Neurotrophins and Trk receptors in the developing and adult ovary of Coturnix coturnix japonica.

NGF, BDNF, NT-3 and their specific receptors TrkA, TrkB and TrkC are known to be involved in the development and maintenance of vertebrates' nervous system. However, these molecules play a role also in non-neuronal tissue, such as in the reproductive system. In this study we investigated the presence and localization of neurotrophins and Trk receptors to unravel their potential role in the developing and adult ovary of Japanese quail, a model species well suited for reproduction studies. Western blotting analysis on ovaries of three month old quails in the period of egg laying showed the presence of pro and mature forms of neurotrophins and splice variants of Trk receptors. Immunohistochemical investigation reported that in embryonic ovaries from the 9th day of incubation to the hatching NGF and NT-3 were observed in the cortical and medullar areas respectively, whereas Trk receptors were observed in both areas. In adult ovary, all NTs were detected in glandular stromal cells, NGF and NT-3 also in the nervous component. Regarding follicle components, NGF and BDNF were observed in oocytes and follicular cells. All TrK receptors were present in nervous components and only TrkA in glandular stromal cells. In follicles, TrkA was present in oocyte cytoplasm and TrkB in theca cells. The results suggest an involvement of the neurotrophin system in the quail ovary physiology, promoting the oocyte development and follicular organization in the embryo, as well as oocyte and follicular maturation in adults.

Expression and distribution of leptin and its receptors in the digestive tract of DIO (diet-induced obese) zebrafish.

The expression and localization of leptin (A and B) and its receptor family in control and diet-induced obese (DIO) adult male zebrafish gut, after 5-weeks overfeeding, administering Artemia nauplii, as fat-rich food, were investigated. Recently, the obese adult zebrafish was considered an experimental model with pathophysiological pathways similar to mammalian obesity. Currently, there are no reports about leptin in fish obesity, or in a state of altered energy balance. By qRT-PCR, leptin A and leptin B expression levels were significantly higher in DIO zebrafish gut than in the control group (CTRL), and the lowest levels of leptin receptor mRNA appeared in DIO zebrafish gut. The presence of leptin and its receptor proteins in the intestinal tract was detected by western blot analysis in both control and DIO zebrafish. By single immunohistochemical staining, leptin and leptin receptor immunoreactive endocrine cells were identified in the intestinal tract either in DIO or control zebrafish. Moreover, leptin immunopositive enteric nervous system elements were observed in both groups. By double immunohistochemical staining, leptin and its receptor were colocalized especially in DIO zebrafish. Thus, our study represents a starting point in the investigation of a possible involvement of leptin in control of energy homeostasis in control and DIO zebrafish.

WITHDRAWN: Neurotrophins and specific receptors in the oviduct tracts of Japanese quail (Coturnix coturnix japonica).

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Ann. Anat., 210 (2017) 112 ­ 120>, http://dx.doi.org/10.1016/j.aanat.2016.04.034. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Neurotrophins and specific receptors in the oviduct tracts of Japanese quail (Coturnix coturnix japonica).

Neurotrophins (NGF, BDNF and NT-3) and their specific receptors (TrkA, TrkB and TrkC) were studied in the oviduct of egg laying quails. Neurotrophins (NTs) are mainly involved in the development and maintenance of neuronal populations in the central and peripheral nervous system, but also in reproductive system. In this survey, we first studied the morphological organization of the quail oviduct, distinguished in infundibulum, magnum, isthmus, uterus and vagina, and then we analyzed the expression and localization of NTs and Trks receptors in the whole tracts. By western blotting we detected that the investigated NTs and Trks receptors are expressed in all oviductal tracts. By immunohistochemistry we were able to define the distribution of NTs and Trks. Specifically, NGF, BDNF and NT3 were localized in lining and ductal epithelial cells, and NGF was also detected in secretory cells of tubular glands and in nervous fibers of vessel wall. TrkA and TrkB were present in the lining and ductal epithelium; TrkA and TrkC were present in nervous fibers of vessel wall in all oviductal tracts. Furthermore, we also observed NGF and BDNF co-localized with TrkA and TrkB in cells of the lining and ductal epithelium, suggesting an autocrine mechanism of action.

GDNF and GFRα co-receptor family in the developing feline gut.

Glial cell-line derived neurotrophic factor (GDNF) and the GFRα co-receptors play a role in the developing enteric nervous system. The co-receptors elicit their action by binding receptor tyrosine kinase RET. This immunohistochemical study reports the presence of GDNF and its specific co-receptor GFRα1 in the cat gastrointestinal apparatus during development, from stage 9 to 22. At stage 9 and 11, immunoreactivity (IR) to GDNF was observed in the cells of mesenchyme of the anterior gut. From stage 14 to 22, GDNF IR was detected in nervous plexuses; moreover, GDNF and GFRα1 IR appeared localized in gastrointestinal endocrine cells. The presence of GDNF in the enteric nervous system and in the endocrine cells suggests an involvement of this neurotrophic factor in the gastrointestinal development. Moreover, the presence of the co-receptor GFRα1 in endocrine cells and its absence in the enteric nervous system seems to indicate a different mode of transduction of GDNF signal. GFRα2 and GFRα3 co-receptors were not detected.

Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle.

Calpains are Ca(2+)-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post-mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post-mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

Reticular groove of the domestic ruminants: histochemical and immunocytochemical study.

The reticular groove mucosa of adult cattle, buffalo and sheep was investigated by histochemical and immunocytochemical techniques. Intense NADPH-d staining was observed in the folds of the epithelium mucosa and at the bottom of the reticular groove in all domestic ruminants studied. The NADPH-d staining showed that the innervations of the tunica muscularis of the reticular groove lip were composed of nerve corpuscles, nerve fibres and nerve cells of the mucosa epithelium. SEM analysis showed an intense nitric oxide synthase (NOS) I immunoreactivity in deep and medium cellular layers. It is interesting to note that the same morphologies were observed in samples of the mucosa epithelium, and of the tunica muscularis processed by NADPH-d and in those processed by immunogold techniques. This study has demonstrated that nitric oxide (NO) is involved in the rumination activity and that it plays a double role in this activity in the reticular groove of all domestic ruminants studied: (1) NO plays a role similar to the one it has in the mucosa epithelium of all the other compartments of the ruminant forestomach, (2) The lip sections of the reticular groove has shown abundant innervations that may indirectly coordinate and control the forestomach motility through the direct activation of the nitrergic (nitroxidergic) nerve cells and nerve fibres.

Structural, histochemical and immunocytochemical study of the forestomach mucosa in domestic ruminants.

The forestomach plays an important role in the digestion physiology of ruminants. The aim of this study is to clarify the morpho-functional role of the mucosa in each of the three compartments of the forestomach in three domestic ruminants species, viz cattle, buffalo and sheep, by means of structural, histochemical and immunocytochemical methods, including transmission electron microscopy, light microscopy and scanning electron microscopy. These methods were chosen to demonstrate the indirect evidence for the presence of nitric oxide (NO) employing NADPHd and nitric oxide synthase I (NOS I). The various cell layers of the forestomach epithelium are described and illustrated in detail. An intense NADPHd staining was observed in the granulosa, spinosa and basal layers of the epithelium, in particular in the cytoplasm over the nucleus. NOS I immunoreactivity was found in all specimens of the forestomach mucosa. The results of this study might reflect a possible role of NO in delaying the onset of cellular apoptosis in the forestomach mucosa of the domestic ruminants, by playing a role in the production of cell energy.

Cellular localization of GDNF and its GFRalpha1/RET receptor complex in the developing pancreas of cat.

Glial cell line-derived neurotrophic factor (GDNF) acts through RET receptor tyrosine kinase and its co-receptor GFRalpha1. In an effort to better understand the possible biological contribution of the GDNF and GFRalpha1/RET complex in pancreatic development, in this study we report the cellular localization of these proteins in the pancreas of domestic cat embryos and fetuses by immunocytochemical methods. In early embryos, GDNF, GFRalpha and RET immunoreactivity (IR) was localized in closely intermingled cells. GDNF and RET immunoreactive cells displayed chromogranin (an endocrine marker) and PGP 9.5 (a neuronal marker) IR, respectively. GFRalpha IR was present in both a few GDNF/chromogranin and RET/PGP 9.5 immunoreactive cells. In elderly fetuses, GDNF and GFRalpha IR were co-localized in glucagon cells and RET IR was detected in few neurons and never co-localized with GFRalpha or GDNF IR. In early embryos, the presence of GDNF IR in chromogranin immunoreactive cells and GFRalpha1/RET complex IR in PGP9.5 immunoreactive cells seems to suggest a paracrine action of GDNF contained in endocrine cell precursors on neuronal cell precursors expressing its receptor complex. The presence in different cell populations of RET and its co-receptor GFRalpha1 IR could be due to independent signaling of GRFalpha1. Thus, the co-presence of GDNF and GFRalpha1 in chromogranin and glucagon cells could lead to the hypothesis that GDNF can act in an autocrinal manner. In fetuses, RET IR was detected only in intrapancreatic ganglia. Because of the lack of GFRalpha1 IR in pancreatic innervation, RET receptor could be activated by other GFR alphas and ligands of GDNF family. In conclusion, these findings suggest that in differently aged embryos and fetuses the GDNF signal is differently mediated by RET and GFRalpha1.

Immunoreactivity to glial cell line-derived neurotrophic factor and its receptors in the trout pancreas: a further endocrine-exocrine relationship?

