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The dogfish shell gland, a histochemical study
J. exp. mar. Bioi. Ecof., 1976, Vol. 23, pp. 267-283; @ North-Holland
Publishing Company
THE DOGFISH SJJELL GLAND, A HISTOCHEMICAL STUDY M. RUSAOUEN Luboratoire de Cytologic, UniversitP Paris VI, Quai St. Bernard, Paris, France Ahstraet: On the basis of previous work and new ~stoche~cal results, the dogfish nidamentat gland is divided into six distinct zones. Several neutra1 or acid mucopolysaccharides are present; the latter are more or less sulphated. Various proteins including a phenolic glycoprotein tanned by a phenolase and a peroxidase, a sulphur containing protein, and collagenons protein are also present. The connection between the zones of secretion and the layers of the egg-shell layers is discussed.
For more than a century a large number of studies of the egg caps&e of selachians (Fig. 1) have been made in an attempt to determine the composition of the secretions of the capsule-making nidamental gland (Fig. 2). Perravex (1884), Henneguy (1893), Widakowich (1906) and Borcea (1906), all demonstrated the lamellar-moulding lamellae in the gland. In a comprehensive study of the scleroproteins, Garrault & Filhol (1937) Fat&-FrtSmiet (1938), FaurBFrimiet & Baudouy (1938), Filhol & Garrault (1938), FaurC-Fremiet & Garrault (1938), attempted to explain the processes which take place in the formation of these eggs. On the basis of their histochemical and biochemical evidence they concluded that a sulphur-containing protein called “prokeratine”, which is chemically closely related to keratin is elaborated in the gland and secreted by its shell-shaping zone. They supposed that before its passage through that zone the egg was first enclosed in albumen secreted by the apical or “albumen secreting” zone of the gland. Twenty years later Threadgold (1957), investigated the problems raised by the formation of nidamental gland of Scyliorhinus canicula basing part of his work on Brown’s (1950, 1952, 1955) studies on quinone tanning systems in the animal kingdom. The evidence for the presence of aphenolic protein and a polyphenoloxydase in the gland led him to conclude that the egg-capsule was formed by quinone tanning. He did not, however, mention the presence of sulphur containing components, previously demonstrated by Faure-Fremiet and his co-workers, but he showed the presence of a PAS positive substance in the apical zone of the gland, possibly due to neutral mucopolysaccharides. The secretion of this zone could then no longer than be regarded as albumen, thus confirmed Borcea’s (1906) doubts as to the albuminoid nature of the secretion. The work of Krishnan (1959) supported and widened the evidence for quinone tanning in the formation of the egg-capsule; his histochemical study of the nidamental gland and egg-capsule of the C~iZoscylZi~~griseum was supported by a chromato267
268
graphic analysis
M.RUSAOUEN
of the capsule components:
he suggested that the capsule protein was
of a collagen type, but makes no reference to the presence of the sulphur-containing protein noted by FaurC-FrCmiet. Vovelle’s work (1965) removed any possible doubt regarding the existence of such a protein in the gland. After having demonstrated the evidence of S-radicals, he concluded that the secretion of a protein containing SH groups is not at all inconsistent with the presence of a phenolic protein and a polyphenoloxydase which account for quinone tanning. We have already applied biochemical and histoautoradiographic techniques to show the presence of collagen in the egg capsule of Scyliorhinus canicula, and to determine which type of the nidamental gland cells gives rise to this secretion (Rusaouen et al., 1975). The present paper endeavours to provide new data regarding other secretory products within the various zones of the shell gland.
MATERIALS AND METHODS
The shell glands of dogfish were removed immediately after killing by beheading. Each gland was split into longitudinal halves which were fixed in neutral formaldehyde, Carnoy’s fluid, Bouin and Gendre’s fluids. Cryostat sections of the gland were also cut. A fragment of the ovary was removed from every sacrificed animal and used to stage the gland as regards its maturity. Three stages were recorded. Each half gland was embedded in paraffin and 7.5 pm sections subjected to the following histochemical tests’. Proteins General reaction, biuret; basic proteins, FCF (Alfert & Geschwind); aromatic radicals, Morel & Sisley’s reaction (Glenner & Lillie); Phenolic groups: azoreaction (diazo sulphanilic acid-fast blue B pH 9.2). Indol and pyrol radicals, Glenner’s rosindol reaction. Sulphydryl and disulphydryl groups, DDD (Barnett & Seligman); RSR (Bennet-Pearse); blocking; sodium iodoacetate or N-ethylmaleimide, reduction by sodium thioglycolate after blocking and followed by DDD reaction; Adams-Slopper’s
reaction.
Carbohydrates: poiysaccharides and acidic or neutral mucopolysaccharides Hotchkiss-MacManus’s reaction; similar test without preliminary oxidation, after amylase digestion, and after dimedone blocking (Bulmer, 1959); metachromatic basophilia with Toluidine Blue (0.1 74) pH 1.O, pH 3.0 and pH 5.0 in Walpole M/50 buffer; Lison, pH 1.0, pH 2.6; Alcian blue colouration (Mowry) with or without Hotchkiss-MacManus’s reaction, with methylation and methylation-saponification controls (Fisher & Lillie); Alcian blue-Alcian yellow staining (Ravetto): aqueous ’ References to well-known manuals (Gabe, 1968; Ganter
histochemical techniques which have been described & Jolk?s, 1969) are not given in the bibliography.
in specialized
THE DOGFISH
269
SHELL GLAND
buffered solution of N,N-dimethyl-p-phenylenediamine several days old (Spicer & Jarrels, 1961).
HCI, freshly prepared
or
Enzymes
Fresh tissue cryostat sections were incubated in DOPA (dihydroxyphenylalanine, Gomori’s variant) or pyrocatechol (Smyth, 1954). In either case controls were made by simultaneous inhibitions with sodium diethyldithiocarbamate (Bunke, 1972). Sections were post-fixed in saline neutral formaldehyde. Peroxidase. Benzidine tests (Wachstein & Meisel, 1964) with inhibitor control of methanol ferricyanure (Straus, 1971). Phenolases.
RESULTS
On the grounds of his own histochemical investigations and previous morphological work, Threadgold (1957) identified five distinct zones in the shell gland: Zone A, albumen zone of Filhol & Garrault (1938); Zone B, metachromatic duct of Filhol & Garrault (1938); Zone C, “Three or four ducts parallel with and following zone B” (Threadgold, 1957); Zone D, prokeratine region of Filhol & Garrault (1938); and Zone E, caudal shell secreting zone of Nalini (1944). To those five zones we shall add a sixth, Zone F (Fig. 4) which runs parallel to Zone E from which it is separated by a conjunctive wall. Subsequently, we shall discuss the significance of this new zone whose tubules lie perpendicular to the tubules of Zone E and open between short lamellae quite similar to those found in Zone A (Fig. 5). In order to study the evolution of the nature and localization of the glandular secretions, three stages of its development have been selected, namely, Stage 1 (Fig. 6a) before the vitellogenesis of ovocyte maturation, Stage 2 (Fig. 6b) after vitellogenesis, and Stage 3 egg-laying activity (Figs 1, 2 and 6~). At first sight some distinction may be drawn between two regions of the gland, the first of which is positive to most carbohydrate tests (Table I) and includes Zones A and B, while the second one reacts to the test for proteins and is made up of Zones C, D, E, and F (Table II). A more detailed analysis of the results shows that in toto the egg-shell gland is much more complex, but for clarity we shall successively deal with results obtained. ZONES
A
AND
B:
CARBOHYDRATES
Hotchkiss-MacManus
reaction
From its early stage, Zone A reacts intensely to PAS (Table I); numerous granules are seen in the apical region of cells (Fig. 7). In Stages 2 and 3, the secretion is present everywhere in the cells (Fig. 8). Zone B gives no reaction to PAS at any stage (Fig. 8).
Fig. Fig. Fig. Fig.
Egg-capsule of the dogfish, Scyliorhinus canicula: c, coils; st, stomium (lines of dehisc :enc:e). Fig. 2. Half nidamenta1 gland, internal view: La, lamellae; Ov, oviduct. T’ransverse section of the egg-shell: Ll, outer layer; L2, second layer; LB, third layer; L4 ,in ner layer. Diagram of a longitudinal section of a half gland showing the different secretory arer 1s: (3v, oviduct; A, B, C, D, E, Fr , F1, F3, F,, the various zones. Longitudinal section of the gland: PAS reaction and picroindigocarmine: D, Zone D: E. Zone E, F, Zone F; la, lamellae; al, alveolae; cw, conjunctive wall; bv, blood vessel.
THE DOGFISH
Fig. 6. Longitudinal
SHELL GLAND
271
section of the gland: a, Stage I; b, Stage II; c, Stage III.
Fig. 7. L,ongitudinal section through Zone A of a gland at stage I: PAS reaction: t, tubule; lu, lu Imen; g, PAS-positive granules. Fig. 8. Longitudinal
section through Zones A and B of a gland at Stage III: t, tubule; lu, luunen; note all cells filled with PAS-positive granules.
The PAS positive secretion of Zone A is no more affected by amylase than it is by saliva {Threadgo~d, 1957). Rimedone controls on para& sections con~rm these results; however, weak but definite evidence of glycogen in Zone A is obtained with cryostat sections after 3-4 h dimedone blocking. Glycogen is present in various amount in all the tubules of Zone A. A difference in the state of the purple cotouration with PAS is observed between the sections subjected to dimedone blocking and those which are not, suggesting that other PAS positive substances are secreted by Zone A, especially neutral or slightly acid mucopolysaccharides which are apparently free since Zone A gives no reaction to tests for proteins. The enzymatic digestion of glycogen by the amyfase may pass unnoticed because of mvcosubstances in large amounts (Table I).
The presence of neutral and more or less acid mucopolysaccharides is demonstrated by Atcian Blue at pH 1.0 and pH 2.6 and Alcian Blue-Alcian Yellow, Toluidine Blue at various pi-I values (Table I). Only the ~otchkiss-~a~~anus reaction (Fig. 9) and TABLE1 Reactions for carbohydrates in Zones A and B at stages J, II, and Hi; -; ~ weak reaction; : . medium positive reaction; $- -t-I-, intense reaction; 0, no reactiort; y, yetlow; bf, blue; me, me&%chromasia; or, orthochromasia; ~g, orange; gr, grey; bk, black; br, brown; Spicer f, newly prepared diamine HCl; Spicer II, several days-old diamine HCJ.
