35
The Histochemical Localization of p-Glucuronidase in the
Digestive Gland of the Roman Snail (Helix pomatia)
By F. BILLETT AND S. M. M C G E E - R U S S E L L
(From the Department of Zoology and Comparative Anatomy, University Museum, Oxford)
With one plate (fig. 4)
SUMMARY
A modification of the histochemical technique for the localization of/?-glucuronidase
originally suggested by Friedenwald and Becker (1948) has been applied to the digestive gland of the gastropod Helix pomatia. In the original technique the ferric 8hydroxyquinoline formed by the enzymic hydrolysis of quinolyl-8-glucuronide, in
a saturated solution of ferric 8-hydroxyquinoline, was converted to Prussian blue.
The Prussian blue conversion is omitted in the technique described in this paper as it
appears to introduce errors in localization. The ferric 8-hydroxyquinoline crystals are
sufficiently characteristic to be used as the end-point of the technique. The results
obtained suggest that /S-glucurcnidase is confined to the digestive cells in the digestive
gland of the snail, and is associated with secretory granules in them.
CONTENTS
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O U T L I N E
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T H E
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O F T H E S U B S T R A T E
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8 - H Y D R O X Y Q U I N O L I N E
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C O U N T E R S T A I N I N G
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F O R T H E M O D I F I E D
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T h e
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T h e
localization
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INTRODUCTION
F
1
ISHMAN (1950) has reviewed the properties of /?-glucuronidase, an
enzyme which catalyses the hydrolysis of /2-glucuronides. The enzyme
appears to be particularly active in the kidney, liver, and spleen of mammals
(Mills, 1946). Far greater activity is displayed by the crop fluid of locusts
(Robinson, Smith, and Williams, 1953), and by the digestive glands of certain
[Quarterly Journal of Microscopical Science, Vol. 96, part 1, pp. 35-48, March 1955.]
6
36
Billett and McGee-Russell—fi-Glucuronidase in the
gastropod molluscs (Utusi, Huzi, Matumoto, and Nagaoka, 1949; Dodgson,
Lewis, and Spencer, 1953). In these invertebrates the enzyme may play some
part in the digestion of plant material; a similar function may be attributed to
the j8-glucuronidase said to be produced by micro-organisms in the digestive
systems of ruminants (Karunairatnam and Lewy, 1950; Marsh, Alexander,
and Levvy, 1952). The function of the enzyme in mammalian tissues is not
clear; it has been implicated in growth and regeneration processes (Lewy,
Kerr, and Campbell, 1948), and in the metabolism of sex hormones (Fishman,
1947; Fishman, Riotton, Farmelant, and Homburger, 1952).
Several methods have been suggested for the histochemical localization of
)3-glucuronidase in tissues. The method originally suggested by Friedenwald
and Becker (1948) appears to be the most satisfactory. A modified form of
this technique has been developed by Burton and Pearse (1952).
Preliminary experiments upon the kidney, liver, and spleen of the mouse
suggested that certain features of the technique required investigation. It was
decided, therefore, to examine the technique critically, stage by stage, using
a tissue known to possess a very high j8-glucuronidase activity, the digestive
gland of the Roman snail, as a test object for localization (Billett, 1954). It was
also of interest to establish the intracellular distribution of the enzyme in the
gland, in view of the possible association between /S-glucuronidase and mitochondria suggested by Campbell (1949), and the probable significance of the
enzyme in the digestive processes of the animal.
MATERIAL
Animals. The Helix pomatia were collected from a colony in a mixed wood
at Seven Springs Hill, Cheltenham, and were maintained in the laboratory in
damp compartments with earth floors, at room temperature, on a diet of
cabbage. Animals in temporary aestivation were not used for the experiments,
but in a number of instances the active animals were starved for a time before
dissection.
Chemicals. Quinolyl-8-glucuronide was prepared according to the method
of Robinson and others (1953). All other chemicals employed were of 'Analar'
or 'Reagent' grade. Chemically clean glassware was used throughout the
procedures described in this paper.
