The Localization of /3-Glucuronidase in the Early
Chick Embryo
by
F. BILLETT 1
and
LEELA MULHERKAR 2
From the Institute of Animal Genetics, University of Edinburgh
WITH ONE PLATE
INTRODUCTION
I N vertebrate tissues there appears to be a connexion between /3-glucuronidase
and the proliferation of cells. This connexion was first noticed by Levvy, Kerr,
& Campbell (1948) when they were investigating the effect of toxic compounds
on mouse-liver glucuronidase. The early stages of embryonic development are
characterized by rapid proliferations of cells and it is, therefore, of some interest
to study the localization of /3-glucuronidase during these stages. Small amounts
of /S-glucuronidase can be detected in the early embryos of Xenopus laevis
(Billett, 1956) and in those of Drosophila melanogaster (Billett & Counce, unpublished). In these embryos no marked increase in the enzyme can be associated with the proliferation of cells. The large amount of yolk in the Xenopus
and Drosophila embryos was a complicating factor in the above experiments.
It was not possible to localize the enzyme in these embryos with a histochemical
method. An embedding technique suited to the nature of the embryos and the
requirements of the histochemical test could not be devised.
It has, however, proved relatively simple to apply a modification of the
Friedenwald & Becker (1948) technique to whole chick embryos cultivated in
vitro. This technique, because of the diffuse nature of the reaction and the
destruction of cytological detail which it entails, can only demonstrate the
presence of /?-glucuronidase in particular cells or groups of cells (Billett &
McGee-Russell, 1956). Intracellular localization of the enzyme is impossible.
The method appears to be well suited to the present purpose, aimed at a histological rather than a cytological localization of the enzyme in the early chick
embryo.
METHODS
The modification of the Friedenwald & Becker technique, used in these experiments, has been described elsewhere (Billett & McGee-Russell, 1955).
1
2
Address: Institute of Animal Genetics, West Mains Road, Edinburgh 9, U.K.
Address: Sir Parashurambhau College, Poona 2, India.
[J. Embryol. exp. Morph. Vol. 6, Part 1, pp. 52-56, March 1958]
/3-GLUCURONIDASE IN CHICK EMBRYOS
53
Chick embryos, which varied in age from the primitive streak to 10 or
more somites, were transferred to culture chambers according to the method
described by New (1955). About an hour after explantation the albumen, which
is in contact with the dorsal side of the embryos, was removed and the embryos
were completely surrounded by a saturated solution of ferric 8-hydroxyquinoline
and quinolyl-8-glucuronide in 0-1 M acetate buffer at pH 4-5. The embryos were
incubated in the substrate mixture for 24 hours at 37° C. Development was
arrested by the substrate, and at the end of incubation the embryonic tissues
appeared to have suffered little distortion. After this treatment the embryos were
removed from the culture chambers, washed in water, and the vitelline membranes removed. The embryos were then washed in oxalate buffer, again in
water, and then placed for half an hour in neutral formaldehyde solution (4 per
cent, w/v). After a final washing in water the embryos were mounted in Farrant's
medium. The formation of brown crystals of ferric 8-hydroxyquinoline, embedded in the tissue, was taken to indicate the presence of the enzyme.
Controls were set up in three ways: (1) substrate mixture was prepared without
the addition of glucuronide; (2) embryos were heated to 80-90° C. for 5 minutes;
(3) substrate was used containing 0001 M potassium hydrogen saccharate. The
latter is a specific inhibitor for the enzyme (Levvy, 1952).
RESULTS
Our findings are summarized in Table 1. Typical preparations are shown in
the Plate. The enzyme could not be detected in the primitive streak stage. In 5
TABLE 1
Glucuronidase reaction
No. of embryos
Stage no. of
Hamburger &
Hamilton (1951)
Description of
stage
Strongly
positive
Weak
reaction
No
reaction
3^
5
6
7-8
8-9
9-12
Primitive streak
Head-process
Head-fold
1-3 pairs somites
4-6 pairs somites
9-12 pairs somites
0
5
1
8
7
6
1
0
0
2
0
0
8
4
9
3
0
2
out of 9 cases the head-process stage gave a strongly positive reaction. At a
slightly later stage, formation of the head-fold, only 1 positive reaction out of
10 was recorded. These observations suggest that the enzyme disappears at this
stage. Alternatively, a technical failure of the histochemical reaction would
account for the result. However, the results for the head-fold embryos were
obtained from four different batches of embryos which contained earlier and
54
F. BILLETT AND L. MULHERKAR
later stages which gave positive results. Once the somites had formed, a strong
positive reaction was usually obtained.
An examination of the preparations gave a general impression that the
enzyme was at first widely diffused on the dorsal side of the embryo, and that
it later became more strictly localized in the mid-dorsal line in the region of the
neural tube and somites. Little or no enzyme could be detected on the ventral
side of the embryo. Crystals were only deposited in the embryos and in the
extra-embryonic tissue immediately surrounding the embryo. At the headprocess stage crystals were deposited in the region of the primitive streak. In the
later stages crystals were rarely deposited in the retreating streak, in marked
contrast with the remainder of the embryo (Plate, figs. B-D).
Some of the treated embryos were embedded in 10 per cent, w/v gelatin and
sectioned. Satisfactory embedding was not achieved because the crystals of
ferric 8-hydroxyquinoline dissolved if left in the aqueous gelatin at 37° C. for
more than 6 hours. Only a short embedding time, of approximately 2 hours,
could be used, and this made sections difficult to cut. Examination of the sections
showed crystals of ferric 8-hydroxyquinoline embedded in the neural folds and
adjacent ectoderm, and in the somites.
