/. Embryol. exp. Morph. 77, 309-322 (1983)
3Q9
Printed in Great Britain © The Company of Biologists Limited 1983
Transplantation of turtle embryonic thymus, into
quail embryo: colonization by quail cells
By J. VASSE 1
From the Institut d'Embryologie du CNRS et du College de France, Nogentsur-Marne
SUMMARY
Turtle (Emys orbicularis L.) embryo thymuses grafted in the somatopleure or onto the quail
embryo chorioallantoic membrane developed in these heterotopic sites for 2-12 days.
When the thymus was removed from embryos at early stages such that no thymocytes were
yet present during normal development, epithelial cells with mitoses were observed in the
explants but no turtle thymocytes developed whatever the duration of explantation. An
extrinsic origin of lymphoid precursor cells can explain such results. Quail lymphoid-like cells
distinguishable from turtle cells by their nuclear structure began entering the explants 5 days
after grafting. Their number increased progressively until the 12th day, when all the grafts
were retrieved.
When an already lymphoid thymus was removed from embryos at later stages, turtle
thymocytes remained fairly abundant in the explants until 5 days. Beyond this period, they
gradually disappeared. After 11-12 days, none were left and only epithelial tissue from the
turtle was present, showing excellent development. Quail lymphoid-like cells entered this
more mature thymus following the same time course as they did in the early rudiment.
Thus we observed no difference between attraction of quail lymphoid cells by the
precolonization thymic epithelium or by the lymphoid thymus.
INTRODUCTION
Thymic lymphoid differentiation can occur only if the thymus rudiment
becomes colonized by extrinsic cells. This has been demonstrated in mammals
(Moore & Owen, 1967; Owen & Ritter, 1969; Fontaine-Perus, Caiman, Kaplan
& Le Douarin, 1981), birds (Moore & Owen, 1965; Le Douarin & Jotereau,
1975), and amphibians (Deparis & Jaylet, 1975; Charlemagne, 1977; Tochinai,
1978; Jaylet & Deparis, 1979; Volpe, Tompkins & Reinschmidt, 1979; Tiirpen,
Knudson & Hoefen, 1981). Various types of experiments have led to this conclusion such as in vitro explantation of the embryonic thymus, parabiosis, grafts
between individuals of different ploidy and interspecific grafts. In explantation
experiments, the thymic anlage, if removed from embryos at an early enough
stage, never becomes lymphoid. In the case of grafts, colonization by host cells
may take place and thus be assessed between individuals of different ploidy
1
Author's address: Institut d'Embryologie du CNRS et du College de France, 49 av. de la
Belle Gabrielle, 94130 Nogent-sur-Marne, France.
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J. VASSE
(amphibians), or between different species (quail and chick). However a mouse
thymus grafted onto a chick chorioallantoic membrane is not colonized by chick
stem cells (Auerbach, 1961; Moore & Owen, 1967).
In reptiles, descriptive work has been carried out on the thymus from adult
(Emys orbicularis and Testudo, Afanassiew, 1877; Van Bemmelen, 1888; Dustin, 1909; Bockman, 1970) and young turtles {Chelydra serpentina, Borysenko
& Cooper, 1972). Various cell categories and the phenomena of seasonal variation with organ involution in winter (Dustin, 1909; Salkind, 1915) were observed. In reptile embryos, earlier authors described the first stages of formation of
the thymus, beginning with a thickening of the branchial pouches which, in
lizards, involves the second and third branchial pouches and, in snakes, the
fourth and fifth ones (Maurer, 1899; Saint-Remy & Prenant, 1904). In the turtle
Chelonia viridis (Van Bemmelen, 1893) and Chrysemys marginata (Shaner,
1921) a constant thickening appears on the third branchial pouch, a variable one
on the fourth pouch and a temporary bud on the fifth one. Pitchappan & Muthukkaruppan recently (1977) carried out a histogenetic study of the thymus of the
lizard Calotes versicolor and observed the emergence of cells with a basophilic
cytoplasm. They suggested that these might be blood-borne precursors of thymic
lymphocytes. However, until now, no experimental analysis had been done in
reptiles on the origin of cells from which thymocytes differentiate.