Dissection. The animal was killed by decapitation and removed from the
shell. As shown in fig. 1, the digestive gland is divided into four lobes occupying two regions, A and B. One lobe, the upper, occupies the extreme tip of the
helical shell and is closely associated with the ovotestis; the other three lobes
overlie the principle viscera and are separated by the looped intestine which
runs through the outer part of the gland. It is difficult to dissect out region A
uncontaminated with ovotestis, and except in one or two experiments in
which the ovotestis was deliberately included in the test material, region B
alone was used. It is easy to remove both the intestine and the mantle epithelium from this region and obtain the digestive gland tissue alone.
Digestive Gland of the Roman Snail
37
OUTLINE OF THE PREVIOUS METHODS
In the original method of Friedenwald and Becker (1948) the enzyme in
fresh tissue sections was allowed to act upon quinolyl-8-glucuronide in the
presence of ferric 8-hydroxyquinoline. Deposits of the latter compound
formed at the presumed site of the enzyme, were converted into Prussian blue.
Seligman and others (1951) found that the jS-glucuronidase of rat liver
upper lobe of
digestive gland
stomach
region A
region B
intestine
anus
mantle
ridge
crop
mouth
FIG. 1. A diagram illustrating the disposition of the digestive gland in the snail Helix pomatia.
withstood formalin fixation at about 40 C. Fixation under these conditions
was used by Burton and Pearse (1952) in their modification of the original
technique. This enabled these workers to cut thinner sections and to obtain
better localization of the Prussian blue deposits. The essentials of the method
described by these authors are as follows:
(1) Fix tissue (rat kidney) in 10 per cent, formalin for 4-48 hours.
(2) Cut frozen sections fio—15/x) into ice-cold o-i M acetate buffer at
(3) Transfer the sections to a mixture of quinolyl-8-glucuronide, ferric
8-hydroxyquinoline, and about o-i M acetate at pH 5-2.
(4) After 5-24 hours' incubation at 370 C. wash in water and transfer to
albumenized slides.
(5) Wash for 15 minutes in 0-5 M oxalate buffer (pH 4-0).
(6) Treat with an acid solution of potassium ferrocyanide to form Prussian
blue, and wash in water.
(7) Counterstain with Mayer's carmalum, or with neutral red. Mount in
DPX after dehydration in alcohols and clearing in xylene.
This technique gave disappointing results when applied to the digestive
gland of Helix pomatia. The Prussian blue deposits were poorly localized and
very much less in amount than the high activity of the tissue would lead one
to expect. Examination of successful preparations in mouse tissue showed that
38
Billett and McGee-Russell—p-Glucuronidase in the
the crystalline precipitate of ferric 8-hydroxyquinoline was sufficiently
characteristic to be used as the end-point of the technique; this appeared to
be important, for upon practical and theoretical grounds the conversion to
Prussian blue seemed to be a stage likely to introduce errors in cytological
localization.
FIXATION
Seligman and others (1951) found that well over 80 per cent, of the /?glucuronidase activity of rat liver remained in the tissue after it had been
fixed in 10 per cent, formalin, buffered to pH 7 with phosphate, at about
40 C. for 24 hours. For rat kidney Burton and Pearse (1952) found the
optimum time of fixation for good histochemical localization to be 4-12 hours
in 10 per cent, formalin at about 40 C.
By a method previously described (Billett, 1954) a number of determinations were made of the jS-glucuronidase activity of the snail digestive gland
before and after fixation in ice-cold formaldehyde-saline. This showed that
after 3 hours' fixation the degree of inactivation of the enzyme varied considerably, ranging from 40 to 80 per cent. Measurements of the activity after
more prolonged fixation suggested that the enzyme is at first rapidly inactivated (1-3 hours) and then more slowly (3-12 hours). Thus the enzyme in the
snail digestive gland appears to be more sensitive to fixation than the enzyme
in rat kidney and liver. Fixation is, however, necessary, for it is impossible to
cut thin sections of the freshly frozen gland satisfactorily. At least 3 hours in
ice-cold formaldehyde-saline is necessary to permit good sectioning at 2Ofi..
