Reversible Conversion among Subtypes of Salivary Gland Duct Cells as Identified by Production of a Variety of Bioactive Polypeptides.

Four major kallikreins (mK1, mK22, mK9, and mK13) were identified in the mouse submandibular gland (SMG). mK1, a true tissue kallikrein, was used as a protein marker to identify different types of SMG granular convoluted tubule (GCT) cells along with epidermal growth factor (EGF), nerve growth factor (NGF), and renin. Kallikrein mK1 was localized in a very small number (~5%) of GCT cells, which were scattered throughout the GCT, indicating that the majority of GCT cells are mK1-negative. Among mK1-positive cells, particularly strong signals were observed in a small number of narrow cells, recognized as slender granular cells (SG cells, Type IV), in the GCT. After postnatal development of the SMG, GCT cells are no longer uniform based on the bioactive substances (mK1, EGF, NGF, and renin) that they produce and secrete. GCT cells were classified into four subtypes, Types I-IV, and it became clear that these subtypes are complicatedly and reversibly converted by the endocrine hormones 5α-dihydrotestosterone (DHT) and triiodothyronine (T3). Duct segments with similar morphology or hormone dependency were recognized in the sublingual and parotid glands. The presence of duct cells with such characteristics is therefore a common feature of the three major salivary glands of rodents.

Expression of a mammalian aquaporin 3 homolog in the anterior pituitary gonadotrophs of the tree frog, Hyla japonica.

Aquaporins (AQPs) are a family of water channel proteins that play a major role in maintaining water homeostasis in various organisms. Several AQPs have been identified in the tree frog, Hyla japonica. Of these, AQP-h3BL, which is expressed in the basolateral membrane of the epithelial cells, is a homolog of mammalian AQP3. Using immunohistochemistry and in situ RT-PCR, we have demonstrated that AQP-h3BL is expressed in the anterior pituitary gonadotrophs of the tree frog but not in the other hormone-producing cells of the anterior pituitary. In gonadotrophs labeled for luteinizing hormone subunit-ß (LHß), AQP-h3BL protein was found to reside in the plasma membrane, the nuclear membrane and the cytoplasm. Double-labeling of AQP-h3BL mRNA and LHß protein revealed that AQP-h3BL mRNA is expressed in the gonadotrophs. Following stimulation by gonadotropin-releasing hormone (GnRH), the label for AQP-h3BL localized in the plasma membrane became more intense, concomitant with the transport of LHß-positive materials to the plasma membrane. These developments coincided with a decrease in the labeling density in the cytoplasm and near the nuclear membrane, suggesting that the latter localizations may function as "storage area" for AQP-h3BL. Immunoelectron microscopy also confirmed these localizations of AQP-h3BL protein. Based on these results, we suggest that AQP-h3BL protein in the frog gonadotrophs is involved in the formation of secretory granules, the swelling and increase in the volume of the granules and exocytosis.

Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland.

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5alpha-dehydrotestosterone (DHT) and triiodothyronine (T(3)), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T(3), and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.

Hormone-induced granular convoluted tubule-like cells in mouse parotid gland.

Most striated duct (SD) cells in the adult mouse parotid gland (PAG) have a few small secretory granules. These granules, however, are usually too small and sparse to be detected using light microscopy. Our serial studies have suggested that these PAG SD cells belong to a group of hormone-responsive granular duct cells, similar to the granular convoluted tubule (GCT) cells found in the submandibular gland. These studies also indicate that and some PAG SD cells may be capable of developing a granular cell phenotype under supraphysiological conditions of androgenic and thyroid hormones, leading to more abundant, and more kinds of GCT-specific secretory polypeptides. Here, the cytology of hormone-modulated SD cells, the immunocytochemistry of their secretory products, and their secretory responses to some autonomic agents are reviewed. Finally, the close similarity of the duct systems of the three major salivary glands in mice is critically emphasized.