Stage .--~-----.~-PAS Schiff reac. Amylase -!-PAS Dimedone I-PAS At&an BJue PR 1
pEf 2,6
Ale. Bit&~;--AIc. YeJIow Methylation$-Ale. Blue Saponificat. aft, Methyl. Toluid. Blue PR 1 PH3 pH 5
_._.” .___.~__..__-_.._.-..-.. A _-_.--_... ._._._^...^__._._._. ._ I II 111 -- _I._-l-.___.. .i { i. _. _; j -1. 0 0 ‘0 .: _i. t ! _,_ j. .!. -+
-‘I-“7 i_2.. y i_bJ me ..: me.). me
: i i ___ y : bl or
4 me
or-+me or 3 me
Spicer I
or
og :-gr
Spicer l’f
gr
hr . gr
-! _ _I
Zone
_...“._ ..-. B
J _~_._. ..____.. 0 0 0
I : . j_ bJ
KJ .._. _”
Ill _.. . 0 0 0
0 0 0
. :. :.
: i I
“bl
bl 0 0
y+bJ 0 0 or 4, me or i- me or ! me
me+ .I -. mc 4 Ij__. me / -‘. i me-J. _i__i me-~ ! ..i. mu !__t_i ,J,e: : me i Ime! 1 :
O&-igr br : bk
bk .I
0
0 hk ‘.
0 bk _ !..?
dicblorhydrate N,N-dietbylparaphenylene diamine fresh solution (Spicer & Jarrels, 1961) give a positive reaction in the outer region of the apicai zone on both sides of the gland so that onfy neutral mu~opo~ysa~charides are secreted by that region which
THE DOGFISH
SHELL GLAND
273
Figs 9 to 20. Drawings of longitudinal section of a half gland at stage III: 111111intense reaction; 1111111 medium positive reaction; 111111weak reaction. Fig. 9. PAS reaction. Fig. 10. Alcian Blue reaction: \\\\\ pH 1.0; ////// pH 2.6. Fig. 11. Toluidine Blue reaction: pH 1.O; metachromasia. Fig. 12. Toluidine Blue reaction: pH 1.0; orthochromasia. Fig. 13. Newly prepared diamine chlorhydrate reaction. Fig. 14. Several days-old diamine.
M. RUSAOUk?N
274
includes the distal ends of the tubules. Yet all tubules in Zone A give this type of secretion, in various amounts, along the whole of their length but it is much more evident in the outer region (Fig. 13). The older the gland, the smaller the main neutral mucopolysaccharide secreting region seems to be. Considering the production of each individual mature tubule on all its length, we find it includes all types of mucopolysaccharide secretion; and the same process of simultaneous secretion applies to acid mucosubstances. On the inner side of the gland near its lumen, the mixture of secretions is obvious; Alcian Blue-Alcian Yellow (Ravetto, 1964) give a distinct green colouration of some cells or cells in the same part of tubule are alternatively coloured yellow and blue. When using Alcian Blue at pH 1.0 and 2.6, changes of localization are noted in the coloured areas (Fig. 10) and similar changes are observed after the orthochromatic and metachromatic reactions of Toluidine Blue at pH 3.0 and 1.O (Figs 11, 12). Methylation-blocking followed by saponification indicates one is dealing here with sulphated acid polysaccharides. They are more or less sulphated, and the variations account for their reactivity to PAS as well as the differences observed in the reactions to Alcian Blue at pH 2.6 (Fig. 10) and Toluidine Blue (Figs 11, 12). Localization of the most strongly sulphated mucosubstances may be precisely determined by the Toluidine Blue test at pH 1.O. A strong metachromasia characterizes the inner fringe of the gland and is still visible, though less intensely. at the centre of Zone A at its inner part (Fig. I I ). It is easier to determine the nature of Zone B secretions since the Hotchkiss-MacManus reaction is absolutely negative (Table I, Figs 8,9) and all further tests confirm the presence of strongly sulphated mucopolysaccharides (Figs lo--14 and Table I). ZONES
C, D, E, F
Proteins These four zones give strongly positive reactions to the Biuret test (Table II). which was not the case for Zones A and B. From the various protein tests we may conclude that Zones C, E, and F, (Fig. 4) give similar reactions; the range of Zone D secretions is more diversified. Aromatic and phenol radicals Zones C, E, F, give an abundant secretion of a phenolic protein with many tyrosine residues. The gland in its early stage shows a strongly positive azoreaction in Zones C. E, F, (Table II) as does Zone D in Stages II and III though the reaction is weaker (Fig. 15). Identical conclusions may be drawn from diazo-sulphanilic acid test though they give a decidedly lighter staining. The presence of tyrosine residues is demonstrated by Morel & Sisley’s reaction which gives intense staining in the tubules of Zones C and E, and distinct ones in the tubules of Zones D and F, (Fig. 16). The localization of phenolic proteins indicates that they are not secreted in all the tubules of Zone D at an early stage; the only region involved appears as an oval-shaped area,
THE DOGFISH
SHELL GLAND
Fig. 1.5. Azoreaction. Fig. 16. Morel & Sisley’s reaction for tyrosine residues. Fig. 17. DDD reaction. Fig. 18. Pyrocatechol reaction. Fig. 19. DOPA reaction. Fig. 20. Peroxidase reaction.
275
M. RUSAOUBN
276
localized in the outer part of Zone D, and which includes the distal ends of several tubules.
Protein detection in Zones C, D, E, and FL at Stages I, II and III. __“-_-----.-~.C -
Stage
i-
0
FCF
. + Azoreaction Morel&Sk. _. i DDD 0 DDD after 0 ethylmal. 0 DDD af. ethyl.
.+.+
-
- .---_-.
Ill
I
II
_~..~
.~._^-- ._... III
j_
++.+
f_“&., +.:
+.i_+-
++-~
.
C
fi-+-
f +- i
:
, _t.
br br
br
0
0
br
0
br
br
o
0
0
0
i-
Ii
_i__.{_ /. .
III
I
, + - -4 : j
‘_
.^ ._~^I.- .FT.-.._ ______.
l_.- _--.
1
0
.-- _- .__ ._ E
i _j_+ .! _+.+ ; _
+_ .j_
$__$ _+ .+_+
Zone
..-_
D
---
Ii
I Biuret
.-..
i.
-++
iI
II
III
~_
.; !
()
:
:
I .j.
: , i_. _++ :_ i ! ! j .j_<_a_ .:_.:.
;.i. :. _-L
0
0
.
0
br br
br br
0’ 0
br hr
br br
/
t
0
hr
br
0
br
br
0
0
& thioglycol. RSR RSR
0
after Na iodoac. Adams-Sloper Rosindole
0 0
0 0
0 0
0
0 0
0
0
0 5
~.
0
tt
0
0
_-
Enzymes Phenolases. The phenolase test gives markedly positive results in Zones C, E, F and
a slight reaction in a localized area of Zone D (Table III). Zone C reacts to DOPA and pyrocatechol (Figs 18, 19); this result is confirmed by sodium diethyldithioTABLE 111
Enzyme reactions at Stage III in Zones C, D, E, F1, F1 and FJ. Zone
._.. .~_.“_-_ Pyrocatechoi. Pyrocatechob -+dieth. thioc. DOPA DOPA+dieth. thioc. Benzidine Eknzidine af. methanol
c _. _.._ : !., 0 1 0 0 0
D
0 0 0 0 0
__.~ -._._. - --. “. E Fz F3 F, _ ___.____ __ - __._ ..i ;.j_ () 0
.--F4 ..-_--. a
0
0
0
0
0
0 0 0
0 a 0
0
(i
0
0 --
0
0
carbamate blocking, and provides further evidence to support Threadgold’s theory (1957) of an “autoquinone tanning” process, involving a phenol&protein and a phenolase secreted by the same cells. Zone D shows a slight reaction to pyrocatechol
THE DOGFISH SHELL GLAND
277
in a limited area which is the site of maximum secretion of the phenolic protein. There is no reaction to DOPA (Figs 18, 19). The whole of Zone E gives a similar positive reaction to pyrocatechol but is only tenuously stained with DOPA (Table ID, Figs 18, 19). Inside Zone F, F, gives a markedly positive reaction to pyrocatechol and a weak reaction to DOPA, while DOPA is highly reactive in F, and F, . Peroxidase. The sites of peroxidase secretion are localized by the reaction of Wachstein & Meisel (1964). These sites only partly coincide with those giving the DOPA reaction. Methanol-blocking-control (Straus, 1971) confirms the presence of peroxidase in Zones F, , F, , F4. No peroxidase is found in Zone C (Table III, Fig. 20). Sulphur groups Sdphydryl radicals. DDD and Mercury Orange were used by Vovelle (1965) to demonstrate SH groups, which we now regard as strictly limited to Zone D. We come to that conclusion for the following reasons. After diethylmaleimide or sodium iodoacetate-blocking, the brown colouration of Zones C, E and F, obtained with DDD is persistent and even more intense. After the SH groups have been blocked, Mercury Orange stains Zones C, E, and F, a bright orange; the reaction occurs irrespective of the type of inhibitor. Only Zone D gives positive results to current staining and controls (Table II). Disulphide radicals. No significant reaction to Alcian Blue after periodic oxidation (Adams-Slopper) is obtained in Zones E and F, (Table II) and the presence there of SS groups is, therefore, questionable. The very slight staining may be due to acid mucopolysaccharides; our interpretation is grounded on a positive Adams-Slopper’s reaction in Zones A and B, on a positive orthochromatic Toluidine Blue reaction in Zones C, D, E and F1 (Table IV, Fig. 13) and on positive reactions by several days-old diamine in Zones A and B, and in Zones C, D, E, F, and F4 (Tables I, IV, V, Fig. 14). Zone D is the only one to give positive reaction to the DDD test after N diethylmaleimide and sodium thioglycolate, which leaves little doubt as to the presence of a limited amount of disulphide groups (Table III). Tryptophan
The Rosindol test (Table II) indicates the presence of indol radicals in the large cells of Zone D tubules (Henneguy, 1893; Nalini, 1944), as well in Zone F, tubules. Though less conspicuous than reactions for tyrosine and sulphur, Rosindol Blue nevertheless involves the same cells in Zone D and is identical to the tyrosine localization in Zone F, . Zones C and E do not stain. We find here a very unusual example -of a secretion common to Zones D and F, , yet lacking in Zones C and E. Carbohydrates Hotchkiss-MacManus reaction. The contrast between Zone A and the other zones (C, D, E, F) is so striking after the reaction to PAS, that the latter could be regarded
1%.RUSAOUEN
278
as giving totally negative results, but closer examination shows that the proximal ends of the tubules in zones C, D and E as well as the alveola-shaped infoldings of Zone D lamella give a positive reaction. The same positive reaction is equally visible in Zone F,, F2, and Fs (Tables IV, V, Fig. 9). These positive PAS tests are obtained in cells and tubule lumen when the gland is in Stage I; later, the reaction seems essentially to affect elements inside the lumen. Even so, positive staining may be seen in the cells of Zone D alveolae in cryostat sections after dimedone blocking so that glycogen is probably present in the alveola cells, while a PAS positive mucopolysaccharide reaction accounts for the positive reactions in the proximal ends of tubules of Zones D and E and on the whole length of the much shorter ducts of Zones F, . F, and F,, TABLEIV
