Histochemical and biochemical Research on the pallial Gland of Lithophaga (Lithodomus) lithophaga L.

Acta histochem. Bd. 55, S. 42-59 (1976)

Institute of Comparative Anatomy and C. N. R. Centre for Histochemistry, University of Pavia. Chair of BiologICal chemistry Faculty of Medicine, University of Milano

Histochemical and biochemical Research on the pallial Gland of Lithophaga (Lithodomus) lithophaga L. By LORENZO BOLOGNANI, ANNA MARIA BOLOGNANI-FANTIN, EMANUELA VIGO and MARIA LAURA CUCCHI With 3 figures (Received May 21, 1975)

Summary Histomorphological staining demonstrated 2 types of cells in the pallial gland of the mantle of LithodomU8 lithophaga: gland cells containing glycolipoprotein and interstitial cells, containing lipid droplets. Biochemical analysis aiming at separate and evaluate lipidIC and peptldIC components were carried out by lIpid extraction, T. L. C. separation and colorimetrIc assays. Phospho- and sulfolipids were separated from non-polar lipids. Sialo-cerebrosides (gangliosides) are missmg. Hydrophobic peptide component was extracted and aminoacid analysis was made. The presence of acid (phosphate) group in the gland secretion is discussed in relatIOn to the function of the secretion (Ca uptake and rock boring). The prominent glycogen storage is considered as an important energy source to be used for osmotic work and to synthesize glycosidIC stones for glycolipo-protein secretion.

It is known that the mantle of LithodomU8 lithophaga has several glands. In the past, workers interested in explaining the rock boring activity of this species have postulated that this may be related to secretions from its mantle glands. The actual mechanism responsible for calcareous rock boring in the species is stilI debated. Some Authors are inclined to believe that it is a mechanical mechanism take place, others suggest that it may be a chemical mechanism, while others again think that a combination of both mechanisms is involved (YONGE 1955, 1963; HODGKIN 1966; KUHNELT 1930; OTTER 1937; CARAZZI 1903). Among more recent papers on the subject HODGKIN (1966) postulates a combined chemical and mechanical mechanisms. Regarding the chemical mechanism, this author states that no free inorganic acids such as HCI or H 2S0 4 are present in the secretion and puts forward the hypothesis that there is an acid secretion due to mucopolysaccharides (glycosaminoglycans) or more complex ion exchange activities between the tissues in the mantle and limestone substrate. A recent paper by JACCARINI et al. (1968) working on the basis of histochemical results supports the idea that the pallial glands produce a neutral mucoprotein which is able to chelate Ca++, as already suggested by NEWELL (1964).

Histochemical and biological research

43

Morphological research has also been carried out on the pallial glands of several Lithophaga species. As early as 1903, CARAZZI first described 3 kinds of gland. One gland (the proto-acid gland) is located in the visceral mass of the animal and 2 other "deutacid" glands. One of these glands (the "anterior acid gland") appeared to be located anterior to the hinge, just dorsal to the anterior adductor muscle, while the other ("the posterior acid gland") was immediately posterior to the hinge in an extreme dorsal position adjacent to the inner surface of the shell valves. All subsequent Authors refer to 2 deutacid glands only. These have been called after CARAZZI, i. e. the anterior and posterior CARAZZI glands. Even so none of them have given any accurate histomorphological description of these glands which have simply been defined as acinous glands. As regards histochemical and biochemical studies, the observations of YOUNGE (1955) are worthy of mention. According to this Author the anterior gland is likely to be responsible for producing acid mucopolysaccharides (Alcian blue positive) which probably starts the rock boring process, while secretion from the "posterior gland" is likely to be used to widen a hole already excavated by secretion from the anterior gland. The paper of JACCARINI et al. (1968) is the only study in which histochemical tests are used to support the concept that there is a neutral secretion of mucoproteins produced by the pallial gland. These Authors have not however exactly determined the chemical nature of this substance. Owing to the lack of any histomorphological data on the pallial glands and to the many hypotheses on the chemical nature of the secretion as well of its mechanism in rock boring, we thought it would be useful to investigate the pallial gland secretion of Lithophaga using histochemical and particularly biochemical procedures with a view to providing a detailed histomorphological description of the glands and a chemical analysis of the main constituents of their secretion. Ultrastructural research was also undertaken to elucidate certain features of the glands and the results will be reported in another paper_

Materials and methods Specimens of Lithophaga lithophaga from Ligurian beaches were used, samples being collected at different seasons durmg the year .

