J. Embryol exp. Morplt. Vol. 61, pp. 221-232, 1981
Printed in Great Britain © Company of Biologists Limited 1981
221
Abnormal overgrowth of chick embryos
treated with p-nitrophenyl p-D-xyloside at early
stages of development
By ATSUHIKO OOHIRA1, HIROSHI NOGAMP AND
YASUO NAKANISHP
From the Department of Embryology, Institute for Developmental Research,
Kasugai, and the Department of Chemistry, Faculty of Science,
Nagoya University, Japan
SUMMARY
The effect of p-nitrophenyl /?-D-xyloside, an inhibitor of proteoglycan biosynthesis, on the
growth of chick embryos was studied by injection of the single dose of 1 -0 mg/egg into
fertile eggs on day 3. Embryos examined on day 10 had systemic edema, and were increased
not only in wet weight (142% of the non-treated embryos) but also in dry weight (125%).
No skeletal malformations were observed in the treated embryos. The glycosaminoglycan
content in the treated embryos began to increase 6 h after treatment and reached the
maximum level (174% of the non-treated) after 3 days, while the DNA and protein content
began to increase 12 h after treatment and reached the maximum level (about 140%) within
3 days. ^-Nitrophenyl a-D-xyloside, />-nitrophenyl /?-D-galactoside, and a mixture of pnitrophenol and D-xylose produced neither the abnormal overgrowth nor the edematous
change of chick embryos.
When fertile eggs were treated with 1 -0 mg/egg of p-nitrophenyl /?-D-xyloside on day 6,
the increase in wet and dry weights was also observed in all surviving embryos. On the
contrary, treatment on day 9 resulted in the slight reduction of embryonic growth in addition
to the systemic edema. Both embryos treated on day 3 and on day 6 contained glycosaminoglycans rich in chondroitin 6-sulfate, whereas the embryos treated on day 9 contained glycosaminoglycans rich in undersulfated chondroitin sulfates. Thesefindingsseem to support the
view that glycosaminoglycans play an important role in the regulation of embryonic growth.
INTRODUCTION
Proteoglycans have been suggested to play an important role in embryonic
growth and development, so that abnormality in the biosynthesis would be
expected to lead to faulty development in embryos (for review, Kochhar &
Larsson, 1977).
1
Author's present address: Department of Biochemistry, University of Washington,
Seattle, Washington 98195, U.S.A.
2
Author's present address: Department of Orthopaedic Surgery, Central Hospital, Kasugai,
Aichi 480-03, Japan.
3
Autlior's address: Department of Chemistry, Faculty of Science, Nagoya University,
Nagoya 464, Japan.
8
EMB 6 l
222
A. OOHIRA, H. NOGAMI AND Y. NAKANISHI
It has already been established that /?-D-xylosides disturb the biosynthesis of
proteochondroitin sulfates (Okayama, Kimata & Suzuki, 1973; Schwartz,
Galligani, Ho & Dorfman, 1974; Robinson et al. 1975; Gibson, Segen &
Audhya, 1977) and heparan sulfate (Johnston & Keller, 1979) by replacing the
need for natural xylosyl protein (so-called core-protein) in the biosynthesis of
normal proteoglycans. Recently Gibson, Doller & Hoar (1978) described
a teratological dwarfism produced by administration of /?-D-xylosides to 9-day
chick embryos. However, it is well-known that proteoglycans were synthesized
by chick embryos at more earlier developmental stages (Abrahamsohn, Lash,
Kosher & Minor, 1975; Solursh, 1976), although the physiological significance
of the proteoglycans in the early stages has not been elucidated to date. We
report here that administration of p-nitrophenyl /?-D-xyloside to chick embryos
at the early stages of development leads to marked alterations in the composition
of glycosaminoglycans and produces marked overgrowth of the embryos.
