/ . Embryol. exp. Morph. Vol. 32, 3, pp. 835-848, 1974
Printed in Great Britain
835
Studies on effects of 5-bromodeoxyuridine on the
development of explanted early chick embryos
By H. LEE,1 A. K. DESHPANDE 2 AND G. W. KALMUS 1
From the Department of Biology, Rutgers University, Camden, N.J.
SUMMARY
Chick embryos were explanted at stages ranging from the definitive streak to one-somite
and cultured for 19-21 h on thin albumen with or without 5-bromodeoxyuridine (BrdU)
(2-12/ig/ml). BrdU primarily inhibited neurulation which was found to be stage dependent.
If exposed to BrdU at the definitive streak stage, it resulted in an open brain region; if treatment was delayed until the head-process stage, it inhibited the closure of the midbrain and,
to some extent, the hindbrain, but the forebrain closure was mostly unaffected. BrdU had
no appreciable effect on later stages. It was also found that BrdU inevitably inhibited somite
formation when exposed to 8 /*g/ml or higher concentrations. The blastodermal expansion,
heart development, and blood island formation were usually not affected.
Cytological changes were observed in adversely affected tissues, particularly the neural
tissue. BrdU produced nuclear enlargement and clumped metaphase, but did not cause
extensive necrosis.
The grafting experiments showed that an increase in the concentration of BrdU from
4 to 12/Ag/ml progressively inhibited the self-differentiation of Hensen's node grafts. However, BrdU (8 /«g/ml) had no significant effect on the inducing capacity of node grafts nor the
competence of the epiblast.
Autoradiographic studies showed that the application of BrdU, at 6 /*g/ml or lower, caused
no obvious variations in the incorporation of [3H]uridine into chick embryonic cells. BrdU,
at 10-12/tg/ml, selectively inhibited the uptake of [3H]uridine in the neural tissue.
INTRODUCTION
The thymidine analogue, 5-bromodeoxyuridine (BrdU = BUdR), has proved
to be a useful tool in the study of cell differentiation (Wilt & Anderson, 1972).
The precise mode of the inhibitory action of BrdU remains unclear, although
the evidence suggests that it is incorporated into DNA (Bischoff & Holtzer,
1970; Stellwagen & Tomkins, 1971; Ostertag et al. 1973; O'Neil & Stockdale,
1974) and/or interferes with the synthesis of carbohydrates necessary for the
formation of some cell membrane constituents (Schubert & Jacob, 1970).
So far BrdU has been shown to inhibit the synthesis of proteins characteristic
of the differentiated state in several developing systems (Abbott & Holtzer,
1968; Miura & Wilt, 1971; Stellwagen & Tomkins, 1971; Weintraub, Campbell
1
Authors'1 address: Department of Biology, Rutgers University, Camden, New Jersey
08102, U.S.A.
2
Author's address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
836
H. LEE, A. K. DESHPANDE AND G. W. KALMUS
& Holtzer, 1972, etc.) and to interfere with the early development of sea urchin
eggs (Gontcharoff & Mazia, 1967; Tencer & Brachet, 1973), tunicate and
amphibian embryos (Tencer & Brachet, 1973). Our previous study (Lee, Deshpande & Kalmus, 1974) showed that BrdU (3 x 10~4 M) selectively inhibited the
morphogenesis of the brain and somites in explanted definitive streak stage
chick embryos. It was also noted that the inhibitory action of BrdU could be
alleviated by subsequent treatment with an equimolar concentration of thymidine, suggesting its incorporation into DNA. However, very little is known
about its effects on other important aspects of early chick development. In view
of this, the present study was undertaken (1) to extend our previous investigation
by including older embryos and cytological observations, (2) to study effects of
BrdU on the self-differentiation and inducing capacity of Hensen's node and
the competence of the epiblast, and (3) to investigate autoradiographically
whether BrdU affects RNA synthesis in chick embryonic cells.
