91

The histochemistry of the saccus vasculosus of Notopteruschitala (Teleostei)

By BANGALORE I. SUNDARARAJ and M. R. N. PRASAD(From the Department of Zoology, University of Delhi, Delhi 6, India)

With one plate (fig. 3)

SummaryThe saccus vasculosus of Notopterus chitala is situated posteriorly to the pituitarygland. It consists of a number of loculi surrounded by blood sinusoids. The loculiopen into a number of collecting channels which unite and ultimately drain into thethird ventricle of the brain. The loculi are lined by a layer of pear-shaped 'coronetcells'. The coronet cell has an apical protrusion provided with hairs, each ending in aglobule. The coronet cells contain glycogen, especially in the apical protrusions andin the globules. Phospholipid, alkaline phosphatase, and mitochondria are concen-trated in the apical protrusion and globules. There are also mitochondria round thenucleus. The Golgi element is not only present in the characteristic Golgi zone butoccasionally round the nucleus as well. It has been stated by other authors that acidmucopolysaccharides are synthesized in the coronet cells of the rainbow trout and'secreted' into the lumen of saccus vasculosus. However, acid mucopolysaccharidesare not histochemically demonstrable in the coronet cells of Notopterus. The observa-tions recorded in this paper indicate that the coronet cells in the saccus vasculosus ofNotopterus are secretory and that glycogen is abundant in them.

IntroductionT H E saccus vasculosus is a highly vascularized protrusion of the caudal in-fundibular wall in the brain of fishes. It consists of a number of loculi eachlined by a layer of pear-shaped 'coronet cells'. Each coronet cell has aprominent apical protrusion provided with a number of hairs, each ending in aglobule. The loculi are surrounded by large blood sinusoids.

The earlier literature on the saccus vasculosus has been reviewed by Dorn(1955). Its function is not clearly understood. Dammerman (1910) suggestedthat the coronet cells act as pressure-perceptive structures. Scharrer (1948)proposed several possible functions for the saccus vasculosus and concludedthat '. . . it is at present unknown whether the sinuses serve as blood depots,or play a role in the secretion of fluid from the blood into the lumen of thesaccus vasculosus, or by changing volume, help equalize differences in intra-cranial pressure during vertical movements'. Kurotaki (1961), on the basis ofelectron microscopical studies, suggested that the coronet cells are similar instructure to the retinal rods and cones. However, the stimuli to which theyrespond are not clear.

A secretory role has also been assigned to the coronet cells. Bargmann (1954)described the saccus vasculosus of a number of elasmobranch and teleostfishes and concluded that the coronet cells are secretory. This was furthersubstantiated by electron microscopical studies (Bargmann and Knoop, 1955,[Quart. J. micr. Sci., Vol. 105, pt. 1, pp. 91-98, 1964.]

92 Sundararaj and Prasad—Saccus vasculosus of Notopterus

1961). Kamer and others (i960) and Jansen and Kamer (1961) demonstratedglycogen in the coronet cells of the rainbow trout, Salmo irideus, and postu-lated that glycogen in the globules is converted into an acid mucopoly-saccharide complex and 'secreted' into the lumen of the saccus vasculosus.Stahl and Seite (i960) demonstrated several types of acid mucopolysaccharidesin the coronet cells of many species of bony fishes. They stated, however, thatalthough a glandular function appears probable, the receptor theory cannotaltogether be ruled out. Zwillenberg (1961) put forward the hypothesis thatcystine is stored in the coronet cells and used as a precursor for acid muco-polysaccharide.

The histochemistry of the saccus vasculosus has not been studied in Indianfishes. The present investigation on the morphology and histochemistry ofthe saccus vasculosus in a freshwater fish, Notopterus chitala, is part of a studyon the histophysiology of the saccus vasculosus in Indian fishes.

Material and methods

N. chitala (Ham.) (order Clupeiformes, suborder Notopteroidei, familyNotopteridae) is a common air-breathing carnivorous fish which occurs in therivers of the Indo-Gangetic plain. The body is laterally compressed and theupper profile of the head is deeply concave while that of the abdomen isconvex. The snout is rather prominent. The sides of the body are silveryand above it is coppery-brown with about 15 transverse silvery bars. Adultsusually range from 30 to 75 cm in length.

