Brain nitric oxides synthase in major pelvic ganglia of aged (LETO) and diabetic (OLETF) rats.

This study was conducted to evaluate the effects of aging and diabetes mellitus (DM) on brain nitric oxide synthase (bNOS) expression in major pelvic ganglia (MPG) of rats. Otsuka Long Evans Tokushima Fatty rats (12, 30, and 70 weeks old), which are genetic models with non-insulin-dependent DM (NIDDM), and age-matched nondiabetic Long Evans Tokushima Otsuka controls were used. The MPG of all rats in this study were subjected to cryo-sectioning and staining with bNOS polyclonal AB and rhodamine-conjugated rabbit IgG. Fluorescence intensities of the stained neurons were assessed in randomly selected fields per each specimen. Animals of both groups revealed significant decline in the staining intensity of their neurons with aging and the progress of DM, but diabetic rats showed more decline than controls. In conclusion, both aging and NIDDM could decrease bNOS expression in rat MPG. However, NIDDM has a more evident effect than aging on that expression. The decrease in bNOS may cause a disturbance in functions of the target pelvic structures of these ganglia under both conditions.

Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord.

Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.

Development changes in long-form leptin receptor expression and localization in rat brain.

The expression and localization of long-form leptin receptor (OB-Rb) were studied immunocytochemically in the brain of fetal and adult rats using a polyclonal antibody that specifically recognized OB-Rb. At 14 days of gestation, immunoreactive cells were observed in the ventricular layer, which contains premature neuronal cells. At 18 days of gestation, they were weakly stained but obvious in the paraventricular nucleus (PVN), and ependymal cells also showed immunoreactivity. At birth, the immunoreactivity of OB-Rb in the PVN seemed to be much lower than that in adult rats and remained low during the suckling period. Considering the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency, these results suggest that the expression of OB-Rb in premature neuronal cells may have some function, and that the regulation of energy balance by leptin through hypothalamic regions, such as PVN, may not yet be developed in the perinatal period.

Influence of serum-free culture conditions on steroid 5alpha-reductase mRNA expression in rat C6 glioma cells.

Immunocytochemical studies previously showed that serum deprivation resulted in the appearance of steroid 5alpha-reductase (5alpha-R) in the cytoplasm of rat C6 glioma cells. To determine whether this increase in cytoplasmic 5alpha-R was due to changes in 5alpha-R gene expression, the effect of serum deprivation on 5alpha-R mRNA expression was examined. No significant change in the mRNA levels was observed in cells grown in serum-free culture medium. Therefore, the appearance of 5alpha-R immunoreactivity in the cell cytoplasm observed under serum-free conditions is probably not due to changes in 5alpha-R gene expression.

Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors.

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.

Identification of prostaglandin F-producing cells in the liver.

Prostaglandin (PG) F synthase forms PGF(2alpha) and 9alpha, 11beta-PGF2 from PGH2 and PGD2, respectively. PGH2 is synthesized from arachidonic acid by cyclooxygenase (COX) and then metabolized to various PGs and thromboxane by specific enzymes. PGD2 is synthesized from PGH2 by PGD synthase. To identify PGF2-producing cells in the rat liver, the occurrence and localization of PGF synthase and COX were studied with immunochemical and immunohistochemical techniques using anti-liver-type PGF synthase and anti-COX antibodies. In Western blot analyses, positive bands of liver-type PGF synthase and constitutive COX-1 were observed at positions approximately 37 kDa and 70-72 kDa, respectively. However, inducible COX-2 was not detected. In the immunohistochemical study, PGF synthase was present in the cytoplasm of the sinusoidal endothelial cells. COX-1 was present on the membranes of the nucleus and endoplasmic reticulum of the endothelial cells and Kupffer cells. Double immunostaining for PGF synthase and COX-1 showed that both enzymes were present in the same endothelial cells. These results suggest that the main site of PGF2 synthesis in the liver is the sinusoidal endothelial cell.

Monoclonal antibody 14F7, which recognizes a stage-specific immature oligodendrocyte surface molecule, inhibits oligodendrocyte differentiation mediated in co-culture with astrocytes.

Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17-26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5- cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development.

Influence of serum-free culture conditions on subcellular localization of steroid 5alpha-reductase in rat C6 glioma cells.

