[Show abstract][Hide abstract] ABSTRACT: The medial preoptic and anterior hypothalamic areas (MPO/AH) are important androgen targets regulating homeostasis, neuroendocrinology and circadian rhythm as well as instinctive and sociosexual behaviors. Although species differences between rats and mice have been pointed out in terms of morphology and physiology, detailed distributions of androgen receptor (AR) have never been compared between the two rodents. In the present study, AR distribution was examined immunohistochemically in serial sections of the MPO/AH and compared for adult rats and mice. Western blotting and immunohistochemistry clearly demonstrated that AR expression in the brain was stronger in mice than in rats and was stronger in males than in females. In addition, we found (1) an "obliquely elongated calbindin-ir cell island" in mice medial preoptic nucleus (MPN) expressed AR intensely, as well as the sexually dimorphic nucleus in the MPN (SDN-MPN) in rats, strongly supporting a "putative SDN-MPN" previously proposed in mice; (2) AR expression in the suprachiasmatic nucleus (SCN) was much more prominent in mice than in rats and differed in localization between the two species; (3) a mouse-specific AR-ir cell cluster was newly identified as the "tear drop nucleus (TDN)", with male-dominant sexual dimorphism; and (4) two rat-specific AR-ir cell clusters were also newly identified as the "rostral and caudal nebular islands", with male-dominant sexual dimorphism. The present results may provide basic morphological evidence underlying species differences in androgen-modified psychological, physiological and endocrinergic responses. Above all, the findings of the mouse-specific TDN and differing AR expression in the SCN might explain not only species difference in gonadal modification of circadian rhythm, but also distinct structural bases in the context of transduction of SCN oscillation. The current study could also serve as a caution that data on androgen-sensitive functions obtained from one species should not always be directly applied to others among rodents.
[Show abstract][Hide abstract] ABSTRACT: The first appearance, distribution and frequency of immunoglobulins (Igs)-positive lymphocytes were investigated in the lymphoid organs of native chicken’s embryos from embryonic day (ED) 8 to ED 20. The tissues from the lymphoid organs were dehydrated in alcohol, cleared in xylene, embedded in different grades of paraffin and 6-micron thick sections were immunostained by the indirect immunoperoxidase method using antichicken immunoglobulins. IgM-positive lymphocytes were first identified in the follicles of bursa of Fabricius at ED 10, in the white pulp of the spleen at ED 14 and in the lamina propria of the cecal tonsil at ED 20. Their frequencies of populations were statistically significant from ED 14 to ED 20. IgG-positive lymphocytes were first appeared in the bursa of Fabricius and spleen at ED 20. In the bursa IgG-positive lymphocytes were located in the medulla and cortical part of the follicles, whereas, in the spleen these immune cells were located around the white pulp. IgA-positive lymphocytes were not observed in any of the developing lymphoid organs of the present study. When the data for bursa of Fabricius, spleen, thymus and cecal tonsil were statistically compared, it was observed that both IgM- and IgG-positive lymphocytes were significantly higher in bursa of Fabricius.
[Show abstract][Hide abstract] ABSTRACT: Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor that is widely expressed as a core molecule of the stigmoid body (a neurocytoplasmic inclusion) in the limbic and hypothalamic regions and has putative protective functions against some neurodegenerative diseases (HAP1 protection hypothesis). Although HAP1 has been reported to be intimately associated with several steroid receptors, HAP1-immunoreactive (HAP1-ir) cells remain to be identified in the hippocampus, which is one of the major steroidal targets. In this study, we determined the distribution of hippocampal HAP1-ir cells in light and fluorescence microscopy and characterized their morphological relationships with steroid receptors, markers of adult neurogenesis, and the GABAergic system in adult male and female Wistar rats. HAP1-ir cells, which were sporadically distributed particularly in the subgranular zone (SGZ) of the dentate gyrus and in the interface between the stratum lacunosum-moleculare and stratum radiatum of Ammon's horn, were identified as the "sporadically lurking HAP1-ir (SLH)" cells. The SLH cells showed no clear association with neural progenitor/proliferating or migrating cell markers of adult neurogenesis, such as Ki-67, proliferating cell nuclear antigen, doublecortin, and glial fibrillary acidic protein in the SGZ, whereas all the SLH cells expressed a neuronal specific nuclear protein (NeuN). More than 90% of the SLH cells expressed nuclear estrogen receptor (ER) α but neither ERβ nor the androgen receptor, whereas glucocorticoid receptor was differently stained in the SLH cells depending on the antibodies. More than 60% of them exhibited GABA immunoreactivity in the SGZ, suggestive of basket cells, but they were distinct from the ones expressing cholecystokinin or parvalbumin. We conclude that SLH cells, which should be stable against apoptosis due to putative HAP1 protectivity, might be involved in estrogen-dependent maturation, remodeling and activation of hippocampal memory and learning functions via ERα and partly through GABAergic regulation.
