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.

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.

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.

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.

Table 1.

Incorporation of [U-14 C]glucose by normal and can/can tissues in vivo

Incorporation of [U-14 C]glucose by normal and can/can tissues in vivo
Incorporation of [U-14 C]glucose by normal and can/can tissues in vivo

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.

Fig. 1.

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.

Fig. 1.

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.

Fig. 2.

can/can tail vertebrae, details as Fig. 1. Note lack of elongation of vertebra and V-shaped growth plate × 63.

Fig. 2.

can/can tail vertebrae, details as Fig. 1. Note lack of elongation of vertebra and V-shaped growth plate × 63.

Fig. 3.

Normal vertebra × 160 showing well-differentiated histology.

Fig. 3.

Normal vertebra × 160 showing well-differentiated histology.

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.

Fig. 4.

can/can vertebra ×l60, showing swollen cells in articular zone, meagre matrix deposition in proliferative zone and poor development of hypertrophic zone.

Fig. 4.

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.

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.

Table 2.

Summary of recent findings in some achondroplasias

Summary of recent findings in some achondroplasias
Summary of recent findings in some achondroplasias
Bargman
,
G. J.
,
Mackler
,
B.
&
Shepard
,
T. H.
(
1972
).
Studies of oxidative energy de-ficiency. 1. Achondroplasia in the labbit
.
Archs Biochem. Biophys
.
150
,
137
146
.
Fell
,
H. B.
&
Grüneberg
,
H.
(
1939
).
The histology and self-differentiating capacity of the abnormal cartilage in a new lethal mutation in the rat (Rattus norvegicus)
.
Proc. R. Soc. Lond. B
127
,
257
277
.
Fraser
,
R. A.
&
Goetinck
,
P. F.
(
1971
).
Reduced synthesis of chondroitin sulphate by cartilage from the mutant’ nannomelia
.
Biochem. biophys. Res. Commun
.
43
,
494
503
.
Hamburger
,
V.
(
1941
).
Transplantation of limb primordia of homozygous and heterozygous chondrodystrophic (‘Creeper’) chick embryos
.
Physiol. Zool
.
14
,
355
364
.
Hamburger
,
V.
(
1942
).
The developmental mechanics of hereditary abnormalities in the chick
.
Biol. Symp
.
6
,
311
334
.
Johnson
,
D. R.
&
Hunt
,
D. M.
(
1974
).
Biochemical observations on the cartilage of achondroplastic (can) mice
.
J. Embryol. exp. Morph
.
31
,
319
328
.
Johnson
,
D. R.
&
Wise
,
J. M.
(
1971
).
Cartilage anomaly (can)‘ a new mutant gene in the mouse
.
J. Embryol. exp. Morph
.
25
,
21
31
.
Kieny
,
M.
&
Abbott
,
U. K.
(
1962
).
L’extrait d’embryon diplopode4 inhibe la croissance des ébauches cartilagineuses normales culturées in vitro
.
C. r. hebd. Séanc. Acad. Sci
.,
Paris
254
,
1520
1522
.
Konyukhov
,
B. V.
&
Ginter
,
E. K.
(
1966
).
A study of the action of the brachypodism-H gene on development in the mouse
.
Folio biol., Praha
12
,
199
206
.
Konyukhov
,
B. V.
&
Paschin
,
Y. V.
(
1967
).
Experimental study of the achondroplasia gene effects in the mouse
.
Acta biol. hung
.
18
,
285
294
.
Konyukhov
,
B. V.
&
Paschin
,
Y. V.
(
1970
).
Abnormal growth of the body, internal organs and skeleton in the achondroplastic mice
.
Acta biol. hung
.
21
,
347
354
.
Noel
,
J. F.
&
Wright
,
E. A.
(
1972
).
The growth of transplanted mouse vertebrae: effects of transplantation under the renal capsule, and the relationship between the rate of growth of the transplant and the age of the host
.
J. Embryol. exp. Morph
.
28
633
645
.
Seegmiller
,
R.
,
Ferguson
,
C. C.
&
Sheldon
,
H.
(
1972
).
Studies on cartilage. VI. A genetically determined defect in tracheal cartilage
.
J. Ultrastruct. Res
.
38
,
288
301
.
Seegmiller
,
R.
,
Fraser
,
F. C.
&
Sheldon
,
H.
(
1971
).
A new chondrodystrophic mutant in mice
.
J. Cell Biol
.
48
,
580
598
.
Shepard
,
T. H.
&
Bass
,
G. L.
(
1971
).
Organ culture studies of achondroplastic rabbit cartilage: evidence for a metabolic defect in glucose utilisation
.
J. Embryol. exp. Morph
.
25
,
347
363
.
Shepard
,
T. H.
,
Fry
,
L. R.
&
Moffett
,
B. C.
(
1969
).
Microscopic studies of achondroplastic cartilage
.
Teratology
2
,
13
22
.