Histochemical research Pallial glands (which could also be clearly seen by the naked eye) were dissected from living animals and cut into 4 portions starting from the mantle nearest the frame. The portions were fixed in 10 % Formol, BourN, CARNOY and REGAUD and then embedded in paraffin. 8!lm thin sections were cut from pieces fixed in the first 3 fixatives; morphological and histochemical sta.ining (see later) was performed to ascertain the features of the gland at different levels, to establish the location of the various glands and to determine whether their secretory ducts were present. The specimens fixed according to REGAUD were cut into 8 !lm slices, immersed in xylene, mounted in balsam and observed directly under the microscope. As is know, this mixture fixes the 4

Acta histochem . Bd. 55

44

L. BOLOGNANI, A. M. BOLOGNANI·FANTIN, E. VIGO and M. L. CUCCHI

reducing substances by dichromate and stain them at the same time. Some pieces were fIxed in cold fixatIves,!. e. alcohol, GENDRE'S flUId, ROSSMAN'S fluid and LILLIE'S mixture to preserve thIS substance for glycogen detection. Fresh frozen sections were tested for lipids and enzymes (hydrolases and oxydoreductases). Histochemical reactions were carried out to evidence ribonucleoproteins, proteins, lipids, polysaccharides (homo and heteropolysaccharides), phenolic substances, ions (Fe+++, Cu++, Zn++, Ca++ and Cl-).

Morphological stains Haematoxylin-Eosine, Ferric haematoxylin and the Azan-MALLORY method.

Reaction for neutral MPS PAS reaction (HOTCHKISS 1948), also after digestion with (X-amylase (LISON 1960), iodine reaction (LANGHANS 1890), specific staining of glycogen with haematoxylin (MURGATROYD et al. 1969).

Reaction for acid MPS Alcian blue 8 GN pH = 3.5 (SPICER 1960), Metachromatic staining with toluidine blue (1: 1000 pH = 3.5; pH = 4.7; pH = 5.8 following the reaction in water.

Reaction for proteins Reaction with bromophenol blue (MAZIA et al. 1953), Fast Green reaction at pH = 1 and pH = 8 ALFERT and GERSCHWIND 1953), BARRNETT and SELIGMAN'S reaction for -COOH, also involving he blocking of -COOH with NaOH, MOREL and SISLEY'S reaction, reaction with DMAB and with osindol for tryptophan, STORTI and SOLDATI'S reaction for arginine, Ninhydrin-ScHIFF reaction YASUMA and ISCHIKAWA 1953), reactions for thiol groups (CHEVREMONT and FREDERIC'S reaction, Performic acid-Schiff reaction according to LILLIE, DDD reaction according to BARRNETT and SELIGMAN, also involving prior blocking of -SH groups with N·ethyl maleImide), Some reactions (Bromophenol blue and Fast Green pH = 1) were also performed after digestion with pepsine. Furthermore these reactions for proteins were performed on cryostat slices from frozen material, also after prior digestion by pepsine and extraction by chloroform-methanol 1: 2_

Reactions for nucleoproteins Azure A (POLLISTER 1950); Methyl green-pyronin following KURNICK'S method; Gallocianinchromallum method (EINARSON 1951) also involving prior digestion by ribonuclease (1 mg/ml).

Reactions for lipids not extracted from tissues by alcohol Sudan Black B method, the Nile blue method and the acid haematein method (BAKER 1945) for phospholipids. All the reactions were also performed by prior extraction with pyridine and chloroform·methanol 1: 2 We also tested the methods for masked lipids using the Burnt Sudan Black B method after BEREMBAUM, Immersion in warm water for 48 h and treatment with phenol.

Reactions for lipids extracted from tissues by alcohol We performed the Sudan Black B method and Nile Blue method on cryostat sections on unfixed material. Both tests were preceded by extraction with chloroform/methanol 1: 2 control or by digestion with pepsine.

Histochemical and biological research

45

Reactions for phenolic substances SCHMORL'S method, MASSON·HAMPERL'S techniques and the diazonium reaction in alkaline solu· tion were used.

Enzymological research The following tests were carried out on cryostat sections: a) Oxydoreductase: Diaphorases (NAD+, NADP+) and Succinic dehydrogenase. Anaerobic de· hydrogenases: lactate, isocitrate, glucose·6·phosphate and malato·dehydrogenase. b) Hydrolytic enzymes: (Na+, K+)·ATPase, Acid and alkaline phosphatases, Arylsulphatases. c) Lyases: Carbonic anhydrase.

Ion detection assays The following ions were tested: Fe+++, Cu++, Zn++, Ca++ and Cl-. The Cu++ test was performed after GABE. Fe+++, Ca++ and Zn++ assays were effected by comparing tests on slices previously fixed in formol and embedded in paraffin with those carried out on spodograms of corresponding slices PEARL'S test was used to detect Fe+++ on normally fixed slices, Zn++ was tested following YOSHINAGA et aI., while both alizarine and haematoxyline tests were used for Ca++. Soluble Ca++ was tested by glyoxal after KASHIVA et aL (1964).

Biochemical methods Biochemical research was aimed at: 1) analysing aqueous homogenates of the glands. The glands were examined in toto and results of these analysis will be described in part 1; 2) analysing chloroform·methanol extracts from glands (the glands were homogenated in chloroform·methanol and polar lipids extracted and analysed). This procedure is reported in part 2; 3) isolating glycogen from alkaline extracts of the gland. Morphological and histochemical indica· tions suggested that glycogen granules are present in the cytoplasma. This extraction procedure and results of the analysis are reported in the part 3.