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting the survival of several neuronal populations in the central, peripheral and autonomous nervous system. Outside the nervous system, GDNF functions as a morphogen in kidney development and regulates spermatogonial differentiation. GDNF exerts its roles by binding to glial cell line-derived neurotrophic factor receptor (GFR) a1, which forms a heterotetramic complex with rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor. In this study we report the presence of GDNF-, RET- and GFRa1-like immunoreactivity in the pancreas of juvenile trout. GDNF immunoreactivity was observed in the islet cells, while GFRa1- and RET- immunoreactivity was observed in the exocrine portion. These findings suggest a paracrine role of GDNF towards exocrine cells showing GDNF receptors GFRa1 and RET. The relationship could reflect physiological interactions, as previously indicated in mammalian pancreas, and/or a trophic role by endocrine cells on exocrine parenchyma, which shows a conspicuous increase during animal growth.

Glial cell line-derived neurotrophic factor expression in the brain of adult zebrafish (Danio rerio).

In mammals, glial cell line-derived neurotrophic factor (GDNF) is a growth factor of many neuronal populations in the central, peripheral and autonomous nervous system. GDNF may also function as a morphogen during kidney development and may regulate spermatogonial differentiation. GDNF has been characterised in zebrafish embryos and was demonstrated experimentally to be critical for the development of the enteric nervous system. However, in adult zebrafish, no data exist regarding GDNF expression and localisation in the brain and in different organs. Thus, the aim of the present study was to investigate the expression of GDNF in the brain of adult zebrafish (Danio rerio). Transcripts of GDNF mRNA were observed in brain extracts by a standard RT-PCR. The presence of the protein in the brain homogenates was confirmed by SDS-PAGE electrophoresis and Western blotting analysis. Immunohistochemistry and in situ hybridization experiments showed that GDNF protein and mRNA were localised in various nuclei of the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata of the zebrafish brain. In conclusion, this study showed that the expression of GDNF was not restricted to developmental periods but it seems that this factor might be involved in adult zebrafish brain physiology, as observed in mammals.

GDNF family ligand immunoreactivity in the gut of teleostean fish.

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRalpha, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.

Artemin-like immunoreactivity in the zebrafish, Danio rerio.

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.

Neurotrophin and Trk receptor-like immunoreactivity in the frog gastrointestinal tract.

Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the neurotrophin family, which is involved in the differentiation, growth, repair, plasticity and maintenance of many neuronal populations. They act through three tyrosin-kinase (Trk) specific receptors: NGF bind to TrkA, BDNF to TrkB and NT3 to TrkC. Despite increasing evidence regarding the presence of neurotrophin and their receptors in many vertebrate species, in amphibians there are very few data concerning them. Thus, the aim of this study was to extend the investigation to the presence of both neurotrophins and their Trk receptors in the gut of an anuran amphibian, Rana temporaria. In the frog gut NT-3- like immunoreactivity (IR) was observed in both the nervous system and endocrine cells of the stomach and intestine, while NGF-like IR was observed only in the enteric nervous system, and BDNF-like IR in the intestinal endocrine cells. TrkA- and TrkB-like IR was detected in both neurons and endocrine cells of the intestine, while TrkC-like IR was observed only in intestinal neurons. No Trk IR was detected in the stomach. The occurrence of the IR to neurotrophins and their receptors in the gut of the frog further confirms the well-conserved presence of this family of growth factors and Trk receptors during the evolution of vertebrates and suggests their complex involvement in the biology of the gastrointestinal neuro-endocrine system.

Neurotrophin-like immunoreactivity in the gut of teleost species.

By means of immunochemistry and immunohistochemistry, we investigated in the gut of teleostean species the presence and localization of three neurotrophins: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin (NT)-3. In all studied species both NGF- and NT-3-like immunoreactivity (IR) were present in the enteric nervous system, while BDNF-like IR was never detected. More in particular, both NGF and NT-3-like IR were detected in neurons of small and large intestine, while only NT3-like IR was also observed in stomach plexuses. Furthermore, Western blot analysis revealed the presence of molecules immunoreactive to NGF and NT-3, which weight were very similar to those of mammalian corresponding neurotrophins. These results extend to teleost species the presence and distribution of NGF- and NT-3-like IR in the enteric nervous system, suggesting a well-preserved presence of these substances in the gut during vertebrate phylogenesis.

Brain-derived neurotrophic factor in higher vertebrate pancreas: immunolocalization in glucagon cells.

