Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior.

In this article, we review studies which have been conducted to investigate the hormonal influence on metamorphosis in bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that controls the secretion of TSH is different from those encountered in mammals is discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion, are mentioned in this article. In addition, the involvement of hormones in eliciting courtship behavior in male red-bellied newts and the discovery of the peptide sex pheromones and hormonal control of their secretion are also discussed in the present article.

Dark Rearing in the Visual Critical Period Causes Structural Changes in Myelinated Axons in the Adult Mouse Visual Pathway.

An appropriate sensory experience during the early developmental period is important for brain maturation. Dark rearing during the visual critical period delays the maturation of neuronal circuits in the visual cortex. Although the formation and structural plasticity of the myelin sheaths on retinal ganglion cell axons modulate the visual function, the effects of dark rearing during the visual critical period on the structure of the retinal ganglion cell axons and their myelin sheaths are still unclear. To address this question, mice were reared in a dark box during the visual critical period and then normally reared to adulthood. We found that myelin sheaths on the retinal ganglion cell axons of dark-reared mice were thicker than those of normally reared mice in both the optic chiasm and optic nerve. Furthermore, whole-mount immunostaining with fluorescent axonal labeling and tissue clearing revealed that the myelin internodal length in dark-reared mice was shorter than that in normally reared mice in both the optic chiasm and optic nerve. These findings demonstrate that dark rearing during the visual critical period affects the morphology of myelin sheaths, shortens and thickens myelin sheaths in the visual pathway, despite the mice being reared in normal light/dark conditions after the dark rearing.

Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies.

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

Pharmacological treatment promoting remyelination enhances motor function after internal capsule demyelination in mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system characterized by remyelination failure, axonal degeneration, and progressive worsening of motor functions. Animal models of demyelination are frequently used to develop and evaluate therapies for MS. We recently reported that focal internal capsule (IC) demyelination in mice with lysophosphatidylcholine injection induced acute motor deficits followed by recovery through remyelination. However, it remains unknown whether the IC demyelination mouse model can be used to evaluate changes in motor functions caused by pharmacological treatments that promote remyelination using behavioral testing and histological analysis. In this study, we examined the effect of clemastine, an anti-muscarinic drug that promotes remyelination, in the mouse IC demyelination model. Clemastine administration improved motor function and changed forepaw preference in the IC demyelinated mice. Moreover, clemastine-treated mice showed increased mature oligodendrocyte density, reduced axonal injury, an increased number of myelinated axons and thicker myelin in the IC lesions compared with control (PBS-treated) mice. These results suggest that the lysophosphatidylcholine-induced IC demyelination model is useful for evaluating changes in motor functions following pharmacological treatments that promote remyelination.

Scanning and transmission electron microscopic observation of femoral head feeding vessels in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are known to show necrosis of the femoral head with a frequency of about 50%. This rat has thus been used as an animal model for necrosis of the femoral head in many studies. In a detailed investigation of feeding vessel disorders that cause femoral head necrosis, we observed changes over time in the feeding vessels using scanning electron microscopy and transmission electron microscopy. In scanning electron microscopy of vascular casts, abnormal findings in feeding vessels of SHRSP with aging from the immature stage included contortion and bending in the lumen with overall narrowing. Under transmission electron microscopy, decreased numbers of smooth muscle cells and increased amounts of collagen fibers were marked, and these changes with hypertrophy of vascular walls might be similar to those of arteriolosclerosis. The structural changes first revealed by transmission electron microscopic observation might cause the friability of the feeding vessels so that contortion and bending occurred, suggesting transient obstruction of blood flow to the femoral head and subsequent induction of femoral head necrosis. These findings should help in understanding the causes of femoral head necrosis in humans, including Perthes' disease.

Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye.

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

Effect of E-cadherin expression on hormone production in rat anterior pituitary lactotrophs in vitro.