Hypophysectomy and hormonal therapy modulate mK1-immunoreactive duct cells in the mice sublingual glands.

The immunocytochemical localization of a true tissue kallikrein, mK1, in mouse sublingual glands (SLGs) was examined following hypophysectomy and hormonal replacement therapy. In the glands of intact mice (14 weeks of age), mK1 was detected in the striated ducts (SDs). Full-fledged granular cells were scattered in the SDs of male mice (but not in those of female mice), showing a cellular mosaic distribution of mK1 with some being positive and others being negative. mK1 was also detected in transitional-type granular cells, though the secretory granules were too small and scarce to be visible by a light microscopy. Hypophysectomy in male mice resulted in the atrophy and loss of secretory granules in many SD cells. Granulation recovered after the repeated injection of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-L: thyronine (T3), and dexamethasone (Dex), given either alone or in combination to the hypophysectomized mice. The concomitant injection of DHT and T3, with or without Dex, resulted in the reappearance of the full-fledged granular cells, only some of which were mK1-positive. Electron microscopy revealed mK1 to be present exclusively in the secretory granules of these mK1-positive cells, and no ultrastructural differences were observed between mK1-positive and mK1-negative full-fledged granular cells. These results show that the differentiation of the granular cell phenotype in the mouse SLG duct system requires the concomitant action of androgen and thyroid hormone and retards mK1 synthesis.

Strain-specific and endocrine control of granular convoluted tubule cells and epidermal growth factor expression in the mouse submandibular gland.

Submandibular glands (SMGs) of 11-week-old mice from four strains, ICR, C57BL/6J, BALB/c, and C3H/HeN were examined by immunohistochemistry for epidermal growth factor (EGF). In addition to sex-related differences in granular convoluted tubules (GCTs), the GCT cells were significantly larger in ICR mice than in other three strains. In males from each of the strains, almost all the GCT cells were strongly positive for EGF. The EGF-positive cells in the females, however, were markedly fewer in number, and were stained weaker in C57BL/6J, BALB/c, and C3H/HeN mice than in ICR mice. The GCT cells and their EGF expression in the F1 progeny from ICR and C3H/HeN strains were approximately intermediate between those of the parent strains of the same sex. T(3) and/or dihydrotestosterone (DHT) enhanced the GCT phenotype in the C3H/HeN mice, and remarkably increased the EGF-positive cells in females. Electron microscopy revealed that gold-labeling of EGF was confined to the secretory granules, and that the GCT cells in females, given T(3) + DHT, had a well-developed Golgi apparatus and net-like RER but few basal infoldings, whereas the equivalent cells in the untreated females had poor RER and prominent basal infoldings. These results suggest that the EGF concentration in SMGs is genetically high in ICR mice and low in other strain mice and that, considering the same response of GCT cells to T(3) and/or DHT between the high and low EGF strains, the low EGF concentrations might be partly caused by a lesser sensitivity of the GCT cells to thyroid hormones.

Repeated androgen and thyroid hormone injection modulates the morphology of hormone-responsive duct cells in the mouse parotid gland.

When the parotid glands of normal male and female ICR mice (12 weeks of age) were examined under a light microscope, no granular cells were seen in the duct system. However, transmission electron microscopy revealed that, in both sexes, many striated duct cells contained a few electron-dense secretory granules in their subluminal cytoplasm and had formed so-called granular striated tubules (GSTs) in some of the striated duct segments. These secretory granules were not large enough to be visible with a light microscope. Fully fledged granular cells, containing large secretory granules visible with a light microscope, could be induced in the GST segments of the glands of males by injection with 5alpha-dihydrotestosterone (DHT), triiodothyronine (T(3)), and dexamethasone (Dex), given alone or in combination every other day for 2 weeks. Dex alone showed no effect on the GSTs in this study. Both DHT and T(3), either individually or with Dex, were moderately effective, inducing a few scattered fully fledged granular cells. A stronger effect was detected after concomitant injection of DHT and T(3), with or without Dex, with more abundant fully developed granular cells appearing in the GST segments. Electron microscopy revealed that these granular cells had abundant large secretory granules in their apical two-thirds, a basal nucleus, and modest basal infoldings. By contrast, the effect of the same hormones was very weak in the glands of females, and even the concomitant injection of DHT and T(3), with or without Dex, rarely induced fully fledged granular cells. These results indicate a close similarity between the ductal systems of the major salivary glands of the mouse, in terms of some of the striated duct segments containing secretory granules, being under the same multihormonal regulation, and being sexually dimorphic.

