Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

Seasonal lectin binding variations of thumb pad in the frog (Pelophylax ridibundus)

The thumb pad is one of the most common secondary sexual characteristics in frogs. Although it is known that amphibian skin has affinity for several lectins, there is no report regarding lectin‐binding affinity of the thumb pad or its structural components. This study investigated localization and seasonal variation of specific carbohydrate moieties of glycoconjugates in both the epidermal and dermal components of the frog thumb pad at the light microscopic level using lectin histochemistry. The study consisted of four seasonal groups of the frog species, Pelophylax ridibundus (Synonym of Rana ridibunda): active, prehibernating, hibernating and posthibernating. Four horseradish peroxidase conjugated lectins were employed. It was found that dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and ulex europaeus (UEAI) gave positive reactions in both epidermal layers and breeding glands. These three lectins bound specific secretory cells in the breeding glands, and the distribution of the cells and epithelial lectin reactions exhibited seasonal changes. In addition, UEA‐I and peanut agglutinin (PNA) showed an affinity in granular glands and the granular zone of mixed glands. Generally, epidermal lectin binding showed dense affinity during the posthibernation period. DBA, UEA‐I, and WGA‐specific cells in the mucous gland decreased gradually until the posthibernation period. These findings suggest that differences of lectin binding in the thumb pad may be related to functional activities and, thus, seasonal adaptations. Moreover, the presence of specific lectin‐binding cells in the breeding glands indicated that they consisted of heterogeneous secretory cell composition or that the cells were at different secretory stages. J. Morphol. 275:76–86, 2014. © 2013 Wiley Periodicals, Inc.     

Comparison of K(+)-p-nitrophenyl phosphatase activity in the urinary bladder of the frog Rana esculenta during hibernation and active life.

We have studied effects of hibernation on the frog urinary bladder, an organ involved in water and ion transepithelial transport and taking part in osmoregulation. We have demonstrated K(+)-p-nitrophenyl phosphatase activity (an enzyme involved in ion and water transport) both in active and hibernating frogs. Most of the reaction product deposition was found on basolateral membranes of granular cells of the urinary bladder epithelium during all seasons. Therefore, it seems likely that this organ, unlike organs studied previously (skin, kidney and lung), maintains its function in the osmoregulatory process during hibernation.     

Localisation by immunofluorescence of thyrotropin-releasing hormone in the cutaneous glands of the frog, Rana ridibunda.

Using the technique of indirect immunofluorescence, thyrotropin-releasing hormone (TRH) has been localised in certain cells of the cutaneous glands of the frog, $\text{Rana ridibunda}$. No specific fluorescence was found in other cells in the dermis, nor were any positive cells found in the epidermis. Previous studies have shown by radioimmunoassay that large amounts of TRH are present in frog skin, particularly in the dorsal region. Since dorsal frog skin contains many more glands than its ventral counterpart, this non-homogeneous distribution of TRH can be explained by its presence in a population of cells found in one type of cutaneous gland.     

Nuclear changes and morphology of the epidermis in the hibernating frog

Cytochemical changes of chromatin and DNA in frog epidermal cells were correlated with some morphological features to investigate the skin physiology during hibernation in comparison with the active period. The epidermal cells of hibernating frogs showed less condensed chromatin in all the layers; a greater loss of DNA was found during the transition from the middle to the superficial layer. In the germinative layer, a lesser frequency of hyperdiploid cells and a remarkably low amount of mitoses were detected; this is accompanied by the increase of epidermal thickness and the presence of two layers of cornified cells. The slowing of tissue differentiation and cell renewal kinetics during hibernation can be related to lowered activity of the frog skin. Further, the smaller intercellular spaces as well as the scarcity of puffed ER and vacuoles may be indicative of a lower ion transport in epidermal cells during hibernation.     

Immunocytochemical localization of serotonin-like immunoreactivities in the ciliated epithelium of the frog palatine mucosa

Immunocytochemical localization of serotonin (5-HT)-like immunoreactivities was studies in the ciliated epithelium of the frog palatine mucosa by the peroxidase anti-peroxidase (PAP) method. 5-HT-like immunoreactivity was found only in the small granular vesicles (100-150 nm in diameter) and not in any mature large secretory granules or in other cell organellae in the goblet cells. No 5-HT-like immunoreactivities were found in any other epithelial and secretory cells in the palatine epithelium. It appears therefore that 5-HT-like immunoreactive granular vesicles have certain physiological effects on the ciliary movement of the ciliated cells or in the goblet cells.     