Part 1: analysis on aqueous homogenates Glands from the same batch to be used for histochemical analysis were weighed and then homogenised in distilled water. The following analyses were made on the homogenate: L Protem determinatlOn (by a modiflCatlOn of the biuret test after GOA). 2. Separation and evaluation of total ammoacids: concentrated hydrochloric acid was added to the aqueous gland homogenates to reach a ratio of I : I by volume. The suspension was again homo· genated and submitted to hydrolysis at + 105 °C for 24 h in vials sealed under Nitrogen. The hydro· lysate was dried using a rotating evaporator. The residue was dissolved in pH = 2.2 citrate buffer and the aminoacids were separated by ion exchange column chromatography (Aminoacid analyzer, Hitachi Perkin·Elmer mod. 034). 3. Determination of Hexosamines (BOLOGNANI, COPPI and ZAMBOTTI 1958) . 4. Determination of Hexoses (RADIN, LAVIN and BROWN 1955). 5. DeterminatlOn of Sialic Acid (SVENNERHOLM 1958). 6. Determmation of total lipids by gravimetric method. 7. The homogenate of the glands in 0.3 N HCl was tested direct for Ca++ by the titrimetric procedure after HENRY et a1. (1957). 4"

46

L. BOLOGNANI, A. M. BOLOGNANI.FANTIN, E. VIGO and M. L. CUCCHI

Part 2: Analysis on lipid extracts and on delipidized residues The glands obtained after dissection were weighed and homogenized in methanol. Two volumes of chloroform were added using the followmg procedure: Homogenization in Methanol (M)

t

AddItion of Chloroform (0) (2 volumes)

t

Extraction by stirring

t

Filtration

t

t

Extract I

Residue

t

Suspended in C/M 2: 1

t

Filtration

I~----

t

Delipidized Residue

Exttact II

t

Crude lipid extract [

Analysis on crude extract

t T. L. C. separation Qualitative - Quantitative

t Analysis of Lipid-boundpeptides

The following analyses were carried out on the crude lipid extract: a) determination of total bpids by gravimetric method. b) determinatlOn of cholesterol (PAREKH and JUNG 1970). c) evaluation of glycolipids (by determining hexoses and hexosamines as an index of lIpid-bound saccharides) . d) determination of gangliosides (tested by sialic acid evaluation). e) determination of phospholipids (BARTLETT 1959). f) determination of sulpholipids (KEAN 1968). g) determination of lipid. bound Ca++. h) thm- and thick-layer chromatograph separation. Precoated Merck plates were used for this separation. Chloroform-methanol-water 70/30/5 was employed as the developer. The spots which appeared after J 2 vapour treatment were marked with a pencil, scraped off, and extracted twice with chloroform-methanol 2/1. Glycobpids were eVIdenced on the plate directly by spraying with anisaldehyde reagent and heating to 150°C in a dry oven for 10 mm. Aminohpids were demonstrated by spraying the plates with ninhydrin reagent. i) lipid-bound peptides were analysed using the following procedure, starting from crude lipid extract:

47

Histochemical and biological research Crude lipid extract

I

t

,~

Evaporation of solvent

Evaporation of solvent

t

t

R esIdue (DLE)

Residue (LE)

t

t

suspended and hydrolyzed in 6NHCI

suspended in water

Separation of amino acids

dialysis against water

I

t

t

I

t

(A)

R etained

t

Dialyzate

t

addition of 12 N HCI

t hydrolysis

t Separation of amino acids (B)

2,3500 g (of wet weight) were extracted by chloroform-methanol 2/1(150 ml) to separate and evaluate total free and bound ammoaClds, 50 ml of crude extract were drIed by rotary evaporation The reSidue was suspensed in 6 N Hel and hydrolysed at 105 °C for 24 h in vmls sealed under Nitrogen, The hydrolysate was filtred through filter paper, the HCI was then removed and the residue dissolved in citrate buffer pH = 2,2 and submItted to IOn exchange separation by column chromatography, The separated aminoacids were estimated by ninhydrin reaction with ammoacld analyser, Both free aminocalds and those bound in lipo-soluble peptides were determined (A), Ammoacids were separated by column chromatography after hydrolysis of "retained" peptIdes (B), The following analyses were carried out on the delipidized residue obtained from the procedure on p , 46; 2c, 1, Determination of he xosamines (non-lipid-bound hexosamme;;, e, g, Glycopeptidic hexosamines); the method of BOLOGNANI-COPPI-ZAMBOTTI was applied after acid hydrolysis 4 N HCI (+ 105°C for 6 h), 2, Determination of hexoses (non-lipid-bound hexoses, e, g, Glycopeptidic hexoses) by anthrone reagent, 3. Separation of aminoacids (bound to peptides and proteins non-soluble in chloroform-methanol) by ion exchange column chromatography of aCId hydrolysate (6 N Hel) of the residue.

