ABSTRACT
The achondroplastic can/can embryo can be distinguished from normal litter-mates at 17 days by reduced protein synthesis. After 3 days post partum protein synthesis is increased to levels well above normal, as is incorporation of glucose into mucopolysaccharides and the levels of uridine diphosphoglucose dehydrogenase and UDP-glucose-4-epimerase.
INTRODUCTION
Although achondroplasia is a common hereditary defect in man, domestic and laboratory animals few investigations exist at other than the gross histo-logical level. Notable exceptions to this are the electron microscope studies on the achondroplastic (ac) rabbit (Shepard, Fry & Moffett, 1969) and the chondro-dystrophic (cho) and cartilage anomaly (can) mouse (Johnson & Wise, 1971; Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon, 1972). The achondroplastic rabbit and the nannomelic chick have also been studied using biochemical techniques (Fraser & Goetinck, 1971 ; Shepard & Bass, 1971 ; Bargman, Mackler & Shepard, 1972). In man cartilage from seven dwarf conditions was examined microchemically by Stanescu, Stanescu & Szirmai (1972).
These contributions suggest that the underlying causes of achondroplasia may be very varied. Seegmiller has identified aberrant cross-banded collagen in the cho mouse; Fraser & Goetinck have noted a decreased uptake of radio-active mucopolysaccharide precursors in the nannomelic chick, whilst the basis of achondroplasia in the ac rabbit seems to be a deficiency in a mitochondrial enzyme.
Because of the paucity of information and because of the diversity of that which exists it was decided to follow up the previous ultrastructural study of the cartilage anomaly mouse by a biochemical investigation.
MATERIAL AND METHODS
Cartilage was isolated from the sternum and uncalcified ribs of can/can mice and normal litter-mates aged 0–5 days. The ribcage was removed by lateral incisions and the ossified portion of the ribs cut off. Musculature was removed by gentle scraping with a scalpel and the individual cartilaginous ribs dissected free and cut into pieces ca. 0·3 mm long. The pieces were then weighed and incubated for 2, 3 or 4 h in a shaking water-bath in Waymouth’s solution, to which had been added 100 i.u. each of streptomycin and penicillin and appro-priate trace amounts of labelled precursors ([l-14C]D-glucosamine, 55 mCi/ mmol; [l-14C]D-galactosamine, 58mCi/mmol; [35S]sodium sulphate, lOOmCi/ mmol; [U-14C]glucose, 285 mCi/mmol; Radiochemical Centre, Amersham). The supernatant medium was discarded and the tissue washed in 2 × 1 ml cold Waymouth’s solution and homogenized in 0·6 ml/mg distilled water. Protein-bound radioactivity was isolated by precipitation with an equal volume of 10 % trichloracetic acid (TCA) followed by centrifugation and two washes in 5 % TCA. Bovine serum albumin (0·0005 g/ml) was added as carrier protein. The precipitate was redissolved in 3N-NH4OH and counted in Unisolve (Koch-Light Ltd.) in a Packard Tricarb liquid scintillation spectrophotometer with external standardization.
Mucopolysaccharide-bound label was measured after digesting protein with papain. Crude papain (0·01 g/ml) was dissolved in 0·1 M-phosphate buffer containing 0·005M-EDTA and 0·005 M-cysteine, and added to the cartilage homogenate. Digestion was allowed to proceed overnight at 70 °C. All muco-polysaccharides were precipitated with 1 % cetylpyridinium chloride (CPC)/ 0-04M-NaCl; hyaluronic acid and chondroitin sulphates were redissolved in 0·1 % CPC/0-4M-NaCl, 0·1 % CPC/l·2M-NaCl respectively and counted in Unisolve.
The action of chondroitinase ABC (Seikaguko Kogyo Co., Tokyo) on protein-bound radioactivity was estimated by incubating labelled cartilage homogenate with chondroitinase ABC in 0·125M Tris/HCl buffer + 0·075 M-sodium acetate (pH 8·0) for 60 min at 37 °C. The reaction was stopped with an equal volume of 10 % TCA.
Activity of the enzymes glucose-6-phosphate dehydrogenase, uridine di-phospho (UDP)-glucose dehydrogenase and UDP-glucose-4-epimerase was assayed fluorimetrically. Cartilage was homogenized in an appropriate buffer (0·lM-Tris, pH 7·6, 0·1 M-glycine, pH 8·7, 0·lM-glycine, pH 8·7) and centrifuged at 10 000 g for 20 min. An aliquot of the supernatant was incubated for 15 min at 37 °C with appropriate substrate (0·025M-glucose-6-phosphate, 0·02M-UDP-glucose, 0·7 mM UDP-galactose plus UDPG-dehydrogenase plus 0·03M-NAD). The formation of NADH was measured at 340 mμ excitation, 460 mμ emission using a standard containing 0·2 μmole/ml NADH. Standards and blanks also contained boiled cartilage supernatant.
Oxidative phosphorylation at site III of liver mitochondria was assayed using Bargman, Mackler & Shepard’s (1972) modification of the technique of Sandai & Jacobs (1960): the reaction was performed in a test tube rather than a War-burg apparatus, and esterified phosphorus only measured.