Reactions for carbohydrates: Zones C, D, E, and F1L _ ^.._..__ C ______
Stage
1 PAS &hit%-reac.
0
.-_. ..-.. ” __I.._.
D
0
1
0
II
0
.-“._._.- -..-..---
III
0
_
E
_ .___-.-_.. _-
11 III
0
Zone
1
0
It
.___..~ Ill I I[ III -_..I. - -...__-
-
0
0
Amylase-+-PAS Alcian Blue pH 1 pH 2.6 Alcian Blue-talc. Yell. Ale. BI. pH 1 aft. methyl. Ale. B1. aft. meth. 8r Sapon. Toluid. Blue pH 5 pH3 PH 1 Spicer 1 Spicer II
FL
-
0 0
0 0
0 0
0
0
0
0
0
(1
0
0
0 0
0
0
0
0
0
0
0 0
?
v
0
4
LI
0
0
or or 0 0 0
or or ar gr gr
or 0 or gr gr
or or 0 0 0
or or or gr gr
or or or sr gr
or or or 0 0
or
or
Or
r,r or
gr bk
0 0
or or or w bk.
*r or or u bk
?
or, 1 EC
or
or
1-
Pdysaccharide components, The whole of the PAS positive components cannot be digested by amylase (Tables IV, V) and an attempt was, therefore, made to determine the nature of carbohydrates present in Zones C, D, E and F, . These zones secrete a small amount of a slightly acid mucopolysaccharide as is evident from reactions with Alcian Blue at p&I 2.6 and pK 1.0 (Table IV, Fig. 10). Saponification after methylation suggests that the mucosubstance is slightly sulphated; it is nevertheless PAS positive and is stained by Alcian Blue at pH 2.6 and by Alcian Yellow (Table IV). Zones F, and F4 give reactions which indicate that the same type of mucosubstances is secreted, but in larger amounts (Table V, Fig. 10). Positive staining in F, becomes intense in F4 to reach its climax near the oviduct. From Zone F, to Zone F4 the increase in sulphation is marked; mucins secreted in F4 are as highly sulphated as the
THE DOGFISH
SHELL GLAND
279
mucosubstances of Zone B. The lamellae goblet-cells lining that zone give the same reactions (Fig. 13). Zone Fs is the only one giving positive results to PAS and it is stained orange by newly prepared diamine (Spicer & Jarrels, 1961) so demonstrating the presence of neutral polysaccharides. Altogether, it seems that secretions which have no distinct localizations in Zone A (Figs. 10, 12) are found in specialized sites in Zone F, namely, neutral mucosubstance in Fz, weakly sulphated mucopolysaccharides in Fs, and highly sulphated mucopolysaccharides in F4 (Table V, Figs TABLEV Reactions for carbohydrates:
F2 Stage
Zones FZ, Fs, and F4.
F4
F3
I
II
III
I
II
III
PAS ++ 0 Schiff reac. AmylasefPAS + Alcian Blue pH 1 0 pH 2.6 0 Alcian Blue+Alc. 0 Yellow Ale. Bl. pH I aft. methylat. Ale. Bl. aft. met. & sapon. Toluid. Blue pH 5 0 pH3 0 pH1 0 Spicer I 0 Spicer II 0
++ 0 i0 0 0
++ 0 + 0 0 0
-I-
+
+
0 + + i-
0
0 + + +
+ + +
y-t-b1 y+bl
0 me+ 0 0 og 0
II
III
0 0 0 i-i-t-
0 0 0
0 0 0
i-t f
y+bl+
+ ySb1-t +
i-f +
0
0
me-l0 0 og 0
y-i-b1
I
0
0
me 0 0 0 0
me++ 0 me 0 0
me++ me me 0
y-kM-i-+
0
me-l0 0 0 0
me-l--i+
me+ 0 0
me+++ me-ime+ 0 gr
10-14). These observations are supported by metachromasia with Toluidine Blue at various pH values (Table V, Fig. 17), but Zones C, D, E and F, (Table IV, Fig. 12) show o~ho~hrom~ia and this coincides with the changes in the content of tyrosine residues (Fig. 16). Zones C, D, E and F, give equally positive reactions to several days old N,N-dimethylparaphenylenediamine chlorhydrate (Spicer & Jarrels, 1961) (Table IV, Fig. 14). A phenolic glycoprotein whose carbohydrate part is probably a sulphated mucopolysa~haride is present. Zone C is stained purple black; Zone D displays colours ranging from grey to deep purple-black (Fig. 14). The darker shade exhibited by the tubule proximal ends fades further from this area whether in the direction of the apical zone of the gland (where the three rows of ducts do not stain) or towards the duct distal ends. Zone E is stained deep purple black. Zone F1 behaves similarly, with some tubule cells becoming extremely black and this clearly shows the presence of sulphomucins. These sulphated mucopolysaccharides do not show metachromasia because of the phenolic protein basic sites (~5 reaction of FCF, Table II), they remain orthochromatic (Ganter & Jollbs, 1969, Vol. 1, p. 482).
280
M.RUSAOUtiN DISCUSSION
The foregoing histochemical investigations provide further evidence of the gland shell complexity. In Zones A and B together with Zones F2, F3, F4 slightly or strongly sulphated acid mucopolysaccharides and neutral mucopolysaccharides are secreted. These are apparently “free” according to Ganter & Jollts (1969) terminology, since this group of zones does not react to protein-tests. The range of various mucosubstances present is definitely wider than Threadgold (1957) was led to suppose. In the absence of a positive protein reaction in Zone A, he eventually dismissed his own hypothesis of an exclusive neutral mucopolysaccharide secretion. Mucosubstances are not, however, necessarily bound to proteins and free mucopolysaccharides could play their part in capsule formation. We must keep in mind the existence of four lines of dehiscence (Fig. 1) which open up when the dogfish embryo reaches 35540 mm in in size (Balfour, 1876; Ouang Te Yio, 1931). The PAS positive reaction of the shell mucous sealing was observed by Collenot (1966) who also noted it was partially stained by Alcian Blue at pH 2.0 and that it reacted in the same way as Zone A secretions. We are, therefore, entitled to suggest that the secretions of Zones A and B perform the sealing function for the apical slits and Zones F?, F, and F, for the proximal ones. A morphological description of the shell has already been given f Rusaoudn et al.. 1976). An attempt is now made to establish a relation between the protein components of the shell walls and the glandular secretions. The component of the shell material and the coiled threads are secreted in Zones C, D. E and F, . The external layer of the shell and the coils probably originate in Zones F, and E. Long thick threads of secretion which undergo a coiling process are often found in the lumen of Zone E tubules. Besides, the only layer in contact with sea water is comparatively thick and will undergo the most intense tanning. it is essentially formed of a glycoprotein which will be tanned by a polyphenoloxidase both sharing the same secreting sites which are also partially responsible for a peroxidase secretion. The latter is most abundant in Zones F, , F, and F,. The action of a peroxidase in such a quinone tanning process has often been demonstrated for invertebrates. It is associated with a phenolase in Lymneu (Timmermans, 1969), in O&zebra (Maschino & Vovelle, 1972), in Syllis rornuta (Michel & Vovelle, 1974). It is found to be the only tanning agent in the gasteropod radula (Ducros, 1967), and in the jaws of Glycera (Michel, Fonzt-Vignaux & Voss-Fucart, 1973). The lamellar structure of the shell middle layer (L,) may be due to the wide range of Zone D secretions, namely, phenolic glycoprotein, sulphated protein and tryptophan. At first sight the recurrent L, laminae seem to be identical. Careful observation, however, indicates changes from the outer region of the layer to the inner zone. The alternation of dark and light bands (Fig. 3), appears as the main pattern, but as the dark ones steadily become thinner, the light ones increase in thickness. When the dark ones are reduced to a mere film, the pattern is dislocated with
THE DOGFISH SHELL GLAND
281
a predominance of the light material. Light bands vary in size but their structure seems to remain constant; this does not apply to the dark ones which soon become subdivided into three strips, namely a dark central one, flanked on either side by lighter ones. The outer border region of the middle layer has granulations which are also found in the external layer; its bottom zone appears to recover some sort of regular pattern near the thin regular dense third layer {Fig. 3, L,).Together with our previous results on the amino-acid content of the shell and histoautoradiography of the gland, this hist~hemical investigation indicates that the middle layer includes at least three secretory components. Amino-acid estimations and shell fragment X-ray di~action have led us to conclude that collagen is one of them. Tritiated proline labelling has demonstrated that collagen synthesis occurs in the very narrow cells of Zone D, E and C tubules. These collagen-producing cells alternate with large prismatic cells in which the presence of SH groups (Zone D) and tyrosine residues (Zones C, D, E and F,) has been shown by histochemical methods (the numerous tyrosine residues are also evident in the amino-acid assays of the shell). The collagenic protein secretion of Zone D could be localized in the dark bands of the middle layer (L2) which would account for their striated appearance; however, collagen may be present elsewhere in the shell since this protein is equally secreted by Zones C and E, though in smaller amounts. Zone D is the site of a phenolic glycoprotein secretion already noted for Zones E and F1 which build the shell outer layer (L,); however, this secretion is less abundant and less tanned. A sulphated protein is also involved in this complex structure (Filhol & Garrault’s, 1938, “prokeratine”). As one of its components, it might be present especially in the light bands of the middle layer (Fig. 3, L,). We have given up the use of the word keratinization suggested to the first workers by the elastic and resistant quality of the structure. In the case of the egg-shell, the sulphated protein is not involved in an intracellular process but contributes to an anhystic steady individual structure. The keratinization which takes place in the vertebrate epidermis is of a totally different character. A similar function for the sulphated protein-phenolic association has, however, been demonstrated in the formation of nematode and ~~~~~s teguments (Lafon, 1943), mussel byssus (Pujol, 1967) and in some fish egg envelopes {Tide-marquis, 1973). No positive statement may be made regarding the localization of tryptophan in the shell. The indol nucleus of that amino acid present in Zones D and F, could be a component of the proteins already mentioned. The origin of the third shell layer (Fig. 3) is far from fully elucidated. From the present work we cannot attribute its existence to a definite zone in the gland but from its dense structure arising from a homogeneous matrix regularly interspaced with fine striations perpendicular to the middle layer bands, a possible hypothesis may be put forward. The upper part of Zone D is the site of collagenous and sulphur-containing protein secretions; it gives no positive reaction to test for phenols or tyrosine residues, and does not react to several days-old diamine, The specialization of that region in Zone D together with our present knowledge of the third layer may be regarded as a
282
M. RUSAOUfN
clue for their possible cannection. Finally, Zone C secretions must constitute the fourth layer. This zone, almost identical in function to Zones E and F, {Tables II, III? IV, Figs 15-19) does not react to tests for peroxidase, so that the inner layer of the shell owes its tanning to phenoloxidase only. The secretions of the dogfish shell gland and the mussel foot and rachis glands display striking similarities; included in the mussel gland secretions are a phenolase (Smyth, 1954), a glycoprotein with tyrosine residues and a sulphated protein (Pujot, 1967), a collageneous protein (Pujol et al., 1970; Pujol, Houvenagel & Bouillon, 1972), neutral and sulphated acid muco~oIysaccharides (Pujol, 1957). in spite of their differences their toughness is remarkably adapted to their common environment,
F. M., 1876. The deveIopment of efasmobranch fishes. J, Anur. P&W., VOL. IO, pp. 517-570. BORCLA,J., 1906. Recherches sur le syst&me urogenital des E~asmobranches. A&s Zaul. ~.xP. g&r., T.J. pp. 1991184. BROWN, c. H., 1950. Quin0ne tanning in the at&‘& kingdom. Nature, .&ond., Vol. 165, p. 275. BROWN. C. H,, 1952. Some structural proteins of Mytififs edulis. p. JI microsc. Sei., Vol. 93, pp. 487 --502. BROWN,c‘. H., 1955. Egg capsule proteins of selachians and trout. Q. JI. micrtw. Sci., Vol. 96, pp. 483.488. BUNKE, D., 1972. Skferotin-Komponenten in den Vitellucyten von ~~~~~~~~~~~ &e&r .~~~r~hi~d~ ITurbeharia), Z. Zellfosch. mikrusk. Anot., Bd 193, 5.383398, COLLENOT,G., 1966. Observations relatives au developpement en laboratoire d‘embryons et d’individus juv&iles de Scyliorhinus canicula L. Cub. Biol. mar., T. 7, pp 319-330. Duc~os, C., 1967. Contribution ii I’etude du tannage de Ia raduXa chez les Gasteropodes. Ann/s ~isfo~hem., T. 12, pp. 243-272. FAURF-FR~MMIET, E., 1938. Structure de la capsure ovuiaire chez yuetques Waciens. Archs Anat. nricrosc. borax. exp.. T. 34, pp. 23-S I. FAW&-FR&XET, E. & C. BAVOOUY,1938. Sur Sovoktratine des SBlaciens. Bdf. SW. Ckitiz. hid.. T. 20, pp. 14-23. FAURZ.!-FR~MIET, E. & H. GARRAIJI;~,1938. Separation d’une prokeratine stcretee par la glande nidamentaire de Raja batis L. Bull. Sue. Chim. biol., T. 20, pp. 24-30. FILHOL,J. & H. GARRAULT,1938. La secretion de la prokeratine et la formation de la capsule ovulaire chez ies Selaciens. Archs Anut. microsc. Morph, exp., T. 34, pp. 105-145. GABE, M., f968. Techigues h~sfo~o~~q~es. Masson et Cie, Paris, f , t 13 pp. G.W’TER,P, & G. JOLL&S,1969, Nisfuc&&? norm& et ~~fbo~o~jq~~. f et if. Gauthier-Viilars, Paris. 1902 pp. GARRA~LT, H. & J. FILHOL, 1937. L’organe nidamentaire des Elasmobranchz~ et son role darts la formation de la capsule de l*oeuf. C. r. S&WC. Sot. Biol., T. 126, pp. 773.-775. HENNEGUY, C. F., 1893. Sur la structure de la glande nidamentaire de l’oviducte des S&aciens. C. r. Sot. Philomafique, 86me stie, T. 5. KRISHNAN,G., 1959. Histochemical studies on the nature and formation of egg capsules of the Shark C~~~osc~~l~u~griseum. Biol. Bull mar. biol. Lab., Woods Hole, Vol. 117,pp. 298-303. LAFON, M., 1943. Sur ia structure et la composition chimique du tegument de ta limuie Xiphosurrr ~#i~~~~~~#~ L. Btdf. Znrf. O&nap. Monaco, No. 850, I t pp. MASCHINO,F. & J. VOVELLE,1972. Comparaison du bourrelet pall&l posterieur et du disque operculigere chez Ocinebra erinucea L., Haliotis, Vol. 2, pp. 187-190. MICWEL,C., FONZ~-VIGNAUX,M.-T%, & M. F, VOS~-FOWART, 1973. Dorm&es nouvelles sur la morphologic, I’histocbimie et la composition chimique des m&&oires de G&vwn conooMa Keferstein f&n&de polychetef. B&i. bioi. Fr. Be&., T. 107. pp. 30I-XI. BALFOCK,
THE DOGFISH
SHELL GLAND
283
MICHEL,C. & J. VOVELLE,1974. Tannage quinonique des soies chez Syllis corm&a R. Bull. Sot. 2001. Fr., T. 99, pp. 175-181. NALINI, K., 1944. Structure and function of the nidamental gland of Chiloscyflium griseum (Miill. et Hen&). Proc. Zool. Znd. Acad. Sci., Vol. 12 B, pp. 189-214. OUANG TE YIO, 1931. La glande de l’tclosion chez les Plagiostomes. Annls. Inst. Oceanogr. Paris, T. 10, pp. 283-370. PERRAVEX,G., 1884. Sur la formation de la coque des oeufs de Scyliium canicula et de Scyllium catulus. C. r. hebd. Seanc. Acad. Sci., Paris, T. 99, p. 1080. PUJOL, J. P., 1967. Le complexe byssogkne des Mollusques bivalves. Histochimie cornparke des &r&ions chez Mytilis edulis L. et Pinna nobilis L. Bull. Sot. linn. Normandie, T. 8, pp. 308-332. PUJOL, J. P., M. ROI.LAND,S. LASRY& S. VINET, 1970. Comparative study of the amino-acid composition of the byssus in some common bivalve molluscs. Comp. Biochem. Physiol., Vol. 34, pp. 193-201. PUJOL, J. P., G. HOLJVENAGHEL SCJ. BOUILLON,1972. Le collag&ne du byssus de Mytilus edulis L. Ultrastructure des cellules secretrices. Archs Zool. exp. gin., Vol. 113, pp. 251-264. RUSAOUBN,M., J. P. PUJOL, J. BOCQUET,A. VEILLARD& J. P. BOREL, 1976. Evidence of collagen in the egg-capsule of the dogfish, Scyliorhinus canicula L. Comp. Biochem. Physiol., Vol. 53, B, pp. 539-543. SMYTH,J. D., 1954. A technique for the histochemical demonstration of polyphenol oxidase and its application to egg-shell formation in helminths and byssus formation in Mytilus. Q. JI. microsc. Sci., Vol. 95, pp. 139-152. SPICER, S. S. & M. H. JARRELS, 1961. Histochemical reaction of an aromatic diamine with acid groups and periodate engendered aldehydes in mucopolysaccharides. J. Histochem. Cytochem., Vol. 9, pp. 368-379. STRAWS,W., 1971. Inhibition of peroxidase by methanol and by methanol nitroferricyanide for use in immuno peroxidase procedures. J. Histochem. Cytochem., Vol. 19, pp. 682-688. THREADGOLD,L. T., 1957. A histochemical study of the shell gland of Scyliorhinus caniculus. J. Histochem. Cytochem., Vol. 5, pp. 159-166. TIBERI-MARQUIS,J., 1973. Quelques aspects histochimiques de l’ovogen&se chez Betta splendens R. (T&Cost&n Anabantidb). Histochemie, T. 33, pp. 139-158. TIMMERMANS, L. P., 1969. Studies on shell formation in Molluscs. Netherlands J. Zool., Vol. 19, pp. 417-523. VOVELLE,J., 1965. Sur la presence des groupes SH et SS dans la s&r&ion de la glande nidamentaire chez Scyliorhinus canicula L. C. r. hebd. SPanc. Acad. Sci., Paris, Vol. 260, pp. 5945-5947. WASCHTEIN,M. & M. MEISEL, 1964. Demonstration of peroxidase activity in tissue sections. J. Histochem. Cytochem., Vol. 12, pp. 538-543. WIDAKOWICH,V., 1906. Uber Bau und Funktion des Nidamental Organs von Scyllium canicula. Z. wiss. Zool., Bd 80, S. l-21.
