An investigation of the prolactin-thyroxine synergism in newt limb regeneration

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.     

Bioengineered human growth hormone supports limb regeneration in the hypophysectomized newt Notophthalmus viridescens

It is well documented that growth hormone (GH) replacement therapy will restore normal limb regeneration to hypophysectomized adult newts. However, it is also known that the GH preparations used in previous reports were contaminated by other pituitary hormones shown to support regeneration when administered free of GH. The recent availability of bioengineered human GH was studied for its ability to restore the regenerative capacity to hypophysectomized newts. Five days posthypophysectomy adult newts were subject to forelimb amputation distal to the elbow. Animals were divided into three groups (n greater than 20). Each received one of three GH preparations: pituitary-derived bovine GH, pituitary-derived human GH, or bioengineered human GH. GH was administered via intraperitoneal injection (0.029 IU/50 microliters) on alternate days for either the first 5 days (total of 3 injections) or for 35 days (total of 18 injections). Pituitary-intact and hypophysectomized control newts were subjected to forelimb amputation and injected with hormone diluent. All newts that received GH demonstrated normal limb regeneration to the early digitiform stage by 35 days postamputation. None of the hypophysectomized control newts showed any evidence of regeneration. We conclude that GH alone can restore the ability to undergo normal limb regeneration to hypophysectomized newts.     

Effects of growth hormone and nutrient on limb regeneration in hypophysectomized adult newts

The effect of hypophysectomy, growth hormone (GH) and an amino acid-glucose mixture on the regenerative ability of the hypophysectomized Triturus pyrrhogaster yielded the following results:

• 1 The survival time of hypophysectomized newts can be prolonged substantially by the sulfamide application.
• 2 Although the limb regeneration in the hypophysectomized newt is retarded as compared with that of the pituitary intact control, it finally completes morphogenetic process under such conditions of prolonged survival.
• 3 The injection of 100 μg of GH restored the speed of regeneration of pituitary-deprived limbs to almost a normal level.
• 4 Injections of the amino acid-glucose mixture also promoted the limb regeneration in hypophysectomized newts. However, initial delay in regeneration to the time of bud appearance was not restored by the nutrients.

     

The effect of hypophysectomy upon cholinesterase activity in the forelimbs of regenerating adult Notophthalmus news

In order to study endocrine influence upon cholinesterase activity during regeneration, adult newts were hypophysectomized either prior to limb transection or during regeneration. Homogenates of limb tissues were assayed for cholinesterase activity during each stage of regeneration.In animals with pituitaries intact, cholinesterase activity in regenerating limb tissues decreases soon after amputation, and then it rises to the level of activity in intact limbs of normal animals, during the period of differentiation. In hypophysectomized newts there seems to be no alteration of this basic pattern of activity, but removal of the pituitary does result in more elevated levels of enzymatic activity. In the intact forelimbs of control newts undergoing regeneration, cholinesterase activity greatly increases as the other transected limb begins to regenerate but it returns to normal as regeneration progresses. If these animals are hypophysectomized, no such increase is observed during the early stages of regeneration. Rather, there is an initial decrease in cholinesterase activity that is followed by an increase in such activity.These data are compatible with the hypothesis that the pituitary modulates cholinesterase activity in the limb tissues of adult newts.     

Limb regeneration and survival of prolactin treated hypophysectomized adult newts

Hypophysectomized adult newts exhibited 98% survival and limb regeneration at 23 days post-hypophysectomy when injected intraperitoneally every other day with prolactin (0.015 U/newt) and kept continuously in aquaria with 1 × 10^?7 concentration of thyroxine. Thyroxine alone was no more effective than saline injections. Prolactin (1.2 U/newt every other day) alone increased survival and limb regeneration, but less effectively than did the prolactin-thyroxine combination.     