Role of thyroid hormone in the initiation of EGF (epidermal growth factor) expression in the sublingual gland of the postnatal mouse.

The effect of triiodo-L-thyronine (T3) and propylthiouracil (PTU) on the initiation of epidermal growth factor (EGF) expression in the sublingual glands (SLGs) of postnatal mice was investigated by indirect enzyme-labeled and immunogold antibody methods for light and electron microscopy, respectively. In normal males, EGF immunoreactivity first appeared in a few scattered granular cells of striated ducts (SDs) at 5 weeks of age, and the immunoreactive cells had increased in number at 6 weeks of age. No EGF expression was observed in the glands of females at any ages examined. When T3 (1 mg/kg body weight) was given to males every other day for 2 weeks before examination, EGF expression began earlier; the immunoreactive granular cells were first detected at 4 weeks of age, and at later ages they were markedly increased in number compared to those of normal males. Moreover, T3 was capable of inducing EGF in the female glands. After T3 was administered to females in the same manner as in males, a few immunoreactive cells were first detected at 5 weeks of age, and increased numbers were detected at later ages. By contrast, when PTU (1 mg/kg body weight) was given to male mice every other day for 2 weeks before examination, the EGF-immunoreactive cells were markedly decreased in number compared to those of normal males of the same age. Electron microscopy revealed that many SD cells contained secretory granules, and that these cells constituted the granular striated tubule (GST) in a portion of SDs, but they were undetectable by light microscopy, because their secretory granules were minimal in size and few in number. Gold-labeling of EGF was confined to the secretory granules of scattered granular cells, whose secretory granules were far larger in size and more abundant than those of the GST cells. These results suggest that thyroid hormone is essential to differentiation of the cellular phenotype of GST precursor cells into typical granular cells (detectable by light microscopy) that express EGF in the mouse SLG, showing a close resemblance to the submandibular granular convoluted tubule cells.

Subcellular redistribution of AQP5 by vasoactive intestinal polypeptide in the Brunner's gland of the rat duodenum.

Aquaporin (AQP)5, an exocrine-type water channel, was detected in the rat duodenum by Western blot analysis, and was localized by immunohistochemistry in the secretory granule membranes as well as in the apical and lateral aspects of the plasma membrane of Brunner's gland cells. Incubation of duodenal slices with vasoactive intestinal polypeptide (VIP) in vitro significantly increased the amount of AQP5 in the apical membrane fraction in a dose- and time-dependent manner with the amount reaching a plateau at 100 nM VIP and becoming near maximal after a 30-s incubation. Protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 50 muM), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; PKA-specific, 1 muM) blocked this increase, but PKC-specific inhibitor calphostin C did not, implying the involvement of PKA but not PKC in this cellular event. Intravenous injection with VIP (40 mug/kg body wt) provoked dilation of the lumen of the Brunner's gland at 2 and 7 min and increased the staining intensity of AQP5 in the apical and lateral membranes. AQP1 (both nonglycosylated and glycosylated forms) was also found to localize in the apical and basolateral membranes of cells of Brunner's gland. VIP, however, did not provoke any significant change in the AQP1 level in the apical membrane, as judged from the results of the above in vitro and in vivo experiments. These results suggest that VIP induced the exocytosis of granule contents and simultaneously caused translocation of AQP5 but not of AQP1 to the apical membrane in Brunner's gland cells.