Publishing Company
THE DOGFISH SJJELL GLAND, A HISTOCHEMICAL STUDY M. RUSAOUEN Luboratoire de Cytologic, UniversitP Paris VI, Quai St. Bernard, Paris, France Ahstraet: On the basis of previous work and new ~stoche~cal results, the dogfish nidamentat gland is divided into six distinct zones. Several neutra1 or acid mucopolysaccharides are present; the latter are more or less sulphated. Various proteins including a phenolic glycoprotein tanned by a phenolase and a peroxidase, a sulphur containing protein, and collagenons protein are also present. The connection between the zones of secretion and the layers of the egg-shell layers is discussed.
For more than a century a large number of studies of the egg caps&e of selachians (Fig. 1) have been made in an attempt to determine the composition of the secretions of the capsule-making nidamental gland (Fig. 2). Perravex (1884), Henneguy (1893), Widakowich (1906) and Borcea (1906), all demonstrated the lamellar-moulding lamellae in the gland. In a comprehensive study of the scleroproteins, Garrault & Filhol (1937) Fat&-FrtSmiet (1938), FaurBFrimiet & Baudouy (1938), Filhol & Garrault (1938), FaurC-Fremiet & Garrault (1938), attempted to explain the processes which take place in the formation of these eggs. On the basis of their histochemical and biochemical evidence they concluded that a sulphur-containing protein called “prokeratine”, which is chemically closely related to keratin is elaborated in the gland and secreted by its shell-shaping zone. They supposed that before its passage through that zone the egg was first enclosed in albumen secreted by the apical or “albumen secreting” zone of the gland. Twenty years later Threadgold (1957), investigated the problems raised by the formation of nidamental gland of Scyliorhinus canicula basing part of his work on Brown’s (1950, 1952, 1955) studies on quinone tanning systems in the animal kingdom. The evidence for the presence of aphenolic protein and a polyphenoloxydase in the gland led him to conclude that the egg-capsule was formed by quinone tanning. He did not, however, mention the presence of sulphur containing components, previously demonstrated by Faure-Fremiet and his co-workers, but he showed the presence of a PAS positive substance in the apical zone of the gland, possibly due to neutral mucopolysaccharides. The secretion of this zone could then no longer than be regarded as albumen, thus confirmed Borcea’s (1906) doubts as to the albuminoid nature of the secretion. The work of Krishnan (1959) supported and widened the evidence for quinone tanning in the formation of the egg-capsule; his histochemical study of the nidamental gland and egg-capsule of the C~iZoscylZi~~griseum was supported by a chromato267
268
graphic analysis
M.RUSAOUEN
of the capsule components:
he suggested that the capsule protein was
of a collagen type, but makes no reference to the presence of the sulphur-containing protein noted by FaurC-FrCmiet. Vovelle’s work (1965) removed any possible doubt regarding the existence of such a protein in the gland. After having demonstrated the evidence of S-radicals, he concluded that the secretion of a protein containing SH groups is not at all inconsistent with the presence of a phenolic protein and a polyphenoloxydase which account for quinone tanning. We have already applied biochemical and histoautoradiographic techniques to show the presence of collagen in the egg capsule of Scyliorhinus canicula, and to determine which type of the nidamental gland cells gives rise to this secretion (Rusaouen et al., 1975). The present paper endeavours to provide new data regarding other secretory products within the various zones of the shell gland.
MATERIALS AND METHODS
The shell glands of dogfish were removed immediately after killing by beheading. Each gland was split into longitudinal halves which were fixed in neutral formaldehyde, Carnoy’s fluid, Bouin and Gendre’s fluids. Cryostat sections of the gland were also cut. A fragment of the ovary was removed from every sacrificed animal and used to stage the gland as regards its maturity. Three stages were recorded. Each half gland was embedded in paraffin and 7.5 pm sections subjected to the following histochemical tests’. Proteins General reaction, biuret; basic proteins, FCF (Alfert & Geschwind); aromatic radicals, Morel & Sisley’s reaction (Glenner & Lillie); Phenolic groups: azoreaction (diazo sulphanilic acid-fast blue B pH 9.2). Indol and pyrol radicals, Glenner’s rosindol reaction. Sulphydryl and disulphydryl groups, DDD (Barnett & Seligman); RSR (Bennet-Pearse); blocking; sodium iodoacetate or N-ethylmaleimide, reduction by sodium thioglycolate after blocking and followed by DDD reaction; Adams-Slopper’s
reaction.
Carbohydrates: poiysaccharides and acidic or neutral mucopolysaccharides Hotchkiss-MacManus’s reaction; similar test without preliminary oxidation, after amylase digestion, and after dimedone blocking (Bulmer, 1959); metachromatic basophilia with Toluidine Blue (0.1 74) pH 1.O, pH 3.0 and pH 5.0 in Walpole M/50 buffer; Lison, pH 1.0, pH 2.6; Alcian blue colouration (Mowry) with or without Hotchkiss-MacManus’s reaction, with methylation and methylation-saponification controls (Fisher & Lillie); Alcian blue-Alcian yellow staining (Ravetto): aqueous ’ References to well-known manuals (Gabe, 1968; Ganter
histochemical techniques which have been described & Jolk?s, 1969) are not given in the bibliography.
in specialized
THE DOGFISH
269
SHELL GLAND
buffered solution of N,N-dimethyl-p-phenylenediamine several days old (Spicer & Jarrels, 1961).
HCI, freshly prepared
or
Enzymes
Fresh tissue cryostat sections were incubated in DOPA (dihydroxyphenylalanine, Gomori’s variant) or pyrocatechol (Smyth, 1954). In either case controls were made by simultaneous inhibitions with sodium diethyldithiocarbamate (Bunke, 1972). Sections were post-fixed in saline neutral formaldehyde. Peroxidase. Benzidine tests (Wachstein & Meisel, 1964) with inhibitor control of methanol ferricyanure (Straus, 1971). Phenolases.
RESULTS
On the grounds of his own histochemical investigations and previous morphological work, Threadgold (1957) identified five distinct zones in the shell gland: Zone A, albumen zone of Filhol & Garrault (1938); Zone B, metachromatic duct of Filhol & Garrault (1938); Zone C, “Three or four ducts parallel with and following zone B” (Threadgold, 1957); Zone D, prokeratine region of Filhol & Garrault (1938); and Zone E, caudal shell secreting zone of Nalini (1944). To those five zones we shall add a sixth, Zone F (Fig. 4) which runs parallel to Zone E from which it is separated by a conjunctive wall. Subsequently, we shall discuss the significance of this new zone whose tubules lie perpendicular to the tubules of Zone E and open between short lamellae quite similar to those found in Zone A (Fig. 5). In order to study the evolution of the nature and localization of the glandular secretions, three stages of its development have been selected, namely, Stage 1 (Fig. 6a) before the vitellogenesis of ovocyte maturation, Stage 2 (Fig. 6b) after vitellogenesis, and Stage 3 egg-laying activity (Figs 1, 2 and 6~). At first sight some distinction may be drawn between two regions of the gland, the first of which is positive to most carbohydrate tests (Table I) and includes Zones A and B, while the second one reacts to the test for proteins and is made up of Zones C, D, E, and F (Table II). A more detailed analysis of the results shows that in toto the egg-shell gland is much more complex, but for clarity we shall successively deal with results obtained. ZONES
A
AND
B:
CARBOHYDRATES
Hotchkiss-MacManus
reaction
From its early stage, Zone A reacts intensely to PAS (Table I); numerous granules are seen in the apical region of cells (Fig. 7). In Stages 2 and 3, the secretion is present everywhere in the cells (Fig. 8). Zone B gives no reaction to PAS at any stage (Fig. 8).
Fig. Fig. Fig. Fig.
Egg-capsule of the dogfish, Scyliorhinus canicula: c, coils; st, stomium (lines of dehisc :enc:e). Fig. 2. Half nidamenta1 gland, internal view: La, lamellae; Ov, oviduct. T’ransverse section of the egg-shell: Ll, outer layer; L2, second layer; LB, third layer; L4 ,in ner layer. Diagram of a longitudinal section of a half gland showing the different secretory arer 1s: (3v, oviduct; A, B, C, D, E, Fr , F1, F3, F,, the various zones. Longitudinal section of the gland: PAS reaction and picroindigocarmine: D, Zone D: E. Zone E, F, Zone F; la, lamellae; al, alveolae; cw, conjunctive wall; bv, blood vessel.
THE DOGFISH
Fig. 6. Longitudinal
SHELL GLAND
271
section of the gland: a, Stage I; b, Stage II; c, Stage III.
Fig. 7. L,ongitudinal section through Zone A of a gland at stage I: PAS reaction: t, tubule; lu, lu Imen; g, PAS-positive granules. Fig. 8. Longitudinal
section through Zones A and B of a gland at Stage III: t, tubule; lu, luunen; note all cells filled with PAS-positive granules.
The PAS positive secretion of Zone A is no more affected by amylase than it is by saliva {Threadgo~d, 1957). Rimedone controls on para& sections con~rm these results; however, weak but definite evidence of glycogen in Zone A is obtained with cryostat sections after 3-4 h dimedone blocking. Glycogen is present in various amount in all the tubules of Zone A. A difference in the state of the purple cotouration with PAS is observed between the sections subjected to dimedone blocking and those which are not, suggesting that other PAS positive substances are secreted by Zone A, especially neutral or slightly acid mucopolysaccharides which are apparently free since Zone A gives no reaction to tests for proteins. The enzymatic digestion of glycogen by the amyfase may pass unnoticed because of mvcosubstances in large amounts (Table I).
The presence of neutral and more or less acid mucopolysaccharides is demonstrated by Atcian Blue at pH 1.0 and pH 2.6 and Alcian Blue-Alcian Yellow, Toluidine Blue at various pi-I values (Table I). Only the ~otchkiss-~a~~anus reaction (Fig. 9) and TABLE1 Reactions for carbohydrates in Zones A and B at stages J, II, and Hi; -; ~ weak reaction; : . medium positive reaction; $- -t-I-, intense reaction; 0, no reactiort; y, yetlow; bf, blue; me, me&%chromasia; or, orthochromasia; ~g, orange; gr, grey; bk, black; br, brown; Spicer f, newly prepared diamine HCl; Spicer II, several days-old diamine HCJ.