THE PREPARATION AND PROPERTIES OF THE SUBSTRATE MIXTURE
The method of preparation recommended by Burton and Pearse is as
follows. Mix 13 ml. of o-oi M quinolyl-8-glucuronide in o-i M acetate
buffer (pH 5-2), 13 ml. of o-oi M 8-hydroxyquinoline in o-i M acetate buffer
(pH 5-2), 9 ml. of 0-03 M ferric sulphate and i-o ml. of M acetate (pH 5-2).
Incubate the mixture for at least 2 hours at 370 C , centrifuge, and store the
supernatant fluid in a refrigerator.
8-Hydroxyquinoline and its glucuronide are not very soluble in water and
in order to achieve a o-oi M solution of these substances it is necessary to
apply heat. On cooling, crystals re-form in the solutions, particularly if these
are placed in a refrigerator. This difficulty was overcome by dissolving the
quinoline and its glucuronide in the same solution of deci-molar acetate
buffer, at the required pH, to make a 0-005 M solution with respect to each
substance (e.g. 14-5 mg. 8-hydroxyquinoline and 37-5 mg. of glucuronide in
20 ml. of acetate buffer). In this way a clear solution which does not crystallize
was obtained and the danger of having a reduced concentration of the substrate avoided. Alternatively, the compounds may be dissolved together in
water and the requisite amount of molar acetate buffer added.
Two reactions appear to occur when the substrate mixture is prepared.
Digestive Gland of the Roman Snail
39
First, the ferric sulphate reacts with the 8-hydroxyquinoline; this gives the
mixture a green colour and results in formation of a precipitate. Secondly, the
ferric sulphate reacts with the acetate forming a precipitate of basic ferric
acetate. Table 1 shows the effect of pH on these reactions. Clearly the
properties of the substrate mixture depend to a certain extent on the pH.
Below 4-5 (added acetate) there is a sudden intensification of the green colour,
presumably due to an increased concentration of ferric 8-hydroxyquinoline
in solution.
TABLE I
The effect of pH on the substrate mixture
pH of added
acetate
pH after 2
hours'
incubation
3-56
4-00
3 06
4-46
4-85
3-85
4-26
5'2O
4-58
Colour of
mixture
Precipitate
Effect of acetate
on Fe 2 (SO 4 ) 3
alone
Very
dark
green
Very
dark
green
Dark green
Light green
None
No
Slight
Precipitate
Pale yellowgreen
Heavy
Heavy
Heavy
precipitate
Heavy precipitate
Heavy precipitate
Table 1 also shows that a marked drop of pH occurs during the preparation
of the substrate. For the snail digestive gland it was necessary to prepare a
substrate mixture close to the optimum pH of the snail enzyme; this is 4-04-2. The pH of the added buffer was therefore 4-5.
Apart from the modifications which have been described, the substrate
mixture was prepared according to the procedure of Burton and Pearse.
It was observed that if a large number of sections was added to the substrate
solution the colour of the solution became appreciably lighter. This suggests
that adsorption of ferric 8-hydroxyquinoline occurs. The degree of adsorption
appears to depend upon the tissue; for instance, it is greater for the snail
digestive gland than it is for mouse kidney. Wigglesworth (1952) has commented on the considerable capacity of tissue sections for taking up iron from
solutions of salts.
The addition of potassium hydrogen saccharate in the concentrations
necessary to inhibit the enzyme also reduces the colour of the substrate
mixture. This is presumably due to the formation of iron saccharate. This
reaction would reduce the concentration of ferric 8-hydroxyquinoline in the
substrate mixture and the concentration of saccharate ions in the control.
THE FORMATION AND PROPERTIES OF FERRIC 8-HYDROXYQUINOLINE
These were studied in the following way. ZO/J. sections of formalin-fixed
tissue were cut into ice-cold saline. Within 10-15 minutes these sections were
40
Billett and McGee-Russell—p-Glucuronidase in the
placed in an incubator at 37° C. in the substrate mixture contained in small
(2-inch) Petri dishes, or in i-inch specimen tubes. The sections were mounted
in distilled water on slides at hourly intervals for observation.
