/. Embryo!, exp. Morph. Vol. 26, 3, pp. 443-458, 1971
443
Printed in Great Britain
Forelimb regeneration in
hypophysectomized adult Diemictylus viridescens
following organ culture and autoplastic
implantation of the adenohypophysis
By RICHARD A. LIVERSAGE 1 AND LUNA LIIVAMAGI 1
From the Ramsay Wright Zoological Laboratories, University of Toronto,
Canada
SUMMARY
After autografting an organ-cultured anterior pituitary gland, maintained in culture for
up to 27 days, into the tail or lower jaw of an hypophysectomized adult Diemictylus viridescens,
the animals recovered and survived readily until fixation at 102 days (129 days posthypophysectomy) and normal bilateral limb regeneration occurred. Also, restoration of
normal skin colour, muscle tone, eating habits and behaviour was identical to control
regenerate cases. In the sham control cases, a muscle fragment from the dismembered portion
of the amputated left forelimb was placed in organ culture one day after hypophysectomy and
then autografted into the host tail 7 days later. The majority of animals lived only up to
28 days post-hypophysectomy; they acquired the gross characteristics of adult hypophysectomized newts; and bilateral forelimb regeneration was thwarted. Newts that were
hypophysectomized only, showed no gross signs of limb regeneration and died within 28 days.
Organ culture and autoplastic implantation of the adenohypophysis permitted a study of
the inhibition and then the concurrent restoration (left forelimb = old amputee) and
initiation (right forelimb) of regenerative activity as well as normal advanced limb
regeneration.
INTRODUCTION
Investigations by Schotte (1926) and Hall & Schotte (1951) have shown that
limb regeneration does not occur following concomitant hypophysectomy and
amputation in the adult European salamander Triton and in the adult newt
Diemictylus viridescens. However, if forelimbs are allowed to regenerate for
varying lengths of time prior to hypophysectomy, there is a 'gradual emancipation' from the influence of the (anterior) pituitary gland and regeneration ensues
(Schotte & Hall, 1952). They proposed that the role of the pituitary is in the
form of a 'trigger-action' commencing at amputation and acting solely upon the
earliest phase of regeneration through the intermediary of ACTH (adrenocorticotrophic hormone) via the adrenocorticosteroid hormones.
1
Authors' address: Ramsay Wright Zoological Laboratories, 25 Harbord Street, University
of Toronto, Toronto 181, Ontario, Canada.
444
R. A. LIVERSAGE AND L. LIIVAMAGI
Schotte & Chamberlain (1955) obtained limb regeneration in hypophysectomized newts following daily injections of ACTH. However, when these
forelimbs were reamputated through more proximal adult tissues and no ACTH
injections were given, regeneration was inhibited. They suggested that their
hypophysectomized animals responded to the exogenous ACTH and, thereby,
the pituitary-adrenal synergism was re-established. The possibility exists,
however, that their ACTH preparations contained trace amounts of other
adenohypophyseal hormones. In this connexion, when Schotte & Bierman
(1956) injected various adrenocorticosteroid preparations, in order to stimulate
forelimb regeneration in hypophysectomized newts, less than half of their cases
regenerated; only a few limbs formed advanced blastemata; and the animals died
by 28 days post-amputation. Apparently, injections of only adrenocorticosteroid
hormones are inadequate to support limb regeneration.
More recently, Wilkerson (1963) induced limb regeneration in adult hypophysectomized newts by injections of somatotrophin (80 % somatotrophic hormone + 20 % thyrotrophic hormone) and also bovine growth hormone. He concluded that it was the absence of growth hormone (STH) that inhibited regeneration. However, Wilkerson's growth hormone preparations contained trace
amounts of ACTH, prolactin and/or TSH (thyroid-stimulating hormone). In
this connexion, Liversage & Scadding (1969) showed that long-term survival
and normal 4-digit regenerates developed when adult hypophysectomized
Diemictylus were given daily injections of adult frog anterior pituitary extracts.