The present report is an experimental study of embryonic thymus development in Emys orbicularis L., which reveals the capacity of the turtle thymic
anlage to develop when grafted onto a quail embryo. Contrary to what occurs in
mammalian avian combinations, cell exchanges occur and may be followed
through the differences between turtle and quail cell nuclei.
MATERIALS AND METHODS
Materials
Freshwater turtles of the species Emys orbicularis L. were collected from the
ponds of the Brenne region, near Chateauroux (France) and eggs were incubated
as previously described (Vasse, 1973, 1974). Twenty-seven stages were distinguished during embryonic development. In the present study, the staging was
based on the age of the embryo (number of days of incubation at 25 °C) (Table
!)•
Fertile Japanese quail eggs (Coturnix coturnix japonica) were obtained from
a commercial source. They were maintained with the long axis vertical and the
air space up, in a damp environment throughout incubation at 37-38°C.
Operating techniques
After 3 to 7 days of incubation (according to the grafting technique), an
opening was made in the upper part of the quail egg at the location of the air
Turtle embryonic
thymus transplanted into quail embryo
311
Table 1. Principal steps of development of the thymus in relation to normal
developmental stages of turtle Emys orbicularis
Stages
Number of days
of incubation
at25°C
Events in the development of the thymus
13
14
17
21
15
16
17
18
24
28
31
35
Purely epithelial anlage
19
38
First entry of lymphocytes precursor cells
20
42
Lobulation
21
22
45
49
Some thymocytes
Numerous thymocytes
23
24
25
26
27 (hatching)
52
59
66
73
80
Augmentation of the size
Thickening of the pharyngeal epithelium
chamber. Albumin was not removed. The turtle thymus was easily isolated
beginning at stage 18; at stage 17, it was not possible to avoid removing the tissues
surrounding the thymus, as well as the thymus itself. Depending on the thymus
size, it was either grafted whole (stages 17 to 18), or divided into fragments (later
stages), with the graft size being around 0-5x0-5x0-5mm. Explants were
marked with carbon before grafting and were thus easily found at dissection.
Two types of grafts were performed: 1) in the somatopleure at the base of the
wing anlage (3- or 4-day quail embryo) and 2) onto the chorioallantoic membrane (CAM) (5-, 6- or 7-day quail embryo). The explant was introduced into
a slit made in the membrane between two vessels.
After grafting, the opening in the quail eggshell was closed with tape, and the
egg was replaced in the incubator in the same orientation as prior to grafting.
Histological techniques
The explants were fixed in Maximow's or Zenker's fluid, dehydrated,
embedded in paraffin, cut into 7-5jum-thick sections and alternately stained by
the May-Griinwald-Giemsa or Feulgen-Rossenbeck techniques. Quail cells
were distinguishable through the particularities of the nucleus (Le Douarin,
1969).
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J. VASSE
RESULTS
Normal thymic development
The main stages of development were found to be the same as observed in
other vertebrates. The turtle thymus is a bilateral organ located in the angle
formed by the subclavian and carotid arteries. A primary thymic anlage initially
forms on each side by a thickening of the pharyngeal epithelium at the level of
the fourth branchial pouches. A second bud forms at the level of the fifth branchial pouches. At that time, the thymus was made up of large epithelial cells with
a light coloured nucleus and large nucleolus. It separated from the pharyngeal
epithelium (stages 13-14). Several basophilic cells were observed in the thymic
bud and its surrounding mesenchyme at stage 19 (Fig. 1). These cells could be
distinguished from epithelial cells by their more deeply coloured nucleus and by
their cytoplasm which appeared as a basophilic rim around the nucleus, after
May-Griinwald-Giemsa staining. Several melanocytes and eosinophil granulocytes were found in the mesenchyme surrounding the organ. At stage 20 the
thymus became divided into several lobes. Basophilic cells were still present but
only in small quantities. Melanocytes and eosinophil granulocytes were observed
in the connective sheath surrounding the organ and in the interlobular connective tissue. The subdividing of the thymus into lobes was even more marked at
stages 21-24 (Fig. 2). Thymocytes were now present in large numbers. Their
nuclei could be clearly identified by small irregularly arranged chromatin clumps
and one or two larger central clumps (Fig. 3). The connective septa subdividing
the organ contained numerous granulocytes and melanocytes. From that point
onwards, as with birds and mammals, a central medulla and a peripheral cortex
richer in thymocytes became distinct in each lobe. From stages 24-27 until
hatching the thymus grew considerably in size. In conclusion, the various cell
types encountered in the turtle embryonic thymus were epithelial cells, basophilic cells, thymocytes, eosinophil granulocytes and melanocytes.