Temporal analysis of the role of growth hormone in the initiation and maintenance of limb regeneration in the hypophysectomized newt Notophthalmus viridescens

This study was designed to investigate and determine for how long, after either hypophysectomy or the third (last) growth hormone injection (to previously hypophysectomized newts), the circulating and now declining titers of endogenous or exogenous hormone remained at a sufficient concentration to permit a morphologically normal forelimb regeneration response in the adult newt Notophthalmus viridescens. To examine the declining levels of endogenous hormone (hormone withdrawal series [HW]), left forelimbs were amputated at specific times following hypophysectomy. Right forelimbs were amputated 5 days prior to hypophysectomy. The declining levels of exogenous hormone (hormone replacement series [HR] were examined in newts whose left forelimbs were amputated at specific times following the last of three consecutive alternate-day growth hormone injections that were initiated 5 days post hypophysectomy. Right forelimbs were amputated immediately following the first hormone injection. All experimental animals were sacrificed when their right forelimbs regenerated to an advanced digitiform regenerate. In both series right forelimbs regenerated normally. In the HW series normal regeneration resulted only when forelimbs were amputated within 48 hours post hypophysectomy, whereas in the HR series normal regeneration occurred in only those newts whose forelimbs were amputated within 12 hours of the last hormone injection. The regeneration response of left forelimbs in both series gradually declined with the time interval between either hypophysectomy or hormone injection and forelimb amputation. As the hormone titer declined, fewer limbs initiated a normal response; they became progressively more hypomorphic and eventually failed to undergo typical regeneration.     

In vitro studies of the influence of prolactin on tail regeneration in the adult newtNotophthalmus viridescens

Summary In vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

The irradiated epidermis inhibits newt limb regeneration by preventing blastema growth. A histological study

It has been established that X-ray irradiation localized to a forelimb or entire irradiation of premetamorphic Pleurodeles larvae prevented limb regeneration. Transplantation of non-irradiated skin, dermis or muscle to limb stumps of locally irradiated newts was sufficient to allow a blastema to develop. Transplantation of the same tissues to limb stumps of entirely irradiated newts yielded different results with the different graft types. Skin graft allowed a normal blastema to be established but dermis or muscle grafts did not. In order to define more precisely the role played by the epidermis in the establishment of a blastema, and in the growth of a regenerate, different combinations of limb tissues, either irradiated or not, were carried out at the level of amputated limb stumps. At four different times (8-10 days; 13-15 days; 20-23 days; 30 days or more) after amputation the stumps were examined in histological longitudinal sections to study the first events of regeneration, that is dedifferentiation and growth. Dedifferentiation occurred in both normal and irradiated tissues of mesodermal origin. The healthy mesenchymal cells began dividing and formed a growing blastema only when associated with a non-irradiated epidermis. Healthy mesenchymal cells covered with an irradiated epidermis exhibited a few mitoses after dedifferentiation, but the mitotic figures became rarer and rarer until the animals died. The lack of dense accumulation of blastemal cells in such limb stumps suggested that the healthy epidermis allows the mesenchymal cells to divide actively to constitute a growing blastema. Hence, X-ray irradiation seems to be responsible for the loss of such an epidermal mitogenic influence on the underlying mesenchymal cells.     

Hormone action in newt limb regeneration: insulin and endorphins

Although several hormones have been linked to newt limb regeneration, a cohesive hypothesis as to how these hormones control the process is yet to emerge. A critical review of the traditional approaches and a reevaluation of currently operative assumptions and interpretations of results precede the data on insulin and beta-endorphin. Results from in vivo and in vitro experiments on insulin are summarized, showing that insulin not only promotes various cellular events but also is essential for the expression of the mitogenic effect of nerves on cultured newt limb blastemata. Furthermore, the strong likelihood that insulin may be the common link in promoting limb regeneration in hypophysectomized newts that received pituitary hormone replacement therapy or a nutritional supplement is discussed. The status of beta-endorphin in regeneration is also explored. Data are presented to show that vertebrates with regenerating capacity (newts, tadpoles) have higher levels of plasma beta-endorphin than that found in species where the capacity to regenerate is either restricted (frogs) or totally lost (mammals). beta-Endorphin-like immunoreactivity has been localized in the epidermis of a regenerating newt blastema, as well as in the intermediate lobe of the pituitary gland of axolotl, newt, and Xenopus. A possible opiate connection in vertebrate limb regeneration, in particular, wound healing, is discussed.     