Part 3 : Extraction and Determination of Glycogen Some glands (2.072 g) were treated with 30% KOH by warming under reflux in a water bath. Methanol was then added and refluxed again for 40 min. After cooling the sediment was collected by centrifugatIOn. It was then washed twice WIth methanol and twice with ether. The ether was evaporated at room temperature to give a white powder. A water solutIOn of this powder was analysed for hexoses with anthrone reagent after RADIN.

48

L.

BOLOGNANI,

A. M.

BOLOGNANI-FAN'l'IN,

E. VIGO and M. L.

CUCCHI

Results a) Histochemical results On observing the mantle of living Lithophaga a cream-coloured shield shaped gland is to be seen located on the mid-line at the anterior and posterior extremities of the pallial isthmus; it encompasses a large part of the mantle. There is only a small strip of mantle without any glands either toward the isthmus or along the edge of the valve. Thin extensions running from the gland towards the pallial isthmus are assumed to be glandular ducts. These extensions are not detectable around the outer edge of the valve. The acinar gland is enveloped by a thin layer of connective tissue, with reticular and collagenous fibres. The isoprismatic epithelium of the mantle, in layers on a basal membrane, covers the connective envelops. The gland is divided into lobes marked by thin connective fibres. The acins are built up by large round or trapezoidal-shaped cells, whose cytoplasm is virtually unstainable by haematoxylin-eosin, but is packed with eosinophilic secretory granules. A round nucleus rich in chromatin is usually located at the base of the cells, but when a large amount of secretion is present inside the cells, most of the nuclei appear to be situated peripherically. No mitoses are found. Granulous mitochondria mostly located at the bases of the cells were observed. The gland appears to be histologically uniform and no ducts can be shown, this being demonstrated by cutting serial sections (Fig. Ia and b). The extensions running from the gland to the isthmus, apparently secretory ducts under by the naked eye inspection, are only extensions of the gland tissue. Amorphous masses of secretion (with scattered vacuoli) between alveoli are noted at several locations in the gland (but not in all slices). Under common morphological stain the gland appears to be constituted by a single cell type only; but after staining with Sudan Black B in fixed and enclosed sections some triangular-shaped cells can be seen between one alveolus and another, with large sudanophilic granules, in their cytoplasm (Fig. I a). This cell type is likely to be the "interstitial cells" described by the early Authors. In the alveolar cells, PAS positive secretion appears to be more intensively stainable on sections cut by cryostat tha n on fixed and enclosed material. This secretion appears to be negative of other usual staining procedures are used for acidic polysaccharides ; when a toluidine- blue stain is used methachromasia never occurs even at the highest pH values (Fig. Ie) . The granules react positively to reactions for protein (Fig. Ie) . Among the reactions employed to demonstrate typical residues for aminoacids, the following tests proved to be positive: BARNETT'S reaction (for -COO H); Ninhydrin-ScHIFF (for -NH2 ), DMAB and Rosindole reactions (for tryptophan) and STORTI-SOLDATI'S reaction (for arginine).

Fig. 1. a) Section of gland fixed in 10 % Formol, embedded in paraffin and stamed with Sudan Black B. It IS possible to see the glandular cells and the "mter~titIaI" cells (arrows). X 200. b) 0.5 flm thick sectIOn stained with Sudan Black B according to MCGEE RUSSELL. X 400. c) Sudan Black B method performed on cryostat sections from frozen matenal. X 1250. d) Like to c) but the test was preceded by digestion with pepsine. X 1250. e) Bromphenol blue reaction performed on cryostat sections from frozen material. X 1250. f) Like to e) but the test was performed after pnor extraction by chloroform-methanol I: 2. X 1250.

50

L. BOLOGNANI, A. M. BOLOGNANI-FANTIN, E. VIGO and M. L. CUCCHI

., ••__ • ....M;d

r:

Zf C

e

)

' 2 @'&i~:;'fI ~

i,

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6

. '

, M~

. . :'''

Olj.. ~" ., ... .".......

A

P

\

"'izrs::., ...-.-

8

Fig. 2. TLC separation of polar lipids extracted from pallIal gland. Developer: chloroform/methanol/ water = 70/30/5. A) Revealed by anysaldehide B) Revealed by ninhydrine. 0 = Origin, F = Solvent front, B

A

1 2 3 4 5 6 7 8

Phosphatidyl serine Unknown Sphyngomielins Phosphatidylcholine Sulpholipids Phosphatidylethanolamine Cholesterol Triglicerides.

I II III IV

Phosphatidylserine Sphyngomielins Phosphatidylcholine Phosphatidylethanolamine

51

Histochemical and biological research

-

". I

."

.. I

-

--1_ . __ _ _ _ _

,...

-

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-.~J"-"""""-;--~:--

-

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_ .L ._._. _---..._.L_. _ . _ .

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--

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Fig. 3. 1. R. spectra of Cerebroside sulphate standard extracted from pig brain (A) and sulfolipid extracted from pallial gland of Lithophaga (B).