Fifty-five adult N. chitala were studied during the period October 1961 toMarch 1962. Specimens collected from the backwaters of the river Jamuna nearDelhi were brought alive to the laboratory. They were killed by decapitationand the saccus vasculosus exposed and fixative poured on to it. After a fewminutes the saccus vasculosus along with the rest of the brain was carefullyseparated from the cranium and dropped entire into the fixative. The follow-ing fixatives were used. The duration of fixation is indicated in parentheses.Bouin's fluid (18 h), Helly's (18 h), formaldehyde-calcium (6 h), 10% neutralformalin (18 h), Aoyama's (4 h), ice-cold Rossman's fluid (24 h), Carnoy's(3 h), chilled acetone (24 h), cold 10% neutral formalin (1 h), weak Bouin's(20 h), Regaud's (96 h), formalin-alcohol-acetic (18 h), Mota's (Mota andothers, 1956) (24 h), 4% basic lead acetate (24 h), and saturated lead nitratesolution (24 h).

Sagittal sections of the saccus vasculosus in paraffin were cut at 5 /M andprocessed to demonstrate polysaccharides, proteins, and lipids (Pearse, 1961;see table 1, p. 93). Mitochondria and the Golgi element were demonstratedby the anilin-fuchsin / methyl green method (Cowdry, 1948) and by Aoyama'smethod (Baker, 1956) respectively. Alkaline phosphatase was localized bythe application of Gomori's technique to frozen sections (Pearse, 1961).General basiphilia was demonstrated by the methyl green / pyronin method(Pearse, 1961).

Sundararaj and Prasad—Saccus vasculosus of Notopterus

TABLE I

Histochemical analysis of the coronet cells in N. chitala*

93

testno.

i

2

3

4

5

6

7

8

9

IO

I I

1 2

1314

IS

16

17

iS

Histochemical test

PAS without oxidationin periodic acid

PASPAS after digestion insaliva at 37° C for i h

Best's carmineBest's carmine after di-gestion in saliva at 3 7° Cfor 1 hr

PASPAS after acetylationPAS after acetylation fol-lowed by deacetylation

P A SPAS after treatment in

5% trichloracetic acidfor 24 h at 40 C

PAS after treatment indistilled water for 24 ha t 4 ° C

P A SPAS after treatment in

5% trichloracetic acidfor zo h at 60° C

PAS after treatment indistilled water for 20 hat 60° C

aldehyde fuchsin

toluidine blue for meta-chromasia

alcian blue

methylene blue extinc-tion, pH 2*62 to 4'93

acridine orange methodfor mucins

Hale's dialysed iron

bi-colcombined dialysed ironPAS technique

toluidine blue after diges-

tion in saliva at 370 Cfor 1 h

alcin blue after digestionin saliva at 370 C for 1 h

Hale's dialysed iron afterdigestion in trypsin at370 C for 1 h

toluidine blue after ex-traction in hot pyridineat 6o° C for 24 h

Fixation

Helly

••

) (

if

Helly orCarnoy

Carnoy

sat. leadnitrate orCarnoyCarnoy

,(

Helly orMota

CarnoyHelly

Carnoy

Helly orCarnoy

Carnoy

Reaction in coront

Cell-body

0

0

4-0

0

+

4-4-

4-

-f0

4-

0

0

0

0

0

0

0

4-

0

0

0

0

Apicalprotrusion

0

0

0

0

4-4-4-

4-4-4-4-4-4-

4-4-4-

4-4-4-0

4-4-4-

0

0

0

0

0

0

0

4-4-4-

0

0

0

0

tcell

Hair andglobule

0

0 /

+ +o)

4-4-1

4- 4-\4- 4-

4-4- '

4- 4-0

4-4-

0

0

0 '

0

0

0

o>4- 4-

°)

oJ0

0

Remarks

free aldehydes ab-sent

glycogen present

glycogen present

1:2 glycol groups

present

Iyoglycogen absent

desmoglycogen

present

sulphatcd acidmucopolysaccha-ride absent

chromotropic sub-stances absent

acid mucopoly-saccharides absent

stained magenta, in-dicating the ab-sence of acid muco-polysaccharides

glycogen not mask-ing acid mucopoly-saccharides

proteins not mask-ing acid mucopoly-saccharides

lipids not maskingacid mucopolysac-charides

94 Sundararaj and Prasad—Saccus vasculosus of Notopterus

TABLE I (cont.)

testno.