Stage .--~-----.~-PAS Schiff reac. Amylase -!-PAS Dimedone I-PAS At&an BJue PR 1
pEf 2,6
Ale. Bit&~;--AIc. YeJIow Methylation$-Ale. Blue Saponificat. aft, Methyl. Toluid. Blue PR 1 PH3 pH 5
_._.” .___.~__..__-_.._.-..-.. A _-_.--_... ._._._^...^__._._._. ._ I II 111 -- _I._-l-.___.. .i { i. _. _; j -1. 0 0 ‘0 .: _i. t ! _,_ j. .!. -+
-‘I-“7 i_2.. y i_bJ me ..: me.). me
: i i ___ y : bl or
4 me
or-+me or 3 me
Spicer I
or
og :-gr
Spicer l’f
gr
hr . gr
-! _ _I
Zone
_...“._ ..-. B
J _~_._. ..____.. 0 0 0
I : . j_ bJ
KJ .._. _”
Ill _.. . 0 0 0
0 0 0
. :. :.
: i I
“bl
bl 0 0
y+bJ 0 0 or 4, me or i- me or ! me
me+ .I -. mc 4 Ij__. me / -‘. i me-J. _i__i me-~ ! ..i. mu !__t_i ,J,e: : me i Ime! 1 :
O&-igr br : bk
bk .I
0
0 hk ‘.
0 bk _ !..?
dicblorhydrate N,N-dietbylparaphenylene diamine fresh solution (Spicer & Jarrels, 1961) give a positive reaction in the outer region of the apicai zone on both sides of the gland so that onfy neutral mu~opo~ysa~charides are secreted by that region which
THE DOGFISH
SHELL GLAND
273
Figs 9 to 20. Drawings of longitudinal section of a half gland at stage III: 111111intense reaction; 1111111 medium positive reaction; 111111weak reaction. Fig. 9. PAS reaction. Fig. 10. Alcian Blue reaction: \\\\\ pH 1.0; ////// pH 2.6. Fig. 11. Toluidine Blue reaction: pH 1.O; metachromasia. Fig. 12. Toluidine Blue reaction: pH 1.0; orthochromasia. Fig. 13. Newly prepared diamine chlorhydrate reaction. Fig. 14. Several days-old diamine.
M. RUSAOUk?N
274
includes the distal ends of the tubules. Yet all tubules in Zone A give this type of secretion, in various amounts, along the whole of their length but it is much more evident in the outer region (Fig. 13). The older the gland, the smaller the main neutral mucopolysaccharide secreting region seems to be. Considering the production of each individual mature tubule on all its length, we find it includes all types of mucopolysaccharide secretion; and the same process of simultaneous secretion applies to acid mucosubstances. On the inner side of the gland near its lumen, the mixture of secretions is obvious; Alcian Blue-Alcian Yellow (Ravetto, 1964) give a distinct green colouration of some cells or cells in the same part of tubule are alternatively coloured yellow and blue. When using Alcian Blue at pH 1.0 and 2.6, changes of localization are noted in the coloured areas (Fig. 10) and similar changes are observed after the orthochromatic and metachromatic reactions of Toluidine Blue at pH 3.0 and 1.O (Figs 11, 12). Methylation-blocking followed by saponification indicates one is dealing here with sulphated acid polysaccharides. They are more or less sulphated, and the variations account for their reactivity to PAS as well as the differences observed in the reactions to Alcian Blue at pH 2.6 (Fig. 10) and Toluidine Blue (Figs 11, 12). Localization of the most strongly sulphated mucosubstances may be precisely determined by the Toluidine Blue test at pH 1.O. A strong metachromasia characterizes the inner fringe of the gland and is still visible, though less intensely. at the centre of Zone A at its inner part (Fig. I I ). It is easier to determine the nature of Zone B secretions since the Hotchkiss-MacManus reaction is absolutely negative (Table I, Figs 8,9) and all further tests confirm the presence of strongly sulphated mucopolysaccharides (Figs lo--14 and Table I). ZONES
C, D, E, F
Proteins These four zones give strongly positive reactions to the Biuret test (Table II). which was not the case for Zones A and B. From the various protein tests we may conclude that Zones C, E, and F, (Fig. 4) give similar reactions; the range of Zone D secretions is more diversified. Aromatic and phenol radicals Zones C, E, F, give an abundant secretion of a phenolic protein with many tyrosine residues. The gland in its early stage shows a strongly positive azoreaction in Zones C. E, F, (Table II) as does Zone D in Stages II and III though the reaction is weaker (Fig. 15). Identical conclusions may be drawn from diazo-sulphanilic acid test though they give a decidedly lighter staining. The presence of tyrosine residues is demonstrated by Morel & Sisley’s reaction which gives intense staining in the tubules of Zones C and E, and distinct ones in the tubules of Zones D and F, (Fig. 16). The localization of phenolic proteins indicates that they are not secreted in all the tubules of Zone D at an early stage; the only region involved appears as an oval-shaped area,
THE DOGFISH
SHELL GLAND
Fig. 1.5. Azoreaction. Fig. 16. Morel & Sisley’s reaction for tyrosine residues. Fig. 17. DDD reaction. Fig. 18. Pyrocatechol reaction. Fig. 19. DOPA reaction. Fig. 20. Peroxidase reaction.
275
M. RUSAOUBN
276
localized in the outer part of Zone D, and which includes the distal ends of several tubules.
Protein detection in Zones C, D, E, and FL at Stages I, II and III. __“-_-----.-~.C -
Stage
i-
0
FCF
. + Azoreaction Morel&Sk. _. i DDD 0 DDD after 0 ethylmal. 0 DDD af. ethyl.
.+.+
-
- .---_-.
Ill
I
II
_~..~
.~._^-- ._... III
j_
++.+
f_“&., +.:
+.i_+-
++-~
.
C
fi-+-
f +- i
:
, _t.
br br
br
0
0
br
0
br
br
o
0
0
0
i-
Ii
_i__.{_ /. .
III
I
, + - -4 : j
‘_
.^ ._~^I.- .FT.-.._ ______.
l_.- _--.
1
0
.-- _- .__ ._ E
i _j_+ .! _+.+ ; _
+_ .j_
$__$ _+ .+_+
Zone
..-_
D
---
Ii
I Biuret
.-..
i.
-++
iI
II
III
~_
.; !
()
:
:
I .j.
: , i_. _++ :_ i ! ! j .j_<_a_ .:_.:.
;.i. :. _-L
0
0
.
0
br br
br br
0’ 0
br hr
br br
/
t
0
hr
br
0
br
br
0
0
& thioglycol. RSR RSR
0
after Na iodoac. Adams-Sloper Rosindole
0 0
0 0
0 0
0
0 0
0
0
0 5
~.
0
tt
0
0
_-
Enzymes Phenolases. The phenolase test gives markedly positive results in Zones C, E, F and
a slight reaction in a localized area of Zone D (Table III). Zone C reacts to DOPA and pyrocatechol (Figs 18, 19); this result is confirmed by sodium diethyldithioTABLE 111
Enzyme reactions at Stage III in Zones C, D, E, F1, F1 and FJ. Zone
._.. .~_.“_-_ Pyrocatechoi. Pyrocatechob -+dieth. thioc. DOPA DOPA+dieth. thioc. Benzidine Eknzidine af. methanol
c _. _.._ : !., 0 1 0 0 0
D
0 0 0 0 0
__.~ -._._. - --. “. E Fz F3 F, _ ___.____ __ - __._ ..i ;.j_ () 0
.--F4 ..-_--. a
0
0
0
0
0
0 0 0
0 a 0
0
(i
0
0 --
0
0
carbamate blocking, and provides further evidence to support Threadgold’s theory (1957) of an “autoquinone tanning” process, involving a phenol&protein and a phenolase secreted by the same cells. Zone D shows a slight reaction to pyrocatechol
THE DOGFISH SHELL GLAND
277
in a limited area which is the site of maximum secretion of the phenolic protein. There is no reaction to DOPA (Figs 18, 19). The whole of Zone E gives a similar positive reaction to pyrocatechol but is only tenuously stained with DOPA (Table ID, Figs 18, 19). Inside Zone F, F, gives a markedly positive reaction to pyrocatechol and a weak reaction to DOPA, while DOPA is highly reactive in F, and F, . Peroxidase. The sites of peroxidase secretion are localized by the reaction of Wachstein & Meisel (1964). These sites only partly coincide with those giving the DOPA reaction. Methanol-blocking-control (Straus, 1971) confirms the presence of peroxidase in Zones F, , F, , F4. No peroxidase is found in Zone C (Table III, Fig. 20). Sulphur groups Sdphydryl radicals. DDD and Mercury Orange were used by Vovelle (1965) to demonstrate SH groups, which we now regard as strictly limited to Zone D. We come to that conclusion for the following reasons. After diethylmaleimide or sodium iodoacetate-blocking, the brown colouration of Zones C, E and F, obtained with DDD is persistent and even more intense. After the SH groups have been blocked, Mercury Orange stains Zones C, E, and F, a bright orange; the reaction occurs irrespective of the type of inhibitor. Only Zone D gives positive results to current staining and controls (Table II). Disulphide radicals. No significant reaction to Alcian Blue after periodic oxidation (Adams-Slopper) is obtained in Zones E and F, (Table II) and the presence there of SS groups is, therefore, questionable. The very slight staining may be due to acid mucopolysaccharides; our interpretation is grounded on a positive Adams-Slopper’s reaction in Zones A and B, on a positive orthochromatic Toluidine Blue reaction in Zones C, D, E and F1 (Table IV, Fig. 13) and on positive reactions by several days-old diamine in Zones A and B, and in Zones C, D, E, F, and F4 (Tables I, IV, V, Fig. 14). Zone D is the only one to give positive reaction to the DDD test after N diethylmaleimide and sodium thioglycolate, which leaves little doubt as to the presence of a limited amount of disulphide groups (Table III). Tryptophan
The Rosindol test (Table II) indicates the presence of indol radicals in the large cells of Zone D tubules (Henneguy, 1893; Nalini, 1944), as well in Zone F, tubules. Though less conspicuous than reactions for tyrosine and sulphur, Rosindol Blue nevertheless involves the same cells in Zone D and is identical to the tyrosine localization in Zone F, . Zones C and E do not stain. We find here a very unusual example -of a secretion common to Zones D and F, , yet lacking in Zones C and E. Carbohydrates Hotchkiss-MacManus reaction. The contrast between Zone A and the other zones (C, D, E, F) is so striking after the reaction to PAS, that the latter could be regarded
1%.RUSAOUEN
278
as giving totally negative results, but closer examination shows that the proximal ends of the tubules in zones C, D and E as well as the alveola-shaped infoldings of Zone D lamella give a positive reaction. The same positive reaction is equally visible in Zone F,, F2, and Fs (Tables IV, V, Fig. 9). These positive PAS tests are obtained in cells and tubule lumen when the gland is in Stage I; later, the reaction seems essentially to affect elements inside the lumen. Even so, positive staining may be seen in the cells of Zone D alveolae in cryostat sections after dimedone blocking so that glycogen is probably present in the alveola cells, while a PAS positive mucopolysaccharide reaction accounts for the positive reactions in the proximal ends of tubules of Zones D and E and on the whole length of the much shorter ducts of Zones F, . F, and F,, TABLEIV