Rat C6 glioma cells are considered to be well characterized, and therefore commonly used as a model system to investigate the function of glial cells. However, recent study has shown that an alteration in the expression of their phenotypic antigens is observed when the cells are maintained under the serum-free conditions, proposing the possibility that various properties of glioma cells can be altered by the growth conditions. To test this possibility, the effects of serum-free culture conditions on the expression of steroid 5alpha-reductase (5alpha-R) type 1 isozyme in glioma cells were examined using immunocytochemical technique. Immunoreactivity of 5alpha-R type 1 was confined to the perinuclear region of glioma cells cultured in serum-containing medium, and observed in the cytoplasmic space as well as the perinuclear region of the cells cultured in serum-free medium. In contrast, serum deprivation failed to affect the expression of phenotypic antigens, glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Further studies showed that the expression of cytoplasmic 5alpha-R immunoreactivity induced by serum deprivation was reversible, and might be attributed to removal of serum proteins rather than biologically active small molecules from culture medium. This alteration in the expression of 5alpha-R immunoreactivity is therefore considered to reflect the translocation of the enzyme from the perinuclear region to the cell cytoplasm rather than the induction of cytoplasmic enzyme, and suggest that the culture conditions cause an alteration in the subcellular localization of 5alpha-R type 1 isozyme without phenotypic change of the glioma cell.

Light and electron microscopic immunocytochemistry on the localization of 17 alpha-hydroxylase/C17,20-lyase (P450c17) in the rat placenta.

The rat placenta is the primary source of androgens during the second half of pregnancy. Androgens are converted to estrogens in the ovaries and contribute to the maintenance of normal pregnancy. We immunocytochemically characterized the cellular and subcellular localization of cytochrome P450 of 17 alpha-hydroxylase/C 17,20-lyase (P450c17), an enzyme responsible for androgen synthesis, in the rat placenta. We also observed the fine structure of the placenta by electron microscopy. The rat placenta had a different structure from the primate, and contained four zones: labyrinth, basal zone, decidua basalis, and metrial gland. The labyrinth had three trophoblastic layers and fetal endothelium, and P450c17 immunoreactivity was homogeneously localized in the three trophoblastic layers but not in the fetal endothelium. In the basal zone, various types of trophoblasts were observed, and the immunoreaction was localized in small basophilic cells and giant cells. The intensity of staining was heterogeneous among these cells. The decidua basalis showed no immunostaining. Subcellular localization of the enzyme was in the cytoplasm, but not in the nucleus or mitochondria. The present study demonstrated a steroidogenic potency in both the labyrinth and the basal zone, although it was shown only in the basal zone in previous studies.

Differentiation of oligodendrocyte occurs in contact with astrocyte.

We have generated stage-specific oligodendrocytes, which are identified as immature oligodendrocytes by the expression of specific markers. These cells were generated by using 3 types of intrinsic and extrinsic factors for influencing the characteristics of oligodendrocyte-lineage cells. The factors included 10% serum in the medium to induce cell proliferation, bFGF in serum-free medium to increase survival and induce differentiation, and 0.25% trypsin to remove nonoligodendrocyte lineage cells. We have also demonstrated that it is possible to supply a huge amount of homogeneous populations of stage-specific immature oligodendrocytes after 35 days' culture. The maturation of these cells is controlled by interacting with astrocytes in vitro, resulting in the expression of MBP isoforms (14.0 and 18.5 kDa) that may participate in mature oligodendrocyte membrane compaction.

Steroid 5 alpha-reductase type 1 immunolocalized in the adrenal gland of normal, gonadectomized, and sex hormone-supplemented rats.

Steroid 5alpha-reductase in the rat adrenal gland is supposed to play a role in the catabolism of adrenal steroids. We showed immunohistochemically the cellular and subcellular localization of 5alpha-reductase in the rat adrenal gland, using a polyclonal antibody against 5alpha-reductase rat type 1. In the adrenal cortex, positive immunoreaction was found in the cells of the zonae fasciculata and reticularis but was absent in those of the zona glomerulosa. The positive staining was restricted to the cytoplasm but not to the nucleus, Golgi complexes or mitochondria. The staining intensity showed a marked change depending on the steroidal milieu. Gonadectomy for 6 weeks increased the immunoreaction, regardless of the sex. Testosterone replacement for the last 2 weeks considerably reduced the immunoreaction in 6-week-castrated males, and estradiol supplement for the last 2 weeks also resulted in the marked reduction of immunostaining in 6-week-gonadectomized females. In the adrenal medulla, the immunoreaction was localized in the supporting cells and the Schwann cells but not in the chromaffin cells. In these cells, the immunoreaction was not affected by steroidal treatments. These findings suggest that the expression of 5alpha-reductase in the rat adrenal cortex is regulated by sex hormones from the gonads, and the enzyme may participate in the conversion of adrenal steroids depending on the steroidal environment, although the functional significance of the enzyme in the adrenal medulla remains unclarified.

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia.

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.

Localization of NADPH diaphorase and vasoactive intestinal polypeptide-containing neurons in the efferent pathway to the rat corpus cavernosum.