Myoid cells and Hassal's bodies, observed by some authors in the young and
Figs 1-3. Turtle thymus normal developmental stages. May-Griinwald-Giemsa staining.
Fig. 1. Stage 19+, the thymus comprises pale-staining epithelial cells. Some basophilic cells (b) are present at the periphery, g, granulocyte; v, vessel. Bar = 50 ^m.
Fig. 2. Stage 24, showing the lobular structure. Bar = 100 jum.
Fig. 3. Part of Fig. 2 enlarged, m, melanocyte; g, granulocyte; t, thymocyte; e,
epithelial cell. Bar = 20|Um.
Figs 4-8. Development of turtle thymus grafts into quail embryos. FeulgenRossenbeck staining.
Fig. 4. Overall aspect of the thymus (th) from a stage-21 embryo grafted for 3 days
onto the quail embryo. The graft has embedded in the chorioallantoic membrane
(cam) and displays a distinct lobulation. The black dots to the left are carbon particles
used to mark the graft. Bar = 200 /an.
Turtle embryonic thymus transplanted into quail embryo
313
adults of other turtle species, were not found in the embryonic stages observed
in the present study.
General behaviour of grafted thymuses
Vascularization of the graft by the quail host's vessels was clearly visible upon
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J. VASSE
dissection and histological study, especially in grafts onto the chorioallantoic
membrane. Grafts had to be sorted out according to their histological condition
(special attention was given to the presence of mitoses in the epithelial tissue).
During the first days of transplantation, a marked decrease in cell density was
sometimes observed, followed 2-4 days later by numerous mitoses in the
epithelial tissue (Figs 4-5). When the transplanted thymus already had a lobular
structure, sometimes some lobes did not develop but became fibrous, while
others developed normally. This was conspicuous in grafts on the chorioallantoic
membrane, since melanocytes, which were maintained, delineated the contour
of each lobe, whether developed or not.
Table 2 shows only those grafts which were retained after histological
examination. These totalled 129; 41 in the somatopleure and 88 on the chorioallantoic membrane. No differences were observed between the results of these
two transplantation techniques in terms of the evolution of turtle thymocytes and
granulocytes and the appearance of quail cells in the grafts.
Grafts of thymuses removed from early embryos (stages 17-20) (Figs 5, 6)
Thymuses which were removed from embryos at stages 17-18 were transplanted in toto (it should be borne in mind that, at these stages during normal
development, neither basophilic cells nor thymocytes were yet present). In the
transplants, epithelial cells with mitoses were clearly observed, but there were
neither turtle thymocytes nor basophilic cells, no matter how long the engraftment period lasted (sometimes up to 11 days). On very rare occasions, turtle
granuloblasts could be observed. These were sometimes found in the mesenchyme surrounding the thymus, if this mesenchyme had been taken at the time
of transplantation.
From stages 19 and 20, only a fragment of the thymus (usually half) was
transplanted. At the end of the grafting period, turtle thymocytes were occasionally seen at the edge of the organ. In normal development at stages 19 and 20,
several basophilic cells were seen. Thus these cells have survived and differentiated into thymocytes. In six cases a few turtle granulocytes were found, at the
periphery of the thymus and in the organ itself.
All explants removed from embryos older than stage 17 (Table 2 and Fig. 6)
were invaded after 6-7 days of engraftment by quail cells, clearly different from
the erythrocytes in the vessels which irrigated the graft and from the granulocytes
distinguishable by their cytoplasmic granulations. Their nuclear structure (a
large central chromatin mass and several small dots adjacent to the nuclear
membrane) was analogous to that described in thymic lymphoid cells of normal
quail. When the grafting period exceeded 7 days, quail lymphoid-like cells
became numerous and were observed in the cortex and the medulla. Quail
basophilic cells were also seen around the thymus and in its most external layer.