The purpose of this investigation was to determine whether or not an organcultured anterior pituitary gland, when autografted, would support long-term
survival and limb regeneration in the adult hypophysectomized newt and, if so,
to study the inhibition and then the initiation as well as advanced limb regeneration in these forms. The following experimental procedures were used: (a)
mechanical removal of the adenohypophysis from an adult newt; (b) organ
culture of the intact gland from 7 to 27 days; followed by (c) ectopic autografting of the previously cultured adenohypophysis.
MATERIALS AND METHODS
This study is based on 83 adult Diemictylus (Triturus) viridescens from
Massachusetts. All animals were kept in individual glass preparation dishes
containing tap water I'm (0-6 cm) deep because hypophysectomized animals
are in a weakened condition and have difficulty raising their heads to respire
when completely submerged. Newts were kept at 20 °C and fed Tubifex worms
twice weekly.
Hypophysectomy
Hypophysectomy was performed according to Liversage (1967) with modifications as described below. Animals were anaesthetized in MS-222 (Sandoz-
Cultured adenohypophysis and regeneration
445
1 g/1 distilled water) and then placed ventral side up on moistened gauze in a
Petri dish. The animal's mouth was washed with a 1 % solution of chloramine
(sodium hypochlorite - Globus, 1970) for 30 sec; then thoroughly rinsed with
glass-distilled water.
Next a bone flap was made in the roof of the mouth by cutting the parasphenoid
complex (see Liversage & Scadding, 1969). This flap was hinged-open contralaterally exposing the intact hypophysis. Then, the hypophysis was separated
from the infundibulum using no. 5 fine watchmaker's forceps, and carefully
placed in culture medium (see below). Any remaining adenohypophyseal cells
and/or cellular debris were cleared from the region by moderate use of suction.
Finally, the bone flap, including its oral epithelium, was replaced closing the
wound.
Culture methods
Following hypophysectomy, the adenohypophysis was rinsed in fresh medium
and placed into organ culture. The basic medium used was CMRL-1415 ATM
(Healy & Parker, 1966), Connaught Medical Research Laboratories, University
of Toronto, and modified in the following manner: (1) 10 % foetal calf serum
was added; (2) the salt concentration was decreased to an osmolarity of 230 ± 5
m-osm/1; (3) 0-02 unit/ml of medium of pure zinc-insulin crystals (beef)
(21-2 units/mg in 10-6 ml glass-distilled H2O acidified with 003 ml 1 N - H C 1 ^
stock solution of 20 units/ml) were added; and (4) 0-5 g/1 sodium bicarbonate
were used instead of 1 g/1 as in CMRL-1415 (Globus, 1970; Vethamany, 1970).
In addition, 100 /ig of penicillin-G potassium and 50 jug streptomycin sulphate
per ml of medium were added just prior to organ culturing. A pH of 7-2 ( ± 0-2)
was maintained (using 2 mg % red phenol indicator) by gassing the cultures
with a mixture of 95 % air and 5 % CO2.
Adenohypophyses were cultured under sterile conditions in 1 ml capacity open
plastic culture cups which were placed into covered Falcon, plastic Petri dishes
(60 x 15 mm). These Petri dishes contained gauze lining saturated with glassdistilled water to ensure moist conditions in the culture chambers. Finally, the
small Petri dishes (culture chambers) were placed inside larger Falcon Petri
dishes (90x25 mm) and then covered and labelled to correspond with the
appropriate hypophysectomized newt.
Cultures were grouped and incubated at 16°C in a sealed glass container
20x21 cm (inside diameter). Every 48-60 h the culture medium was removed
and fresh medium added. The adenohypophyses were cultured for varying
periods (from 7 to 27 days).
Amputation
Left forelimb amputations were performed through the distal stylopodium 2
or 3 days following hypophysectomy in the experimental animals. Care was taken
to amputate the remaining protruding bone, after the soft tissues retracted, and
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R. A. LIVERSAGE AND L. LIIVAMAGI
to trim away any skin fragments covering the lateral edges of the amputation
surface.
Implantation of the adenohypophysis
After organ culture, the hypophysis was ectopically autografted into the host.
There were two implantation sites: (a) mid-way in the dorsal tail fin or dorsal
musculature; or (b) into a subcutaneous pocket in the lower jaw region.