MATERIALS AND METHODS
Cultivation and treatment of chick embryos
Fertile White Leghorn eggs were incubated at 37-5 °C for varying periods of
time to obtain embryos at stages ranging from the definitive streak to 1-somite
(stages 4-7; Hamburger & Hamilton, 1951). The embryos were cultured by
New's (1955) technique. A stock solution of BrdU (Sigma Chemical) was prepared in Pannett-Compton (PC) saline at a concentration of 1 mg/ml (3-3 x
10~3 M) and stored in the dark at 4 °C. The desired amounts of stock solution
were added to thin albumen immediately before use. Similarly, equal volume
of PC saline was added to thin albumen for the control series. Explanted
embryos were exposed to BrdU before incubation according to the procedure
described by Billett, Bowman & Pugh (1971). Unless otherwise stated, embryos
were grown for 19-21 h on thin albumen with or without BrdU.
Grafting procedure
The grafts used in this study were obtained from the node area (size = 0-3 x
0-3 mm) of stage 3 + embryos (average length of streak = 1-32 mm). Each graft
was inserted between the epiblast and hypoblast in the area pellucida of a host
embryo at stage 4 following the technique described by Waddington (1932).
The host embryos were grown for 24 h on thin albumen with or without BrdU.
Histological preparations
At the end of the incubation period, some embryos were fixed in Bouin's
fluid, stained with Delafield's hematoxylin, and kept as whole mounts. Others
were fixed in Bouin's fluid or 10% neutral formalin, embedded in paraffin,
sectioned at 6 ju,m, and stained with Delafield's hematoxylin and eosin or methyl
green-pyronin.
Effects of 5-bromodeoxyuridine on chick embryos
837
Autoradiographic techniques
Stage 4 embryos were grown for different lengths of time on thin albumen
containing 5 /tCi/ml [3H]uridine (New England Nuclear). This labelling process
did not interfere with the subsequent development of control embryos. After
the incubation period, the blastoderms were washed several times in PC saline,
fixed in absolute ethanol-glacial acetic acid (3:1), embedded in paraffin, sectioned at 4 /tm, and mounted on slides. The slides were deparaffinized, divided
into 4 groups, and were treated for 1 h at 37 °C with one of the following: (1) distilled water, (2) 0-8 mg/ml RNase (Worthington Biochem.) boiled at 100 °C
for 30 min to inactivate DNase, (3) 0-1 mg/ml DNase-RNase free (Worthington
Biochem.), and (4) 0-8 mg/ml RNase plus 0-1 mg/ml DNase. All the slides were
covered with Kodak NTB-2 liquid photographic emulsion using the dipping
technique of Kopriwa & Leblond (1962) and exposed for 10 days at 4 °C. The
emulsion was developed for 6 min in Kodak D-19 at 20 °C and fixed for 10 min
in Kodak acid-fixer. The sections were stained with hematoxylin-eosin, dehydrated through a graded ethanol series, and mounted in Permont. For quantitative evaluation of autoradiographs, the following procedure was used. For
example, a section through the tip of the notochord of each embryo was initially
located. Grain counts were then made over nuclei and cytoplasm of the cells
in the neural fold region (unit area = 40x40/mi). Similar procedure was
followed to study the distribution of labelled materials in other tissues such as
notochord, epidermis, and somite mesoderm. Background counts were made
near the section to which the reading would apply.
RESULTS
Effects ofBrdU on the development of early chick embryos
In this series of experiments, chick embryos at stages ranging from 4 to 7
were exposed to different concentrations of BrdU for 19-21 h. It was found that
BrdU, at a concentration of 1 /*g/ml, caused a growth retardation of stage 4
embryos, but had no obvious effect on older embryos. Concentrations of 16 /tg/
ml or higher caused extensive disintegration of all embryos. Four categories
were used to record the degree of development: (1) little or no development embryos at the same stage of development as when explanted; (2) very abnormal
- severely malformed embryos (Fig. 1 A); (3) abnormal - embryos with one or
more discernible abnormalities (Fig. 1B, C, D), and (4) normal (Fig. 1E). The
results are summarized in Table 1.