J9

20

2 1

212

23

24

25

26

27

28

29

Histochemical test

toluidine blue after sul-phation in cone, sul-phuric acid

mercuric bromphenolblue

ninhydrin/Schiff

Sudan black B

Sudan black after extrac-tion in pyridine at 60 Cfor 24 h

acid haematein

acid haematein after ex-traction in pyridine at6o° C for 24 h

calcium-cobalt methodfor alkaline phospha-tase

anilin-fuchsin / methylgreen method for mito-chondria

Aoyama's method forGolgi

methyl green / pyronin

Fixation

Carnoy

,,

Helly orCarnoy

formal-dehyde-calcium

formal-dehyde-calcium

weakBouin

coldacetone

Helly orRegaud

Aoyama

Carnoy

Reaction in coronet cell

Cell-body

0

+

0

-f

0

-f-

0

-|-

+ + +

+

+ +

Apicalprotrusion

0

+

0

-)-

0

-f

0

4.4-

+ + +

0

+

Hair andglobule

0

-)-

0

1°

+ 1

0)

+ +

+ +

0

+

Remarks

neutral mucopoly-saccharides absent

proteins present

reactive NH2 groupsnot present in suf-ficient quantities

lipids present

pbospholipidpresent

alkaline phosphat-ase present

mitochondria pre-sent

Golgi element pre-sent

RNA present

* The histochemical tests were carried out in accordance with the methods given by Pearse (1961):+ - H - = strong reaction; + + = medium reaction; + = weak reaction ; o = no reaction.

ObservationsMorphology. The saccus vasculosus of N. chitala is a small oblong body

situated ventrally in the brain behind the pituitary gland (fig. i) and is coveredover by the dura mater, which is continuous over the pituitary (fig. 2). It ismade up of a number of loculi surrounded by large blood sinusoids (fig. 3, E).The lumina of the loculi open into a number of collecting channels which

FIG. 3 (plate). Sections of the saccus vasculosus of N. chitala.A, low-power view of a sagittal section, showing the confluence of the collecting channels

and their drainage into the third ventricle. Note the continuity of some of the loculi with thecollecting channels (arrows). Helly, PAS/haematoxylin.

B, alkaline phosphatase reaction in the coronet cells. Arrows indicate the intense reactionin the apical protrusion and in the globules. Cold acetone, Gomori's technique.

c and D, demonstration of glycogen by the PAS reaction before and after saliva treatment.C, note the intense PAS reaction in the apical protrusions in control sections (arrows) and itsremoval in the adjacent section, D, after saliva treatment (arrows). Helly, PAS/haematoxylin.

E, low-power view showing a few loculi surrounded by blood sinusoids. Note the PASreaction in the apical protrusions of some coronet cells, reflecting variations in the physio-logical activity of the cells. Helly, PAS/haematoxylin.

F, distribution of mitochondria in the coronet cells. Arrows indicate the presence of mito-chondria in the apical protrusions and globules in addition to perinuclear distribution. Helly,anilin-fuchsin / methyl green.

I'"-

FIG. 3

B. J. SUNDARARAJ and M. R. N. PRASAD

Sundararaj and Prasad—Saccus vasculosus of Notopterus 95

ultimately drain into the third ventricle of the brain (figs. 2; 3, A). Theloculi are lined by pear-shaped coronet cells with the so-called supportingcells between them (fig. 3, c). The coronet cell has a nucleus situated basallyand the distal part of the cell is drawn into an apical protrusion (fig. 3, c)provided with a number of hairs each ending in a globule.

inferiorlobe

optic chiasma

optic lobe

FIG. 1. Diagram of the brain of N. chitala in ventral view, showingthe saccus vasculosus in relation to the pituitary gland.

anterior end •diencephalon

alpars distalis

FIG. 2. Camera lucida drawing of the mid-sagittal section of the brain ofN. chitala, showing the saccus vasculosus and pituitary gland in relation to

the third ventricle.