(a) Dorsal tail. A tunnel was made mid-way in the tail fin or musculature in
the following way: two small slits were made on one side, approximately 4 mm
apart. Closed tines of a no. 5pair of Swiss (IREX) forceps were inserted into the
anterior slit and extended through the fin mesenchyme or muscle to the posterior
slit. Next, the tines were separated slightly as they were being withdrawn in
order to enlarge the tunnel. The anterior pituitary was removed from the
culture medium by suspending it in a drop of medium held between slightly
opened tines. Finally, the forceps were inserted into the anterior slit and then
slowly withdrawn; thus, the pituitary was deposited mid-way in the tunnel. The
wider, anterior slit was closed using a single suture (no. 7 fine - Ethicon Inc.,
Somerville, N.J.).
(b) Lower jaw region. A small subcutaneous pocket was made beneath the
lower jaw and the pituitary was implanted between the intact muscle and the
skin flap. The incision was closed with a no. 7 fine Ethicon suture.
Two or three days after adenohypophysis implantation, the right forelimb
was amputated through the distal stylopodium.
Control series
(a) Sham controls (muscle culture). The dismembered portion of an amputated
left forelimb from a newt, hypophysectomized 1 day previously, was immersed
in 1 % chloramine for 30 sec; then it was rinsed twice in sterile glass-distilled
water. Next, a muscle fragment from this forelimb, approximately the size of the
anterior pituitary (0-8 mm3), was dissected out in culture medium and rinsed in
three baths of medium. After 7 days in culture, a muscle fragment, instead of an
animal's adenohypophysis, was autografted into the dorsal tail musculature or
fin in a manner identical to the procedures used in the experimental series. Right
forelimbs were amputated at the time of muscle implantation, instead of 2-3
days later.
(b) Control hypophysectomy. Our surgical techniques were tested using animals
in which only hypophysectomy and right forelimb amputation were performed.
Forelimbs were amputated 2-3 days after the pituitary was removed and
discarded.
(c) Control regenerates. Right forelimbs were amputated in these cases at
the same time, level and in an identical manner to the limbs of the experimental animals in order to test and compare the rate and degree of regeneration.
Cultured adenohypophysis and regeneration
447
Histological preparation
Heads, left and right forelimbs, and tail segments and lower jaws containing
the anterior pituitary or muscle autografts, were preserved in G-Bouin's solution
(Liversage, 1967). Tissues were rinsed in 50% ethanol; decalcified in Jenkin's
solution (Pearse, 1968) for 15-20 days; cleared in methyl salicylate; infiltrated
in paraffin (under vacuum); sectioned at 8/tm; and stained with Delafield's
haematoxylin with orange-G-eosin as a counterstain.
RESULTS
This study is based on gross morphological and histological observations of
40 experimental, 8 sham control, 17 control hypophysectomy and 18 control
regenerate animals.
The stages of normal limb regeneration in adult Diemictylus viridescens (20°C)
have been described by Liversage & Scadding (1969) and graphically illustrated by
Schotte & Liversage (1959). Therefore no description of regeneration is included.
After our experimental animals were hypophysectomized, a 'recuperation
period' (Liversage, 1959) of 2-3 days followed prior to left forelimb amputation.
Presumably, this period helped to lower the amount of surgical stress on the
animals, and to reduce the residual circulatory titres of anterior pituitary
hormones that remained following hypophysectomy.
The experimental data in Series I, Table 1, consists of repeated experiments
grouped according to the number of days (7-27) the adenohypophysis remained
in organ culture. Various culture intervals were employed to determine the
optimum period for culturing the gland.
In Series II, muscle was chosen as the sham control tissue. This experiment
was designed to determine whether or not: (a) the presence of a previously cultured
adenohypophysis could promote recovery and support long term survival as
well as limb regeneration in adult hypophysectomized newts as opposed to
another type of cultured tissue or organ; (b) if our culture and implantation
techniques were adequate to maintain a functional adenohypophysis; and (c) if,
in some way, our techniques, and not the cultured adenohypophysis, were
responsible for survival and regeneration. In Series III, 17 animals lived for
17-28 days in absence of the adenohypophysis (as observed also by Liversage &
Scadding, 1969). These animals were used to test our surgical technique and to
determine longevity in our hypophysectomized animals. In Series IV, 18 animals
had only their right forelimbs amputated; these cases constituted the control
regenerate series.