MATERIALS AND METHODS
Fertile eggs (White Leghorn) weighing 52 g to 58 g were obtained from
Hattori Chicken Farm Co., Nagoya, and were incubated in a moist atmosphere
at 38 °C. p-Nitrophenyl /?-D-xyloside (PNB-/?-xyl), p-nitrophenyl a-D-xyloside
(PNP-a-xyl), /7-nitrophenyl /?-D-galactoside (PNB-/?-gal), chondroitinase-ABC,
chondroitinase-AC II, and glycosaminoglycan standards for electrophoresis
(hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate,
heparan sulfate, keratan sulfate and heparin) were obtained from Seikagaku
Kogyo Co., Tokyo. Eagle's minimum essential medium (MEM), p-nitrophenol
(PNP) and D-xylose (Xyl) were obtained from Nakarai Chemicals, Ltd, Kyoto.
Animal experiments
PNP-/?-xyl was dissolved into MEM at the concentration of 4 mg/ml, and
125 /i\ of the solution was injected into the egg white through a pin hole in the
shell with a Hamilton No. 725 microsyringe. PNP-/?-gal was dissolved in MEM
at the concentration of 12 mg/ml. A mixture of 6 mg PNP and 6 mg Xyl was
dissolved in 1 ml of MEM. An aliquot (125 fi\) of the solutions was also administered to each fertile egg. PNP-a-xyl was suspended in MEM at the concentration of 12 mg/ml, and 125 ju\ of the suspension was injected with a 18G
needle.
Each egg received a single injection on designated day, and the embryo
was removed, weighed and examined externally on day 10. About half of the
living embryos were lyophilized and weighed, and the rest were fixed in 10 %
formalin and stained with methylene blue by the method of Noback (1916).
Abnormal embryonic overgrowth produced by fi-D-xyloside 223
Chemical determinations
Chemical determinations were performed on portions of homogenates of
whole embryos. The wet embryos or the lyophilized embryos were homogenized
in a tight-fitting Potter homogenizer in ice-cold 4 M guanidine HC1-005 M
Tris-HCl, pH 7-5, at the volumes of 0-1 ml/embryo for 3- and 4-day-old
embryos, 0-5 ml/embryo for 6-day-old embryos, 2-0 ml/embryo for 9-day-old
embryos and 10 ml/embryo for 12-day-old embryos.
Protein was estimated by the method of Lowry, Rosenbrough, Farr & Randall
(1951), after precipitation from portions of the homogenates with 95 % ethanol1*3 % potassium acetate as described previously (Oohira & Nogami, 1978). For
the determinations of DNA and hexuronate, portions of the homogenates were
subjected to the sequential treatments of 95 % ethanol-1-3 % potassium acetate
and Pronase-P (Kaken Kagaku Co., Tokyo) as described previously (Oohira &
Nogami, 1978). DNA was measured by the method of Burton (1956), using
aliquots of the pronase-digests. The rest of the digests was further treated
sequentially with 0-3 N-NaOH, 5 % trichloroacetic acid and precipitation with
ethanol in order to obtain crude glycosaminoglycans by the method of Oohira
et al. (1977). Hexuronate in the final precipitates was determined by the method
of Bitter & Muir (1962).
Glycosaminoglycan analysis
Two-dimensional electrophoresis of glycosaminoglycan preparations was
carried out on cellulose acetate film (Fuji Film Co., Tokyo) by the method of
Hata & Nagai (1972), using the buffer systems of 0-1 M pyridine-0-47 M formic
acid for the first run and 0-1 M barium acetate for the second. The relative
amounts of isomeric chondroitin sulfates in glycosaminoglycan preparations
were estimated by the chondroitinase-digestion method described by Saito,
Yamagata & Suzuki (1968).
RESULTS
Gross effects
Our preliminary experiment (Oohira et al. 1979) showed that the mortality of
the chick embryos treated with PNP-/?-xyl during the period between 0 and
4 days of development roughly depended on the dosage and time of administration; the treatment with high dosage at the early stages of embryonic development resulted in high rates. Common external malformations produced in
day-10 embryos, which had been treated with PNP-/?-xyl at the early stages,
were closely similar to those, namely systemic edema, described by Gibson et al.