Additive and/or synergistic action (downregulation) of androgens and thyroid hormones on the cellular distribution and localization of a true tissue kallikrein, mK1, in the mouse submandibular gland.

We investigated the effects of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-l-thyronine (T(3)), and dexamethasone (Dex) on the expression of mK1 in the granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of hypophysectomized (Hypox) male mice by indirect enzyme-labeled antibody and immunogold antibody methods for light and electron microscopy. Hypox resulted in considerable atrophy of the GCT cells, which were always immunoreactive for mK1, and the cells were characterized by apical small dense secretory granules labeled with gold particles suggesting the presence of mK1, small Golgi apparatus, sparse rough endoplasmic reticulum (RER), and developed basal infoldings. Each of the hormones, DHT, T(3), and Dex, enhanced the GCT phenotype to various degrees in Hypox male mice. Both DHT alone and T(3) alone moderately inhibited mK1 synthesis by increasing the number of mK1-immunonegative GCT cells in Hypox males, but Dex alone had no inhibitory effect on mK1 synthesis. A significant trophic effect on GCT cells was induced by combined injection of DHT and T(3) or of all three hormones, and was reflected in the appearance of abundant large secretory granules, well-developed Golgi apparatus and RER, and reduced basal infoldings. Only a few such GCT cells were immunopositive for mK1, and the pattern of immunopositive and immunonegative cells very closely resembled the mosaic pattern seen in normal male GCTs. These findings suggested that the sexual dimorphism of mK1 expression and the morphological appearance of GCT cells can be induced by treatment with two hormones, DHT and T(3), but not by either of them alone. T(3) appears to have a permissive effect on committed GCT cells that results in downregulation of mK1 expression in these cells.

Immunocytochemical localization of mK1, a true tissue kallikrein, in the mouse parotid gland: sexual dimorphism and effects of castration and hypophysectomy.

In the normal parotid glands of mice at 12 weeks of age, mK1, a true tissue kallikrein, was detected at the apical rim of the striated ducts (SDs). Sexual dimorphism in the immunostaining intensity in parotid glands was seen, i.e., immunostaining was more intense in males than in females. Under electron microscopy, secretory granules, being small in size, and condensed at the subluminal cytoplasm, were labeled with immunogold particles showing the presence of mK1. These secretory granules were rather abundant and large in males. Castration in males reduced the immunoreactivity of mK1 in the SD cells because of a decrease in the number and size of secretory granules as revealed by electron microscopy. Hypophysectomy in male mice resulted in considerable loss of immunoreactivity for mK1, which was characterized under electron microscopy by complete disappearance or significant reduction of secretory granules in many SD cells. These results suggest that mK1 expression in the SD cells of murine parotid glands is regulated by pituitary-dependent hormones, and sexual dimorphism of mK1 expression is regulated by androgens.

Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice.

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo- l-thyronine (T3) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T3 (1 mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T3-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.

Immunocytochemical localization of prohormone convertases PC1 and PC2 in the mouse thyroid gland and respiratory tract.

We examined immunocytochemical localization of the prohormone convertases, PC1 and PC2, in the thyroid gland and respiratory tract of the adult mouse using the indirect enzyme- and immunogold-labeled antibody methods for light and electron microscopy, respectively. In the thyroid gland, PC1- and/or PC2-immunoreactive cells were cuboidal, scattered in the follicular epithelium and in the interfollicular spaces. When serial sections were immunostained with anti-calcitonin, anti-PC1, anti-calcitonin-gene-related-peptide (CGRP), and anti-PC2 sera, respectively, localization of both PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells. In the respiratory tract, only PC1 immunoreactivity was observed in the basal granulated neuroendocrine cells, which were scattered in the tracheal epithelium. Consecutive sections immunostained with anti-PC1 and anti-CGRP sera showed that a subpopulation of these PC1-immunoreactive cells contained CGRP. Double immunogold electron microscopy of the thyroid parafollicular cells revealed that calcitonin- and/or CGRP-immunopositive secretory granules were also labeled with both PC1 and PC2. These findings suggest that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.

