ABSTRACT
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 post-hypophysectomy) 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 hypophy-sectomized 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 Schotté (1926) and Hall & Schotté (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 (Schotté & 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 (adreno-corticotrophic hormone) via the adrenocorticosteroid hormones.
Schotté & Chamberlain (1955) obtained limb regeneration in hypophy-sectomized 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 Schotté & 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 hypophy-sectomized newts by injections of somatotrophin in (80 % somatotrophinic 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 organ-cultured 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 auto-grafting 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 in (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-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 contra-laterally 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 0·03 ml 1 N-HCl-> 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, 100μg of penicillin-G potassium and 50μg 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 × 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 (90×25 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 20×21 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 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.
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 8μm; 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 Schotté & 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.
Extent of forelimb regeneration in hypophysectomized adult Diemictylus viridescens following organ culture and then ectopic autografting of the anterior pituitary 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.
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.
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 (N) remain following hypophysectomy. Anterior is to the right.
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 (N) remain following hypophysectomy. Anterior is to the right.
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 (D); absence of remnant anterior pituitary cells; fibroblasts (F) which have invaded the cavity; and the presence of red blood cells (R). Anterior is to the right.
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 (D); absence of remnant anterior pituitary cells; fibroblasts (F) which have invaded the cavity; and the presence of red blood cells (R). Anterior is to the right.
Morphological observations
(1) 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-hypophy-sectomy 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 1). 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 the host did not survive; usually newts die within 15—25 days after hypophy-sectomy. 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.
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.
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.
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 Schotté & Liversage (1959) and Liversage & Scadding (1969).
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 cytoplasmic vacuolation or granulation or any nuclear pycnosis (compare with intact pituitary - Fig. 1).
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).
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 is a section of an adenohypophysis from Series I, implanted sub-cutaneously 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 are intact, round and heavily stained with haematoxylin. Also, no cytoplasmic granulation or vacuolation and no nuclear pycnosis were observed.
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 (H) 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.
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 (H) 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.
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.
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.
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.
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 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.
Photomicrograph of a longitudinal section through the left forelimb cone regenerate of case PC 9 (Series I) 45 days post-amputation. Following hypophy-sectomy, 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 (G) 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.
Photomicrograph of a longitudinal section through the left forelimb cone regenerate of case PC 9 (Series I) 45 days post-amputation. Following hypophy-sectomy, 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 (G) 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 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.
Photomicrograph of a longitudinal section through the left forelimb regenerate of case PC 27 (Series I). This forelimb regenerated 4 digits by 127 days post-amputation. 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.
Photomicrograph of a longitudinal section through the left forelimb regenerate of case PC 27 (Series I). This forelimb regenerated 4 digits by 127 days post-amputation. 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.
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.
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.
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.
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; Schotté & Talion, 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 post-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 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 pituitary-adrenal synergism (see Schotté, 1961). Also, somatotrophinin (STH) is involved in adult Diemictylus forelimb regeneration (Wilkerson, 1963; Vethamany, 1970). In addition, Richardson (1945), Schotté 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-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.
ACKNOWLEDGEMENTS
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.