(1978), except that the treated embryos were much larger than the non-treated
(Fig. 1). No skeletal malformations were observed in the treated embryos
examined by staining of the cartilaginous skeletons.
8-2
224
A. OOHIRA, H. NOGAMI AND Y. N A K A N I S H I
Fig. 1. Ten-day treated and control embryos. Left, an embryo treated with 1 -0 mg
of PNP-/?-xyl on day 3; right, a control embryo. Note that the treated embryo with
systemic edema is larger in size than the control.
Table 1. Effect of PNP-fi-xyl and the related compounds administered
on day 3 on growth of chick embryos
Compound
(Dose)
No.
eggs
No. live Mortality Wet weight Dry weight
(g±s.D.)
(mgts.D.)
embryos
(%)
None
45
43
MEM (125 /tl)
19
19
PNP-/?-xyl(l-0mg)
36
17
PNP-a-xyl(l-5mg)
15
15
8
2-24 ±0-35
(II = 43)
(100)*
PNP (0-75 mg)
+ Xyl(0-75mg)
26
25
PNP-^-gal(l-5mg)
21
21
53
2-31 ±0-35
(II = 19)
(103)
3-17±0-18f
(n = 17)
(142)
2-20 ±0-22
(» = 15)
(98)
2-23 ±0-25
(« = 25)
(100)
2-13±0-22
(n = 21)
(95)
150±12
(n = 20)
(100)*
157±26
(n = 10)
(105)
187±17f
(n = 10)
(125)
152±14
(n = 10)
(101)
149 ±18
(« = 10)
(99)
149115
(n = 10)
(99)
Water
93-3
93-2
94-1
931
93-3
930
* The values in the parentheses are expressed as the percentages of the non-treated.
t Significantly different from the non-treated (P < 0-05 by /-test).
Abnormal embryonic overgrowth produced by fi-D-xyloside 225
Table 2. Time course of changes in glycosaminoglycan, DNA and protein contents
of the chick embryo after administration of 1 -0 mg PNP-fi-xyl on day 3
Time after
treatment
0
6 hours
Treatment
None
None
PNP-/?-xyl
12 hours
None
PNP-/?-xyl
24 hours
None
PNP-/?-xyl
3 days
None
PNP-A-xyl
7 days
None
PNP-A-xyl
G lycosami noglycan*
DNAf
(nmol/embryo)
(nmol/embryo)
ProteinJ
(mg/embryo)
12-5±0-9
16-2±l-3
(100)
15-9±l-5
( 98)
28-6 ±2-3
41-0 + 3-9
(100)
38-5 ±3-7
(94)
0-362 ±0034
0-496 ±0047
(100)
0-471 ±0045
(95)
23-4+1-8
(100)
290±2-5§
(124)
63-6 ±5-9
(100)
65-5 ±6-4
(103)
0-753 ±0071
(100)
0-745 ±0072
(99)
45-6±3-9
(100)
66-6 ± 61 §
(146)
128 + 10
(100)
179+14§
(140)
1-41 ±011
(100)
1-78 +0-14§
(126)
297 + 22
(100)
517±49§
(174)
641 ± 57
(100)
84O±77§
(131)
8-37 ±0-76
(100)
ll-5±0-95§
(137)
6110±586
(100)
7576 ±751
(124)
3700 ±314
(100)
4884±426§
(132)
74-4 ±6-8
(100)
80-4 + 8-0
(108)
The data represent mean of triplicate samples from two separate experiments. The values
in the parentheses are expressed as the percentage of the non-treated.
* Expressed as glucuronic acid (Bitter & Muir, 1962).
t Expressed as deoxyadenosine-5'-monophosphate (Burton, 1956).
t Expressed as bovine serum albumin (Lowry et al. 1951).
§ Significantly different from the non-treated (P < 005 by /-test).