Morphologic changes in the granular convoluted tubule cells of the mouse submandibular gland following hypophysectomy and hormonal replacement.

Morphological changes in the granular convoluted tubule (GCT) cells of the male mouse submandibular gland (SMG) were examined following hypophysectomy and hormonal replacement. Semithin sections stained with Heidenhain's iron hematoxylin showed that hypophysectomy severely regressed the GCT phenotype. Although only a few dispersed cells containing secretory granules were observed in the GCT segments under a light microscope, electron microscopy revealed that many regressed cells continued to constitutively elaborate apical secretory granules (although they were very small) and contained a euchromatic nucleus at the center of the cell, poor rough endoplasmic reticulum (RER) and Golgi apparatus in the perinuclear region, and well-developed basal infoldings. These findings suggest that hypophysectomy resulted in atrophy of GCT cells, but that they retained evidence of being secretory cells. 5 alpha-Dihydrotestosterone (DHT); 3,5,3'-triiodo- l-thyronine (T(3)); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T(3) in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T(3) alone induced a normal female-like GCT phenotype, with considerably abundant secretory granules and the usual short basal infoldings, in hypophysectomized male glands. Furthermore, Dex was found to synergistically enlarge secretory granules when administered with T(3) and/or DHT, although it was only weakly effective in enhancing the GCT phenotype when used alone. Taken together, the above findings confirmed that the GCT phenotype of the mouse SMG is regulated by the synergistic action of pituitary-dependent hormones.

Fine structures on the surface of nuptial pads of male hylid and rhacophorid frogs.

Nuptial pads, secondary sexual characteristics of male frogs, develop on the first digit of the hand of Hyla japonica in the family Hylidae and of Rhacophorus schlegelii in the family Rhacophoridae, and on both the first and second digits of the rhacophorids Buergeria buergeri and B. japonica. By scanning electron microscopy (SEM), it was seen that numerous mounds covered the surface of the pads. Mounds were similarly hemispherical in R. schlegelii and B. buergeri and flat in B. japonica. The mounds of H. japonica were irregular in shape and size and some of them stood in rows. Fine columnar protuberances were present all over the surfaces of the mounds in this species. Numerous peg-like protuberances projected radially upward from the top of the mounds in B. buergeri and B. japonica. Irregular-shaped, leaf-like protuberances bearing knobby or rod-shaped apical protrusions were present on the top of the mounds in R. schlegelii. In pads observed by transmission electron microscopy, the outermost epithelial monolayer of the mounds was generally thick, especially at the top, compared to that of the rest of the skin. Epidermal cells in this layer were well keratinized, devoid of organelles, and contained closely packed, fine filaments within a dense matrix. Apical accessory protuberances projecting from the outermost cells were also packed with dense filamentous materials, showing rigid comb-like structures. © 1994 Wiley-Liss, Inc.

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog, Xenopus laevis.

The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.

Denervation Effects on Limb Regeneration in Postmetamorphic Xenopus laevis: (regeneration/denervation/Xenopus/limb).

The effects of denervation on limb regeneration of postmetamorphic Xenopus laevis in the early to late stages of regeneration were studied. Limbs that were denervated immediately after amputation did not show any signs of regeneration. Moreover regenerating limbs denervated 20, 30, 40 and 60 days after amputation showed significant regression of regenerates. After this regression of regenerates, the regenerative capacity was not restored during a long observation period. Denervation reduced both protein synthesis (3 H leucine autoradiography) and mitotic activity of regenerating limbs even in later stages of regeneration. These results on the role of the nerve in limb regeneration of Xenopus were compared with results in Xenopus and urodeles reported previously.