Histological verification of hypophysectomy
Heads of hypophysectomized animals from Series I, II and III were examined
microscopically for remnant groups of anterior pituitary cells; heads with
remnant pituitary cells were not included in the data.
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R. A. LIVERSAGE AND L. LIIVAMAGI
Table 1. Extent offorelimb regeneration in hypophysectomized adult
Diemictylus viridescens following organ culture and then ectopic autografting
of the anterior pituitary gland
Series
and case
no.
Experimental series - 1 (anterior pituitary culture)
Degree of right limb
Implanta- No. days post-amputation
tion site and
(at
fixation)
regeneration*
A
No. days
no. days
,
*
^ ,
^
pituitary
pituitary
Left
Right
C or
in culture implanted
limb
limb
WH A
B
D
PC 1-15
PC 16
PC 17-19
PC 20-22
PC 23-30
PC 31-37
PC 38
PC 39, 40
7-17
21
21
21
21-24
25
27
27
Tail 13-62
Jaw 6
Jaw 80
Tail 62
Jaw 81-105
Tail 14-55
Tail 7
Tail 13, 14
26A-76D*
24WH
98 D
80D
103D-127D
35A-76D
32WH
38A, 39A
12-60
3
77
60
78-102
12-53
4
10,11
Total 40
Series
and case
no.
-
10
1
10
18
= 40
Sham control series - II (muscle culture)
No. days
No. days
No. days newts survived
muscle
muscle fragment
,
*
x
fragment
implanted in
Left limb
Right limb
in culture
tail
(no regeneration) (no regeneration)
9
27
1
8
17
21
MCI
MC2
MC3
MC4
MC5
MC6-8
16
34
8
15
24
28
9
27
1
8
17
21
Total 8
Control hypophysectomy series III
No. newts
4
3
Total 17
No. days newts
survived
17-18
20-25
26-27
28
no regeneration
no regeneration
no regeneration
no regeneration
Control regenerate series IV
No. newts
No. days post-amputation
(at fixation)
6
3
3
3
3
MOD*
33P
24C
22B
15A
Total 18
* Letters = extent of left limb regeneration: epidermal wound healing = WH; accumulation blastema = A; blastema formation = B; cone or palette = C or P ; digit formation = D.
Cultured adenohypophysis and regeneration
449
Fig. 1 is a photomicrograph of a median longitudinal section through the
head of a control regenerate case from Series IV. The intact pituitary gland is
residing in the sella turcica and is attached to the infundibulum which protrudes
posteriorly from the floor of the diencephalon. The pre-optic nuclear tract and
the neurohypophysis are clearly discernible. Fig. 2 is a median sagittal section
through the head of a newt from control hypophysectomy Series III. The
cavity in which the adenohypophysis was located has been partially invaded by
fibroblasts; no remnant pituitary cells are present; and the infundibulum is
intact.
Fig. I. Photomicrograph of a median longitudinal section through the head of an
animal from Control Regenerate Series IV. The intact anterior pituitary gland {A) is
situated in the sella turcica (arrows) and attached to the infundibulum at the base of
the diencephalon. The pre-optic nuclear tract (PO) and usually the neurohypophysis
(JY) remain following hypophysectomy. Anterior is to the right.
Fig. 2. Photomicrograph of a median sagittal section through the head of a
hypophysectomized newt from Control Hypophysectomy Series III. The cavity (C)
is the result of adenohypophysis removal. Note: sella turcica (S); base of
diencephalon (£)); absence of remnant anterior pituitary cells; fibroblasts (F)
which have invaded the cavity; and the presence of red blood cells (/?). Anterior is to
the right.
Morphological observations
(I) Experimental series. Within a week after hypophysectomy, a visible change
in skin pigmentation occurred and when the animals were handled the muscle
was limp (lacked muscle tone). Normally, adult newt skin is olive green and the
epidermis undergoes periodic moulting. However, by 10 days post-hypophysectomy the epidermis became crusty and abnormally black due to the build-up
of unmoulted epidermis.
