent ional electron microscopy has proven to be a useful approach when investigating the anatomical relation- ships of the ampul la caudalis with adjacent structures. F o r pioneer work in this area full credit should be given to Hofer et al. (1984). These authors have shown that in am- mocoetes of Petromyzon planeri wide intercellular spaces and large openings in the dorsal wall of the ampul la cauda- lis establish a direct communicat ion between the ampul la and large cavities or lacunae. These, in turn, appear to be in open communicat ion with the lumen of b lood vessels. We have made similar observations in larvae of Geotria australis (for further details, see Peruzzo et al. 1986). On the other hand, at variance with Hofer et al. (1984), we find junct ional complexes between the ependymal cells lin- ing the latero-ventral walls of the ampul la caudalis. The cells lining the dorsal wall, however, not only lack junc- tional complexes but are also devoid of a support ing basal lamina. These features place these cells in a very unique situation, also with respect to their layer-forming capacity. Since this is the only region of the ampulla caudalis in close spatial association with the massa caudalis, it is tempt- ing to speculate on a possible interaction between the massa caudalis and the adjacent ependymal differentiations. Note- worthy in this connection is also the absence of a basal lamina from the capillaries located close to the ampulla caudalis (see also Peruzzo et al. 1986).

The presence in the lacunae and the lumina of capillaries of a fibrillar material resembling that in the massa caudalis

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led Hofer et al. (1984) to the suggestion that R F material finally gains access to the blood stream via the extended lacunae. By means of conventional electron microscopy we can confirm this observat ion in another lamprey species. However, the p roof that this " f ibr i l la r ma t t e r " is indeed immunoreact ive RF-mater ia l requires immunocytochemi- cal studies at the ul trastructural level (see Peruzzo et al. 1986).

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Accepted June 24, 1986