Brain-derived neurotrophic factor (BDNF) is a growth factor that belongs to the group of neurotrophins. Its amino acid sequences are well conserved during vertebrate phylogenesis. Pancreatic tissue has recently been reported to be one of the physiological sources of BDNF in humans and mice. In this study we investigated the presence and localization of BDNF immunoreactivity (IR) in the pancreas of three species of higher vertebrates: mouse, duck and lizard. BDNF IR was present in the islets and in single cells scattered in the exocrine parenchyma of all three species examined. Using double staining, BDNF IR was seen to be colocalized with glucagon IR in all the species studied. There was a total overlap of BDNF and glucagon IR in duck and lizard pancreas, and partial overlap in mouse pancreas. Our findings suggest that, as well as the primary structure, the presence and pattern of distribution of BDNF in higher vertebrates is also well conserved. Moreover, the abundance of BDNF IR in the pancreas of the species studied leads us to the suggestion that these neurotrophins could regulate the function of pancreatic innervation and/or act on pancreatic cells in a paracrine/autocrine fashion.

Localisation of neurotrophin - containing cells in higher vertebrate intestine.

Neurotrophins are structurally related proteins that regulate the development, differentiation and maintenance of many neuronal populations. In higher vertebrates (reptiles, birds and mammals) four neurotrophins have been found: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3 and NT4/5. In the gut, experimental data and the occurrence of neurotrophin receptors in intestinal neurons and endocrine cells suggest neurotrophin involvement in intestinal physiology. However, very few data are available regarding the cellular localization and distribution of neurotrophins in the gut. In this study we report the presence of NGF, BDNF and NT3 in neurons and endocrine cells of mouse, duck and lizard intestine. In particular, immunoreactivity to NGF was observed: (a) in both endocrine and nerve cells of mouse and duck intestine, (b) in endocrine cells of lizard gut. Immunoreactivity to BDNF was seen: (a) in nerve cells of mouse intestine, (b) in very few endocrine cells of mouse and duck intestine. Immunoreactivity to NT3 was detected: (a) in nerve cells of the mouse intestine, (b) in endocrine and nerve cells of duck and lizard gut. Our results, together with data previously reported, on the distribution of specific neurotrophin receptors, seem to suggest a possible paracrine/autocrine mechanism of neurotrophin action in both the enteric nervous system and endocrine cells.

TrkA and TrkB neurotrophin receptor immunoreactivity in the teleost (Scorpaena porcus) endocrine pancreas.

Mammalian-like and specific neurotrophins, as well as their cognate tyrosine kinase Trk-like receptors have been identified in teleosts. They are mainly distributed in neuronal tissues, but evidence suggests that some non-neuronal tissues also express Trks. In this study we used immunohistochemistry to investigate the occurrence and cell distribution of Trks in the pancreas of teleosts (Scorpaena porcus). Immunoreactivity for TrkA and TrkB, but not for TrkC receptors, was found in the scorpionfish pancreas. TrkA-like positive cells were exclusively observed within pancreatic islets, among insulin- and glucagon-containing cells, but apparently did not co-localize with these hormones. TrkB immunostaining was found in islet cells, presumably colocalized with glucagon, as well as in some cells of the exocrine portion. These data provide a morphological basis for a role of the TrkA and TrkB ligands in the endocrine pancreas of teleosts.

Trk-neurotrophin receptor-like immunoreactivity in the gut of teleost species.

Neurotrophins, acting through their high-affinity signal-transducing Trk receptors, are involved in the development, differentiation and maintenance of discrete neuron populations in the higher vertebrates. Furthermore, the presence of Trk receptors in some non-neuronal tissues, including the endocrine cells of the gut, could indicate an involvement of neurotrophins also in these tissues. Recently, neurotrophins and neurotrophin receptor proteins have been identified in the lower vertebrates and invertebrates, whose amino acid sequences are highly homologous with those found in mammals. The present study investigates the occurrence and distribution of Trk-like proteins in the neurons and gut endocrine cells in five species of teleost. Single and double immunolabeling was carried out on fresh and paraffin-embedded tissue using commercially available antibodies against sequences of the intracytoplasmic domain of the mammalian Trk. Western-blot analysis, carried out on samples of stomach and intestine of bass, identified proteins whose estimated molecular masses (140 kDa, 145 kDa and 143-145 kDa) were similar to those reported for full-length TrkA, TrkB and TrkC in the higher vertebrates. TrkA-like immunoreactivity was found in the enteric nervous system plexuses of three fish species. Trk-like immunoreactivity was observed in the endocrine cells as follows: sparse TrkA-like immunoreactive endocrine cells were detected only in the intestine: TrkB-like immunoreactive cells were detected only in the stomach; and TrkC-like immunoreactive cells were found both in the intestine of the carp and in the stomach of the bass, where they also showed TrkB-like immunoreactivity. These findings confirm the occurrence and distribution of Trk-like proteins in teleosts. These proteins are closely related to the Trk neurotrophin receptors of mammals. The functional significance of Trk-like proteins in both neuronal and non-neuronal cells of teleosts is still not clear.