The effect of potassium hydrogen saccharate on the development of the embryos
Although a 0 001 M solution of potassium hydrogen saccharate prevented
the formation of ferric 8-hydroxyquinoline in the histochemical test, a similar
concentration of the inhibitor did not prevent the normal development of
embryos explanted at a primitive streak stage. This observation suggests that
the enzyme is either inactive or inaccessible in the intact embryo. In this connexion it is to be noted that Karunairatnum & Levvy (1949) failed to influence
liver regeneration and growth in mice by the administration of large doses of
saccharic acid.
DISCUSSION
Using the method described above, /?-glucurinodase cannot be detected in
chick embryos until the tissues have begun to differentiate. These results do not
support a general hypothesis that the enzyme is specifically associated with the
proliferation of cells.
Hollinger & Rossiter (1952), studying Wallerian degeneration of nerve, observed that an increase in /3-glucuronidase occurred after cellular proliferation
had taken place in the degenerating tissue. In the liver of the rat, regenerating
after sub-total hepatectomy, the peak of /3-glucuronidase activity also occurs
after the phase of rapid cellular proliferation (Mills et al., 1950).
These results for regenerating tissue, and the present findings for the chick
embryo, suggest that /?-glucuronidase is often associated with tissues which are
in the process of differentiation following an initial phase of cellular proliferation. Changes in /3-glucuronidase activity may in fact reflect changes in cell-
0-GLUCURODINASE IN CHICK EMBRYOS
55
type, as was suggested by Mills & Smith (1951). Raised /3-glucuronidase levels,
such as occur in certain neoplasms (Fishman et ah, 1947, 1950) may indicate a
type of tissue whose cells have reverted to, and remain in, a state which is characteristic of those found in tissues in the early stages of differentiation.
SUMMARY
The technique for the localization of /3-glucuronidase, based on the precipitation of ferric 8-hydroxyquinoline from quinolyl-8-glucuronide has been
applied to explanted chick embryos. When the enzyme is first detectable, at the
head-process stage, it appears to be widely diffused in the dorsal side of the
embryo and in the adjacent area opaca. In later stages the histochemical reaction is confined to the region of the developing somites and neural tube.
ACKNOWLEDGEMENTS
We wish to thank Professor C. H. Waddington for the help and advice he has
given us in our work. The work was assisted by a grant from the Advisory Committee for Medical Research for Scotland. One of us (L. M.) is indebted to the
Indian Government for an overseas scholarship.
REFERENCES
BILLETT, F. (1956). The /?-glucuronidase activity of extracts from various stages of Xenopus
embryos. Proc. roy. pliys. Soc. Edinb. 25, 21-23.
•—— & MCGEE-RUSSELL, S. M. (1955). The histochemical localisation of /3-glucuronidase in
the digestive gland of the Roman snail. Quart. J. micr. Sci. 96, 35-48.
-(1956). The histochemical localisation of /S-glucurodinase in the liver of the newt.
Quart. J. micr. Sci. 97, 155-9.
FISHMAN, W. H., & ANYLAN, A. J. (1947). The presence of high /3-glucuronidase activity in cancer
tissue. J. biol. Chem. 169, 449-50..
—— & BIGELOW, R. (1950). A comparative study of the morphology and glucuronidase activity
in 44 gastrointestinal neoplasms. / . Nat. Cancer Inst. 10, 1115-22.
FRIEDENWALD, J. S., & BECKER, B. (1948). The histochemical localization of /3-glucuronidase.
/. cell. comp. Physiol. 31, 303-10.
HAMBURGER, V., & HAMILTON, M. L. (1951). A series of normal stages in the development of the
chick embryo. / . Morph. 88, 49-92.
HOLLINGER, D. M., & ROSSITER, R. J. (1952). Chemical studies of peripheral nerve during Waller ian degeneration. 5. ^-glucuronidase. Biochem. J. 52, 659-63.
KARUNAIRATNUM, M. C , & LEVVY, G. A. (1949). The inhibition of ^-glucuronidase by saccharic
acid and the role of the enzyme in glucuronide synthesis. Biochem. J. 44, 599-604.
LEVVY, G. A. (1952). The preparation and properties of j3-glucuronidase. 4. Inhibition by sugar
acids and their lactoses. Biochem. J. 52, 464-72.
KERR, L. M. M., & CAMPBELL, J. G. (1948). /3-glucuronidase and cell proliferation.
Biochem. J. 42, 462-8.
MILLS, G. T., & SMITH, E. E. B. (1951). The /3-glucuronidase activity of chemically induced rat
hepatoma. Science, 114, 690-2.
•
STARY, B., & LESLIE, I. (1950). The behaviour of ^-glucuronidase and nucleic acids in
rat liver during growth. Biochem. J. 47, xlviii.
NEW, D. A. T. (1955). A new technique for the cultivation of chick embryos in vitro. J. Embryol.
exp. Morph. 3, 326-31.
56
F. B I L L E T T A N D L. M U L H E R K A R
E X P L A N A T I O N OF P L A T E
The photographs are of whole chick embryos seen from the dorsal side. The crystals of ferric
8-hydroxyquinoline appear as black spots. The preparations are unstained.
FIG. A. Head-process. Test. Deposits of ferric 8-hydroxyquinoline in the dorsal side of embryo
and in the adjacent area pellucida.
FIG. B. Three pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline in dorsal side of
embryo and in area pellucida on each side of the embryo.
FIG. C. About ten pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline concentrated in
mid-dorsal line, in neural tube and somites.
FIG. D. About twelve pairs of somites. Similar to fig. C.
(Manuscript received 9: iv: 57)
J. Embryol. exp. Morph.
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F. BILLETT and L. MULHERKAR