Cadherins are a family of transmembrane glycoproteins that mediate cell-to-cell adhesion. A change in cadherin type in cells, i.e., cadherin switching, induces changes in the character of the cell. Recent studies of the developing rat adenohypophysis found that primordial cells co-expressed E- and N-cadherins, but that hormone-producing cells lost E-cadherin and ultimately possessed only N-cadherin. In the present study, we examined the roles of cadherin switching in cytogenesis of anterior pituitary cells by observing prolactin mRNA and protein expression in lactotrophs that were transformed with an E-cadherin expression vector. In hormone-producing cells that were transfected with a pIRES2-ZsGreen1 plasmid with a full-length E-cadherin cDNA (rE-cad-IZ) insert in primary culture, we detected E- and N-cadherins on plasma membrane and E-cadherin in cytoplasm. In these rE-cad-IZ-transfected cells, in situ hybridization revealed prolactin mRNA signals that were at a level identical to that in control cells, while prolactin protein was barely detectable using immunocytochemistry. The mean signal intensity of prolactin protein in rE-cad-IZ-transfected cells was approximately one fourth that in intact cells and in null-IZ-transfected cells (P<0.01). These results suggest that the expression of E-cadherin does not affect prolactin mRNA transcription; rather, it reduces prolactin protein content, presumably by affecting trafficking of secretory granules.

Estrogen receptor alpha regulates retinaldehyde dehydrogenase 1 expression in rat anterior pituitary cells.

Retinoic acid (RA) plays a critical role in embryonic development, growth, and reproduction. RA is synthesized from retinoids via oxidation processes, and the oxidation of retinal to RA is catalyzed by the retinaldehyde dehydrogenases (RALDHs). We previously reported that RALDH1 mRNA was expressed in the anterior pituitary glands of adult rats and suppressed by administration of 17beta-estradiol in vivo. However, little is known about the mechanism regulating pituitary RALDH1 expression. In order to characterize the mechanism of estrogen-induced RALDH1 reduction, we examined the effect of 17beta-estradiol on the regulation of pituitary RALDH1 gene expression and protein production both in vivo and in vitro. Using quantitative real-time PCR and immunoblot analysis, we found that levels of RALDH1 gene expression and protein production markedly decreased after 1-week treatment with 17beta-estradiol in male rats. In immunohistochemical analysis, RALDH1-immunoreaction was observed in prolactin cells and folliculo-stellate cells. In 17beta-estradiol-treated rats, RALDH1-immunoreactivity was lower in prolactin cells, but not in folliculo-stellate cells. Treatment of isolated anterior pituitary cells with 17beta-estradiol (10(-14) - 10(-8) M) decreased expression of RALDH1 mRNA in a dose-dependent manner. Estradiol-induced suppression of RALDH1 expression was completely blocked by the estrogen receptor (ER) antagonist ICI 182, 780. The ERalpha-selective agonist propylpyrazole triol (10(-8) M) mimicked the effect of 17beta-estradiol on RALDH1 expression, but the ERbeta-selective agonist diarylpropionitrile (10(-8) M) did not. These results strongly suggest that RALDH1 mRNA expression is suppressed by 17beta-estradiol through ERalpha, and that estrogen regulates the expression of RALDH1 and production of RA in the anterior pituitary gland.

Beneficial Effects of Cocoa in Perivascular Mato Cells of Cerebral Arterioles in SHR-SP (Izm) Rats.

As previously reported, the cerebral arterioles are surrounded by unique perivascular Mato cells. They contain many inclusion bodies rich in hydrolytic enzymes, and have strong uptake capacity. They are thus considered scavenger cells of vascular and neural tissues in steady-state. In this study, employing hypertensive SHR-SP (Izm) rats, the viability of Mato cells was investigated. In hypertensive rats, the capacity for uptake of horse radish peroxidase (HRP) and the activity of acid phosphatase (ACPase) of Mato cells were markedly reduced, and on electron-microscopic examination Mato cells were found to include heterogeneous contents and appeared electron-dense and degenerated. Vascular cells exhibited some signs of pathology. However, in hypertensive rats fed chow containing 0.25% cocoa, the uptake capacity and ACPase activity of Mato cells for HRP were enhanced, and on electron-microscopic examination Mato cells appeared healthy, with mitochondria with nearly normal profiles. Signs of pathology in vascular cells were also decreased. Superoxides may impair Mato cells and vascular cells.

Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells.

Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anterior pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.

Postnatal development of septal projections to the midbrain central gray in female rats: tract-tracing analysis with DiI.

The neural projection of the lateral septum (LS) to the rostral mesencephalic central gray (MCG) is sexually dimorphic and plays an important role in inhibiting female reproductive behavior. In this experiment, development of the LS-MCG connection from birth to 15 days after birth was examined in female rats by a tract-tracing method with DiI. On the birth day (D1 rat), and 5, 10 or 15 days after birth (D5, D10 or D15 rat, respectively) or 8 weeks after birth (adult), the brain was fixed by perfusion of a mixture of 4% PFA and 0.1% glutaraldehyde. DiI was pasted on the coronally cut-surface of the LS and the sample was incubated in PFA at 40 degrees C for up to 4 months. After incubation, 200-microm frozen parasagittal sections were prepared and observed by fluorescence microscopy. As a result, numerous DiI labeled fibers were found in the preoptic area, the anterior and posterior hypothalamus, and the MCG in adult rats. In D1 rats, several labeled axons extended caudal to the anterior hypothalamic area. In D5 rats, a few labeled fibers reached the MCG. Some labeled fibers were observed in the rostral MCG of D10 rats. In D15 rats, a considerable number of labeled fibers were seen to reach the rostral MCG and relative density of the fibers was comparable to that of adult. These results suggest that the neural pathway from the LS to the rostral MCG develops acutely during the period from 5-10 days up to more than 15 days after birth.

[Study on the distribution of collagen fibers in the bifurcating area of cerebral arterioles of Wistar rats].

It was previously reported that Mato cells (Mato's fluorescent granular perithelial cells) were frequently localized in the bifurcating areas of cerebral arterioles and occasionally, collagen fibers appeared close to Mato cells of aged rats. It has also been established that Mato cells were scavenger cells in the cerebral tissue and provided with MHC-class II antigen. The present paper deals with the relationship between the distribution of collagen fibers and Mato cells in the bifurcating area of cerebral arterioles. 6 Wistar rats (3 rats aged 4 months and 3 rats aged 16 months) were employed for this electron microscopical study. They were perfused with the mixture of paraform-glutaraldehyde solution, and cerebral cortices were excised and fixed with 1% osmic solution and embedded in Epon 812. In order to observe the bifurcating area of cerebral arterioles, serial semithin sections cutting with the diamond knife were stained with toluidine blue, and checked under the light microscope. After obtaining available regions, serial thin sections were stained with uranyl acetate and lead nitrate, and observed with Hitach H7600 electron microscope. In order to survey the distribution of collagen fibers, 2 groups of the specimens of 4 months old (2 rats) and 5 groups of specimens of 16 months old rats (2 rats) were prepared. Each group consisted of serial 10 thin sections. Other rats of young and aged were used for complementary use. From the observation, it is confirmed that in the cerebral arteriole, collagen fibers are localized only in the interstices around Mato cells, and the fibers appear in a small quantity in the rats aged 4 months, while a certain amounts of the fibers are arranged sporadically in the rats aged 16 months. However, no collagen fibers can be detected in the subendothelial space and in the interstices among smooth muscle cells of cerebral arteriole. If Mato cells lack in some regions of cerebral arterioles, collagen fibers cannot be recognized. The following is also to be stressed that no fibroblastoid cells do appear in any serial sections of cerebral arterioles. From these findings, it seems possible that Mato cells play a principal role in the formation of collagen fibers in the cerebral arterioles, and associate with the sclerosis of cerebral arterioles.

Folliculostellate cell interacts with pericyte via TGFß2 in rat anterior pituitary.

