/ . Embryol. exp. Morph. Vol. 31, 2, pp. 313-318,1974
Printed in Great Britain
313
The in vivo behaviour of achondroplastic cartilage
from the cartilage anomaly (can/can) mouse
ByD. R.JOHNSON1
From the Department of Animal Genetics, University College London
SUMMARY
Explanted cartilage from can/can mice aged 3 days shows increased protein and mucopolysaccharide 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 suggesting 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 achondroplastic 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 %; undine 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 canjcan 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 canjcan cartilages into a favourable environment,
beneath the kidney capsule of normal histocompatible hosts for a period exceeding
their normal life-span.
1
Author's address: Department of Animal Genetics, Wolfson House, Stephenson Way,
London NW1 2HE, U.K.
314
D. R. JOHNSON
MATERIAL AND METHODS
Three-day-old can/can mice and normal litter-mates were injected intraperitoneally with lO/^Ci [U-14C]glucose (230mCi/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 - N H 4 O H and counted in Unisolve (KochLight 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 vertebrae 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 //m 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 jean mice in the uptake of protein-bound [U-14C]glucose
into mucopolysaccharides in any of the tissues assayed.
Table 1. Incorporation of[U-uC]glucose by normal and
can/can tissues in vivo
Tissue
Cartilage
Liver
Muscle
Brain
Normal
4306 ±286
4708 ±671
3018 ±397
2549 ±334
can/can
(6)
(6)
(6)
(6)
4508 ±660
3856 ±188
3673 ±515
2360 ±195
(4)
(4)
(4)
(4)
Results are given in dpm/g tissue:dpm/ml serum ±S.E.M. Numbers in parentheses indicate
number of mice used. Animals (3 days old) received 10/iCi [U-14C]glucose i.p. 1 h before death.
In all, vertebrae from ten can/can and ten normal litter-mates were transplanted. 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
Behaviour of achondroplastic cartilage
315
Fig. 1. Normal tail vertebrae maintained for 2 weeks in kidney capsule of 3-monthold normal sib. /, Intervertebral disc; A, articular zone; P, proliferative zone;
H, hypertrophic zone of cartilage; B, bone spicules; M, marrow, x 63.
Fig. 2. can/can tail vertebrae, details as Fig. 1. Note lack of elongation of vertebra
and V-shaped growth plate x 63.
Fig. 3. Normal vertebra x 160 showing well-differentiated histology.
Fig. 4. can/can vertebra xl60, showing swollen cells in articular zone, meagre
matrix deposition in proliferative zone and poor development of hypertrophiczone.
316
D. R. JOHNSON
vertebrae closely resembled the illustrations by Noel & Wright (1972) of 7- and
8-day-old mouse vertebrae transplanted for 3 weeks.
can/can vertebrae grew less well (Figs. 2, 4). Intervertebral discs were indistinguishable 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.
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 (en I en) 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 (bpHlbpH) mouse, also with suppression of chondrocyte
hypertrophy. Similar results have been obtained in the chick (Cp, Hamburger
1941,1942; dp*, Kieny & Abbott, 1962) and in the rat (Fell & Griineberg, 1939).
However, these apparent similarities between achondroplasias may be misleading. 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 achondroplastic 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 I can mouse is different
yet again (Johnson & Hunt, 1974) and all these differ from Seegmiller's chondrodystrophy (Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson &
Sheldon, 1972). We must not lose sight of the fact r that the underlying defects
in achondroplasia are both complex and poorly understood.
Behaviour of achondroplastic cartilage
317
Table 2. Summary of recent findings in some aehondroplasias
Organism
Source
Main findings
Shepard et al. (1969)
ac rabbit
EM*
EM
Shepard & Bass (1971)
ac rabbit
B
Bargmane/a/. (1972)
ac rabbit
B
Fraser & Goetinck (1971)
nm chick
B
Seegmillere/ al. (1971,1972)
cho mouse
EM
Johnson & Wise (1971)
can mouse
EM
Johnson & Hunt (1974)
can mouse
B
Dead chondrocytes, reduced
matrix
Increased utilization of
[14C]glucose, [14C]galactose
Defective oxidative
phosphorylation
Decreased incorporation of
[14C]glucose, [35S]sulphate
Banded collagen, reduced
matrix
Defective chondrocytes,
reduced matrix
Reduced protein synthesis up to
3 days post par turn, increased
protein and mucopolysaccharide synthesis thereafter
* EM, electron microscopy; B, biochemical techniques.
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{Received 11 June 1973, revised 12 October 1973)