Protein synthesis was measured by incubating cartilage or liver with [U-14C]-glycine (114 mCi/mmol) as previously described. After washing the tissue was homogenized in 0·lM-Tris buffer (pH 7·2) containing 0-lM-CaAc. An aliquot was incubated with 100 units of collagenase (Sigma Biochemicals type III) for 30 min at 37 °C. Remaining protein was precipitated with TCA and counted as described above. The collagenase was shown to be protease-free by repeating the above experiment with [U-14C]tryptophan (45 mCi/mmol) as substrate. The collagenase failed to remove any counts in this experiment.
Litters of 17- and 18-day-old embryos were obtained by priming female offspring from matings segregating for can ( of which are +/can) with 3 i.u. serum gonadotrophin followed after 40 h by 3 i.u. chorionic gonado-trophin (Folligon and Chorulon, Organon Labs.). The females were then mated to known +/can ♂ ♂. Cartilage preparations from 17- and 18-day-old embryos were made as described above.
RESULTS
The cartilaginous matrix has as its main components collagen and muco-polysaccharides. The mucopolysaccharides are attached to a protein backbone after synthesis. Techniques for the estimation of polysaccharides fall into two groups, those which degrade the protein backbone and measure total poly-saccharides and those which precipitate the protein-bound polysaccharides and disregard the remainder.
In the neonatal rat epiphysis (Handley & Phelps, 1972) the mucopoly-saccharides are of two main kinds, hyaluronic acid (4 %), a repeating polymer of glucuronic acid linked to N-acetylglucosamine, which is unsulphated and contains no galactose, and the chondroitin sulphates (chondroitin 4-sulphate 65 %, chondroitin 6-sulphate 15 %) which have alternating glucuronic acid and A-acetylgalactosamine residues and are sulphated. Keratan sulphate (2 %) is also present.
Protein-bound radioactivity is incorporated at identical rates by + and can/can newborn cartilage, whether the precursor is glucosamine, galactosamine or sulphate (Table 1). At three days the incorporation of glucosamine residues is significantly higher in can/can than in + ; this has returned to normal by 5 days. Incorporation of galactosamine and sulphate is normal throughout.
The increase in glucosamine is due to increased uptake rather than increased turnover: in a chase experiment (Table 2) there was no increased breakdown in can/can mucopolysaccharides in the 2 h period during which the tissue was exposed to unlabelled medium. The increased incorporation represents increased synthesis of mucopolysaccharides susceptible to the action of chondroitinase ABC (Table 3).
Incorporation of [1-14C]D-glucosamine into can/can and normal cartilage at 3 days old. Chase experiment
![Incorporation of [1-14C]D-glucosamine into can/can and normal cartilage at 3 days old. Chase experiment](https://cob.silverchair-cdn.com/cob/content_public/journal/dev/31/2/10.1242_dev.31.2.319/3/m_develop_31_2_319tb2.png?Expires=1742302156&Signature=aFNdXBpExVWnuoHlbNj~ync6mtGy9djw6Lf6rIJw~edAh2mkNM9PtsiPN7wWPMymBvXJra2qBubjMGgKWaMruSfLWubBxS5gA7aUnYHXieds5a6cFWMiu129HX5mnTS5e~W6BGShRNgqsvXp9lGKVdXm-tye1p6HypmtkANhJ5zcxybzBeHcK6TWp1G0WGZY~5q5FQS51hCxChnxDKtn~92Fzp73kwkVgS2~COK7QsvGtTNGpnYMwec9hFPfbj8lWsnTLZcoACzOAGAwCxlTObXymo4NqvCvHZ7WYljCHngBvsR7ZsKUQOP975Xvr32KMMlzNaSEGOw12wBFtriROA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Non-protein-bound radioactivity follows a similar but distinct pattern (Table 4). At 3 days the incorporation from glucosamine into hyaluronic acid is significantly raised, and that into chondroitin sulphate increased, but not significantly so. At 5 days both fractions contain significantly more 14C label. The incorporation of 35S is normal throughout, although very variable in 3-day can/can chondroitin sulphate. Trace amounts of sulphate label ( < 5 % of the total) were also found in the hyaluronic acid fraction, presumably as a con-taminant.
This increase in 14C incorporation is limited to cartilage. Samples of + and can/can skin treated in a similar way showed no such difference (Table 5). It should be noted that the ‘hyaluronic acid’ fraction of skin contained large amounts of 35S label. This is thought to be due to the wide range of acid muco-polysaccharides found in skin (Hardingham & Phelps, 1968, 1970a, b) which were not fully resolved by the technique used.
Two cartilage-specific enzyme systems, UDP-glucose dehydrogenase and UDP-glucose-4-epimerase, both concerned with mucopolysaccharide synthesis, were found to be elevated in 5-day-old can/can cartilage (Table 6). The non-specific glucose-6-phosphate dehydrogenase was not elevated in can/can at either 3 or 5 days.