All the reactions for thiol groups were negative. It should be pointed ou t that the granules take on a deeper colour if the staining procedures for proteins are made on sections previouosJy extracted by chloroformmethanol for 24 h at room temperature (Fig. 1 f). The secretion appears to be negative to other staining procedures such as those for indolic compounds and no fluorescence can be seen. If REGAUD ' S fixative is used (a reagent which fixes and stains reducing substances at the same time) the secretion appears to be unstained. The procedure for lipids ga ve the following results: Fixed and enclosed sections were positive; "interstitial cells" are stainable with Sudan Black B and Nile Blue sulphate (pink granules); the secretion granules are only slightly stained. These results are unchanged even if unmasking procedures are used before staining, but no positivity occurs after extraction with pyridine or chloroform-methanol 1/2 for 24 h. Slices of formol-fixed material cut by cryostat do not result in any increase of Sudan reactivity in the secretion granules (Fig. I c), but the number of lipid droplets in the interstitial cells is considerably increased. It is worth noting that large sudano-

52

L.

BOLOGNANI,

A. M.

BOLOGNANI-FANTIN,

E.

VIGO

and M. L.

CUCCHI

philic granules also appear in the glandular cells which are quite different from the usual secretion granules, if the pieces are fixed in dichromate instead of formol and then cut by cryostat. The most interesting results are achieved when the cryostat slices are digested with pepsine before staining with Sudan Black B. If this procedure is used several secretion granules showing a clear nucleus surrounded by a black sudanophilic ring are evident amongst unstained granules and large sudanophilic droplets (Fig. 1 d). These results should be compared with those obtained using the double staining procedure (Sudan Black B-Naphthol Yellow) carried out on the same slices. Apart from fine granules stainable with naphthol yellow only 01' droplets revealed by Sudan Black only, there are many droplets showing a "nucleus", stainable with naphthol yellow, surrounded by a sudanophilic ring. Secretion granules are not stainable by acidic haematein. No positivity for ribonucleoproteins was observed in the acinar cells. Enzyme activity tests carried out on the same cells showed NADH diaphorase activity only. This test reveals a few circular mitochondria located near the basal membrane of the acinar cells. We failed to observe any positivity in other enzyme tests, e. g. ATPase, acid and alkaline phosphatases, arylsulphatases nor were we able to demonstrate carbonic anhydrase. Ions Zn++, Cu++ and Fe++ were demonstrated only in the epithelial cells on the inner surface of the mantle. In the exterior epithelium and in the glandular cells some very thin granules, attributable to Cl- are evident. A large number of fine glycogen granules were seen in the gland cells. Characteristic histochemical reactions and digestion by amylase are support the idea that glycogen is likely to be present in these cells. Biochemical extraction (see later on) proved the chemical nature of this homopolysaccharide. It should be noted that glycogen was demonstrated on slices fixed by GENDRE'S and ROSSMAN'S fluid. The glycogen is lost when slices are fixed in 10 % formol or BOUIN'S mixture. The "interstitial" cells contain droplets which react positively only to the lipid staining procedures (including the acid haematein reaction); the number and reactivity of these droplets does not change even if unmasking treatments are carried out beforehand or chemical hydrolysis and enzymatic digestion is effected. b) Biochemical results Part 1: Analysis of the gland in toto gave the following results. Aminoacids 9 to 11 % Lipids 13.8 % Hexoses 4.8 % Hexosamines 0.43 % Sialic Acid absent Calcium 0.0735 %

53

Histochemical and b iological research

Higher values of protein are apparently detectable if tested by biuret, but this is due to interference of unknown origin. T otal aminoacids (values refer to wet tissue) are about 9 to 11 %: 25 % of them being represented by basic aminoacids, with lysine prevailing (1.7 % ). Glutamic and aspartic acids are about 1/3 of total aminoacids. An acid aminoacid of unknown nature was detected in our chromatographs. The histogram 1 summarizes aminoacids concentration as %referred to wet weight. Part 2: Results of biochemica l analysis of "crude lipid extract" and of " delipidized residue". A) Ana l ys i s of c rude extract Samples of gland batches collected in various different seasons and treated with chloroform-methanol gave a delipidized residue ranging from 23 .9 to 28 % depending by seasonal sampling. The crude lipid extract submitted to chemical analysis gave the following results: 13 % 4.20% 0.82% 0.24%

Lipid concentration Cholesterol Lipid-bound phosphate Lipid-bound hexosamines Lipid-bound hexoses Sialic acid Sulpholipids

0.67

%

absent 0 .051 to 0.154 %

Further analysis carried out on glands from batches of Lithol)haga collected at different seasons proved the sulpholipid concentration changes during the year. The lowest sulpholipid value (0.051 %) \vas observed in the specimens collected in N ovem bel', while during th e spring concentration reaches a value 3 times higher (April and May 0 .154 %). TLC chromatograph separation of lipid extracts using chloroform·methanol-wat.er 70/30/5 as the developer reveals ten fractions as reported in fig . 2. Preliminary qualitative t ests reveal the presence of glycolipids, phospholipids and aminolipids: some polar lipids show Rfs very close to those of sulpholipids. No true gangliosides were found.