BrdU (8/tg/ml or higher) inhibited somite formation, regardless of the
developmental stage of embryos at treatment. The blastodermal expansion,
heart development, and blood island formation, except in severely malformed
embryos, were not significantly affected. In contrast, the magnitude of the
inhibitory action of BrdU (2-12/^g/ml) on the brain development was stage
838
H. LEE, A. K. DESHPANDE AND G. W. KALMUS
Effects of 5-bromodeoxyuridine on chick embryos
839
Table 1. Effects ofBrdU on the development of chick embryos explanted
at stages 4-7 and cultured for 19-21 h
% of embryos
Cone, of
BrdU (/tg/ml)
0
(Control)
2-4
6-8
10-12
Stage(s)
treated
No. of
embryos
4
5
6-7
4
5
6-7
4
5
6-7
4
5
6-7
28
24
24
33
28
30
38
20
21
25
24
21
Little or no
Very
development abnormal Abnormal
0
0
0
0
0
0
0
0
0
8
0
0
0
0
0
6
4
3
11
15
10
20
25
0
14
12
8
64
50
27
89
65
47
72
67
62
Normal
86
88
92
30
46
70
0
20
43
0
8
38
dependent: if treatment was begun at stage 4, it resulted in an open brain region
(Fig. IB); if treatment was delayed until stage 5, it inhibited the closure of the
midbrain and, to some extent, the hindbrain, but the forebrain closure was
usually unaffected (Fig. 1 C, 2 A). However, BrdU had no apparent effect on
the brain development of embryos treated at stages 6-7 (Fig. 1D). Furthermore,
in many malformed embryos, particularly those treated at stages 5-7, large
vesicles were found along the posterior axis (Fig. 1 C, D). These vesicles appeared
to be formed from the disintegration of somite mesoderm and accumulation of
fluid between ectoderm and endoderm.
One of the most conspicuous changes seen in transverse sections of BrdUtreated embryos was a marked enlargement of the dorsal aortae (compare
Fig. 1A with Fig. IB).
FIGURE 1
(A) Embryo explanted at stage 4 and cultured for 21 h on thin albumen with
12/ig/ml BrdU. Scale line = 0-63 mm.
(B) Embryo explanted at stage 4 and cultured for 21 h on thin albumen with
8 /ig/ml BrdU. Scale line = 0-63 mm.
(C) Embryo explanted at stage 5 and cultured for 19 h on thin albumen with
8 /tg/ml BrdU. Scale line = 0-63 mm.
(D) Embryo explanted at stage 6 and cultured for 19 h on thin albumen with
10 /ig/ml BrdU. Scale line = 0-63 mm.
(E) Embryo explanted at stage 4 and cultured for 21 h on thin albumen. Scale
line = 0-63 mm.
840
H. LEE, A. K. DESHPANDE AND G. W. KALMUS
B1
Fig. 2. (A) Transverse section through forebrain region of embryo explanted at
stage 5 and cultured for 19 h on thin albumen with 8/*g/ml BrdU (scale line = 009
mm). (B) Transverse section through forebrain region of embryo explanted at
stage 5 and cultured for 19 h on thin albumen (scale line = 009 mm). (C) Neural
tube cells of embryo explanted at stage 4 and cultured for 21 h on thin albumen with
8 /Mg/ml BrdU (scale line = 16-7 /*m). (D) Neural tube cells of embryo explanted at
stage 4 and cultured for 21 h on thin albumen (scale line = 16-7 /mi).
Cyto logical effects of BrdU
The neural tube of malformed embryos showed various types of abnormal
cells depending largely on the concentration of BrdU used. Treatment with
2-4 /£g/ml BrdU increased the number of cells with nucleus larger than normal.
Nuclear enlargement, whether slight or pronounced, appeared to be the first
sign of the nucleotoxic effect of BrdU. The enlarged nuclei stained diffusely with
methyl green, showing a barely recognizable network of chromatin. At 6-12 jag/
ml, some neural tube cells showed, besides the nuclear enlargement, pycnosis
and chromosomal abnormalities (compare Fig. 2C with Fig. 2D). Most cells
in division were at metaphase, although examples of other mitotic phases were
scattered throughout the adversely affected neural tissue. Chromosomal abnormalities such as clumped metaphase ('star' metaphase), C-metaphase, and
chromosomal bridge were frequently observed (Fig. 2 C).
In some severely malformed embryos the head mesenchyme was represented
Effects of 5-bromodeoxyuridine on chick embryos
841
Table 2. Effects of different concentrations of BrdU on the self-differentiation
and inducing capacity of Hensen's node grafts
Total % of grafts
Cone, of
Total
Differentiated
RrHI I
Undifferentiated
A
(/'g/ml)
grafts
Induction
No induction
Induction
0
(control)
4
8
12
18
72-2
111
111
5-6
12
12
12
33-3
33-3
250
8-3
00
00
500
500
500
8-3
16-7
250
No induction
by fewer cells than usual. These cells were large compared to the controls, often
rounded, and lost the usual spindle-shaped character.