All adenohypophyses were ectopically autografted after 7-27 days in culture
(Table I). If a newt remained hypophysectomized for a more extensive period
(more than 27 days) the animal died before the adenohypophysis could be
implanted, or the newt became so weak that even after pituitary implantation
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R. A. LIVERSAGE AND L. LIIVAMAGI
the host did not survive; usually newts die within 15-25 days after hypophysectomy. Thus, the optimum culture period was considered to be between 21
and 24 days.
Right forelimbs were amputated 2-3 days following gland implantation
to allow the pituitary to become vascularized and functional. Within 5 days
after implantation, we observed sloughing of the old, thickened, black epidermis
and a gradual appearance of the olive green pigmented dermis beneath the new,
normally thin layered epidermis. By 7-10 days after pituitary implantation, the
skin pigmentation was normal and regular moulting ensued. Also, the animal's
muscle tone, eating habits and activity became more like the controls.
In the 40 experimental cases, the left limb showed no gross morphological
indications of regeneration at the time the cultured anterior pituitary was
ectopically autografted; however, regeneration resumed after gland implantation. Regeneration of the right forelimb was initiated immediately after
amputation in the presence of the newly implanted pituitary. As seen in Fig. 3,
left limb regeneration is more advanced than that seen in the right limb.
Animals were fixed at various stages of regeneration. The left limbs ranged in
ages from 24 to 127 days and the right limbs from 3 to 102 days post-amputation.
By 30 days following adenohypophysis autografting, the experimental animals
exhibited skin colour, muscle tone, eating habits, and behaviour similar to
control regenerate newts. Also, the morphogenetic patterns of the regenerated
left and right limbs (Fig. 3) were identical to the control regenerates of Series IV
and to those of Liversage & Scadding (1969).
(2) Sham control series (muscle culture). Organ culturing then ectopic
autografting of a muscle fragment, instead of the animal's pituitary, had no
effect on these eight hypophysectomized newts; that is, they retained the
characteristics of hypophysectomized newts and bilateral forelimb regeneration
was thwarted. After 9-29 days post-hypophysectomy, seven out of eight animals
became very weak and, therefore, were fixed. Ages of the left forelimb stumps at
fixation ranged from 8 to 28 days; whereas, ages of the right limb stumps
ranged from 1 to 21 days post-amputation.
(3) Control hypophysectomy series. These 17 animals displayed the characteristics of hypophysectomized adult newts. Furthermore, they survived for only
17-28 days at which time they showed no morphological signs of right forelimb
regeneration.
(4) Control regenerate series. The right forelimbs of these 18 newts were
amputated at the same time and level as those in the experimental animals
for comparative purposes. Histo- and morphogenetically, the experimental
regenerates were identical in their degree (and rate) of regeneration when
compared to our control regenerates and to those of Schotte & Liversage (1959)
and Liversage & Scadding (1969).
Cultured adenohypophysis and regeneration
451
Fig. 3. Photograph of the dorsal view of case PC 18 from Series I. Following
hypophysectomy, the anterior pituitary gland was placed in organ culture; 3 days
later the left forelimb was amputated (lower arrow). No gross morphological
indications of a blastema were observed when the pituitary was ectopically
autografted into the lower jaw after 21 days in culture. Three days after implantation, the right forelimb was amputated (upper arrow). Whereupon, regeneration
in the left and right forelimbs progressed to an advanced 4-digit stage by fixation
101 days post-hypophysectomy. Note: normal pigmentation and smooth epidermis
on limbs and lateral body skin.
Histological observations of ectopic autografts and forelimb regenerates
Photomicrographs of anterior pituitary gland and muscle fragment autografts
in the lower jaw and dorsal tail regions are shown below. Figure 4 is a section
showing the adenohypophysis embedded among dorsal tail tissues (Series I).
The gland retained its shape indicating that it did not undergo degeneration in
organ culture or following implantation, and that it was not under undue physical
pressure from the tail tissues surrounding it. The gland cells are intact and are
stained heavily with haemotoxylin. Also, the cells do not show signs of cytoplasm ic vacuolation or granulation or any nuclear pycnosis (compare with
intact pituitary - Fig. 1).