These had the characteristic nucleus of quail lymphoid precursors, i.e. a large,
irregular, light-red-staining central heterochromatin mass.
Turtle embryonic
thymus transplanted into quail embryo
315
Table 2. Repartition of lymphoid cells in turtle embryonic thymus grafted into
quail embryos
Stage
length of
of
engraftment
explantation period in days
Quail lymphoid
cells
Turtle lymphoid
cells
0
+
++ + + +
0
Number
of
grafts
3
17
9-11
3*
3
4
7-10
1
4
1
18
19
2-3
7-9
1
2
3
4
20
3-5
6
7-11
1
1
1
7
21
3-4
5
22
4
5
7
23
7-11
24
8-12
25
4
5
7-9
26
27
3
1
1
4
1
5
4
6
5
6
2
8
4
6
20
6
1
3
6
1
20
3
1
1
2
20
6
3
1
1
3
4
3
4
4
5
2
3
4
7
1
4
2
3
5
4
3
7
2
3-4
5-6
6
3
7-8
12
2
1
9
4
2
9
5
1
2
8
9
9
4
1
4
3
10
13
14
0:No cells
+ : Several cells visible on a section
+ + :Frequent cells (quail cells = Fig. 6)
+ + + :Very numerous cells (quail Fig. 8; turtle Fig. 7)
* Number of grafts in each category.
Grafts ofthymuses obtained from embryos at advanced stages (21-27) (Figs 7, 8)
At these stages, the normal thymus was lobed with numerous thymocytes and
granulocytes. Only a fragment of the organ was grafted. Turtle thymocytes were
fairly abundant until 5 days of engraftment. Beyond that period, they gradually
disappeared with various timings from one explant to the next. Five to 9 days
after transplantation, turtle thymocytes were still found in the explants, but they
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J. VASSE
were about twice as many on the 5th day as on the 7th day. Finally after 11-12 days,
they had disappeared and only epithelial tissue from the turtle remained, showing
excellent development (Fig. 8). Turtle granulocytes could still be observed at 5 to
Turtle embryonic thymus transplanted into quail embryo
317
7 days after transplantation (with several per lobe); they became very scarce later
(at 8-9 days).
Beginning on the 7th day of engraftment, quail lymphoid-like cells were found
in great quantities. Their number increased continuously until 8-12 days (Table
2 and Fig. 8). The explants, comprising turtle epithelial cells and quail lymphoidlike cells, resembled those developing from stage 19-20 rudiments (compare
Figs 6 and 8).
The kinetics of invasion of the graft by quail cells were studied at different
stages. After 4-5 days in graft, quail basophilic cells were observed in the vessels
at the periphery of the thymus; 5-6 days after grafting, several quail lymphoidlike cells became visible within the thymus itself, in the cortex. It was only after
7 days that these cells became fairly numerous; they were of two types, which
could be differentiated by their nuclear structure and cytoplasm. The nucleus of
cells in the first category had a central mass of DNA characteristic of quail cells,
but no perinuclear cluster (or only a very minute one). In the second category,
the nucleus had a structure typical of quail lymphocytes, with a thick central mass
of heterochromatic DNA and five or six clearly discernible perinuclear clusters.
Up to the 7th day only cells of the first category, which exhibited a basophilic
cytoplasm when the May-Griinwald-Giemsa technique was applied, were observed. From day 8 onwards, cells of the second category became increasingly
numerous; cells of the first category, however, were still present at that point, no
matter from what stage the thymus had been obtained.
DISCUSSION
Normal development of the turtle thymus
This is very similar to that described in amphibians, birds and mammals, one
important feature being the appearance of basophilic cells around and among
cells of the epithelial rudiment. In other vertebrates these basophilic cells are
regarded as lymphoid precursors. At the next stage, cells with a characteristic
nuclear structure appeared; numerous chromatin dots, a typical feature of
Fig. 5. Thymus from a stage-19 embryo grafted for 2 days into the quail embryo
somatopleure. The graft consists of epithelial cells and a few turtle lymphoid cell$.
No quail cells are yet visible. Bar = 20 jum.