OBJECTIVE: We examined the coexistence of nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP) in the efferent pathway to the rat corpus cavernosum. METHODS: We used NADPH diaphorase (ND) histochemical staining, a specific marker of neuronal NOS, in combination with retrograde axonal transport of True Blue (TB) and VIP immunohistochemistry. RESULTS: Many neurons were labeled by TB in the cavernous nerve and the body of the major pelvic ganglion (MPG), and fewer neurons in the pelvic nerve. In the cavernous nerve, about 90% of TB-labeled efferent neurons were ND positive, and about 80% in the body of the MPG and pelvic nerve. Besides, 60-80% of TB-labeled efferent neurons projecting to the corpus cavernosum showed VIP immunoreactivity. VIP was colocalized with NOS in 70-80% of neurons. CONCLUSIONS: These findings suggest that most of the ND-positive nerves and terminals in the corpus cavernosum come from the MPG via the cavernous nerve and that NO plays an important role; it may act in combination with NO because most NOS-containing neurons simultaneously showed VIP immunoreactivity.

Steroid 5 alpha-reductase type 1 immunolocalized in the anterior pituitary of intact and castrated male rats.

The localization of 5 alpha-reductase was immunohistochemically studied in the anterior pituitary of male rats, using a polyclonal antibody against 5 alpha-reductase rat type 1. The immunoreactive cells were concentrated in the central region and on the border of the intermediate lobe in the anterior pituitary, but not in the intermediate or posterior lobe. The immunoreaction was located mostly in the cytoplasm and occasionally in the cell nuclei. The immunoreactive cells showed alterations in size and number and in the intensity of the immunoreaction after gonadectomy. One week after castration, the cells became larger and the immunoreactivity increased. Two weeks after castration, the number of immunoreactive cells increased. Double immunostaining using anti-luteinizing hormone beta-subunit or anti-follicle stimulating hormone beta-subunit antibody revealed that most of the cells containing 5 alpha-reductase were gonadotrophs. Electron microscopically, the immunoreactive cells showed lamelliform rough endoplasmic reticulum and a depletion of secretory granules 1 week after castration. One week later, the rough endoplasmic reticulum was developed and dilated and the number of secretory granules increased. These results suggest that 5 alpha-reductase is located in the gonadotrophs of rat anterior pituitary and that it is involved in the feedback regulation of gonadotropin secretion by androgens.

Immunohistochemical presence of 5 alpha-reductase rat type 1-containing cells in the rat brain.

We showed immunohistochemically the localization of 5 alpha-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5 alpha-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38-53 of 5 alpha-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29,000 Da. The immunoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5 alpha-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.

Immunocytochemical localization of 5 alpha-reductase type 1 in the prostate of normal and castrated rats.

We immunohistochemically studied the localization of 5 alpha-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5 alpha-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5 alpha-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5 alpha-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5 alpha-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5 alpha-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain.

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal, division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.

Presence of estrogen receptors in aromatase-immunoreactive neurons in the mouse brain.

The present study was undertaken to demonstrate estrogen receptors in aromatase-immunoreactive neurons showed the immunoreactivity for estrogen receptors in the cell nuclei in the medial subdivision of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus, but a few did in the medial preoptic area. These results suggest that aromatase-immunoreactive neurons in the former two areas are regulated by steroids through estrogen receptors in their cell nuclei.

Coexistence of nitric oxide synthase, tyrosine hydroxylase and vasoactive intestinal polypeptide in human penile tissue--a triple histochemical and immunohistochemical study.

Recently, nitric oxide (NO) has been believed to act as a neuronal messenger to mediate penile erection. In the present study using human penile tissue, we investigated the coexistence of neuronal NO synthase (NOS), tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) by a triple staining method using NADPH diaphorase (ND) staining, a specific histochemical marker of neuronal NOS, and immunohistochemical staining for TH and VIP. Numerous ND-positive nerve fibers and TH-containing fibers were seen in axon bundles, but their distributions were different. Only a few axons in the bundles showed VIP immunoreactivity. Abundant fine varicose nerve terminals innervating cavernous smooth muscles and deep and helicine arteries were observed. The proportion of fibers showing TH-immunoreactivity in ND-positive terminals in the cavernous space was about 25%, and that of VIP was about 40%. Vasoactive intestinal polypeptide may act as a coworker in these fibers both in cavernous trabeculae and around arteries, as about 40% of NOS-containing fibers also showed VIP immunoreactivity. The physiological significance of the colocalization of TH and NOS is unclear, and further studies are required to know the physiological significance of the colocalization of NOS and other neurotransmitters in penile tissue.