The anterior pituitary gland comprises five types of endocrine cells plus non-endocrine cells including folliculostellate cells, endothelial cells, and capillary mural cells (pericytes). In addition to being controlled by the hypothalamic-pituitary-target organ axis, the functions of these cells are likely regulated by local cell and extracellular matrix (ECM) interactions. However, these complex interactions are not fully understood. We investigated folliculostellate cell-mediated cell-to-cell interaction. Using S100ß-GFP transgenic rats, which express GFP in folliculostellate cells, we designed a three-dimensional cell culture to examine the effects of folliculostellate cells. Interestingly, removal of folliculostellate cells reduced collagen synthesis (Col1a1 and Col3a1). Because pericytes are important collagen-producing cells in the gland, we stained for desmin (a pericyte marker). Removal of folliculostellate cells resulted in fewer desmin-positive pericytes and less desmin mRNA. We then attempted to identify the factor mediating folliculostellate cell-pericyte interaction. RT-PCR and in situ hybridization revealed that the important profibrotic factor transforming growth factor beta-2 (TGFß2) was specifically expressed in folliculostellate cells and that TGFß receptor II was expressed in pericytes, endothelial cells, and parenchymal cells. Immunocytochemistry showed that TGFß2 induced SMAD2 nuclear translocation in pericytes. TGFß2 increased collagen synthesis in a dose-dependent manner. This action was completely blocked by TGFß receptor I inhibitor (SB431542). Diminished collagen synthesis in folliculostellate cell-deficient cell aggregates was partially recovered by TGFß2. TGFß2-mediated folliculostellate cell-pericyte interaction appears to be essential for collagen synthesis in rat anterior pituitary. This finding sheds new light on local cell-ECM interactions in the gland.

Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats.

The neural control systems for the ovulatory cycle and lordosis behavior are sexually differentiated by estrogen during the perinatal period in rats. In the present study, the effects of a single neonatal injection with the phytoestrogen, coumestrol, on female reproductive functions were investigated. Female rats were injected subcutaneously with 1 or 3mg coumestrol (CM1, CM3), 1mg genistein (GS1), 1mg estradiol (E2), or oil at day 5 after birth (birth day=day 1) and an estrous cycle check and lordosis behavior test were performed. As a result, vaginal opening was advanced in CM1-, CM3- or E2-treated females. A vaginal smear check indicated that oil- or GS1-treated females showed a constant 4- or 5-day estrous cycle, whereas CM1-, CM3- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovary weights in the CM1-, CM3- or E2-treated groups were lower than those in the oil- and GS1-treated groups and no corpora lutea were found in any rats of these three groups, except for two E2-treated rats. Behavioral tests were carried out after implantation of E2-tubes. All rats in the CM1-, GS1-treated groups showed a high lordosis quotient (LQ), being comparable to that in the oil-treated females. On the other hand, LQs in the CM3, E2 or male groups were lower than that in the control female group. These results suggest that a single neonatal injection of 3 mg coumestrol was effective in suppressing the functions of ovulation-inducing mechanisms and the induction of lordosis, but 1mg coumestrol was effective in only the estrous cycle of female rats.

Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

Changes in fine structure of pericytes and novel desmin-immunopositive perivascular cells during postnatal development in rat anterior pituitary gland.

Pericytes are perivascular cells associated with capillaries. We previously demonstrated that pericytes, identified by desmin immunohistochemistry, produce type I and III collagens in the anterior pituitary gland of adult rats. In addition, we recently used desmin immunoelectron microscopy to characterize a novel type of perivascular cell, dubbed a desmin-immunopositive perivascular cell, in the anterior pituitary. These two types of perivascular cells differ in fine structure. The present study attempted to characterize the morphological features of pituitary pericytes and novel desmin-immunopositive perivascular cells during postnatal development, in particular their role in collagen synthesis. Desmin immunostaining revealed numerous perivascular cells at postnatal day 5 (P5) and P10. Transmission electron microscopy showed differences in the fine structure of the two cell types, starting at P5. Pericytes had well-developed rough endoplasmic reticulum and Golgi apparatus at P5 and P10. The novel desmin-immunopositive perivascular cells exhibited dilated cisternae of rough endoplasmic reticulum at P5-P30. In addition, during early postnatal development in the gland, a number of type I and III collagen-expressing cells were observed, as were high expression levels of these collagen mRNAs. We conclude that pituitary pericytes and novel desmin-immunopositive perivascular cells contain well-developed cell organelles and that they actively synthesize collagens during the early postnatal period.