The mean wet weight of day-10 embryos which had been treated with 1-0 mg/
egg of PNP-/?-xyl on day 3 (approximately stage 20 of Hamburger & Hamilton,
1951) increased significantly (142 % of the non-treated, P < 0-05). The mean
dry weight was also markedly higher than that of the non-treated (Table 1),
suggesting that the treatment causes overgrowth of chick embryos in addition
to edematous change. Higher doses of the structural analogues, such as PNP-axyl, PNP-/?-gal, and a mixture of PKP and Xyl, cause neither the overgrowth
nor the edematous change of chick embryos (Table 1).
226
A. OOHIRA, H. NOGAMI AND Y. NAKANISHI
Table 3. Effect of PNP-/3-xyl administered on various days on embryonic growth
Wet weight*
Day of
treatment Treatment (g/embryo)
DNAf
GlycosaminoglycanJ
Dry weight*
(mg/embryo) (/*mol/embryo) (/*mol/embryo)
3
None
0-293 + 0026
(100)
PNP-/?-xyl 0-434 ±0051
(148)
17-2+1-8
(100)
23-9±5-6
(139)
0-641 ±0017
(100)
0-840 ±0041
(131)
0-297 ±0-005
(100)
O-517±O-OO8
(174)
6
None
1-35 ± 0 0 5
(100)
l-84±0-18
(136)
90-3 + 2-7
(100)
108 ±10
(120)
1-43 ± 0 0 5
(100)
1-67 ±002
(117)
1-24 ±003
(100)
1-68 ±006
(135)
3-83 + 0-22
(100)
4-29 ±0-21
(112)
339+16
(100)
322 ±44
(95)
413 +0-40
(100)
3-80 ±019
(92)
607 ± 0 1 0
(100)
5-95 +014
(98)
PNP-/?-xyl
9
None
PNP-yff-xyl
PNP-/#-xyl (l-0mg/egg) was administered to fertile eggs on the designated day and the
embryos were removed and examined 3 days after administration. The values in the parentheses are expressed as the percentage of the non-treated.
* The data represent mean of 10 surviving embryos with an S.D.
t The data are expressed as deoxyadenosine-5'-monophosphate (Burton, 1956), and
represent mean of triplicate samples with an S.D.
% The data are expressed as glucuronic acid (Bitter & Muir, 1962), and represent mean of
triplicate samples with an S.D.
Chemical composition
The increased dry weight of the treated embryos indicated that the chemical
composition of the treated embryos differed from that of the non-treated. In
fact, our preliminary experiments (Oohira et al. 1979) demonstrated that the
DNA and protein contents in the treated embryos as well as the glycosaminoglycan content increased by 30 % or more within 24 h after treatment. Table 2
shows the time course of changes in chemical composition of the embryos
treated with 1-Omg/egg of PNP-/?-xyl on day 3. The embryonic growth was
inhibited slightly during the first 6 h after administration. During next 6 h, the
amount of glycosaminoglycans increased rapidly whereas the amounts of DNA
and protein were nearly the same as those of the non-treated. The glycosaminoglycan content continued to increase thereafter, reached the maximum level
(174 % of the non-treated) around 3 days after treatment, and then gradually
decreased. The amounts of DNA and protein increased at the most rapid rate
during the period from 12 h to 24 h after treatment.
The present results described above are not consistent with those indicating
that the administration of /?-D-xylosides into amniotic sac of 9-day-old embryos
causes marked dwarfism in chick embryos (Gibson, et al. 1978). The discrepancy
may be attributed to the difference of time when the reagent was injected into
Abnormal embryonic overgrowth produced by fi-D-xyloside 227
fertile eggs. To test this idea, PNP-/?-xyl was administered at the dose of 1-0 mg/
egg on various days of incubation. After 3 days, the embryos were removed and
examined. The activation effect of the reagent on embryonic growth, measured
by dry weight of embryos, decreased in rate with advance of embryonic development (Table 3). When administered on day 9, the reagent showed tendency to
repress embryonic growth. Both DNA and glycosarninoglycan contents in the
embryos treated on day 9 were slightly lower than those in the non-treated,
while those in the embryos treated at earlier developmental stages were significantly higher than those in the non-treated (Table 3). These observations suggest
that the discrepancy is attributed mainly to the difference of administration
time, but do not exclude the possibility that the discrepancy is related in part to
the different methods of administration.