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region although stained strongly by HRP-SBA, did not react with HRP-UEA 1 indicating that few, if any, fucose residues were present in the ventral epidermis. These findings, as suggested in the discussion, indicate that different glycoconjugate patterns in dorsal and ventral skin may be associated with the regulation of water balance in the frog.     

Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae)

The granular glands of nine species of dendrobatid frogs were examined using light and electron microscopy. The glands are surrounded by a discontinuous layer of smooth muscle cells. Within the glands proper the secretory cells form a true syncytium. Multiple flattened nuclei lie at the periphery of the gland. The peripheral cytoplasm also contains mitochondria, rough surfaced endoplasmic reticulum, the Golgi apparatus, and an abundance of smooth endoplasmic reticulum. Centrally, most of the gland is filled with membrane-bound granules surrounded by amorphous cytoplasm. Few other organelles are found in this region. Early in the secretory cycle, the central part of the gland is filled with flocculent material which appears to be progressively partitioned off by membranes to form the droplet anlage. As granules form, the structure of the contents becomes progressively more vesicular. Dense vesicles, which bud off from the Golgi apparatus, fuse with the granular membrane during the development of granules, and might contain enzymes involved in toxin synthesis. The granules at this point resemble multivesicular bodies. Their structure is similar in all species of dendrobatid frogs even though the different frogs secrete substances of different chemical structure and toxicity.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

Integumentary structure and its relationship to wiping behaviour in the common Indian tree frog, Polypedates maculatus

Skin of the Indian tree frog, Polypedates maculatus (Rhacophoridae), was studied in the context of self-wiping behaviour which functions to expel and distribute cutaneous secretions recently shown to retard evaporative water loss. The secretions contain both mucus and lipids and are derived from a common gland considered to be homologous with characteristic anuran mucous glands. The glands are bipotent and secrete both mucus and lipoid products which are evidently mixed within the glandular lumen. Another type of gland resembling characteristic anuran serous (or granular) glands is found in dorsal but not ventral skin, whereas the lipid-secreting mucous glands are found in skin associated with all body surfaces. There is no distinct, lipid-secreting gland present in the skin of this species other than the mucous glands. These histochemical data complement the earlier finding that resistance to evaporative water loss in this species is relatively small compared with phyllomedusine 'waterproof frogs which also exhibit wiping behaviour associated with secretion of lipids. Thus, wiping behaviour may have evolved in association with mucous secretions before dominant lipoid secretions resulted from strong selection for water conservation.     

Co-existence of enkephalin and adrenalin in the frog adrenal gland

Summary By means of immunohistochemistry combined with formaldehyde-induced fluorescence microscopy, metenkephalin-like immunoreactivity was found to occur in the adrenalin-cells of frog adrenal glands. Immunoelectron microscopy demonstrated that the immunoreactive material is confined to the secretory granules. These findings suggest the concomitant release of enkephalin and adrenalin by exocytosis.     

Immunohistologic studies of the distribution of proliferative compartments in the appendages of adult human skin

Both, calmodulin (CaM) as well as the antigen Ki67 show a close relationship to cell proliferation. By means of specific antibodies against them, it has become possible to study the spatial distribution of proliferative compartments in tissues. We performed an indirect immunofluorescence study on unfixed frozen sections of human adult skin to gain more informations about the spatial distribution of immunoreactive CaM and Ki67 in skin appendages, i.e. anagen hair follicle, sebaceous and eccrine sweat gland. Two major patterns of immunoreactivity were seen: Type (1) or epidermis-like, which was present in the interfollicular epidermis and the pilosebaceous unit. Type (2) or sweat gland type, which was seen in eccrine sweat glands. Both types disclosed significant differences in the relative number of proliferative cells in S-phase, which might be a consequence of a quiet different tissue architecture. Furthermore, myoepithelial cells of secretory coils were likely to represent mainly SQ-cells. Their immunoreactivity in human skin was quiet different from other parts of eccrine sweat glands suggesting another ontogenetic pathway.     