[Paraneoplastic cerebellar degeneration with bladder cancer confirmed the existence of an anti-neural antibody: a case report].

A 66-year-old man was diagnosed with bladder cancer at our urology department. Three months later, he developed subacute progressive cerebellar limb ataxia and truncal oscillation. Analysis of cerebrospinal fluid showed pleocytosis and increased concentrations of protein, while brain magnetic resonance imaging revealed no abnormalities. Based on the presence of the bladder cancer, the etiology of subacute cerebellar ataxia could be a paraneoplastic neurological syndrome. Four months later, the patient underwent transurethral resection of the bladder tumor, which was identified as urothelial cancer on the basis of pathological examinations. However, this procedure failed to improve his neurological symptoms. Serum paraneoplastic markers such as anti-Yo, anti-Hu, anti-Tr, and other antibodies were not detected. Immunohistochemical staining of mouse cerebellum using the patient's serum revealed coarse granular staining in the cytoplasm of Purkinje cells and diffuse staining in the neuropil of the molecular layer, suggesting the presence of an unknown antibody. Subsequently, one-dimensional electrophoresis western blotting using the patient's serum revealed several bands including a strong positive band of approximately 45 kDa in mouse cerebellum lysates but not in liver lysates. These bands have never been detected in sera derived from healthy donors. These results suggested the presence of a novel antibody in the patient's serum that might recognize the approximately 45 kDa protein related to paraneoplastic cerebellar degeneration. Cases of paraneoplastic neurological syndrome associated with bladder cancer have rarely been reported. We concluded that the present case may be categorized as paraneoplastic neurological syndrome caused by an unknown antibody.

Transmission and scanning electron microscopy study of the characteristics and morphology of pericytes and novel desmin-immunopositive perivascular cells before and after castration in rat anterior pituitary gland.

Pericytes are perivascular cells associated with microcirculation. Typically, they are localized close to the capillary wall, underneath the basement membrane, and have sparse cytoplasm and poorly developed cell organelles. However, the specific properties of pericytes vary by organ and the conditions within organs. We recently demonstrated that pericytes in rat anterior pituitary gland produce type I and III collagens. The present study attempted to determine the morphological characteristics of these pituitary pericytes. Castrated rats were used as a model of hormonal and vascular changes in the gland. Pericytes, as determined by desmin immunohistochemistry, were more numerous and stained more intensely in castrated rats. Transmission electron microscopy revealed that pituitary pericytes displayed the typical characteristics of pericytes. In pituitary sections from castrated rats, the Golgi apparatus of pericytes was well developed and the rough endoplasmic reticulum was elongated. Additionally, scanning electron microscopy revealed four pericyte shapes: oval, elongate, triangular, and multiangular. As compared with normal rats, the proportion of oval pericytes was lower, and the proportions of the other three shapes were higher, in castrated rats. These results suggest that pericytes change their fine structure and cell shape in response to hormonal and vascular changes in the anterior pituitary gland. In addition, a novel type of perivascular cell was found by desmin immunoelectron microscopy. The morphological properties of these cells were dissimilar to those of pericytes. The cells were localized in the perivascular space, had no basement membrane, and contained dilated rough endoplasmic reticulum. This new cell type will require further study of its origin and characteristics.

Expression of the proteoglycan syndecan-4 and the mechanism by which it mediates stress fiber formation in folliculostellate cells in the rat anterior pituitary gland.

Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.