Figure 5 is a section of an adenohypophysis from Series I, implanted subcutaneously beneath the lower jaw. The difference in shape of this gland compared to the autograft in Fig. 4 is probably due to the tone of the jaw muscles
and the positioning of the gland during implantation. Nevertheless, the cells
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R. A. LIVERSAGE AND L. LIIVAMAGI
are intact, round and heavily stained with haematoxylin. Also, no cytoplasmic
granulation or vacuolation and no nuclear pycnosis were observed.
The histological observations above are supported by the results of the assay,
namely, following implantation of an organ, cultured pituitary: the animals
recovered and survived readily; the forelimbs developed normal 4-digit
regenerates; and regular moulting and normal pigmentation ensued.
Figure 6 shows a section of a forearm muscle fragment autografted into the
tail musculature of a sham control case from Series II. Prior to implantation, the
grafted muscle fragment remained in organ culture for seven days. Sections on
the first few slides show only 'local' teased host tail muscle and connective
tissues in the tunnel area. Upon progressive examination, a distinct island of
autografted intact forearm muscle bundles can be seen in the centre of the
tunnel, supported by 'local' tail cells and tissues. Upon further observation, the
muscle bundles of the implant gradually disappear until only 'local' cells and
tissues are again visible.
The effects of cultured adenohypophyseal autografts on limb regeneration in
hypophysectomized newts are seen in Figs. 7-9. Fig. 7 is a median sagittal
section through the left forelimb of case PC 9 (Series I) in which regeneration
Fig. 4. Photomicrograph of a cross-section through the tail segment of case PC 22
from Series I showing the adenohypophysis (A) embedded in tail musculature. The
gland remained in this ectopic site for 62 days. The anterior pituitary has retained
its shape and can be seen in relation to its immediate environment. Note: anterior
pituitary is surrounded by cross-sections of intact, newly regenerated muscle bundles
(B), as compared with adult intact muscle (M) above, and some connective tissue (C).
Figure 5. Photomicrograph of a median sagittal section through the adenohypophysis
(A) of case PC 28 from Series I. This gland remained in organ culture for 24 days
before being subcutaneously autografted into the musculature (M) of the lower jaw for
105 days. The cartilaginous hyoid bone (//) is above the pituitary, whereas the
epidermal (left arrow) and dermal (right arrow) layers of the skin are beneath the
implant. Note: the nuclei of the pituitary gland are large, round and stained heavily
with haematoxylin.
Cultured adenohypophysis and regeneration
453
has progressed to the cone stage. Before pituitary implantation, some dermal
wound healing and cicatrix formation occurred; presumably, this is the reason
that the regenerate shows characteristics of a non-regenerating limb.
Fig. 8 is a photomicrograph of a section from the left forelimb of case PC 27
(Series I). Four digits are well developed and considerable ossification, characteristic of advanced bone development, is evident along the shafts of the long
bones.
Finally, Fig. 9 shows a section from the right forelimb of case PC 27. There
appears to be approximately a 12-day delay in the rate and degree of limb
regeneration compared to the left limb regenerate, above. That is, this regenerate
is smaller; less ossification has occurred along the shafts of the long bones; and
only some muscle is present at the elbow. However, in both left and right limb
regenerates a normal morphological limb pattern has developed.
Since forelimb regeneration was inhibited in the experimental series prior to
pituitary implantation and also in the sham muscle and hypophysectomy control
series, it appears that the normal regeneration observed in the experimental
limbs was due to the presence of the previously cultured adenohypophysis. These
techniques permitted us to control, concurrently, the restoration and initiation
of regenerative activity as well as normal advanced regeneration.
DISCUSSION
In the present work, 40 experimental animals were hypophysectomized and
their anterior pituitaries were placed in organ culture. The left forelimbs were
amputated 2 or 3 days later, but failed to form regeneration blastemata in
absence of the pituitary. After a culture period of 7-27 days, (optimum period
21-24 days), the anterior pituitary gland was autografted into the dorsal tail
fin or musculature in 28 cases; in the remaining 12 newts the pituitary was
implanted subcutaneously into the lower jaw region. The right forelimbs were
amputated 2-3 days following implantation. Normal bilateral forelimb regeneration ensued and the animals survived readily even though the autografts were
organ cultured before being implanted some distance from the hypothalamus.
