The effect of p-nitrophenyl β-D-xyloside, an inhibitor of proteoglycan biosynthesis, on the growth of chick embryos was studied by injection of the single dose of 1.0mg/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. p-Nitrophenyl α-D-xyloside, p-nitrophenyl β-D-galactoside, and a mixture of p-nitrophenol 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 β-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. These findings seem to support the view that glycosaminoglycans play an important role in the regulation of embryonic growth.

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).

It has already been established that β-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 β-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 β-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.

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 β-D-xyloside (PNB-β-xyl), p-nitrophenyl α-D-xyloside (PNP-α-xyl), p-nitrophenyl β-D-galactoside (PNB-β-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-β-xyl was dissolved into MEM at the concentration of 4 mg/ml, and 125 μl of the solution was injected into the egg white through a pin hole in the shell with a Hamilton No. 725 microsyringe. PNP-β-gaI 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 μl) 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 μl 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).

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 HC1-0·05 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 % ethanol-1·3 % 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).

Gross effects

Our preliminary experiment (Oohira et al. 1979) showed that the mortality of the chick embryos treated with PNP-β-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-β-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.

Fig. 1.

Ten-day treated and control embryos. Left, an embryo treated with 1·0 mg of PNP-β-xyl on day 3; right, a control embryo. Note that the treated embryo with systemic edema is larger in size than the control.

Fig. 1.

Ten-day treated and control embryos. Left, an embryo treated with 1·0 mg of PNP-β-xyl on day 3; right, a control embryo. Note that the treated embryo with systemic edema is larger in size than the control.

The mean wet weight of day-10 embryos which had been treated with 1·0 mg/ egg of PNP-β-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-α-xyl, PNP-β-gal, and a mixture of PNP and Xyl, cause neither the overgrowth nor the edematous change of chick embryos (Table 1).

Table 1.

Effect of PNP-β-xyl and the related compounds administered on day 3 on growth of chick embryos

Effect of PNP-β-xyl and the related compounds administered on day 3 on growth of chick embryos
Effect of PNP-β-xyl and the related compounds administered on day 3 on growth of chick embryos

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 1·0 mg/egg of PNP-β-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.

Table 2.

Time course of changes in glycosaminoglycan, DNA and protein contents of the chick embryo after administration of 1·0 mg PNP-β-xyl on day 3

Time course of changes in glycosaminoglycan, DNA and protein contents of the chick embryo after administration of 1·0 mg PNP-β-xyl on day 3
Time course of changes in glycosaminoglycan, DNA and protein contents of the chick embryo after administration of 1·0 mg PNP-β-xyl on day 3

The present results described above are not consistent with those indicating that the administration of β-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 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 glycosaminoglycan 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.

Table 3.

Effect of PNP-β-xyl administered on various days on embryonic growth

Effect of PNP-β-xyl administered on various days on embryonic growth
Effect of PNP-β-xyl administered on various days on embryonic growth

Glycosaminoglycan 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 electro-phoretogram of the glycosaminoglycan markers is shown in Fig 2a), 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, Bâck-Strôm, Jansson & Hallén, 1973). Of the three, the spot of chondroitin sulfates obtained from the treated sample seemed to be larger than that from the control (Figs. 2b and 2c).

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-β-xyl on day 3 (b), and from the control (c). Electrophoretograms of the preparations from day-12 embryos which had been treated with 10 mg of PNP-β-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.

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-β-xyl on day 3 (b), and from the control (c). Electrophoretograms of the preparations from day-12 embryos which had been treated with 10 mg of PNP-β-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.

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 μmol 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 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.

Glycosaminoglycan composition in embryonic chicks 3 days after treatment with 1·0 mg/egg of PNP-β-xyl

Glycosaminoglycan composition in embryonic chicks 3 days after treatment with 1·0 mg/egg of PNP-β-xyl
Glycosaminoglycan composition in embryonic chicks 3 days after treatment with 1·0 mg/egg of PNP-β-xyl

Table 4 also shows that treatment with PNP-β-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 morphogenesis (Toole, 1973), the affected metabolism of hyaluronic acid may be involved partially in the production of the abnormality.

It is of interest that the effect of PNP-β-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 protein-linked glycosaminoglycans (Kato et al. 1978). Since higher doses of several structural analogues which were proved to have less activity for the disturbance of proteoglycan biosynthesis (Robinson et al. 1975) could not cause the overgrowth of the treated embryos (Table 1), it is reasonable to conclude that PNP-β-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 & Cássaro, 1977). The administration of the reagent at later developmental stages resulted in less stimulation of embryonic growth (Table 3) and in higher content of under. sulfated 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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