Results. After incubation times varying from 1 hour to 12, crystals appeared
in the test sections. Control sections, inhibited by saccharate, showed no
development of crystals. In both test and control sections there was a general
darkening of the tissue associated, probably, with an uptake of iron from the
substrate. This darkening of the tissue was greater in the test than in the
control but it did not appear to be directly related to the subsequent development of the crystalline deposits.
The size, shape, and abundance of the crystals formed varied, apparently
TABLE 2
Reagent
Distilled water (about 19° C.)
Distilled water (about 60° C.)
Oxalate buffer (pH 4-0)
o-i M acetate buffer (pH 4^5)
Ethanol
Acetone
Glycerol
Observation
Appearance of the crystals remains unchanged for at least
7 days
Crystals dissolve in 4-5 hours
Crystals remain unchanged for
at least 24 hours
Crystals remain unchanged for
at least 24 hours
Some crystals dissolve in 5
minutes, most have disappeared within 30 minutes
Crystals dissolve within 30
seconds
Crystals unchanged after 7 days
Conclusion
Insoluble
Soluble
Insoluble
Insoluble
Fairly soluble
Very soluble
Insoluble
in accordance with a number of factors including the initial activity of the
tissue, the nature of the substrate (governed by the care taken in its preparation), and the time of incubation. Under optimum conditions a rapid decomposition of the substrate occurs and results in the intracellular production
of small crystals of ferric 8-hydroxyquinoline after 1-2 hours' incubation.
These crystals are irregularly rounded bodies about 1-3 /x in diameter. The
deposits grow rapidly with continued incubation, and after 4-5 hours are
exceedingly heavy. If conditions favour slow decomposition of the substrate the
initial crystal size tends to be large, the crystals are relatively few in number,
and they appear to be formed irrespective of cell boundaries. Crystals formed
in this way are needle-shaped (about 10/1 long), short rods (about 5^), and
hexagonal plates (3-4/x in diameter). Whatever their shape and size, the
crystals are characteristically brown and anisotropic.
We examined the solubility of the crystals formed within the sections of the
tissue in a number of solvents. Sections were transferred to solid watch
glasses containing the solvent and the condition of the crystals was followed
by using a low-power microscope. The observations are summarized in
table 2. The rate of dissolution of the crystals is probably influenced by such
Digestive Gland of the Roman Snail
41
factors as their size and their site of formation in the tissue. Attention was also
paid to the reaction of the crystals with the reagents employed to convert
them to Prussian blue in the techniques of the previous authors. These
observations are summarized in table 3.
TABLE 3
Reagent
Observation
Conversion of crystal deposits is incomplete
after 30 minutes. Colour formed is diffuse
in distribution. There is some fragmentation of the crystals
Equal volumes of i N HC1 and i per Complete conversion to Prussian blue in
cent, (w/v) K4Fe(CN)e at 6o° C.
15 minutes. Colour is diffuse
Equal volumes of i N HC1 and 2 per Conversion to Prussian blue is incomplete
after 30 minutes
cent, (w/v) K4Fe(CN)6
Equal volumes of 2 N HC1 and 1 per Conversion to Prussian blue is incomplete
cent, (w/v) K4Fe(CN)6
after 30 minutes
1 per cent, (w/v) K4Fe(CN)6
Crystals appear unchanged after 24 hours
1 NHC1
Little change in the appearance of the crystals
after 1 hour. Crystals disappear after 12
hours
1 per cent, (w/v) K4Fe(CN)0 at 60° C. Most crystals dissolve in 4—5 hours. (Solubility in hot water)
1 N HC1 at 60° C.
Crystals dissolve within 15 minutes
Equal volumes of i N HC1 and i per
cent, (w/v) K4Fe(CN)6
THE USE OF OXALATE BUFFER
After the formation of ferric 8-hydroxyquinoline, Burton and Pearse
recommend washing the sections in half-molar oxalate buffer (pH 40) for
15 minutes. In table 2 it may be seen that prolonged washing in the buffer has
no appreciable effect on the crystals. The effect of the oxalate washing is said
to be the removal of ferric hydroxide, and it is noticeable that the treatment
removes the general dark background coloration previously mentioned.