The somite mesoderm did not show such conspicuous cytological changes as
the neural tissue, although its differentiation into dermatome, myotome, and
sclerotome was inhibited where the neural tube was very degenerate. Nuclear
enlargement and metaphase clumping were occasionally found in severely
affected somite mesoderm.
The notochordal, cardiac, and endodermal cells were not noticeably affected
by BrdU, at concentrations of 12/tg/ml or lower. The blood island formation
was also unaffected.
Effects of different concentrations of BrdU on self-differentiation
and inducing capacity of Hensen's node
Stage 3 embryos (average length of streak = 0-76 mm) were grown on thin
albumen with or without BrdU. The node area was excised from the embryos
which had advanced to stage 3 + (average length of streak = 1-32 mm), washed
several times in PC saline to ensure removal of unbound BrdU, and grafted on
to untreated host embryos at stage 4 (average length of streak = 1-86 mm). The
hosts were grown for 24 h on thin albumen. Four categories were used to record
the results: (1) the graft had differentiated and had caused induction (Fig. 3 A);
(2) the graft had differentiated but had not caused induction (Fig. 3B); (3) the
graft had not differentiated but had caused induction (Fig. 3 C); (4) the graft
had neither differentiated nor caused induction. The results are summarized in
Table 2. Fig. 4 clearly shows that the inhibitory effect of BrdU on the graft
differentiation is concentration dependent. In contrast, BrdU had no appreciable
effect on the capacity of Hensen's node to induce a secondary neural tissue in
the epiblast of host embryos.
842
H . LEE, A. К. D E S H P A N D E
HNT
j _
A N D G. W . K A L M U S
INT
G
А
HNT
G
в
.
, с"
Effects of 5-bromodeoxyuridine on chick embryos
843
Differentiated grafts
Induced neural tissue
Concentration of BrdU 0<g/ml)
Fig. 4. Effects of different concentrations of BrdU on the self-differentiation and
inducing capacity of Hensen's node grafts.
Effects of BrdU on the competence ofepiblast
Embryos serving as the hosts were explanted at stage 3, grown for 3-5 h on
thin albumen with or without 8 ^g/ml BrdU (a concentration found to inhibit
strongly the morphogenesis of stage 3 embryos), and then washed several times
in PC saline. The node areas of untreated stage 3 4- embryos were grafted on to
the hosts which had advanced to the stage 3 + . The hosts were grown for 24 h on
thin albumen. Three categories were used to record the results: (1) strong
induction - in which the graft had induced in the host epiblast a neural tube of
FIGURE 3
(A) Transverse section through embryo with 4/*g/ml BrdU-treated node graft.
Note neural tissue and differentiated node graft. G, graft; HNT, host neural tissue;
INT, induced neural tissue. Scale line = 007 mm.
(B) Transverse section through embryo with 4/*g/ml BrdU-treated node graft.
Note differentiated node graft. G, graft; HNT, host neural tissue. Scale line =
007 mm.
(C) Transverse section through embryo with 8/*g/ml BrdU-treated node graft.
Note induced neural tissue and undifferentiated node graft. G, graft; HNT, host
neural tissue; INT, induced neural tissue. Scale line = 007mm.
844
H. LEE, A. K. DESHPANDE AND G. W. KALMUS
Table 3. Effect of 8 figjml BrdU on the competence of the epiblast
% of host embryos
A
Group
Control
Treated
No. of host
embryos
Strong
induction
Weak
induction
No
induction
16
24
62-5
58-3
18-7
16-7
18-7
250
considerable dimensions; (2) weak induction - in which the graft had caused in
the host epiblast the formation of thickened neural ectoderm; (3) no induction.
The results are summarized in Table 3. It can be seen that BrdU (8 /*g/ml) had
no appreciable inhibitory effect on the competence of the epiblast.
Effects of BrdU on the uptake of[3H]uridine
Autoradiography was employed to study whether BrdU had any effect on
RNA metabolism of chick embryonic tissues.