Enhancement of insulin secretion by human chorionic somatomammotropin and related hormones.

Hypophysectomized rats were treated for 6 days with 200 mug per day of either human chorionic somatomammotropin, human pituitary growth hormone, plasmin-modified human pituitary growth hormone, or ovine prolactin. All hormone preparations except ovine prolactin enhanced the ability of the pancreases of hypophysectomized rats to secrete insulin in the isolated pancreas perfusion system.     

Tolerance, limb regeneration and collagen fibrogenesis under high oxygen concentrations by the adult newt, Diemictylus viridescens

Adult newts placed in an atmospheric environment of 85% oxygen, saturated humidity, and at a temperature of 20 ± 1°C survived particularly well a 44-day test period. They did not succumb to “oxygen toxicity” as has been frequently reported for other vertebrate species. Having established the newt's tolerance of high oxygen atmosphere, the effect of oxygen on growth and development in the regenerating newt limb was investigated. Under the atmospheric conditions described above, and under 92% oxygen, the regeneration of adult newt limbs appeared to be retarded during the first 25 days after amputation when compared with regenerating limbs of control animals kept under a normal atmosphere of 21% oxygen (air). Thereafter, little or no difference could be discerned between the regeneration of experimental and control limbs. It is known that molecular oxygen participates directly in the hydroxylation of proline to hydroxyproline in the synthesis of collagen. Sectioned regenerates stained specifically for collagen were examined to determine if collagen synthesis was induced in experimental animals. Two regeneration-inhibited limbs of oxygenated newts showed cicatrical repair of the apical limb stump 25 days after amputation. However, the majority of the experimental animals revealed no obvious increase in collagen fibers. These results contraindicate any marked “oxygen toxicity” affecting the life of the newts, or regeneration of their limbs. It is suggested that a change in collagen fiber type might have been induced by the high-oxygen atmosphere. Investigations to test this hypothesis are currently underway.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

Lysosomal enzymes in the rat harderian gland are altered by either bromocriptine treatment or hypophysectomy and hormone replacement therapy

Four groups of adult male hypophysectomized rats were injected subcutaneously twice daily between 0800-0900 hr and 1600-1700 hr with either saline diluent, 150 micrograms sheep prolactin and/or growth hormone (GH); intact rats received either saline or 150 micrograms bromocriptine twice daily. After 4 days of treatment, lysosomal enzyme assays revealed significant elevations in both acid phosphatase and alpha-mannosidase enzyme activities in the Harderian glands of saline-injected hypophysectomized rats compared to those in intact controls. beta-Glucuronidase levels were depressed and hexosaminidase activity unaffected by hypophysectomy treatment alone compared to intact controls. Lysosomal enzyme activities in hypophysectomized animals treated with prolactin were not different from the hypophysectomized control animals. However, treatment with GH alone or in combination with prolactin had a significant inhibitory effect on beta-glucuronidase, hexosaminidase, and alpha-mannosidase enzyme activities in the Harderian gland of hypophysectomized animals. Bromocriptine treatment in intact rats only elevated acid phosphatase activity. In summary, the patterns of responses did not reveal a role for prolactin in the control of Harderian gland lysosomal enzyme activities by the pituitary. However, some of the influence on this target system may be exerted by growth hormone.     

Cell kinetics of the growth plate in hypophysectomized rats treated with growth hormone and thyroxine

Summary The cell production in the growth plate of the proximal tibia was calculated in hypophysectomized rats given growth hormone and/or thyroxine from values of longitudinal bone growth determined with oxytetracycline and the size of degenerative cells in the growth plate.The changes in longitudinal bone growth induced by thyroxine and growth hormone in hypophysectomized rats were found to be predominantly caused by changes in the cell production, whereas the changes in the size of the degenerative cells were minor. The stimulation of cell production by growth hormone was dependent on the dose and the administration period. Thyroxine was found to stimulate the cell production up to an optimum dose of thyroxine.     