Sections are very much 'cleaner' and more transparent after washing for an
hour or longer in the buffer.
THE CONVERSION OF FERRIC 8-HYDROXYQUINOLINE TO PRUSSIAN BLUE
Ferric 8-hydroxyquinoline may be converted into Prussian blue by the
action of an acid solution of potassium ferrocyanide. Burton and Pearse used
a mixture of equal volumes of 1 N hydrochloric acid and 1 per cent, (w/v)
potassium ferrocyanide, applied to the sections for 15 minutes. The reaction
probably occurs in two stages: the acid breaks down the ferric 8-hydroxyquinoline, and the ferrocyanide reacts with the liberated iron.
The conversion of the crystals formed in our technique was readily observed under the microscope. Twenty fi sections were incubated in the
substrate mixture until heavy deposits of crystals had formed. After being
washed in oxalate and rinsed in distilled water these sections were subjected
42
Billett and McGee-Russell—fi-Glucuronidase in the
to the ferrocyanide hydrochloric acid mixture, and to the separate components
of this reagent. The reactions were observed with the sections in solid watch
glasses, or mounted on slides beneath coverslips. The effects of a number of
factors were studied. The results show that the acid ferrocyanide mixture
effects a relatively slow conversion of the ferric 8-hydroxyquinoline into
Prussian blue. The rate of conversion can be accelerated by using higher
concentrations of acid and ferrocyanide, and by carrying out the conversion
at 60° C. However, at the higher temperature the errors in localization liable
mitochondria
crystalline
deposit
outward diffusion
during formation
of prussian blue
colourless secretory
granule
FIG. 2. A diagrammatic representation of the appearance of crystals of ferric 8-hydroxyquinoline deposited within cytoplasmic vacuoles of digestive cells in association with secretory
granules.
to occur because of diffusion are likely to be greater than at the lower (laboratory) temperature. The Prussian blue deposits appear to bear only a general
resemblance to the localization of the crystals, the site of which is surrounded
by a diffuse blue colour. Apparently Prussian blue is as easily taken up by the
tissues as iron. Observation of the conversion of single crystals with a 2 mm.
oil-immersion objective shows that there is considerable diffusion of the blue
colour away from the decomposing crystals; the colour is taken up heavily
on the elements of the surrounding tissue. Thus for a crystal embedded in the
tissue, apparently within one of the cytoplasmic vacuoles, the situation illustrated in fig. 2 results. The Prussian blue formed diffuses outwards from the
crystal and colours the wall of the vacuole and any granular material which
is in the vacuole. Despite repeated observations, we have been unable to
ascertain whether the crystalline material is deposited around or merely close
to the granular material in the vacuoles.
Thus the ferrocyanide treatment may give rise to two sources of error.
First, during the conversion of the crystals to Prussian blue, considerable
redistribution by diffusion may occur. Secondly, owing to the slow rate of
Digestive Gland of the Roman Snail
43
conversion, both Prussian blue and ferric 8-hydroxyquinoline may be present
when the section is carried through the mounting procedure. Any such unconverted ferric 8-hydroxyquinoline will be rapidly removed by alcohol
(table 2). There is no reason to suppose that the conversion to Prussian blue
begins first at sites of highest activity, where the greatest number of crystals
occur: in fact the microscopical observations suggest rather the contrary.
Thus considerable misinterpretation may arise through the use of acid ferrocyanide, and it appears both desirable and practical to dispense with this
treatment altogether. Modified in the way described, the technique also
avoids the use of alcoholic dehydration, the sections being mounted directly
in an aqueous medium. This also appears to be advantageous, for when
sections of the digestive gland are passed from water into alcohol they undergo
a marked visible shrinkage and distortion. Such an effect may also give a false
disposition of the Prussian blue deposits, or of any crystals which survive the
alcohol treatment. In the modified technique, which is given in detail below,
the acid ferrocyanide treatment is omitted; we consider that the ferric
8-hydroxyquinoline crystals alone are sufficiently identifiable, and indicative
of the enzyme site, provided that rapid precipitation and small crystal size
are achieved.