Histochemistry. The histochemical methods applied to demonstrate thevarious inclusions in the coronet cells are listed in table 1 (p. 93). Theapical protrusions and globules contain primarily glycogen (fig. 3, c, D). Notall the cells in a loculus show the same intense PAS reaction (fig. 3, E). Acidmucopolysaccharides are not present in any part of the coronet cell (tests7 to 13). Masking of acid mucopolysaccharides by glycogen, protein,and lipids was excluded by appropriate treatments (tests 15 to 18). Thesaccus vasculosus and other control material known to contain acid

96 Sundararaj and Prasad—Saccus vasculosus of Notopterus

mucopolysaccharides (the intestinal mucosa of N. chitala and test cells ofascidian eggs (Seshachar and Rao, 1963)) were fixed and processed simul-taneously to demonstrate acid mucopolysaccharides (tests 9 and 12). Apositive reaction was noticed in control sections regardless of the fixativesused, while the saccus vasculosus was uniformly negative. This, besidesconfirming the specificity of our staining methods for acid mucopolysac-charides, also confirmed the absence of this material in the coronet cell.

The apical protrusions and globules contain a small amount of phospholipid(tests 22 to 25). Alkaline phosphatase was localized in the apical protrusion,globules, and cell-body; the reaction was more intense in the apical protrusionand the globules than in the cell-body (fig. 3, B). Mitochondria are concen-trated around the nucleus in the cell-body, in the apical protrusion, and alsoin the globules (fig. 3, F). The Golgi element is present not only in the charac-teristic Golgi zone but also occasionally around the nucleus.

DiscussionOur results indicate that the coronet cells of N. chitala are secretory. The

secretory activity was noticed even without chemical stimulation of the typeused by Kamer and his colleagues (i960), who injected pilocarpine to stimulatethe secretory process in the coronet cells and noticed greater activity in thosecells of the ventral wall of the saccus vasculosus that lay above wide sinuses.In Notopterus, secretory activity is greatest in the cranial and caudal extremi-ties of the gland. The significance of this is not clear.

Histochemical studies of the saccus vasculosus of Notopterus suggest thatthe coronet cells contain glycogen in the so-called bound form (desmogly-cogen), both in the apical protrusions and in the globules. Stetten and Stetten(i960), however, are of the opinion that the classification of glycogen into twosubdivisions, free and bound, is inadequate and arbitrary. Acid mucopoly-saccharides are not demonstrable in any part of the cell.

Kamer and others (i960), Stahl and Seite (i960), Jansen and Kamer (1961),and Zwillenberg (1961) have noticed PAS-positive substance in the apicalprotrusion of the coronet cells in a number of fishes. Kamer and others (i960)and Stahl and Seite (i960) observed a mild PAS reaction in the globules andin the cavity of the saccus vasculosus after treatment with saliva and amylaserespectively. They did not, however, indicate the nature of this substance.The PAS reaction after saliva and amylase digestion might be due to glyco- ormucoprotein or glycolipid and not to glycogen or acid mucopolysaccharide(compare Hooghwinkel and Smits, 1957; Leblond and others, 1957; Seshacharand Rao, 1963).

Kamer and others (i960) and Jansen and Kamer (1961), on the basis ofaldehyde fuchsin (AF) staining, concluded that the glycogen in the apicalprotrusion is converted into an acid mucopolysaccharide. Brilliant stainingof the test cells of ascidian eggs (Seshachar and Rao, 1963), processed simul-taneously with the saccus material, indicates the specificity of our stainingprocedure.

Sundararaj and Prasad—Saccus vasculosus in Notopterus 97

Jansen and Kamer (1961) noticed glycogen granules in the apical pro-trusions, hairs, and globules of the coronet cells of the trout. They furtherdemonstrated that the basal corpuscles in the coronet cell are PAS-positiveas well as metachromatic. We have noticed glycogen granules in the apicalprotrusions and globules of the coronet cells of Notopterus; these are notmetachromatic. We feel that the PAS-positive reaction in the apical pro-trusions below the origin of the hairs is due to highly polymerized glycogengranules in various stages of transport from the apical protrusion to theglobules (Sundararaj and Prasad, 1963).