In the first series, stage 4 embryos were grown on thin albumen containing
5 /*Ci/ml [3H]uridine (control group) or on thin albumen containing both
5 ^Ci/ml [3H]uridine and BrdU (experimental group) until the majority of the
control embryos had reached the stage 6 or 7. The application of BrdU, at
6 /tg/ml or lower, caused no obvious variations in the incorporation of [3H]uridine into chick embryonic cells. In both control and experimental groups,
treatment with RNase resulted in the loss of 60-70 % of the labeled materials,
the remainder were predominantly found in the nuclei and were removable with
DNase. At 8 /^g/ml, the incorporation of [3H]uridine into the nuclei, but not
into the cytoplasm, of neural tube cells was significantly affected (P < 001).
BrdU, at 10-12 /*g/ml, strongly inhibited the uptake of [3H]uridine in both the
nucleus and cytoplasm of neural tube cells. The incorporation of [3H]uridine
into DNA of neural tube cells was similarly inhibited by BrdU. However,
BrdU, at the concentrations used, had no appreciable effect on the incorporation
of [3H]uridine into other tissues.
In the second series, stage 4 embryos were grown for 4 h on thin albumen
containing 5 ^Ci/ml [3H]uridine, washed several times in PC saline, and subcultured on thin albumen containing 8 /*g/ml BrdU (experimental group) or on
thin albumen (control group) until the majority of the control embryos had
reached the stage 6 or 7. In the case of BrdU-treated embryos, cells of various
tissues had about 8 % less cytoplasmic labeling than the controls. This finding
indicates that BrdU has very little inhibitory effect on the transfer of labeled
materials from the nucleus to the cytoplasm.
In the third series, stage 4 embryos were grown for 4 h on thin albumen
containing 10 /tg/ml BrdU (experimental group) or on thin albumen (control
group), washed several times in PC saline, and subcultured on thin albumen
Effects of 5-bromodeoxyuridine on chick embryos
845
3
containing 5 /iCifml [ H]uridine until the majority of the control embryos had
reached the stage 6 or 7. In the neural tissue, 88-92 % of the nuclei in the control
series were labeled, whereas only 12-24% of the nuclei in the experimental
series were labeled. However, no marked difference was noted in other tissues.
This finding indicates that BrdU penetrates neural plate cells and inhibit the
the uptake of [3H]uridine.
DISCUSSION
The present study showed that BrdU (8/tg/ml or higher) inhibited somite
formation, regardless of the developmental stage of embryos at treatment. The
blastodermal expansion, heart development and blood island formation, except
in severely malformed embryos, were usually unaffected. In contrast, the magnitude of the inhibitory action of BrdU (2-12 /tg/ml) on the brain development
varied from one stage to another, i.e. a critical time of administration is required
for BrdU to inhibit different primary divisions of the neural tube. This particular
pattern of abnormalities is not restricted to BrdU, because a number of other
chemical agents, e.g. actinomycin D (Heilporn-Pohl, 1964; Lee & Poprycz,
1970; Billett et al. 1971; Lee, Auslander & Kalmus, 1973) can produce a similar
picture. However, BrdU differs from actinomycin D in that (1) it has no apparent
effect on the blastodermal expansion; (2) it does not cause extensive necrosis
and cell death in adversely affected tissues.
Microscopic studies showed that the effects of BrdU on the chick embryo
was directed mainly to the developing neural tissue. At lower concentrations
(2-4 /fcg/ml), the affected cells showed either slight or severe nuclear enlargement.
The severely enlarged nuclei stained diffusely with methyl green, showing a
barely recognizable network of chromatin. These observations imply that the
cells were arrested at the early prophase and there might have been an inhibition
of DNA synthesis. At higher concentrations (6-12/ig/ml), the affected cells
showed, in addition, pycnosis and chromosomal abnormalities. Similar observations have been made in other developing systems, e.g. sea urchin embryos
(Mazia & Gontcharoff, 1964) and amphibian embryos (Tencer & Brachet,
1973). Hsu & Somers (1961) reported that BrdU preferentially damaged certain
adenine-thymine rich regions in mammalian chromosomes. Whether this is also
true in avian chromosomes needs further investigations. Neural tube closure
was inhibited in many malformed embryos, particularly those treated at stage 4.