Regeneration of the left forelimbs was always more advanced than that seen
in the right limbs. This is because limited soft tissue dedifferentiation, including
muscle fragmentation (Hay, 1959), and some cell accumulation commenced in
the left limb following hypophysectomy, but then ceased. However, after the
adenohypophysis was autografted, regenerative activity in the left limb resumed
(see also Liversage, 1964; Schotte & Tallon, 1960), even in the presence of a
developing cicatrix, which began forming in absence of an adequate hormonal
background (Liversage & Scadding, 1969).
Digit formations were apparent in 25 out of 40 experimental animals at
fixation; the remaining 15 cases were sacrificed prior to the digit stage. Of the
25 cases, six animals survived readily up to fixation at 126-129 days post29-2
454
R. A. LIVERSAGE AND L. LIIVAMAGI
Cultured adenohypophysis and regeneration
455
hypophysectomy; our animals probably could have survived for considerably
longer periods in the presence of adenohypophyseal implants (see Dent, 1970;
Masur, 1962; Pasteels, 1960).
After adenohypophysis implantation, left limb regeneration was not initiated,
it merely resumed. Right forelimb regeneration, however, was initiated at the
time of amputation. Thus, regeneration is dependent upon the presence of the
anterior pituitary gland (hormones) for the resumption and also the initiation of
regeneration as well as for differentiation (Vethamany, 1970). In this regard, one
of the major activities of adenohypophyseal hormones is the stimulation of
RNA synthesis (Stackhouse, Chetsanga & Tan, 1968; see Schmidt, 1968) and
the control of protein, carbohydrate and fat metabolism (Korner, 1970;
Schmidt, 1968; Turner, 1966).
In the sham control cases the left forelimbs were amputated 1 day after
hypophysectomy and a small forearm muscle fragment was organ cultured for
Fig. 6. Photomicrograph of a cross-sectional portion of a newt's tail from Sham
Control case MC 8, Series II, showing an implanted fragment of intact forearm
muscle. This autograft was dissected from the dismembered portion of the animal's
amputated left forelimb and placed in organ culture for 7 days before being
implanted into the host dorsal tail musculature. A distinct island of autografted intact
forearm muscle bundles (implant) can be seen in the centre of the tail tunnel area
(arrows). The muscle implant is surrounded by 'local' tail connective tissue and
regenerated tail muscle (M) (as a result of tunnel formation). The tail tissues
locked the implanted muscle in place. No limb regeneration occurred in this
case.
Fig. 7. Photomicrograph of a longitudinal section through the left forelimb cone
regenerate of case PC 9 (Series 1) 45 days post-amputation. Following hypophysectomy, the pituitary was placed in organ culture (for 15 days); two days later the left
forelimb was amputated. In absence of the pituitary, limb regeneration was
inhibited. However, after gland implantation regeneration progressed to the cone
stage. Note: dermal skin glands (C) encroaching into the blastema area; abortive
cicatrix (connective tissue) formation (C) and non-dedifferentiated muscle (M)
which are characteristics of non-regenerating limbs; and an accumulation of a
homogeneous population of blastema cells (P) as a result of the restoration of
regenerative activity. Stump bone (B) and a large intact nerve trunk (T) are clearly
visible.
Fig. 8. Photomicrograph of a longitudinal section through the left forelimb regenerate of case PC 27 (Series 1). This forelimb regenerated 4 digits by J27 days postamputation. The adenohypophysis was in culture for 24 days and then remained
implanted in the lower jaw for 105 days. Note: muscle differentiation throughout
the length of the limb regenerate; and the normal, advanced differentiation of the
skeletal pattern.
Fig. 9. Photomicrograph of a longitudinal section through the right forelimb
regenerate from case PC 27 (Series 1). There is approximately a 12 day delay in the
rate of limb regeneration compared to the left limb in Fig. 8. This is due, primarily,
to the interval between amputation of the left and right forelimbs, during which
time the anterior pituitary was in organ culture. The rate and degree of right
forelimb regeneration is normal as compared to control regenerates 102 days after
amputation. Arrow designates amputation level.
