横須賀 誠

In an attempt to define the role of the pineal secretory melatonin anti an analogue, 6-hydroxymelatonin (6-OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)-induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6-OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague-Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co-treatment of CP-treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the CrSH/GSSG ratio was prevented. Treatment of CP-enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH-Px), decreased as a result of CP administration, which was restored to control levels with melatonin co-treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP-treated rats; these changes were prevented by melatonin treatment. 6-OHM has been shown to have some antioxidative capacity, however, the protective effects of 6-OHM against CP-induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co-treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH-Px activating property, and/or to its regulatory activity for renal function.

In dogs, embryo transfer (ET) techniques such as induciton of excessive ovulation and synchronization of estrus have not progressed well. Therefore, using embryos at various developmental stages, ET was investigated in dogs from a beagle colony in which the ovulation dal's were close, as estimated by the progesterone level. Embryos were recovered 8-11 days after ovulation (4-9 days after mating) by excising the oviducts and uteri (excision method) in 16 animals and by surgical flushing of the uteri at laparotomy (surgical method) in 3 animals. In 24 dogs with -4 to +2 days of difference in the timing of ovulation between donor and recipient dogs, 1-10 embryos at the 8-cell to blastocyst stages were transferred per animal. The mean embryo recovery rate by the excision method (97.1%) was significantly higher than that by the surgical method (42.5%) (p<0.01). Twelve(57.1%) of 21 animals with -1 to +2 days difference in ovulation day became pregnant after the transfer of 8-cell to blastocyst stage embryos. Although 3 dogs with 3 to -2 days of difference of ovulation day underwent ET of morula or compacted morula, none of these dogs became pregnant. The mean ratio of the number of newborns to the number of transferred embryos was only 51.9%. The mean duration of the period between ovulation and delivery in the pregnant recipients was 65.8 days, which tended to be longer than that in natural mating. These results demonstrate that pregnancy can be induced by ET at the 8-cell to blastocyst stage in dogs with -1 to +2 days difference in ovulation day.

To identify the site(s) of NPY Y5 receptor (Y5R) mediation of NPY-induced feeding, we employed c-Fos immunostaining and a selective Y5R antagonist (Y5R-A), CGP71683A, in adult male rats. Intracerebroventricular (icv) administration of NPY stimulated feeding and c-Fos-like immunoreactivity (FLI) in the dorsomedial hypothalamus, supraoptic nucleus and the two subdivision of the hypothalamic paraventricular nucleus (pPVN), the parvocellular (pPVN), and magnocellular (mPVN). YSR-A on its own, injected either intraperitoneally or icy, neither affected feeding nor FLI in hypothalamic sites. However, YSR-A pretreatment suppressed NPY-induced food intake and FLI serectively in the mPVN. Taken together with our previous similar finding of YIR involvement, these results suggest that NPY receptor sites concerned with feeding behavior reside selectively in the mPVN and Y1 and Y5 receptors are either coexpressed or expressed separately in those target neurons that promote appetitive drive. (C) 2001 Elsevier Science Inc. All rights reserved.