CONTROLS
In the experiments described in this paper the controls contained potassium
hydrogen saccharate, added to the substrate mixture so as to give a final
concentration of the inhibitor of 0-0005 M. This concentration completely
inhibits the reaction occurring in the tests with the snail tissue. Additional
controls were made as follows. In some cases sections were incubated in
substrate which did not contain quinolyl 8-glucuronide, in others sections
were heated to 8o°-ioo° C. and then incubated in the substrate mixture under
normal conditions.
COUNTERSTAINING
A wide range of stains was tested, the majority of which were unsuitable
because of their solubility in glycerol mounting media. Satisfactory staining
was achieved with nuclear fast red (G. Gurr Ltd., batch no. 3569) for about
1 hour; or Mayer's haemalum for 5 minutes, followed by blueing in tap water;
or methyl green/acetic for 24 hours. The acidity of the last stain did not affect
the crystals.
SCHEDULE FOR THE MODIFIED TECHNIQUE
The following schedule was adopted in the light of the observations described and discussed above. Throughout the procedure the sections are
handled with glass needles.
(1) Kill a snail by decapitation and remove the digestive gland.
(2) Place the tissue in ice-cold formaldehyde-saline and leave it in the
refrigerator for 3 hours.
44
Billett and McGee-Russell—fi-Glucuronidase in the
(3) Wash tissue in ice-cold saline (0-9 per cent, w/v) for 20 minutes (three
changes).
(4) Cut sections at 20 /z on a freezing microtome into chilled saline.
(5) Place sections into freshly prepared substrate mixture:
test: 2-0 ml. substrate mixture with o-i ml. distilled water;
control: 2-0 ml. substrate mixture with o-i ml. o-oi M potassium
hydrogen saccharate.
(6) Examine the sections hourly by removing and mounting one on a slide
(float it on with distilled water), until heavy deposits of crystals are
formed.
(7) Rinse the sections in distilled water and wash in oxalate buffer for
1 hour.
(8) Rinse again in distilled water and counterstain.
(9) Float sections on to glass slides with distilled water and mount in
Farrant's medium.
RESULTS
The results of this investigation may be considered under two headings :
first, the findings with respect to the bases of the technique, and secondly, the
results of the technique upon the selected tissue, the digestive gland of the
snail. The former have been largely set out above; the latter involves some
account of the structure of the digestive gland.
The histology of the digestive gland. The results of a detailed study of the
histology of this tissue will be summarized shortly; a longer account is in
preparation.
The digestive gland of the snail is a diverticulum of the gut, composed of a
highly modified secretory epithelium grouped into blind-ended tubules. The
tubules are loosely bound together by connective tissue, and by a ramifying
system of haemocoelic ducts. Histologically the organ is complex, but it shows
no differentiation into locally specialized areas, the numerous cell-types
occurring everywhere throughout the tissue.
Previous authors have distinguished three principal cell types in the
secretory epithelium: the Kalkzellen or calcium cells, which are the most
characteristic; the Fermentzellen, Keulenzellen, or ferment cells; and the
Leberzellen, Karnerzellen, or liver cells (Barfurth, 1883; Frenzel, 1885;
Cuenot, 1892). More recent authors (Wagge, 1951; Fretter, 1953) have not
distinguished between the last two types named. Fretter (personal communication) considers that the digestive cells may be regarded as having both
secretory and excretory functions, and we agree with this view. Krijgsman
(1925; 1928) detected a secretory cycle in both the salivary glands and the
digestive gland. He also distinguished only calcium cells and digestive cells
in the digestive gland epithelium. We are in substantial agreement with this
point of view, but would point out that the category denoted by 'digestive cell'
includes a number of cell-types of different form, including those distinguished by earlier authors as Fermentzellen and Leberzellen, which probably
Digestive Gland of the Roman Snail
45
represent stages in two principal cycles of excretion and secretion (McGeeRussell, in preparation). Fig. 3 illustrates the four forms of cell which we can
detect in the digestive epithelium fixed by this technique: three (A, Bv and
B2) are digestive cells in what are presumed to be different stages of cell
activity; the fourth is the characteristic calcium cell (C).