Glycosarninoglycan composition
An aliquot (10 nmol of hexuronate) of each glycosaminoglycan preparation
shown in Table 3 was subjected to two-dimensional electrophoresis on cellulose
acetate film (Fig. 2). Both glycosaminoglycan preparations obtained from
embryos treated on day 3 (Fig. 2b) and from the control (Fig. 2c) were separated
into three distinct components. A major component appeared as a broad spot
with the mobility corresponding to the chondroitin sulfate marker (an electrophoretogram of the glycosaminoglycan markers is shown in Fig 2 a), and was
susceptible to degradation with chondroitinase-ABC. A compact spot with the
mobility corresponding to the hyaluronate marker was susceptible to degradation not only with chondroitinase-ABC but also with hyaluronidase from
Streptomyces hyalurolyticus. The minor, broad spot with the mobility corresponding to the heparan sulfate marker was proved not to be digested with
either of these enzymes but to be degraded with nitrous acid (Lindahl, BackStrom, Jansson & Hallen, 1973). Of the three, the spot of chondroitin sulfates
obtained from the treated sample seemed to be larger than that from the control
(Figs. 2 b and 2 c).
The samples treated on day 9 (Fig. 2d) and the control (Fig. 2e) were both
separated into two spots; a major, chondroitin sulfate spot and a minor,
hyaluronate spot. However, the spot of chondroitin sulfates obtained from the
treated sample was broader and extended from the chondroitin sulfate zone to
the hyaluronate zone (Fig. 2d). This suggests the occurrence of undersulfated
chondroitin sulfates. The electrophoretogram of the sample treated on day 6
was shown to be intermediate between those treated on day 3 and on day 9 (data
not shown).
To determine the relative amounts of the glycosaminoglycans, another
aliquot (0-3 fimo\ of hexuronate) of each glycosaminoglycan preparation shown
in Table 3 was digested with chondroitinases and assayed for unsaturated
disaccharide products (Table 4). The glycosaminoglycan preparation obtained
from the embryos treated on day 9 gave large amount (approximately 40 % of
228
A. OOHIRA, H. NOGAMI AND Y. NAKANISHI
iHP
CS
\
OS
HS
KS
iHA
1st
t
2nd
I
(c)
\
(rf)
(e)
I
Abnormal embryonic overgrowth produced by fi-D-xyloside 229
Table 4. Glycosaminoglycan composition in embryonic chicks 3 days after
treatment with 1 -0 mg/egg of PNP-ft-xyl
Glycosaminoglycan (%)
Day of
treatment Treatment
3
6
9
None
PNP-/?-xyl
None
PNP-/?-xyl
None
PNP-/?-xyl
A-unit
B-unit
C-unit
2
3
4
12
10
13
9
9
4
6
10
7
18
26
38
34
32
29
O-unit
13
14
18
25
23
41
HA-unit
32
21
14
10
13
7
Others
26
27
22
13
12
3
The glycosaminoglycan preparations shown in Table 3 were digested separately either with
chondroitinase-ABC or with chondroitinase-AC and assayed for the unsaturated disaccharide
products (Saito, Yamagata & Suzuki, 1968). The nomenclature used are: A-unit, ADi-4S
produced by digestion with only chondroitinase-AC; B-unit, ADi-4S which cannot be
produced by digestion with chondroitinase-AC but can be produced with chondroitinaseABC; C-unit, O-unit and HA-unit are ADi-6S, ADi-OS and ADi-OS(HA), respectively,
produced by digestion with chondroitinase-ABC (for the structure and abbreviation of the
disaccharides, see Oohira et al. 1977). Others are components resistant against digestion with
chondroitinase-ABC and proved to be composed mainly of heparan sulfate (see Fig. 2).
total hexuronate) of O-unit, namely the product derived from unsulfated chondroitin by the lyase reaction, while the control gave less amount (about 20 %)
of O-unit. These findings are closely similar to those described by Gibson, Segen
& Doller (1979), and consistent with the results shown in Fig. 2d. On the
contrary, treatment on day 3 increased the content of C-unit, namely the
product derived from chondroitin 6-sulfate, rather than the content of O-unit
in the glycosaminoglycans of the embryos. The composition of glycosaminoglycans in the embryos treated on day 6 seemed to be intermediate between
those treated on day 3 and on day 9 (Table 4).