The morphology of the mucous gland and its responses to prolactin in the skin of the red-spotted newt

The mucous gland of the red-spotted newt, Notophthalamus viridescens viridescens, Rafinesque was examined by histochemical and ultrastructural techniques and its cytological responses to various hormonal conditions were studied. Its secretory epithelial cells produce and release in merocrine fashion a neutral, unsulphated mucosubstance. The secretory epithelium is bounded peripherally by a thin, but apparent non-functional, myo-epithelium. The duct of this mucous gland consists of a single keratinized tubular cell that extends from the neck region of the gland to the surface of the epidermis. Mucous secretion is absent or greatly reduced on the skins of newts maintained under laboratory conditions for a few weeks but reappears after injection of ovine prolactin. Mucous glands in laboratory conditioned animals show a 4-fold increase in volume brought about by the engorgement of their epithelial cells with secretory granules. Ovine prolactin reduces the volume of the glands to unconditioned levels with a corresponding reduction in granular content, suggesting that prolactin functions in the release of the granules. This view is reinforced by the findings that autotransplantation of the pituitary gland prevents the conditioning effect and that glandular volume increases in auto-transplanted animals given ergocornine. Granular accumulation begins also in hypophysectomized newts but ceases after a week, indicating the need for some hypophyseal factor in the synthesis as well as the release of the granules. Ovine prolactin restores mucous glands of hypophysectomized newts to the unconditioned state. Contrary to earlier findings, ovine prolactin induces a reduction in the volume of the mucous gland in thyroidectomized newts.     

Protein synthesis in the brain of newts undergoing limb regeneration.

The nervous system plays an important role during the process of amphibian limb regeneration. However, the molecules that are involved in such a control of regeneration are largely unknown. We have attempted to map protein synthesis in the brains of intact newts and from newts undergoing limb or tail regeneration. Our results show unique protein synthesis in the brain of newts undergoing limb regeneration. Such an analysis can lead to the identification and characterization of these proteins.     

Role of the hypophysis in thyroid regeneration after partial thyroidectomy.

The role of the hypophysis in thyroid regeneration was investigated by measuring the mitotic activity of the thyroid remnant in hemithyroidectomized rats as well as the blood levels of thyroid hormone at various time-intervals after hemithyroidectomy. Mitotic activity underwent a significant increase to reach a peak (a 5- to 8- fold increase) 2 days after hemithyroidectomy. The thyroid hormone level in blood was lower than in controls. Histologically, the thyroid gland showed signs of an elevated rate of functional activity, as indicated by losses of colloid and cell hypertrophy. In a second approach, the mitotic activity of the thyroid remnant was estimated in hypophysectomized and in thyroxine treated rats. Both hypophysectomy and thyroxine injection prevented occurrence of the mitotic peak at 2 days. The regeneration of the thyroid after hemithyroidectomy, as it occurred in the present work, may be explained by a release of thyroid stimulating hormone from the pituitary, brought about by the low level of circulating thyroid hormone, itself resulting from a loss of thyroid tissue.     

Forelimb regeneration in thyroidectomized adult newts following organ culture and autografting of the thyroid glands

Summary Thyroidectomy and organ culture of adult newt thyroid glands three days prior to forelimb amputation was followed by autografting the glands subcutaneously into the animal's lower jaw region 9, 18 or 25 days postamputation (GC^{9, 18, 25} day series). This was an attempt, utilizing 515 animals, to elucidate further the role of the thyroids in regeneration. Amputated limbs of the thyroidectomized (Thx) and autografted muscle explant (MC = sham) cases underwent stumping or were significantly delayed in their regeneration rate and displayed abnormal morphogenesis compared with control regenerates. In the GC⁹ series newts, regenerates were identical to controls 45 days postamputation. However, regenerates of the GC¹⁸ series cases exhibited delayed and abnormal development at 45 days; but they were not as delayed and had fewer abnormalities than those cases in the Thx and MC groups. Results of the GC²⁵ series newts were similar to those of the Thx group. Within 5 days of autografting the thyroids, epidermal moulting resumed and long-term survival ensued. We conclude that normal limb regeneration in the adult newt is thyroid hormone(s) dependent, specifically the later stages of growth, differentiation and morphogenesis.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.