[Show abstract][Hide abstract] ABSTRACT: The preoptic and anterior hypothalamic areas (PO/AH) are important sex-steroid responsive regions regulating intrinsic programs relevant to homeostatic, neuroendocrinergic and instinct behavioral functions. Although related morphological and physiological data obtained from knockout mice have often been referred to data previously accumulated in rats, detailed distributions of sex-steroid receptors have never been examined between the two rodents. In the present study, expressions of androgen receptor (AR) in the PO/AH were immunohistochemically compared between the adult male Wistar rats and C57BL/6 mice, using 4% paraformaldehyde-fixed serial frozen brain sections. In general, the AR expression was much stronger in the mouse than the rat. In particular, AR-immunoreactive (AR-ir) cells were more prominent in the OVLT, anterior part and periventricular zone of medial preoptic area (MPO), posterodorsal preoptic nucleus (PDPN), anterodorsal junction area of the AH and suprachiasmatic nucleus (SCN) of the mouse brain, while in the sexually dimorphic nucleus (SDN) of the MPO and periventricular zone of the AH, more highly concentrated in the rat brain. In addition, we found a distinct AR-ir neuronal cluster in the mouse AH as the “tear drop nucleus” (TDN), which is not present in the rat. The TDN is also identified just dorsal to the SCN in Nissl staining like its dorsal extension, and the AR-ir cells in both nuclei are localized at the same rostrocaudal levels. The AR-ir cell cluster in the TDN, however, resembles those of the rat SDN or mouse putative SDN in terms of presence of the prominent calbindin-ir cells rather than the SCN where calbindin-ir cells are scarcely seen. Species difference in AR expression shown in our current results might give warning that data related to androgen-sensitive functions obtained from the two different rodents might not directly be applied each other.
Neuroscience Research 09/2011; Volume 71, Supplement:e266. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Huntingtin-associated protein 1 (HAP1) is a core component of the stigmoid body (STB), which is a neurocytoplasmic inclusion with protective functions against neurodegeneration in polyglutamine diseases. In the present study, we found that HAP1 was expressed in the subgranular zone (SGZ) of the rat dentate gyrus, where neurogenesis persists in adulthood. To clarify the possible relationship of HAP1-immunoreactive (HAP1-ir) cells with adult neurogenesis in the SGZ, we performed single- and double-labeling immunohistochemistry in light or fluorescence microscopy in adult male Wistar rat using the antibody against HAP1 in combination with molecular markers for neurogenesis including glial fibrillary acidic protein (GFAP), a proliferating cell marker Ki-67, proliferating cell nuclear antigen (PCNA), doublecortin (DCX) and neuron-specific nuclear protein (NeuN, a mature neuronal marker). HAP1-ir cells with STB or without STB were always found to exist near the cluster of Ki67- or PCNA-ir cells but showed no clear coexpression with neural progenitor/proliferating cell markers, while almost all the HAP1-ir cells expressed NeuN. For further characterization, we also examined the relationships of HAP1 with gamma-aminobutyric acid (GABA) and estrogen receptor α (ERα) that has also been reported to be expressed in the SGZ. It was revealed that 91.04% of HAP1-ir cells expressed ERαwhereas 63.4% of HAP1-ir cells exhibited GABA immunoreactivity. These results suggest that HAP1-ir cells in the SGZ are distinct group of the mature neurons sensitive to estrogen via ERα and at least half of these neurons are GABAergic although there might some possibilities of HAP1 to play a transient role in adult neurogenesis just after proliferation or migration stage. It can be concluded that HAP1/STB modifies estrogen-mediated neuronal activities and it may also relate to GABAergic function in the SGZ.