456
R. A. LIVERSAGE AND L. LIIVAMAGI
7 days before being autografted into the dorsal tail region. Seven out of eight
newts lived up to 21 days with an autografted muscle fragment in the tail - up
to 29 days post-hypophysectomy. Forelimb regeneration was thwarted in these
cases and the animals retained the characteristics of adult hypophysectomized
newts. The autografts, however, had intact muscle bundles. Since regeneration
was inhibited in these cases it must not have been merely the physical presence
of a piece of cultured, autografted tissue that was responsible for regeneration
in the experimental newts, but rather the hormones released by the pituitary
implants (see Dent, 1966, 1970; Masur, 1962; Pasteels, 1960).
Our hypophysectomy technique was tested using animals in which only
pituitary extirpation and forelimb amputation were performed. These animals
lived up to 28 days; no limb blastemata formed; and all cases exhibited the gross
characteristics of hypophysectomized adult newts. The control regenerate series
involved forelimb amputation at the same time and level as the experimental
limbs. Regeneration in the experimental limbs was comparable to the control
regenerates.
Normally, newts have olive green skin and periodically moult the stratum
corneum layer of the epidermis as a single piece. However, when the adult newt
is hypophysectomized, thyroid activity is drastically reduced (Dent, 1966, 1970).
As a consequence, by 10 days after hypophysectomy, the epidermis becomes
crusty, black and moults irregularly in small pieces. However, within 5 days
after adenohypophysis implantation the crusty black epidermis disappears and
normal olive green skin appears due to the resumption of regular moulting.
Also, when Connelly, Tassava & Thornton (1968) treated hypophysectomized
newts with thyroxine, the skin appeared normal and the epidermis moulted. It
is likely that the implanted adenohypophysis supplied TSH which stimulated
the newt thyroid to resume production of thyroid hormones required for
moulting (see Dent, 1966, 1970). The observed pigmentation changes in our
experimental newts were probably due to decreases in the adrenocorticosteroid
and intermediary lobe melanocyte stimulating hormones (Schmidt, 1968).
ACTH is probably implicated in limb regeneration through the pituitaryadrenal synergism (see Schotte, 1961). Also, somatotrophin (STH) is involved
in adult Diemictylus forelimb regeneration (Wilkerson, 1963; Vethamany, 1970).
In addition, Richardson (1945), Schotte and Washburn (1954), and Theodosis
(1968) have shown that poor limb regeneration ensues in thyroidectomized
newts. But, Richardson (1945), Wilkerson (1963), Connelly et al. (1968), and
Tassava (1969) showed that thyroxine or TSH treatments only are completely
ineffective in promoting limb regeneration in adult hypophysectomized
Diemictylus.
According to Connelly et al. (1968) and Tassava (1969), prolactin and prolactin plus thyroxine effectively support limb regeneration and survival in
hypophysectomized adult newts. More recently, Vethamany (1970) showed
conclusive evidence of a direct involvement of insulin as well as STH, hydro-
Cultured adenohypophysis and regeneration
457
cortisone and thyroxine upon cartilage differentiation in adult urodele tail
blastemata in vitro.
The present work shows that if the anterior pituitary of adult Diemictylus is
ectopically autografted into the animal following 21-24 days in organ culture,
recovery and long term survival of the animal ensues. The cultured intact
pituitary also supports concomitant restoration (left forelimb = old amputee)
and initiation (right forelimb) of regenerative activity as well as normal advanced
limb regeneration. Our organ culture procedures represent a method of controlling limb regeneration in the adult newt.
We would like to express our appreciation to Dr R. G. Romans, Connaught Medical
Research Laboratories, Dufferin Campus, University of Toronto, for supplying the pure
crystalline zinc-insulin and to Mr Tihamer Hellenyi, Ramsay Wright Zoological Laboratories,
University of Toronto for his valuable technical assistance. This paper was prepared from a
portion of an M.Sc. thesis submitted to the Department of Zoology, School of Graduate
Studies, University of Toronto. The investigation was supported by grant no. A-1208 from
the National Research Council of Canada.
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{Manuscript received 13 May 1971)