~r~O~g 0 Il. If)

«

W

~

I-

Cl

a: W If)

D0

0

Cl

::J

> ...J

...J

...J

C>

0

a:

Il.

C>

« «

.....

Cl c

If)

...J

>- « u >-

....W ~

0

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::J W

>-

...J

...J

a:

W

l-

ll.

~

0 If)

>...J

c

0

If)

C>

~

«

a:

Histogram 1. Aminoacid concentration (ref. to wet weight) of total ammoaclcls of an aCHlJC hydrolysate of aqueous state.

54

L. BOLOGNANI, A. M. BOLOGNANI-FANTIN, E. VIGO and M. L. CUCCHI

Thick layer chromatograph enables lipid fractions to be separated for quantitative lipid-bound-phosphate and lipid-bound sulphate analysis. The results are summarized in the next table. Table 1. Chromatograph separation by TLC of polar lipids extracted from the pallial gland. (Thick layer chromatography). Fraction No. Rf 1 2 3 4 5 6 7 8 9 10 11

0.03 0.10 0.21 0.25 0.32 0.38 0.49 0.56 0.63 0.72 0.95

Phospholipids (0)

4.3 3.4 5.6 19.4

Sulpholipids (0)

1.4 1.5 1.5 1.5 5.5 4.5

11.5

Ninhydrin

+ ++ +++ +++ + + + +++

1.5

(0) ,umol x 10- 2 •

Sulpholipids are present in considerable amounts in fractions 7 and S. Smaller scattered amounts are detectable in other fractions. Phospholipids are present in at least 5 fractions. The highest concentration of phospholipids appears in fraction 7 and 9. On the basis of their Rf standard phospholipids indicate that the following compounds are likely to be present: phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. It is worth mentioning that both sulpholipids and phospholipids are present in fraction 7. It is not possible to evaluate the hexose-sulphate ratio owing to the existence of phospholipids in this fraction and to the fact that sulpholipids in our material do not show any 1. R. profiles similar to those reported in the literature for cerebrosidosulphates. 1. R. spectra are reported in fig. 3 as compared with those of cerebrosidosulphate standards. The determination of aminoacids in the lipid extract (free and bound aminoacids) gave the results reported in the histogram 2. In histogram the values are reported as p,g/100 mg of wet tissue. The ninhydrin test on the crude lipid extract gave O.SO mg of extractable aminic Nitrogen from 100 mg of wet gland. Of the profile of extractable aminoacids more than 2/3 are glycine and alanine. A ninhydrin positive peak also appears far before aspartic acid. It was not accounted for by the usual aminoacids. A pK value of lower than 3, the absence of sulphate and the presence of phosphate (S.S to 10 p,g of P to mg/l00 g) led us to conclude that it was not cysteic acid or taurine. It is more likely to be a phosphorated amino· acid.

55

Histoch emical a n d biological research

pg/100mg wet tissue

D. L. E.

5 4 3

2 0

Do

~D 00

____ 0

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,p gI100mg wet tissue 43

l. E.

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Histogram 2. Determination of aminoac id s i n the lipid ex tract: D. L. E. retained fraction ; L. E d ia lyzed fraction.

Ilg/100 m g of de lipidized residue

10 8 6

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H istogram 3. Determination of aminoacids in the delipidized tissue.

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>-

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56

L.

BOLOGNANI,

A. M.

BOLOGNANI-FANTIN,

E.

VIGO

and M. L.

CUCCHI

An analysis carried out on 50 ml of the same crude extract after dialysis and used to evaluate free aminoacids and those bound to dialyzable peptides indicate that 0.36 mgll00 g of aminic Nitrogen extractable in chloroform-methanol 2 : 1 is retained after prolonged dialysis, while 0.44 mgllOO g dialyzes (histogram 2). On examining aminoacids bound in peptides of DLE (retained fraction) it appears that proline, valine, cysteine and methionine are lacking. By comparing with the LE results it is evident that these aminoacids were free or linked to dialysable oligopeptides. Most of the alanine and glycine was also loosed after dialysis. An appreciable amount of aminoacids appear however to be linked to non-dialyzable peptides. Serine is retained in higher amounts. Most of the unknown ninhydrin positive peak also appear to be retained. Less than 1/10 of the usual total extractable aminoacids appear to be retained as nondialysable peptides. This apparently contrasts with the ninhydrin results, but we should add the ninhydrin value of the unknown peak to that of the retained aminoacids. B) Analysis of delipidized residue Analyses of the delipidized residue gave the following results: Hexosamines 0.9 % Aminoacids 31.7% Hexoses 25 % The residue is 23 to 28 % of wet weight. The absence of sialic acid was confirmed. Less than 1 % of hexosamines are likely to be bound in glycopeptides. About 1/3 ofthe aminoacids consisted oflysine, aspartate and glutamate (histogram 3). Hexoses are in part linked to proteins and in part are present in the form of hexose polymers. The next section reports data supporting evidence for glycogen. Part 3: Extraction and determination of glycogen. Large amount of hexose in the delipidized residue and the histochemical findings suggested the presence of glycogen. This was confirmed by alkaline extraction. About 23.5 mg of a white precipitate was obtained (0.8 %) from 2.872 g of glands. It appears as a white powder soluble in water. Chemical tests gave p03itivity for antrone reagent and chromatographic evidence for glucose.