This defect did not appear to be caused by mitotic failure in the tissue itself,
since cells in various phase of mitosis were often found in the vicinity of degenerating cells. It appears that cell processes associated with the neural tube
closure are more sensitive to BrdU than are those essential to growth and cell
division. There is little or no evidence that the early chick embryo has differential
regional resistance to penetration of BrdU, whereas there is a large body of
evidence demonstrating that there are regional metabolic differences in the
early chick embryo (Spratt, 1952; Duffey & Ebert, 1957; etc.). Furthermore, it
54
E M B 32
846
H. LEE, A. K. DESHPANDE AND G. W. KALMUS
is at stage 4 that the process of neural differentiation is initiated (England,
1973) and it is precisely at this stage that the presumptive neural tube tissue is
highly susceptible to BrdU. These considerations suggest that the interruption of
metabolic pathways and/or induction process may account for the observed
neural tube defects. Importance of microtubules in neurulation has been pointed
out by Waddington & Perry (1966). It is of interest to know whether microtubules of the chick neural plate cell are susceptible to BrdU. This could help
us elucidate the effect of BrdU on the morphogenesis of the neural tube. Our
autoradiographic studies showed that high concentrations of BrdU (10-12 jug/
ml) selectively inhibited the incorporation of [3H]uridine into the neural tissue.
Whether or not BrdU may suppress or alter the synthesis of tissue specific
nuclear RNA is presently not known. If we assume that in our experiments
BrdU is incorporated into DNA and inhibits the transcription of certain genes
into mRNA as suggested by Stellwagen & Tomkins (1971), then the results
would indicate that a change in the pattern of transcription of genetic information might be involved during the initial phases of brain development. Ingram
et ah (1974) reported that BrdU lowered the rate of uptake of adenosine into
the ATP pool during the chick erythropoiesis. The differential susceptibility of
the neural tissue to BrdU suggests some disturbances in energy supply mechanisms. In this respect the BrdU effects resemble those of cyanide (Spratt, 1952)
and 2,4-dinitrophenol (Bowman, 1967). This view is supported by the observation that BrdU, at a concentration of 6 /^g/ml, had a selective inhibitory effect
on the neural tissue, but had no obvious effect on the incorporation of [3H]uridine into RNA. Alterations in oxidative phosphorylation, whether immediate
consequences of BrdU treatment or secondary results of the interference with
nucleic acid synthesis, may have led to the observed neural tube defects.
This study also showed that the brain development often became insensitive
to BrdU, if treatment was delayed until the embryo had reached stages 6-7.
BrdU-insensitive processes are not uncommon in developing systems, e.g.
echinochrome synthesis by sea urchin embryos (Gontcharoff & Mazia, 1967),
haemoglobin synthesis by erythrocytes (Miura & Wilt, 1971), chondroitin
sulphate synthesis by chondrocytes (Abbott, Mayne & Holtzer, 1972), etc.
Weintraub et ah (1972) suggested that normal differentiation involves the
institution of a programme that is resistant to BrdU. It does not, however,
explain the reason why BrdU selectively inhibits the differentiation of certain
tissues and/or cell functions.
Hensen's node of the stage 4 chick embryo is found to be active in cell proliferation and RNA synthesis (Lee, 1972). It is, therefore, expected that BrdU
has a marked inhibitory effect on the differentiation of Hensen's node grafts.
Our grafting experiments did show that an increase in the concentration of
BrdU from 4 to 12/*g/ml caused a significant decrease in the degree of graft
differentiation which was, however, not accompanied by a proportionally
decreased frequency of neural inductions. These findings suggest that the vital
Effects of 5-bromodeoxyuridine on chick embryos
847
activity of the inductor is not prerequisite to elicit typical neural induction in
the chick epiblast. Furthermore, BrdU (8 y^g/ml) did not impair the competence
of the epiblast, although the host embryo proper showed characteristic syndromes of BrdU treatment. The observations that BrdU is incorporated into
DNA (Wishnow, Feist & Glowalla, 1974) and inhibits the uptake of [3H]uridine
suggest that DNA-mediated RNA synthesis required for initiating the differentiation of the competent epiblast is insensitive to BrdU or it occurs prior to
treatment period (stage 3).
This study was supported by a grant from the Rutgers University Research Council No.
07-2189.
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{Received 26 February 1974, revised 30 May 1974)