Stahl and Seite (i960), Jansen and Kamer (1961), and Zwillenberg (1961),on the basis of alcian blue staining, identified the alcian-blue-positive materialin the coronet cell and in the lumen of saccus vasculosus as acid mucopoly-saccharide. We have not been able to demonstrate acid mucopolysaccharidein any part of the coronet cell of Notopterus by using specific histochemicaltests other than alcian blue. Zwillenberg (1961) suggested that cystine mightbe involved in the conversion of glycogen into acid mucopolysaccharide. Theacceptance of this view would depend on findings of radio-isotope studies.

Stahl and Seite (i960), Jansen and Kamer (1961), and Zwillenberg (1961)have demonstrated metachromasia in the cytoplasm of the coronet cells, butthey did not indicate the type of metachromasia. The metachromasia mightbe due to a number of substances including nucleic acids, especially RNA(Seshachar, 1953: Hale, 1957; Pearse, 1961). Non-chromotropic substancessuch as mucoid secretion, starch, and glycogen would stain metachromaticallyafter pretreatment with periodic acid, chromic acid, or other substances(Bergeron and Singer, 1958).

Kamer and others (i960) put forward the hypothesis that ' . . . the glycogencontent of the apical protrusions is used up in the formation of an acid muco-polysaccharide containing both 1:2 glycol groups and sulfonic acid groups,which is stored in the globules and ultimately secreted into the ventricle1.Jansen and Kamer (1961) suggested that '. . . the synthesis of acid muco-polysaccharide is initiated in the cell body and the formed product will betransported to the basal corpuscles'. They further suggested the transport ofthis material through the hollow hairs into the globules '. . . where it is storedbefore it is secreted into the cavity of the organ'. There is an apparent sug-gestion of the existence of more than one pathway of acid mucopolysaccharidesynthesis in the coronet cells of the trout, but there is no definite statementas to whether either or both pathways are equally operative.

Since these authors have not excluded the possibility of metachromasia dueto RNA, their conclusions that the metachromatic substance in the coronetcells of the trout is acid mucopolysaccharide, is based on incomplete histo-chemical evidence. Hence the validity of their hypothesis of acid mucopoly-saccharide synthesis and release should be subjected to further histochemicalverification. Moreover, they do not indicate whether the secretory productsreleased into the lumen of the saccus vasculosus of fishes treated with pilo-carpine are similar to those released in fishes not so treated. Appropriate

98 Sundararaj and Prasad—Saccus vasculosus of Notopterus

control material rich in acid mucopolysaccharide does not appear to have beenused to determine the specificity of their staining procedures.

While subscribing to the secretory hypothesis of Bargmann (1954), Barg-mann and Knoop (1955, 1961), Kamer and others (i960), Stahl and Seite(i960), Jansen and Kamer (1961), and Zwillenberg (1961), we differ from theobservations of Kamer and others and of Jansen and Kamer in regard to theproduct formed in the cell body and released from the globules into the lumenof the saccus vasculosus. Our histochemical studies of the saccus vasculosusof N. chitala indicate that the substance present in the coronet cells is primarilyglycogen. In addition, traces of phospholipid have been noticed in the apicalprotrusion and globules. Acid mucopolysaccharide was not demonstrable inany part of the coronet cell of N. chitala. Experiments using S35 labelledsulphate are in progress to confirm the above observations. Since glycogen isthe major constituent of the coronet cells of N. chitala, further experiments arebeing designed to determine the role of glycogen in these cells.

The authors wish to express their appreciation to Dr. Ragnar Olsson,Zoological Institute, University of Stockholm, Sweden, for initiating us intothis field and for the loan of literature, to Prof. B. R. Seshachar andDr. S. R. V. Rao for helpful criticisms, to Mr. R. B. Raizada and Mr.R. N. Chaturvedi of the Fisheries Department, Delhi State, for help in thecollection of specimens, and to the C.S.I.R., New Delhi, for financial sup-port.

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