Conclusions A smgle type of gland is present in the mantle of Lithophaga on the basis of histomorphologlCal staining. This contrasts with the findings of previous Authors who have suggested that 2 glands are present without however giving any histological data to support this theory. Serial sections of the mantle demonstrated that a single gland type is present and that its secretion inside the cells invariably appears to be same from a histochemical viewpoint, supporting the statement that there is a single gland only. The absence of gland ducts in several sections also appears to be in contrast with previous investigations. Instead of ducts, we observed amorphous masses Of

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secretion (sometimes showing inside several vacuolae) located m spaces betwe en the cells. It seem that the secretlOn produced by acinar cells may be released into the intercellular spaces and hence it may escape through the epithelium. At the present time we obtained some information from electron microscope pictures supporting that secretion granules pass outside through the epithelium. 2 types of cell are present in the gland, i. e. the gland cells and the "interstitial" cells. The trio angular "interstitial" cells are relatively less numerous; inside them only lIpid droplets can be seen, partly made up of "simple" lipids and partly of complex lipids (phospholIpids) not extracted from tissues by alcohol and paraffin. The secretion from the gland cells, which takes the form of granules with no constant dimensions (as already mentioned), was characterized on the basis of histochemical reactions and biochemical analysis, as being constituted by glycolipoproteins and not by glycoproteins, as might appear from the first analytical approach. The lack of positivity for acid MPS reaction would seem to indICate that these compounds are absent. This appears to be confirmed by the lack of metachromatic staining with toluidine blue even at high pH values. The lack of MPS observed by us also confirms the previous observations of JACCARINI et al. Biochemical data support the hypothesis that lipoproteins are characterized by an high percentage of lysine (1.7 % of all the aminoacid concentration) and an high concentration of aspartic and glutammic acid. The ninhydrin-Schiff reaction for -NHz groups and BARRNETT and SELIGMAN'S procedure for -COOH groups contribute to identifying the location of lipoproteins and to characterizing the composition of the granules. Tyrosine and arginine are present in appreciable amount, aE' also borne out by histochemical tests. Sulphur containing aminoacids is present only in small amounts (cystine and methionine). This is in agreement with the lack positivity for specific histochemical reactions. The lipid fraction is quite prominent (13.8 % referred to wet weight). This fraction consists of glycerides, cholesterol and polar lipids, including sulpholipids and phospholipids which are present in quite large amount. We found some glycolipids which apparently behave like gangliosides on thin layer chromatography, but do not contain sialic acid. It is worth pointing out that sulpholipid concentration vanes according to the season in which the specimen was collected, showing a maximum at the end of the spring and a minimum during October and November. Infra-red profiles appear unlike those found in cerebrosidosulphates of higher animals. Contrary to the conclusions presented by us in an earlier paper, the protein and lipid fraction appear in some way to be bound together, at least in some granules. There are however secretion granules, which are olopr'?teic, some others that are ololipidic. Data supporting this conclusion are set out below: 1. the percentage distribution of some hydrolysable aminoacids in delipidized residue is higher than in the total gland; 2. cryostatic sections show an increase in positivity for lipid droplets after peptic digestion, or an increase in positivity for protein granules after extraction with chloroform-methanol respectively; 3. using suitable Iprocedure we managed to obtain a simultaneous positive stain for some granules: outside for Sudan Back B (lipids), inside for Naphthol Yellow (proteins). As regards the function of the secretion, it seems to have a Ca ++ binding activity (this property is probably due to a lipoprotein). Accordingly the secretion may be responsible for rock boring activity. Some other mechanisms that have been proposed are not demonstrable by histochemical or biochemical tests. For example, rock boring by an aCl d , such H 2S0 4 , ruled out since arylsulphatase was not present in any appreciable amounts. Likewise the mechanism postulated on the basis of carbon anhydrase activity or ATPase involvement in osmotic work involved in rock boring, is equally unlikely. We failed to demonstrate any Na+ and K+ ATPase activity on our sections. Moreover few mitochondria are present in the gland cells and oxoreductase activity is low. The requisites for active ion transport, which depends by strong oxoreductase and Na+ and K+ ATPase activity have therefore been demonstrated by us as being absent.

