ABSTRACT
Explanted cartilage from can/can mice aged 3 days shows increased protein and mucopoly-saccharide synthesis. Injection of [U-14C]glucose into 3-day-old can/can mice failed to duplicate the increased mucopolysaccharide formation. Transplanted can/can tail vertebrae grown in the renal capsule of normal sibs grew less well than those from normal litter-mates’ also suggest-ing that the increased metabolism seen in vitro is not a feature of in vivo development.
INTRODUCTION
Cartilage anomaly (can) is a recessive gene which displays a classic achondro-plastic phenotype and which is lethal at about 10 days after birth (Johnson & Wise, 1971). Recent biochemical studies of explanted cartilage (Johnson & Hunt, 1974) revealed a decrease in protein synthesis as early as the 17th day of gestation (before normal and can/can mice can be distinguished externally). Later, on or after the third day post partum, this is reversed, and levels of protein synthesis, incorporation of [14C]glucosamine into mucopolysaccharides and the activity of certain enzymes on the mucopolysaccharide biosynthetic pathway all increase to values well above the levels seen in normal litter-mates (incorporation of [14C]glycine into protein 168 % normal; incorporation of [14C]glucosamine into protein-bound mucopolysaccharides 199 %; uridine diphosphoglucose (UDPG)-dehydrogenase activity 138 % ; UDPG-4-epimerase activity 188 %).
It was a matter of interest to see whether these in vitro changes also take place in vivo, and if so, whether they represent some kind of compensatory mechanism for previous poor growth which might allow can/can cartilage to achieve ultimate normality. The experiment was performed in two ways, first by exposing whole mice to [14C]glucose and assaying its incorporation into mucopolysaccharides and secondly by transplanting can/can cartilages into a favourable environment, beneath the kidney capsule of normal histocompatible hosts for a period exceeding their normal life-span.
MATERIAL AND METHODS
Three-day-old can/can mice and normal litter-mates were injected intra-peritoneally with 10 μCi [U-14C]glucose (230 mCi/mM, The Radiochemical Centre, Amersham). One hour later they were killed by decapitation, and samples taken of sternal cartilage, sternal musculature, liver, brain and blood. The solid tissues were weighed, homogenized in 0·6 ml/mg distilled water and protein bound radioactivity isolated by precipitation with an equal volume of 10 % trichloracetic acid (TCA) followed by centrifugation and two washes with 5 % TCA. Bovine serum albumin (0·0005 g/ml) was added as carrier protein. The precipitate was redissolved in 3 N-NH4OH and counted in Unisolve (Koch-Light Ltd.) in a Packard Tricarb liquid scintillation spectrophotometer with external standardization. The blood was centrifuged, and an aliquot of serum counted in Unisolve.
Tail vertebrae (6–8) from 3-day-old can/can and normal litter-mates were transplanted into the renal capsules of 3-month-old normal male sibs, following the technique of Noel & Wright (1972) except that the ‘injection’ of tail verte-brae into the kidney capsule was performed using a 4 cm 16 gauge sternal puncture needle. The host animals were killed and the implanted vertebrae dissected free 14 days after implantation. The implants were fixed in Bouin’s fluid, decalcified, wax-embedded, sectioned longitudinally at 8 μm and stained with haematoxylin and eosin.
RESULTS
Because of small inaccuracies in injection volumes (unavoidable in very small mice) results of the uptake experiment are expressed as the ratio dpm/g tissue : dpm/ml serum (Table 1). It is clear that no significant difference exists between normal and can/can mice in the uptake of protein-bound [U-14C]glucose into mucopolysaccharides in any of the tissues assayed.
In all, vertebrae from ten can/can and ten normal litter-mates were trans-planted. Normal vertebrae grew well, elongated, and after 14 days showed clearly discernible intervertebral discs, articular, proliferative and hypertrophic cartilage zones, bone spicules and haemopoetic marrow (Figs. 1, 3). These vertebrae closely resembled the illustrations by Noel & Wright (1972) of 7- and 8-day-old mouse vertebrae transplanted for 3 weeks.
Normal tail vertebrae maintained for 2 weeks in kidney capsule of 3-month-old normal sib. I, Intervertebral disc; A, articular zone; P, proliferative zone; H, hypertrophic zone of cartilage; B, bone spicules; M, marrow, × 63.
can/can tail vertebrae, details as Fig. 1. Note lack of elongation of vertebra and V-shaped growth plate × 63.
can/can tail vertebrae, details as Fig. 1. Note lack of elongation of vertebra and V-shaped growth plate × 63.
Can/can vertebrae grew less well (Figs. 2, 4). Intervertebral discs were indis-tinguishable from normal, but the growth plate acquired a characteristic V shape. The articular zone was pronounced, with many large cells. The proliferative zone had little matrix and the hypertrophic zone was represented only by a few cells. Little bone had been laid down.
can/can vertebra ×l60, showing swollen cells in articular zone, meagre matrix deposition in proliferative zone and poor development of hypertrophic zone.
The epiphyseal regions had increased in width, but as a result of meagre bone development had not moved apart appreciably. In some cases a small amount of bone with marrow separated the epiphyses, but this was often abnormal, with spicules perpendicular to the long axis of the vertebra (Fig. 2). In others the diaphysis had been invaded by host tissue and the epiphyses were physically separated from each other.
DISCUSSION
It is clear that the potential displayed by can/can cartilage in vitro under near ideal conditions is not realized in vivo. This finding is born out by the results of the transplantation studies.
The performance of can/can cartilage transplanted into normal sibs is typical of achondroplastic cartilage from other sources. Konyukhov & Paschin (1967) reported that ulna, radius and humerus of achondroplastic (cn/cn) mice grew less well than those of normal litter-mates when transplanted into normal recipients, and Konyukhov & Paschin (1970) noted a reduced hyperplastic zone in this mutant. Konyukhov & Ginter (1966) described a similar transitory effect in the brachypod (bpH/bpH) mouse, also with suppression of chondrocyte hypertrophy. Similar results have been obtained in the chick (Çp, Hamburger 1941,1942; dp4, Kieny & Abbott, 1962) and in the rat (Fell & Grüneberg, 1939).
However, these apparent similarities between achondroplasias may be mis-leading. In recent years a number of studies have been made using a combination of ultrastructural and biochemical methods (Table 2) and these indicate that the underlying causes beneath the superficially similar phenotype may be very varied. Evidently achondroplasia is not a disease : it is a symptom, and suggests no more than the name implies— the failure of cartilage to grow. In the achondro-plastic rabbit (Bargman, Mackler & Shepard, 1972) the underlying cause seems to be concerned with a defect of oxidative phosphorylation in the mitochondria; the nannomelic chick (Fraser & Goetinck, 1971) produces fewer chains of chondroitin sulphate than normal litter-mates. The can/can mouse is different yet again (Johnson & Hunt, 1974) and all these differ from Seegmiller’s chon-drodystrophy (Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon, 1972). We must not lose sight of the fact that the underlying defects in achondroplasia are both complex and poorly understood.