The nerves in frog skin

In the epidermis of frog skin, most nerves are situated at the top of the basal layer. More superficial nerve fibres are usually adjacent to flask cells; it is concluded that this is not a functional association, but a consequence of the pattern of moulting. There are nerve fibres in the walls of the granular glands; mucous glands appear to have no intrinsic innervation although nerves pass within a short distance of their walls. The smooth muscle bundles of the dermis are innervated, and have a physical attachment to the overlying epidermis.     

Localization of 28-kDa peroxiredoxin in rat epithelial tissues and its antioxidant properties

Peroxiredoxins are a novel family of antioxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. The localization of a member of the mono-cystein subfamily of peroxiredoxins, the 28-kDa protein, in different rat tissues and its antioxidant properties were investigated. By immunoblotting, the 28-kDa peroxiredoxin was found to be most highly concentrated in olfactory epithelium and present in all tissues tested (skin, lung, trachea, kidney, womb, and brain). Immunostaining with rabbit polyclonal antibody raised against the 28-kDa peroxiredoxin revealed the particularly high level of the 28-kDa peroxiredoxin immunoreactivity in air-contacting areas (apical regions and mucus of the olfactory and respiratory epithelium and skin epidermis), which are continually exposed to numerous air-borne reactive oxygen species. In the apical regions of the olfactory and respiratory epithelium, the 28-kDa-peroxiredoxin immunogold labeling outlined microvilli and cilia and was mainly located in sustentacular cells and in respiratory and goblet cells, as electron-microscopic analysis revealed. In skin epidermis, the 28-kDa peroxiredoxin immunoreactivity was confined to the granular layer and specifically concentrated in sebaceous glands of hair follicle. In situ hybridization with 33P-labeled antisense RNA probe revealed the expression of the 28-kDa peroxiredoxin mRNA in tissues with a high level of the 28-kDa peroxiredoxin immunoreactivity. Immunodepletion of the 28-kDa peroxiredoxin profoundly decreased the antioxidant activity of the olfactory tissue extract.     

Nerve Stimulation and Electrical Properties of Frog Skin

The suitability of frog skin glands as a model for the study of secretory mechanisms in exocrine glands was explored. Periodic voltage clamp was used to determine continually the short-circuit current, chord conductance, and electromotive force of frog skin during neural and pharmacological activation of the skin glands. Both the chord conductance and the short-circuit current increased with glandular activation; the temporal dissociation of these increases suggests that there are at least two separate components to the secretory response. The sensitivity of the secretory electrical changes to changes in the ionic composition of the bathing solutions supports the notion of electrogenic chloride active transport as being basic to the activity of the exocrine glands.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig

Summary Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1–2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.     

Distribution and characterization of endozepine-like immunoreactivity in the central nervous system of the frog Rana ridibunda.

The localization of endozepine-like immunoreactivity in the brain of the frog Rana ridibunda was investigated by indirect immunofluorescence, using an antiserum against synthetic rat octadecaneuropeptide (ODN). A specific immunoreaction was detected in ependymal cells lining the ventricular system of the brain and in circumventricular organs. Numerous immunoreactive cells were found covering the walls of the lateral ventricles in the telencephalon, as well as in the diencephalic and mesencephalic ventricles. In the hypothalamus, both the preoptic nucleus and the infundibular region showed numerous immunopositive cells. Ependymal cells lining the rhomboencephalic fourth ventricle and the central canal of the spinal cord were also immunoreactive. The concentration of endozepine-like immunoreactivity was measured in various regions of the brain using a sensitive and specific radioimmunoassay for rat ODN. The highest levels of ODN-like immunoreactivity were found in the infundibulum, cerebellum and preoptic area. Reverse phase high performance liquid chromatography and radioimmunoassay quantification were used to characterize endozepines in the frog brain. The elution profiles of the different brain regions revealed four major immunoreactive peaks. The present results demonstrate the presence of peptides immunologically related to the endozepine family in the central nervous system of the frog. The localization of immunoreactive endozepines in ependymal cells suggests that these peptides play important neuromodulatory functions in the amphibian brain.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.