Reactions for carbohydrates: Zones C, D, E, and F1L _ ^.._..__ C ______
Stage
1 PAS &hit%-reac.
0
.-_. ..-.. ” __I.._.
D
0
1
0
II
0
.-“._._.- -..-..---
III
0
_
E
_ .___-.-_.. _-
11 III
0
Zone
1
0
It
.___..~ Ill I I[ III -_..I. - -...__-
-
0
0
Amylase-+-PAS Alcian Blue pH 1 pH 2.6 Alcian Blue-talc. Yell. Ale. BI. pH 1 aft. methyl. Ale. B1. aft. meth. 8r Sapon. Toluid. Blue pH 5 pH3 PH 1 Spicer 1 Spicer II
FL
-
0 0
0 0
0 0
0
0
0
0
0
(1
0
0
0 0
0
0
0
0
0
0
0 0
?
v
0
4
LI
0
0
or or 0 0 0
or or ar gr gr
or 0 or gr gr
or or 0 0 0
or or or gr gr
or or or sr gr
or or or 0 0
or
or
Or
r,r or
gr bk
0 0
or or or w bk.
*r or or u bk
?
or, 1 EC
or
or
1-
Pdysaccharide components, The whole of the PAS positive components cannot be digested by amylase (Tables IV, V) and an attempt was, therefore, made to determine the nature of carbohydrates present in Zones C, D, E and F, . These zones secrete a small amount of a slightly acid mucopolysaccharide as is evident from reactions with Alcian Blue at p&I 2.6 and pK 1.0 (Table IV, Fig. 10). Saponification after methylation suggests that the mucosubstance is slightly sulphated; it is nevertheless PAS positive and is stained by Alcian Blue at pH 2.6 and by Alcian Yellow (Table IV). Zones F, and F4 give reactions which indicate that the same type of mucosubstances is secreted, but in larger amounts (Table V, Fig. 10). Positive staining in F, becomes intense in F4 to reach its climax near the oviduct. From Zone F, to Zone F4 the increase in sulphation is marked; mucins secreted in F4 are as highly sulphated as the
THE DOGFISH
SHELL GLAND
279
mucosubstances of Zone B. The lamellae goblet-cells lining that zone give the same reactions (Fig. 13). Zone Fs is the only one giving positive results to PAS and it is stained orange by newly prepared diamine (Spicer & Jarrels, 1961) so demonstrating the presence of neutral polysaccharides. Altogether, it seems that secretions which have no distinct localizations in Zone A (Figs. 10, 12) are found in specialized sites in Zone F, namely, neutral mucosubstance in Fz, weakly sulphated mucopolysaccharides in Fs, and highly sulphated mucopolysaccharides in F4 (Table V, Figs TABLEV Reactions for carbohydrates:
F2 Stage
Zones FZ, Fs, and F4.
F4
F3
I
II
III
I
II
III
PAS ++ 0 Schiff reac. AmylasefPAS + Alcian Blue pH 1 0 pH 2.6 0 Alcian Blue+Alc. 0 Yellow Ale. Bl. pH I aft. methylat. Ale. Bl. aft. met. & sapon. Toluid. Blue pH 5 0 pH3 0 pH1 0 Spicer I 0 Spicer II 0
++ 0 i0 0 0
++ 0 + 0 0 0
-I-
+
+
0 + + i-
0
0 + + +
+ + +
y-t-b1 y+bl
0 me+ 0 0 og 0
II
III
0 0 0 i-i-t-
0 0 0
0 0 0
i-t f
y+bl+
+ ySb1-t +
i-f +
0
0
me-l0 0 og 0
y-i-b1
I
0
0
me 0 0 0 0
me++ 0 me 0 0
me++ me me 0
y-kM-i-+
0
me-l0 0 0 0
me-l--i+
me+ 0 0
me+++ me-ime+ 0 gr
10-14). These observations are supported by metachromasia with Toluidine Blue at various pH values (Table V, Fig. 17), but Zones C, D, E and F, (Table IV, Fig. 12) show o~ho~hrom~ia and this coincides with the changes in the content of tyrosine residues (Fig. 16). Zones C, D, E and F, give equally positive reactions to several days old N,N-dimethylparaphenylenediamine chlorhydrate (Spicer & Jarrels, 1961) (Table IV, Fig. 14). A phenolic glycoprotein whose carbohydrate part is probably a sulphated mucopolysa~haride is present. Zone C is stained purple black; Zone D displays colours ranging from grey to deep purple-black (Fig. 14). The darker shade exhibited by the tubule proximal ends fades further from this area whether in the direction of the apical zone of the gland (where the three rows of ducts do not stain) or towards the duct distal ends. Zone E is stained deep purple black. Zone F1 behaves similarly, with some tubule cells becoming extremely black and this clearly shows the presence of sulphomucins. These sulphated mucopolysaccharides do not show metachromasia because of the phenolic protein basic sites (~5 reaction of FCF, Table II), they remain orthochromatic (Ganter & Jollbs, 1969, Vol. 1, p. 482).
280
M.RUSAOUtiN DISCUSSION
The foregoing histochemical investigations provide further evidence of the gland shell complexity. In Zones A and B together with Zones F2, F3, F4 slightly or strongly sulphated acid mucopolysaccharides and neutral mucopolysaccharides are secreted. These are apparently “free” according to Ganter & Jollts (1969) terminology, since this group of zones does not react to protein-tests. The range of various mucosubstances present is definitely wider than Threadgold (1957) was led to suppose. In the absence of a positive protein reaction in Zone A, he eventually dismissed his own hypothesis of an exclusive neutral mucopolysaccharide secretion. Mucosubstances are not, however, necessarily bound to proteins and free mucopolysaccharides could play their part in capsule formation. We must keep in mind the existence of four lines of dehiscence (Fig. 1) which open up when the dogfish embryo reaches 35540 mm in in size (Balfour, 1876; Ouang Te Yio, 1931). The PAS positive reaction of the shell mucous sealing was observed by Collenot (1966) who also noted it was partially stained by Alcian Blue at pH 2.0 and that it reacted in the same way as Zone A secretions. We are, therefore, entitled to suggest that the secretions of Zones A and B perform the sealing function for the apical slits and Zones F?, F, and F, for the proximal ones. A morphological description of the shell has already been given f Rusaoudn et al.. 1976). An attempt is now made to establish a relation between the protein components of the shell walls and the glandular secretions. The component of the shell material and the coiled threads are secreted in Zones C, D. E and F, . The external layer of the shell and the coils probably originate in Zones F, and E. Long thick threads of secretion which undergo a coiling process are often found in the lumen of Zone E tubules. Besides, the only layer in contact with sea water is comparatively thick and will undergo the most intense tanning. it is essentially formed of a glycoprotein which will be tanned by a polyphenoloxidase both sharing the same secreting sites which are also partially responsible for a peroxidase secretion. The latter is most abundant in Zones F, , F, and F,. The action of a peroxidase in such a quinone tanning process has often been demonstrated for invertebrates. It is associated with a phenolase in Lymneu (Timmermans, 1969), in O&zebra (Maschino & Vovelle, 1972), in Syllis rornuta (Michel & Vovelle, 1974). It is found to be the only tanning agent in the gasteropod radula (Ducros, 1967), and in the jaws of Glycera (Michel, Fonzt-Vignaux & Voss-Fucart, 1973). The lamellar structure of the shell middle layer (L,) may be due to the wide range of Zone D secretions, namely, phenolic glycoprotein, sulphated protein and tryptophan. At first sight the recurrent L, laminae seem to be identical. Careful observation, however, indicates changes from the outer region of the layer to the inner zone. The alternation of dark and light bands (Fig. 3), appears as the main pattern, but as the dark ones steadily become thinner, the light ones increase in thickness. When the dark ones are reduced to a mere film, the pattern is dislocated with
THE DOGFISH SHELL GLAND
281
a predominance of the light material. Light bands vary in size but their structure seems to remain constant; this does not apply to the dark ones which soon become subdivided into three strips, namely a dark central one, flanked on either side by lighter ones. The outer border region of the middle layer has granulations which are also found in the external layer; its bottom zone appears to recover some sort of regular pattern near the thin regular dense third layer {Fig. 3, L,).Together with our previous results on the amino-acid content of the shell and histoautoradiography of the gland, this hist~hemical investigation indicates that the middle layer includes at least three secretory components. Amino-acid estimations and shell fragment X-ray di~action have led us to conclude that collagen is one of them. Tritiated proline labelling has demonstrated that collagen synthesis occurs in the very narrow cells of Zone D, E and C tubules. These collagen-producing cells alternate with large prismatic cells in which the presence of SH groups (Zone D) and tyrosine residues (Zones C, D, E and F,) has been shown by histochemical methods (the numerous tyrosine residues are also evident in the amino-acid assays of the shell). The collagenic protein secretion of Zone D could be localized in the dark bands of the middle layer (L2) which would account for their striated appearance; however, collagen may be present elsewhere in the shell since this protein is equally secreted by Zones C and E, though in smaller amounts. Zone D is the site of a phenolic glycoprotein secretion already noted for Zones E and F1 which build the shell outer layer (L,); however, this secretion is less abundant and less tanned. A sulphated protein is also involved in this complex structure (Filhol & Garrault’s, 1938, “prokeratine”). As one of its components, it might be present especially in the light bands of the middle layer (Fig. 3, L,). We have given up the use of the word keratinization suggested to the first workers by the elastic and resistant quality of the structure. In the case of the egg-shell, the sulphated protein is not involved in an intracellular process but contributes to an anhystic steady individual structure. The keratinization which takes place in the vertebrate epidermis is of a totally different character. A similar function for the sulphated protein-phenolic association has, however, been demonstrated in the formation of nematode and ~~~~~s teguments (Lafon, 1943), mussel byssus (Pujol, 1967) and in some fish egg envelopes {Tide-marquis, 1973). No positive statement may be made regarding the localization of tryptophan in the shell. The indol nucleus of that amino acid present in Zones D and F, could be a component of the proteins already mentioned. The origin of the third shell layer (Fig. 3) is far from fully elucidated. From the present work we cannot attribute its existence to a definite zone in the gland but from its dense structure arising from a homogeneous matrix regularly interspaced with fine striations perpendicular to the middle layer bands, a possible hypothesis may be put forward. The upper part of Zone D is the site of collagenous and sulphur-containing protein secretions; it gives no positive reaction to test for phenols or tyrosine residues, and does not react to several days-old diamine, The specialization of that region in Zone D together with our present knowledge of the third layer may be regarded as a
282
M. RUSAOUfN
clue for their possible cannection. Finally, Zone C secretions must constitute the fourth layer. This zone, almost identical in function to Zones E and F, {Tables II, III? IV, Figs 15-19) does not react to tests for peroxidase, so that the inner layer of the shell owes its tanning to phenoloxidase only. The secretions of the dogfish shell gland and the mussel foot and rachis glands display striking similarities; included in the mussel gland secretions are a phenolase (Smyth, 1954), a glycoprotein with tyrosine residues and a sulphated protein (Pujot, 1967), a collageneous protein (Pujol et al., 1970; Pujol, Houvenagel & Bouillon, 1972), neutral and sulphated acid muco~oIysaccharides (Pujol, 1957). in spite of their differences their toughness is remarkably adapted to their common environment,
F. M., 1876. The deveIopment of efasmobranch fishes. J, Anur. P&W., VOL. IO, pp. 517-570. BORCLA,J., 1906. Recherches sur le syst&me urogenital des E~asmobranches. A&s Zaul. ~.xP. g&r., T.J. pp. 1991184. BROWN, c. H., 1950. Quin0ne tanning in the at&‘& kingdom. Nature, .&ond., Vol. 165, p. 275. BROWN. C. H,, 1952. Some structural proteins of Mytififs edulis. p. JI microsc. Sei., Vol. 93, pp. 487 --502. BROWN,c‘. H., 1955. Egg capsule proteins of selachians and trout. Q. JI. micrtw. Sci., Vol. 96, pp. 483.488. BUNKE, D., 1972. Skferotin-Komponenten in den Vitellucyten von ~~~~~~~~~~~ &e&r .~~~r~hi~d~ ITurbeharia), Z. Zellfosch. mikrusk. Anot., Bd 193, 5.383398, COLLENOT,G., 1966. Observations relatives au developpement en laboratoire d‘embryons et d’individus juv&iles de Scyliorhinus canicula L. Cub. Biol. mar., T. 7, pp 319-330. Duc~os, C., 1967. Contribution ii I’etude du tannage de Ia raduXa chez les Gasteropodes. Ann/s ~isfo~hem., T. 12, pp. 243-272. FAURF-FR~MMIET, E., 1938. Structure de la capsure ovuiaire chez yuetques Waciens. Archs Anat. nricrosc. borax. exp.. T. 34, pp. 23-S I. FAW&-FR&XET, E. & C. BAVOOUY,1938. Sur Sovoktratine des SBlaciens. Bdf. SW. Ckitiz. hid.. T. 20, pp. 14-23. FAURZ.!-FR~MIET, E. & H. GARRAIJI;~,1938. Separation d’une prokeratine stcretee par la glande nidamentaire de Raja batis L. Bull. Sue. Chim. biol., T. 20, pp. 24-30. FILHOL,J. & H. GARRAULT,1938. La secretion de la prokeratine et la formation de la capsule ovulaire chez ies Selaciens. Archs Anut. microsc. Morph, exp., T. 34, pp. 105-145. GABE, M., f968. Techigues h~sfo~o~~q~es. Masson et Cie, Paris, f , t 13 pp. G.W’TER,P, & G. JOLL&S,1969, Nisfuc&&? norm& et ~~fbo~o~jq~~. f et if. Gauthier-Viilars, Paris. 1902 pp. GARRA~LT, H. & J. FILHOL, 1937. L’organe nidamentaire des Elasmobranchz~ et son role darts la formation de la capsule de l*oeuf. C. r. S&WC. Sot. Biol., T. 126, pp. 773.-775. HENNEGUY, C. F., 1893. Sur la structure de la glande nidamentaire de l’oviducte des S&aciens. C. r. Sot. Philomafique, 86me stie, T. 5. KRISHNAN,G., 1959. Histochemical studies on the nature and formation of egg capsules of the Shark C~~~osc~~l~u~griseum. Biol. Bull mar. biol. Lab., Woods Hole, Vol. 117,pp. 298-303. LAFON, M., 1943. Sur ia structure et la composition chimique du tegument de ta limuie Xiphosurrr ~#i~~~~~~#~ L. Btdf. Znrf. O&nap. Monaco, No. 850, I t pp. MASCHINO,F. & J. VOVELLE,1972. Comparaison du bourrelet pall&l posterieur et du disque operculigere chez Ocinebra erinucea L., Haliotis, Vol. 2, pp. 187-190. MICWEL,C., FONZ~-VIGNAUX,M.-T%, & M. F, VOS~-FOWART, 1973. Dorm&es nouvelles sur la morphologic, I’histocbimie et la composition chimique des m&&oires de G&vwn conooMa Keferstein f&n&de polychetef. B&i. bioi. Fr. Be&., T. 107. pp. 30I-XI. BALFOCK,
THE DOGFISH
SHELL GLAND
283
MICHEL,C. & J. VOVELLE,1974. Tannage quinonique des soies chez Syllis corm&a R. Bull. Sot. 2001. Fr., T. 99, pp. 175-181. NALINI, K., 1944. Structure and function of the nidamental gland of Chiloscyflium griseum (Miill. et Hen&). Proc. Zool. Znd. Acad. Sci., Vol. 12 B, pp. 189-214. OUANG TE YIO, 1931. La glande de l’tclosion chez les Plagiostomes. Annls. Inst. Oceanogr. Paris, T. 10, pp. 283-370. PERRAVEX,G., 1884. Sur la formation de la coque des oeufs de Scyliium canicula et de Scyllium catulus. C. r. hebd. Seanc. Acad. Sci., Paris, T. 99, p. 1080. PUJOL, J. P., 1967. Le complexe byssogkne des Mollusques bivalves. Histochimie cornparke des &r&ions chez Mytilis edulis L. et Pinna nobilis L. Bull. Sot. linn. Normandie, T. 8, pp. 308-332. PUJOL, J. P., M. ROI.LAND,S. LASRY& S. VINET, 1970. Comparative study of the amino-acid composition of the byssus in some common bivalve molluscs. Comp. Biochem. Physiol., Vol. 34, pp. 193-201. PUJOL, J. P., G. HOLJVENAGHEL SCJ. BOUILLON,1972. Le collag&ne du byssus de Mytilus edulis L. Ultrastructure des cellules secretrices. Archs Zool. exp. gin., Vol. 113, pp. 251-264. RUSAOUBN,M., J. P. PUJOL, J. BOCQUET,A. VEILLARD& J. P. BOREL, 1976. Evidence of collagen in the egg-capsule of the dogfish, Scyliorhinus canicula L. Comp. Biochem. Physiol., Vol. 53, B, pp. 539-543. SMYTH,J. D., 1954. A technique for the histochemical demonstration of polyphenol oxidase and its application to egg-shell formation in helminths and byssus formation in Mytilus. Q. JI. microsc. Sci., Vol. 95, pp. 139-152. SPICER, S. S. & M. H. JARRELS, 1961. Histochemical reaction of an aromatic diamine with acid groups and periodate engendered aldehydes in mucopolysaccharides. J. Histochem. Cytochem., Vol. 9, pp. 368-379. STRAWS,W., 1971. Inhibition of peroxidase by methanol and by methanol nitroferricyanide for use in immuno peroxidase procedures. J. Histochem. Cytochem., Vol. 19, pp. 682-688. THREADGOLD,L. T., 1957. A histochemical study of the shell gland of Scyliorhinus caniculus. J. Histochem. Cytochem., Vol. 5, pp. 159-166. TIBERI-MARQUIS,J., 1973. Quelques aspects histochimiques de l’ovogen&se chez Betta splendens R. (T&Cost&n Anabantidb). Histochemie, T. 33, pp. 139-158. TIMMERMANS, L. P., 1969. Studies on shell formation in Molluscs. Netherlands J. Zool., Vol. 19, pp. 417-523. VOVELLE,J., 1965. Sur la presence des groupes SH et SS dans la s&r&ion de la glande nidamentaire chez Scyliorhinus canicula L. C. r. hebd. SPanc. Acad. Sci., Paris, Vol. 260, pp. 5945-5947. WASCHTEIN,M. & M. MEISEL, 1964. Demonstration of peroxidase activity in tissue sections. J. Histochem. Cytochem., Vol. 12, pp. 538-543. WIDAKOWICH,V., 1906. Uber Bau und Funktion des Nidamental Organs von Scyllium canicula. Z. wiss. Zool., Bd 80, S. l-21.