Table 4 also shows that treatment with PNP-/?-xyl decreases the relative
amount of HA-unit, namely the disaccharide product derived from hyaluronic
acid. Since hyaluronic acid is considered to play an important role in the
Fig. 2. Two-dimensional electrophoresis on cellulose acetate of glycosaminoglycan
preparations with reference glycosaminoglycan standards, (a) Electrophoretogram
of authentic mixture which consists of hyaluronic acid (HA), chondroitin sulfates
(CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS) and heparin
(HP). Electrophoretograms of the preparations from day-6 embryos which had
been treated with 1 -0 mg of PNP-/?-xyl on day 3 (b), and from the control (c). Electrophoretograms of the preparations from day-12 embryos which had been treated with
10mg of PNP-/tf-xyl on day 9 (d), and from the control (e). Electrophoretic
systems; 0-1 M pyridine-0-47 M formic acid at 1 ma/cm for 1 -2 h in the first dimension
and 0-1 M barium acetate at 1 ma/cm for 4-5 h (a, b and c) or 5-5 h (d and e) in the
second.
230
A. OOHIRA, H. NOGAMI AND Y. NAKANISHI
morphogenesis (Toole, 1973), the affected metabolism of hyaluronic acid may
be involved partially in the production of the abnormality.
DISCUSSION
It is of interest that the effect of PNP-/?-xyl on embryonic growth varies with
stage when the reagent was administered. The reagent has a stimulative effect
on embryonic growth when administered to embryos at early developmental
stages (Table 3). This reagent has been shown to stimulate the synthesis of
protein-free glycosaminoglycan chains and to inhibit the synthesis of proteinlinked glycosaminoglycans (Kato et ah 1978). Since higher doses of several
structural analogues which were proved to have less activity for the disturbance
of proteoglycan biosynthesis (Robinson et ah 1975) could not cause the overgrowth of the treated embryos (Table 1), it is reasonable to conclude that
PNP-/?-xyl alters the proteoglycan synthesis of the embryos and then the
alteration induces stimulation of embryonic growth. In fact, after treatment with
the reagent on day 3, the glycosaminoglycan content in the treated embryos
increased first followed by increase in DNA and protein contents (Table 2).
Of the glycosaminoglycans, the amount of chondroitin 6-sulfate increased at the
highest rate by the treatment (Table 4). These findings support the hypothesis
that chondroitin 6-sulfate promotes the growth by the stimulation of cell
division (Takeuchi, 1968; Dietrich, Sampaio, Toledo & Cassaro, 1977). The
administration of the reagent at later developmental stages resulted in less
stimulation of embryonic growth (Table 3) and in higher content of undersulfated chondroitin sulfates in the treated embryos (Table 4). Undersulfated
chondroitin sulfates were proved to occur in animals with heritable dwarfism
(Orkin, Pratt & Martin, 1976) and with experimentally induced dwarfism
(Seegmiller & Runner, 1974; Hjelle & Gibson, 1979). Taken together, one can
postulate that undersulfated chondroitin sulfates have growth-inhibiting
activity. All the results presented above seem to emphasize the possibility that
glycosaminoglycans play an important role in the regulation of embryonic
growth.
We would like to thank Professor Sakaru Suzuki, Dr Masahiro Tsuji, Mr Shigemi Kato
and Mr Noboru Tomiya, Nagoya University, for their criticisms, suggestions, and kindnesses
offered during the course of this work. This work was supported by a grant from Aichi
Prefecture (S-53-A-7).
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{Received 25 April 1980, revised 24 July 1980)