Fig. 6. Thymus from a stage-19 embryo grafted for 7 days onto the quail embryo
cam. The cell population is composed of turtle epithelial cells (e) and quail lymphoidlike cell (q) with characteristics chromatin clumps. Bar = 20 jum.
Fig. 7. Thymus from a stage-21 embryo grafted for 4 days onto the quail embryo
cam. Turtle thymocytes and epithelial cells are present but no quail lymphoid-like
cell. Bar = 20jwm.
Fig. 8. Thymus from a stage-24 embryo grafted for 12 days into the quail embryo
somatopleure. Quail lymphoid like cell (q) have become very numerous (compare
with Fig. 6). Bar = 20|Um.
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J. VASSE
thymocytes, allows one to distinguish the nuclei from those of the epithelial cells
of the thymus.
Survival and growth of the thymus rudiment transplanted into a quail host
Emys orbicularis L. embryos normally develop at temperatures ranging between 20 °C and 32 °C. The present study demonstrates that an isolated organ
survives, grows and differentiates at 38 °C, which is the incubation temperature
for quail embryos. Medullar atrophy was sometimes observed in the first days
after transplantation, followed 2-3 days later by mitoses in the epithelial tissue
in much larger numbers than in normal development. This might be a reaction
to the higher incubation temperature, which would decrease the duration of the
mitotic cycle. Alternatively it might be a regeneration phenomenon following
distress before the graft becomes vascularized by the host; the various authors
who have performed allogeneic and syngeneic mouse adult organ grafts (subcutaneously or under the renal capsule) tend to accept this hypothesis (Metcalf
& Wakonig-Vaartaja, 1964; Dukor, Miller, House & Allman, 1965; Blackburn
& Miller, 1967; Cheers, Leuchars, Wallis & Davies, 1972).
Results of the present study show that turtle embryonic thymuses taken at
various stages beginning at stage 17 are capable of developing in a heterotopic
site of the xenogeneic host such as the base of the wing anlage or the chorioallantoic membrane of a quail embryo. A lobular structure develops in a thymus
removed at an early stage (17-18), before this structure is present. Granulocytes, melanocytes, basophilic cells and thymocytes remain present for several
days, but are no longer encountered after 11 days in the quail host. Nonetheless the excellent histological appearance of the epithelial tissue testifies to the
good development of the graft at the end of this period. Since thymocyte
depletion in the thymus was reported in experiments on other materials, it is
likely that, in the turtle case also, this is a physiological event; this is discussed
below.
Origin and fate of turtle thymocytes
In normal development, the thymus of an embryo which has reached stage
17-18 does not yet have basophilic cells. After explantation onto quail embryo,
turtle epithelial cells with numerous mitoses are clearly discernible, but even
after 11 days no turtle basophilic cells are visible. The normal thymus of stage19 to -20 embryos has very few basophilic cells, and turtle thymocytes may
occasionally, but rarely, be observed up to 11 days after explantation. If the
thymus is transplanted at stages 21 to 27, turtle thymocytes may be found until
9 days after the transplantation. But 11-12 days after grafting, they are no
longer present. This was also the case for thymuses obtained from 6-month-old
turtles (unpublished results). Mature thymocytes appear to leave the thymus in
the same manner as in normal development in other species. Thymocyte
depletion in a transplanted thymus has been previously described for mammals
Turtle embryonic thymus transplanted into quail embryo
319
(Weissman, 1967; Scollay, Butcher & Weissman, 1980) and amphibians
(Charlemagne, 1980).
An extrinsic origin of basophilic cells differentiating into thymocytes can explain such results. At stage 18, basophilic cells are sometimes observed in the
peripheral mesenchyme. They reach the thymus itself at stages 19-20. In the
thymus, which is already lymphoid at the time of transplantation (stages 21-27),
since precursor cells no longer reach it, thymocytes cease to renew. This is in
accordance with the generally accepted conclusion of an extrinsic origin for
thymocytes in various groups of vertebrates.