diqestive cell
typeB,
digestive ce
type A
connective tissue
fibrocytes
cells surrounding the_
hoemocoelic space
haemocoelic
space
FIG. 3. A diagram of the structure of the digestive gland tubules of Helix pomatia cut in
transverse section.
The digestive cells are all elongate club-shaped or rounded cells varying in
height from 50 to 100/x in fixed preparations. There is a basal nucleus about
5 to 10/i, in diameter, usually with a single nucleolus which is not prominent.
The cytoplasm is greatly vacuolated, and the vacuoles contain different sorts
of granules. These granules are the chief differences between types A, B lt
and B2. Type A contains yellow irregular granules in basal vacuoles around
and above the nucleus, and colourless more rounded granules in vacuoles
distal to the nucleus, occupying the border of the cell that abuts on the lumen
of the tubule. Type Bx contains colourless granules of the same nature in
vacuoles throughout the cytoplasm, and few if any of the yellow granules.
Type B2 has a single vacuole, or very few extremely large vacuoles occupying
the greater part of the cell cytoplasm. These contain large, dark brown,
rounded bodies. In life the round brown bodies are suspended in a yellow
liquid within the vacuole. Type B2 is distinguished as Fermentzellen by the
earlier authors; type A corresponds to Leberzellen. Cuenot (1892) regards
type B2 as cellules excretrices and type A as cellules hepatiques. The results with
the histochemical technique described in this paper seem to indicate that this
46
Billett and McGee-Russell—fi-Glucuronidase in the
is the correct interpretation, types B1 and A being chiefly concerned in
secretion.
The localization of fi-glucuronidase in the digestive cells. After incubation in
accordance with the schedule given, crystals of ferric 8-hydroxyquinoline
appear in two principal sites within the cells of the tubule epithelium. The
first is in the distal tips of the digestive cells where they abut on the lumen of
the tubule, and the second is in the body of the digestive cells, extending down
around the basal nuclei, and also out into the tips of the cells. This may be
seen in the photomicrographs (fig. 4), which show the heavy nature of the
deposit and the complete absence of response in the control section. Careful
study of a large number of sections from the experiments shows that there is
no constant relationship between the distribution of the crystals and that of
the brown bodies of cell type B2, or that of the yellow irregular granules of
cell type A; but the distribution in the sites given above is constant and
typical of the tissue. At the highest magnifications it can be seen that the
crystals are embedded in the tissue, and often appear to have been formed
within the cytoplasmic vacuoles, either around or in close association with
the colourless granules. This accords with the gross distribution of the crystals
in two principal sites, since it would arise from association with the colourless
granules of cell type A and the colourless granules of cell type Bv
Many crystals appear to be deposited in an irregular manner upon the cut
surface of the section, and a large number of these may float off during the
various washings. However, crystals which have developed within the tissue
consistently show the characteristic distribution. Crystals do not appear to
develop in any consistent association with the calcium cells, or with the
connective tissue, or the cells of the haemocoelic system. The crystal deposits
do not form in all the tubules of a section, which may indicate that different
tubules are in different phases of secretory activity.
The histology of the digestive gland of the closely related species Helix
aspersa is identical with that of H. pomatia, and the distribution of the crystal
deposits after incubation is also the same.
DISCUSSION
/J-glucuronidase appears to be associated with the digestive processes of a
number of herbivorous animals. In some it has been suggested that it is the
product of intestinal micro-organisms (Marsh and others, 1952). Such organisms have been suggested as responsible for the high activity of the enzyme
in the crop fluid of locusts (Robinson and others, 1953). Even higher activities
occur in gastropod molluscs, particularly in H. pomatia, but the evidence
presented in this paper indicates that the enzyme is produced by the tissues
Fie. 4. A, a test section of the digestive gland of Helix pomatia after 4 hours' incubation.