58

L. BOLOGNANl, A . M. BOLOGNANl-FANTIN, E. VIGO and M. L . CUCCHI

Finally, we should draw attention to the large glycogen concentration (0.8 % of wet tissue). Glycogen appears easy removed by fixing or alcohol dehydratation. It is well preserved on histological slices if fixed cold in alcohol fixative mixtures. The large amount of glycogen probably represents a reservoir of energy for rock boring work. In conclusion, if a remote energy supply could be found in the large glycogen granules stored in the gland, the boring mechanism could be attributed to some Ca-binding lipoproteins, which act as chelating agents in physiological conditions.

Literature BARTLETT, G. R., Phosphorous assay in column chromatography. J. BioI. Chem. 234,466 - 468 (1959). BOLOGNANI FANTlN, A. M., BOLOGNANI, L., VIGO, E., e CUCCHI, M. L., Dati istochimici e ultrastrut· turali sulla ghiandola palleale di LithodomU8 lithophaga. Atti X Congresso della Soc. It. I stoch . Ferrara, Riv. Istoch. norm. pat. 17, 305 (1971). - - - La ghiandola palleale di LithodomU8 lithophaga. Boll. Z XL Conveguo U. Z. 1. Garda 1971.

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38, 496-497 (1971). Atti

and VIGO, E., Dati ultrastrutturali sulla ghiandola palleale di LithodomU8 lithophaga. Atti 41 0 Convegno U. Z. 1., Trieste 1972. BOLOGNANI, L., COPPI, G., and ZAMBOTTI, V., Sulla determinazione delle esosamine. Boll . S. 1. B . S. 34, 1950 - 1951 (1958). CARAZZI, D., Contributo all'istologia e alIa fisiologia dei Lamellibranchi. 3. Come si scavano il nicchio i Lamellibranchi perforanti? Int. Mschr. Anat. Physiol. 20, 57 -73 (1903). GABE, M., Techniques. Histologiques. Masson & Cie, Paris 1968. GANTER, P., et Jolles, G., Histochimie normale et pathologique. Vol. 1,2. Gauthier VIllars, ParIs 1969{1970. HODGKIN, N . M., Limestone boring by the Mytilid Lithophaga. Vehger 4, 123-129 (1966). JACCARINI, V., BANNISTER, W. H., and MICALLEF, H., The pallial glands and rock boring in L ithophaga lithophaga (Lamellibranchia, Mytilidae). J. Zool. London 154, 397-401 (1968). KAsmvA, H. K., and MARSHALL, H. C., Jr., The glyoxal bis (2-hydroxyanil)method modified for localizing insoluble calcium salts. Stain Technol. 39, 359 - 367 (1964). K EAN, E. L., Rapid sensitive spectrophotometric method for quantitative determination of sulphatides. J. Lipid Res. 9,319-327 (1968). KUHNELT, W ., Bohrmuschelstudien. 1. Paleobiologica 3, 53-91 (1930). LISON, L., Histochimie et cytochimie animals. Gauthier Villars, Paris 1960. MlffiGATROYD, L. B., and HOROBIN, R. W., Specific staining of glycogen with haematoxylin and certain anthraquinone dyes. Stain Technol. 44,59-62 (1969). NEWELL, G. E., Physiological aspects of the ecology of intermedial Molluscs. In Physiology of Mol· lusca 1,59-81. Wilbur E. M. and Yonge C. M. (eds.) Academic Press, New York 1964. OTTER, G. W., Rock destroying organisms in relation to coral reefs. Sci. Repts Gr. Barnier Feef Exp. 1,323-352 (1937). PAREKH, A. C., and JUNG, D. H., Cholesterol determination with Ferric Acetate -Uranium Acetate and Sulfuric Acid-Ferrous Sulfate reagents. Anal. Chern. 42, 1423-1427 (1970). PEARSE, A. G. E., Histochemistry theoretical and applied. Vol. 1 J. & A. Churchill Ltd., London 1968. RADIN, N . S., LAVIN, F. B ., and BROWN, J . R., D ctermination of cerebrosides. J. BioI. Chem. U7, 789-796 (1955). SPICER, S. S., A correlative study of histochemical properties of rodent acid mucopolysac~ charides. J. Histochem. Cytochem. 8, 18-36 (1960).

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SVENNERHOLM, L., Quantitative estimation of sialic acids. III. An anion exchange resin method. Acta chern. scand. 12, 547-554 (1958). YONGE, C. M., Adaptation to rock boring in Botula and Lithophaga (Lamellibranchia, Mytilidae) with a d,scuSSIOn on the evolution of the habIt. Quart. J. Microsc. Sci. 96, 383-410 (1955). Rock·Bormg Organisms. Da: Mechanisms of Hard T,ssue DestructIOn. ed. by Reidar F. Am. Ass. Advanc. Sc. n. 75, Washington 1963. Adress: Prof. LORENZO BOLOGNANI, Istituto Chimica BiologICa, Facolta di Medicina, Via Saldini 50, Milano, (Italy), and Prof.ssa ANNA MARIA BOLOGNANI FANTIN, Istituto Anatomia Com· parata, piazza Botta 10, Pavia, Italy.

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Acta histochem. Bd. 55