Neuroscience Research 09/2011; Volume 71, Supplement:e241. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Native chickens (Gallus domesticus) of Bangladesh are scavenging in nature. Data regarding morphology of lymphoid organs in prenatal stages are lacking. H&E staining method was performed to study the development of the bursa of Fabricius, thymus, spleen and cecal tonsil from embryonic day (ED) 10 to 20. The budding of thymus was seen on ED 10. At ED 12, fiber network of thymus was formed to create a basement of cells of thymus and on ED 14 these cells began to organize to form cortex and medulla. But the cortex and medulla of thymus could not be differentiated before ED 20. The plicae of bursa Fabricius started to develop on ED 10. From ED 12 the plicae became shorter and wider to form bursal follicles and these follicles were clearly organized into cortex and medulla on ED 20. At ED 10, very thin capsule was seen in embryonic spleen. During ED 12, only a few white pulps were observed, while on ED 14, purple colored white pulp and pinkish red pulp were easily visible. At ED 20, the thickness of capsule was increased and pulps were more distinguishable. All the lymphoid organs showed major development during the later incubation period, indicating that the immune system in that period is being prepared to face the scavenging environment after hatching.
[Show abstract][Hide abstract] ABSTRACT: The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.
Experimental Cell Research 07/2011; 317(12):1689-700. DOI:10.1016/j.yexcr.2011.05.004 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.
Human Molecular Genetics 03/2011; 20(7):1274-84. DOI:10.1093/hmg/ddq568 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Huntingtin-associated protein 1 (HAP1) is an essential component of the stigmoid body (STB) and known as a possible neuroprotective interactor with causative proteins for Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 17 (SCA17), and Joubert syndrome. To clarify what other causative molecules HAP1/STB could interact with, we cloned normal causative genes for several neural disorders from human brain RNA library and evaluated their subcellular interaction with HAP1/STB by immunocytochemistry and immunoprecipitation after cotransfection into Neuro2a cells. The results clearly showed that HAP1/STB interacts with the normal ataxin-3 through Josephin domain and polyglutamine-expanded mutants derived from SCA3 as well. The findings suggest that HAP1/STB could modify the physiological function of normal ataxin-3 and pathogenesis of SCA3 attributable to the mutant ataxin-3.
[Show abstract][Hide abstract] ABSTRACT: The stigmoid body (STB) is a neurocytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and its formation is induced by transfection of HAP1-cDNA into cultured cells. Although STB is believed to play a protective role in polyglutamine diseases, including Huntington's disease and spinal and bulbar muscular atrophy, by sequestering the causative proteins, huntingtin and androgen receptor, respectively, its physiological function and formation remain poorly understood. Therefore, STB is occasionally confused with another cytoplasmic inclusion observed in polyglutamine diseases, the aggresome. Here we examined the subcellular dynamics of STB and compared it immunohistochemically and cytochemically with the aggresome in the rat brain and COS-7 or HeLa cells transfected with HAP1 and/or polyglutamine disease-associated genes. In time-lapse image analysis of HAP1-transfected cells, the HAP1-induced STB is formed from multiple fusions of small HAP1 inclusions characterized by vigorous cytoplasmic movement. In HAP1-transfected cells treated with a microtubule-depolymerizing drug, although the formation of small HAP1 inclusions was not affected, their fusion was critically inhibited. Immunohistochemistry and cytochemistry revealed the absence of association between STB and aggresomal markers, such as ubiquitin/proteasome, intermediate filaments, and the centrosome. Taken together, we concluded that STB is formed by a two-step process comprising microtubule-independent formation of small HAP1 inclusions and microtubule-dependent fusion of these inclusions, and that STB is distinct from pathological aggresomes.