Unstained. Note the heavy deposits of ferric 8-hydroxyquinoline.
B, a control section. Unstained; the lack of transparency of the tissue is sufficient for
photography.
Digestive Gland of the Roman Snail
47
of the animal itself, an adaptation probably directly related to the efficient
herbivorous digestion by terrestrial snails. It would be interesting to know
whether more detailed studies of the locust would demonstrate a similar
intrinsic synthesis of the enzyme.
The crop fluid of H. pomatia has long been known to contain a variety of
enzymes (Biedermann, 1911). Holden and others (1950) review some thirty
of the enzymes which have been found in the fluid. It is secreted by two
principal secretory organs, the salivary glands and the digestive gland. The
greater volume of the fluid appears to be derived from the digestive gland,
within which the j8-glucuronidase is produced. Recent authors have employed
the crop fluid as a useful reagent for the maceration of plant tissue, for there
is almost no proteolytic activity in it (Faberge, 1945; Holden, Pirie, and
Tracey, 1950). The cellulolytic activity of the fluid is usually attributed to a
complex of enzymes termed 'cytase'. At present it is reasonable to suggest
that /J-glucuronidase is part of this complex. Marsh and others (1952) have
suggested hemicellulose as a possible substrate for the enzyme. It is likely
therefore that rich sources of /3-glucuronidase may be found in wood-boring
organisms such as the mollusc Teredo, and beetle larvae. Biochemical and
histochemical information on such organisms would be valuable.
The modifications in technique given in the paper have the advantages of
greater simplicity and increased consistency of results. The principal step of
omitting the stage of conversion to Prussian blue appears to give localization
as good as, or better than, the original method. This advantage is somewhat
offset by the impossibility of making preparations in Canada balsam, but our
preparations mounted in Farrants's medium keep well, and avoid the disadvantages of alcoholic dehydration.
Campbell (1949) has suggested that jS-glucuronidase is closely associated
with the mitochondria of certain cells. This does not appear to be the case
with the cells of the snail digestive gland. In view of the large crystal size, it
does not appear to us to be possible to demonstrate such an association with
the technique as it stands. In any case the conditions of the test, involving the
use of acetate buffer, make the survival of mitochondria problematical.
Furthermore, cytological detail may be destroyed by autolytic enzymes which
have not been completely inactivated by the fixation procedure. The aim of
the fixation, in common with other techniques for the localization of enzymes
in tissues, is to leave the enzyme as active as possible rather than to preserve
cytological detail.
During the course of the experiments with the snail digestive gland it was
found that the addition of sodium chloride and potassium sulphate to the
substrate mixture produced a more rapid and more intense reaction. This may
follow from one of two causes: either the salts may exert a true activating
effect on the enzyme, or they may produce a salting out effect, causing a more
rapid precipitation of the ferric 8-hydroxyquinoline.
We have applied the technique described in this paper to other tissues.
The results obtained confirm the observations made with the snail tissue, but
48
Billett and McGee-Rtissell—fi-Glucuronidase in the Snail
they demonstrate that variable factors in the tissues, other than the enzyme
content, may affect the results of the technique. Different tissues vary in their
capacity to adsorb ferric iron. Under certain conditions this factor and the
reaction of saccharate with ferric 8-hydroxyquinoline can combine to produce
invalid controls (unpublished observations).
We wish to thank Professor A. C. Hardy, F.R.S., for the facilities which he
provided for us within his Department. We are most grateful to Dr. J. R.
Baker and Dr. P. C. J. Brunet for their interest and valuable criticism. This
work was carried out during the tenure of a personal grant from the Medical
Research Council (F. B.), and of a Christopher Welch Scholarship in the
University of Oxford, with support from the Department of Scientific and
Industrial Research (S. M. M.-R.).
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