Protein synthesis from [14C]glycine is significantly reduced in can/can cartilage at birth and at 3 days (Table 7): by 5 days it is significantly higher than in normal litter-mates, reflecting the increased mucopolysaccharide synthesis and enzyme levels seen at this age. These changes in protein synthesis are not seen in the liver.
Little of the protein synthesized by 3-day-old can/can mice is removed by collagenase (Table 8): in 5-day-old mice both collagen and non-collagen moieties are significantly increased.
If any of the abnormalities so far described were near the root cause of the can achondroplasia it might be expected that they would be present before the embryos can be classified externally. As a test of this, cartilage from 10 litters of embryos aged 17 and 18 days was incubated randomly with either [U-14C]-glucose or [U-14C]glycine. The litters were derived from known + /can fathers and mothers whose genotype was either or
. Hence
of the embryos were expected to be can/can. The results pre-sented as frequency distributions (Figs. 1, 2) show that the mucopolysaccharide incorporation peak is unimodal whilst that for protein synthesis is bimodal, with 1/5 1/6 (not significantly different from 1/6) embryos comprising the lower peak. The rates of incorporation into protein obtained also correspond well with those seen in newborn can/can mice and normal litter-mates.
Incorporation of [U-14C]glucose in 17- and 18-day-old embryos from litters segregating for can.
DISCUSSION
The earliest abnormality seen in can/can mice is a decrease in the rate of protein synthesis in cartilage. This was seen in 17-day-old embryos which could not be externally classified as abnormal. This decreased synthesis persists until 3 days post partum: at this time the amount of collagen being synthesized is significantly lower than normal. The period between 3 and 5 days seems to be something of a landmark for the can/can mouse: protein synthesis (including collagen synthesis) increases markedly, as does the incorporation of glucos-amine into mucopolysaccharides and the levels of two mucopolysaccharide synthetic enzymes. Perhaps all these represent a belated compensatory mechan-ism for previous inadequacies.
There are apparent anomalies in the data presented; the difference in total glucosamine incorporation between normal and can/can is large at 5 days, but incorporation into the protein-bound fraction is normal. One is tempted to suggest that the synthetic mechanisms for the protein backbone(s) of hyaluronic acid and chondroitin sulphate cannot keep pace with this abnormally high synthesis, especially as the highest can/can rate (6 ·47 nmol/h at 3 days) is similar to the highest normal rate seen (5 ·92 nmol/h at 5 days). This seems unlikely, however, as the total rate of protein synthesis in can/can cartilage is high at this time.
In 3-day-old can/can mice, although total protein synthesis and collagen synthesis are depressed (Table 8) collagenase-insoluble protein is increased. Is this a portent of the 5-day situation with collagen synthesis lagging behind ?
The ratio of glucosamine : galactosamine incorporation is very high through-out. As chondroitin sulphate contains equal amounts of glucose and galactose derivatives one would expect a priori something nearer to 1:1. Handley & Phelps (1972) showed that the specific radioactivities of UDP-glucosamine and UDP-galactosamine were identical after exposure to [U-14C]glucose, suggesting rapid epimerization. However, glucosamine seems to be preferred, and Heyner (1960) found that galactose failed to support the growth of foetal cartilage in the absence of glucose.
The situation in can contrasts with that reported by Fraser & Goetinck (1971) in the embryonic nannomelic chick. Here the amount of protein-bound 14C label incorporated from glucosamine was significantly less than in normal chicks; nannomelic chicks also incorporated 35S at a lower rate than normal. As the chondroitin sulphate produced was of normal molecular weight, Fraser & Goetinck conclude that the nannomelic chick produces fewer chains of chon-droitin sulphate than its normal sibs.
In the achondroplastic (ac) rabbit utilization of glucose and galactose is increased (Shepard & Bass, 1971). Bargman, Mackler & Shepard (1972) later showed that the increased utilization of glucose compensates for a lack of phosphorylation at the cytochrome oxidase region of the terminal oxidase system of ac mitochondria. In can/can the increased uptake of glucose is into mucopolysaccharides: in addition the oxidative phosphorylation of can/can liver mitochondria is normal (Table 9).
Seegmiller et al. (1971, 1972) described the ultrastructural appearance of cartilage from the epiphyses and trachea of chondrodystrophic (cho) mice. They observed a lack of metachromatic staining (common to all achondroplasias) and an aggregation of collagen into banded fibres with a 640 Å (64 nm) repeat.
Seegmiller suggests that the reduced mucopolysaccharide content implied by lack of metachromia allows the tropocollagen to polymerize into banded ‘native’ collagen fibres.
Stanescu et al. (1972) looked at cartilage samples from seven distinct dwarfing conditions in man : their main finding was a marked increase in collagen content in the cartilage of a 4 · 5-day-old achondroplastic boy. This may be equivalent to the post 5-day-old can/can mouse which might be expected to show a fibrotic matrix.
It is clear that the achondroplastic phenotype can be the end result of a variety of very different first causes and that can represents a type which differs fundamentally from those previously described. It is also clear that our knowledge of these first causes is minimal and that the achondroplastic phenotype presents a field in which there is much scope for further research.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Central Research Fund University of London. We thank Mrs P. Beveridge and Mrs S. Mansfield for technical help.