[Show abstract][Hide abstract] ABSTRACT: Wolfram syndrome is a rare genetic disorder accompanying diabetes insipidus, sensorineural hearing loss, neurological complications, and psychiatric illness. This syndrome has been attributed to mutations in the WFS1 gene. In this study, we made a detailed histochemical analysis of the distribution of Wfs1 mRNA in the brain of developing mice. There were three patterns of change in the strength of Wfs1 mRNA signals from birth to early adulthood. In type 1, the signals were weak or absent in neonates but strong or moderate in young adults. This pattern was observed in the CA1 field, parasubiculum, and entorhinal cortex. In type 2, the signals were of a relatively constant strength during development. This pattern was seen in limbic structures (e.g. subiculum and central amygdaloid nucleus) and brainstem nuclei (e.g. facial and chochlear nuclei). In type 3, the signals peaked in the second week of age. This pattern was observed in the thalamic reticular nucleus. Thus, Wfs1 mRNA was widely distributed in the normal mouse brain during postnatal development. This evidence may provide clues as to the physiological role of the Wfs1 gene in the central nervous system, and help to explain endocrinological, otological, neurological, and psychiatric symptoms in Wolfram syndrome patients.
Neuroscience Research 07/2009; 64(2):213-30. DOI:10.1016/j.neures.2009.03.005 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death.
We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone.
Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development.
Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.
[Show abstract][Hide abstract] ABSTRACT: Confocal laser scanning microscope image of double immunostaining for Wfs1 protein (green) and for glial fibrillary acidic protein (GFAP; an astrocyte marker; red) in the normal mouse retina and optic nerve. Cell nuclei are labeled in blue. The Wfs1 protein, also called wolframin, is the Wolfram syndrome 1 (Wfs1) gene product. Wfs1 is the disease gene for Wolfram syndrome, a neurodegenerative disorder defined by insulin-dependent diabetes mellitus and progressive optic atrophy. This image shows that Wfs1 immunoreactivity is observed in the GFAP-rich part of the optic nerve immediately behind the eye (central part of the image), and in most of the retinal layers (lower part of the image). J. Comp. Neurol. 510:1-23, 2008. (c) 2008 Wiley-Liss, Inc.
The Journal of Comparative Neurology 09/2008; 510(1):SPC1. DOI:10.1002/cne.21797 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuronal aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is involved in brain sexual differentiation, the regulation of reproductive behavior, and gonadotropin secretion. We have previously reported that aromatase P450 (AromP450) protein expression is enhanced by both androgens and estrogens in the principal nucleus of the bed nucleus of the stria terminalis (prBST) and posterodorsal part of the medial amygdaloid nucleus (pdMAm) of the adult rat but is not altered in the central amygdaloid nucleus (CeAm) even after sex-steroid withdrawal or supplementation. Here, we have evaluated, via in situ hybridization with digoxigenin-labeled cRNA probes, the sex-steroidal regulation of brain AromP450 mRNA in the prBST, pdMAm, and CeAm of orchidectomized and adrenalectomized adult male rats treated with sesame oil, testosterone (1 mg/rat/day), dihydrotestosterone (1 mg/rat/day), or 17beta-estradiol (2 microg/rat/day) for 6 days. AromP450-mRNA expression in the prBST and pdMAm was markedly reduced in orchidectomized/adrenalectomized rats treated with sesame oil but strongly enhanced by testosterone or dihydrotestosterone and significantly reinstated by 17beta-estradiol. These results are essentially consistent with those of AromP450 protein expression and thus indicate that enhanced AromP450-protein expression in the prBST and pdMAm reflects transcriptional upregulation and/or post-transcriptional stabilization of its mRNA by sex steroids. In the CeAm, despite moderate AromP450-protein expression, the mRNA has never been detected with or without sex-steroidal manipulations, indicating that the putative sex-steroid-insensitive AromP450 mRNA in the CeAm may be distinct from that in the prBST and pdMAm or, if it occurs at all, expressed at much lower levels.
Cell and Tissue Research 07/2008; 332(3):381-91. DOI:10.1007/s00441-008-0606-8 · 3.57 Impact Factor