The heterogeneous structure of the accessory olfactory bulb (AOB) has been demonstrated immunocytochemically. In this study, we analyzed the expression of an immediate-early gene protein, c-Fos, as a marker of neuronal activity in response to chemonsensory cues was analyzed. The number of c-Fos-immunoreactive (Fos-ir) cells was measured in the rostral and caudal zones of the AOB in male ICR mice after exposure to the soiled bedding of female mice. The results revealed no significant difference in the number of Fos-ir cells in the caudal zone of the AOB between exposure to the soiled bedding of female ICR mice (ICR group) and exposure to that of female Balb mice (Balb group). In the rostral zone, however, the number of Fos-ir cells in the glomerular layer and granule cell layer was larger in the ICR group than in the Balb group. The difference in the expression of c-Fos in response to different pheromonal stimuli between the rostral and caudal zones in the mouse AOB has been shown for the first time in this study. These results strongly suggest that the heterogeneous structure of the AOB has an important role in the perception and processing of pheromones. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Previous studies have indicated that the ventral part of the premammillary nucleus (PMv) of rodents is involved in the regulation of aggressive and male mating behavior, although the precise physiological function of the PMv is still unclear. To analyze the physiological role of the PMv in male mating behavior, the effects of exposure to bedding soiled by female mice on Fos immunoreactivity (Fos-ir), an early marker of neuronal activation, were studied in the PMv and some sex-related nuclei. We observed that exposure to female-soiled bedding induced Fos-ir expression in the PMv of the male mouse. Although Fos-ir positive cells were found in the posterodorsal part of the medial amygdaloid nucleus and in the posteromedial cortical amygdaloid nucleus, which are terminals of the neuronal projections from the main and accessory olfactory bulbs, the numbers of Fos-ir cells in those nuclei were not affected by exposure to female-soiled bedding. Moreover, Fos-ir was not detected in the ventromedial hypothalamic nucleus. It is well established that soiled bedding is useful as a source of chemosensory substances, which include "pheromones," Thus, our findings, in agreement with previous behavioral and anatomical data, suggest that the PMv plays a role in initiating male copulative behavior that is induced by a female mice pheromone(s). (C) 1999 Elsevier Science Inc. All rights reserved.

Neuropeptide Y (NPY) is one of the important endogenous orexigenic peptides. In these studies we employed c-Fos immunostaining and a selective NPY Y-1 receptor antagonist to identify the site of action of NPY in the hypothalamus. The results showed that intracerebroventricular administration of NPY stimulated feeding and increased immunostaining of c-Fos, a product of the immediate early gene c-fos, in several hypothalamic sites, including the dorsomedial nucleus, the supraoptic nucleus, and the two subdivisions of the paraventricular nucleus (PVN), the parvocellular PVN, and magnocellular PVN (mPVN). Intracerebroventricular administration of 1229U91, a selective NPY Y-1 receptor antagonist, affected neither food intake nor c-Fos-like immunoreactivity (FLI) in these hypothalamic sites. Coadministration of NPY and NPY Y-1 receptor antagonist inhibited NPY-induced food intake by 48%, but failed to affect NPY-induced FLI in the supraoptic nucleus, dorsomedial nucleus, and parvocellular PVN. However, this combined treatment decreased FLI by 46% in the mPVN (P < 0.05). These results showed that whereas NPY can stimulate FLI in several hypothalamic sites, the selective NPY Y-1 antagonist suppressed NPY-induced FLI only in the mPVN. Thus, these findings lend credence to the view that a subpopulation of Y-1 receptor-containing neurons in the mPVN in Dart mediate stimulation of feeding by NPY.

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase, The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle. (C) 1999 Harcourt Publishers Ltd.

Intracerebroventricular (i.c.v.) injection of leptin, the adipocyte hormone, inhibits neuropeptide Y (NPY)-induced feeding in the rat. To identify the neural substrate for leptin and NPY interaction in the hypothalamus, we evaluated the expression of c-fos-like immunoreactivity (FLI), an early marker of neuronal activation, in response to icy administration of leptin, NPY and leptin plus NPY. As expected, leptin significantly decreased NPY-induced feeding in leptin plus NPY-treated rats. A comparative evaluation of the number of FLI-positive neurons in hypothalamic sites showed that both leptin and NPY activated FLI in the parvocellular subdivision of the paraventricular nucleus (pPVN), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). NPY also augmented the FLI response in the magnocellular PVN (mPVN) and supraoptic nucleus (SON), two sites where leptin alone was ineffective. Combined leptin and NPY treatment significantly decreased the number of FLI-positive neurons in the magnocellular PVN but increased their number in the dorsomedial nucleus as compared to the number of FLI-expressing neurons in these sites after NPY and leptin alone. Because there is morphologic evidence of a link between magnocellular PVN and dorsomedial nucleus, these results suggest the functional involvement of leptin plus NPY responsive elements in these sites in reduction of NPY-induced feeding by leptin. (C) 1998 Elsevier Science Inc.