Agreement is far from unanimous, however, on the question of which organ
harbours precursor cells, and what is the pathway of their migration. In the
reptile embryo, according to a histological study of the various stages of the lizard
Calotes versicolor thymus development, Pitchappan & Muthukkaruppan (1977)
suggested that thymocyte precursors were blood-borne stem cells. In our study
of Emys orbicularis L. embryo, at the stages preceding those of thymus colonization, only the blood islands of the yolk sac show marked haematopoietic activity
(Vasse & Beaupain, 1981). Nonetheless, we did observe a budding of the cardiac
endothelium, a thickening of the aortic endothelium with basophilic cells, and,
in the mesentery, an accumulation of granulocytes and basophilic cells. Such
discrete foci of haematopoiesis, as observed by early authors (Jordan, 1917) can
be compared to the para-aortic foci in birds (Dieterlen-Lievre & Martin, 1981).
At the beginning of thymic colonization (stage 19), when the thymus becomes
connected to the general circulation (Fig. 1), cells moving toward the thymus
may be blood borne and emanate from the yolk sac and the foci of intraembryonic haematopoiesis.
The arrival of basophilic cells in the thymus is probably continuous, as suggested by the fact that the turtle thymus attracts quail cells at all stages. Insofar as
the chronology of colonization is concerned, this cannot be deduced from that
of experimental colonization by quail cells as established in this study, for two
reasons: a) the incubation temperature, which is not the normal development
temperature for turtle embryos, and b) the time needed for the onset of quail
vascularization in the turtle thymus.
The exit pathway of turtle thymocytes has not been precisely determined in the
present study, though they appear to leave by the quail vessels. In the experimental conditions used here, it is not possible to know whether the exit time in
the graft corresponds to the rate of maturation and exit of thymocytes in normal
development.
Origin of turtle granulocytes
These become very scarce in the thymus after 8 days in graft, and disappear
by 11 days. Thus, as for turtle thymocytes, it is strongly suggested that there is
an extrinsic origin for these cells, their disappearance in the grafts being explained by the lack of renewal of precursor cells.
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J. VASSE
Entry and differentiation of quail lymphoid-like cells into turtle thymus
Quail cells begin to be seen in the grafts after 5-6 days. They consist mainly
of basophilic cells and are probably carried by the blood to the graft after the host
vessels have already reached it. Upon leaving the vessels, they are found directly
inside the thymus. Thus, while in the beginning they are found at the graft
periphery, they can later be found directly in the thymus. The number of quail
cells increases the longer the period in graft; these cells which probably arise
from the basophilic cells seen after 5 days, take on an appearance which is typical
of quail lymphocytes. The conclusion that quail precursors entering the turtle
thymus rudiment indeed undergo lymphoid differentiation needs confirmation.
A further study will address this question by using antibodies specific for quail
lymphocyte surface markers. From stage 18 onwards, the turtle thymus behaves
in the same manner at all stages of embryonic development (so does that of the
young turtle, unpublished results): the quail cells always invade it following an
identical time course. Le Douarin & Jotereau (1975), in a quail-chick experimental system, found a very precise time pattern: the embryonic thymusattracting ability ceased during a refractory period; they suggested this was
because it was filled by precursors from a first colonization wave. After differentiation of these cells into thymocytes, and their subsequent exit towards the
peripheral organs, the thymus recovered its ability to attract and was able to
receive cells of extrinsic origin again. In the present study, we observed no
difference between attraction by the thymic epithelium alone (stages 18-19) or
attraction by a lymphoid thymus (stages 21-27; young turtle), nor any difference
in the timing of entry of quail cells related to the turtle's thymus age.
It was interesting to find that quail cells could invade the turtle thymus, despite
the class difference. Recognition processes cannot occur between avian
haemopoietic precursors and mammalian stromal cells (Auerbach, 1961; Moore
& Owen, 1967; Fontaine-Perus etal. 1981). In vitro, peripheral attraction by the
thymic anlage may take place, but colonization does not occur (Pyke & Bach,
1979). Our histological observation of the evolution of the nuclear characteristics
of quail cells suggests a slow maturation of these cells in the turtle thymus after
it has remained in the host for a fairly long period. By further following the
appearance of surface markers on quail precursors, we hope to monitor their rate
of evolution and possible differentiation into lymphocytes, in response to the
micro-environment of the turtle thymus.
I am much indebted to Dr F. Dieterlen for critical reading of the manuscript.
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{Accepted 24 June 1983)