Localization of neuronal nitric oxide synthase-immunoreactivity (nNOS- IR) in the median eminence of female rats (n = 4) was examined by electron microscopy to explore the possibility that nitric oxide is involved in the terminal regulation of neurosecretory peptides such as GnRH. Under light microscopy, a dense distribution of nNOS-IR was observed in this region. Electronmicroscopically, nNOS-IR was found in glial elements and nerve terminals containing dense-core vesicles. We also found a few nNOS- immunopositive synapses, in which intense immunoreactivity was found on the postsynaptic density and mitochondrial membrane. The localization of nNOS-IR in nerve terminals and glial elements in the median eminence might indicate that nNOS plays a role in regulating the release of neurosecretory peptide.

Estrogen has been considered as a key substance that induces sexual differentiation of the brain during fetal and neonatal life in the rat. Thus, to define the brain regions involved in the brain sexual differentiation, we examined the regions where the estrogen receptor (ER) is located in the developing rat brain. We examined immunohistochemical distribution of the cells containing estrogen receptor-alpha (ER-alpha) in the preoptic region, the diencephalon, and the amygdala in male and female rats on postnatal days 1-35 (PD1-PD35). The antibody used recognizes ER-alpha equally well for both occupied and unoccupied forms. ER-alpha immunostaining was restricted to the cell nuclei of specific cell groups. In PD1 rats, ER-alpha-immunoreactive (ER-IR) signals were detected in the lateral septum, the organum vasculosum lamina terminalis, the medial preoptic nucleus (MPN), the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, the lateral habenula, the posterodorsal part of the medial amygdala nucleus, the posterior part of the cortical amygdala nucleus, the hypothalamic ventromedial nucleus (VMH), the hypothalamic arcuate nucleus, and the posterior hypothalamic periventricular nucleus. The distribution pattern of ER-IR cells in the newborn rat was much the same as that in the adult in the preoptic-hypothalamic and amygdala regions. Moreover, the signals in the MPN and the VMH were stronger in the female than in the male, perhaps reflecting the ability of estrogen generated by aromatization of testosterone in the male to down-regulate the ER signal. Thus, the brain regions showing sex differences may be sites of sexual differentiation of the brain by aromatizable androgen during the neonatal period. (C) 1997 Wiley-Liss, Inc.

The distribution of the neuronal nitric oxide synthase (nNOS), androgen receptor (AR), estrogen receptor (ER) and aromatase (ARO) was studied in the dorsal and ventral premammillary nuclei (PMd and PMv) of the newborn rat by immunohistochemistry. In the intact male pups, nNOS immunoreactivity (-IR) was present both in the PMd and the PMv, while AR-IR was detected only in the PMv. On the other hand, ER-IR and ARO-IR were scarcely encountered in the both PMd and PMv. By double immunostaining of nNOS and AR, all the nNOS-IR cells in the PMv were revealed to contain AR-IR. In the intact female pups, nNOS-IR was present in the both PMd and PMv, but neither ER-, nor ARO-LR were detected in the PM region. In the PMv of the intact female rat, no AR-IR was detected at 6 days of age, while it was detected as only a faint staining within 12 h after birth. When the male pups were castrated neonatally, no AR-IR was detected in the PMv. Subcutaneous injections of Scr-dihydrotestosterone (DHT) induced strong AR-IR in the castrated male and the intact female pups. On the contrary, the intensity of nNOS-IR stayed unchanged among these animals. Neonatal androgen and nitric oxide has been considered important to brain development. Moreover, involvement of the PMv in aggressive and mating behavior of male animals has been reported. Together with the fact that the AR-IR and nNOS-IR were found in the same neurons in the PMv, involvement of this nucleus in masculinization of the brain by non-aromatizable androgen is postulated. (C) 1997 Elsevier Science B.V.

To analyze the importance of premammillary region in male behavior such as aggression and mating, distribution of androgen- and estrogen-receptors (AR and ER), aromatase (ARO) and neuronal nitric oxide synthase (nNOS) was studied in the dorsal and ventral premammillary nuclei (PMd and PMv) of the rat by immunohistochemistry. The nNOS-immunoreactivities (-IR) were present both in the PMd and the PMv, while AR-IR were detected only in the PMv. AR-IR became undetectable after orchidectomy but they recovered by an injection of 5 alpha-dihydrotestosterone (DHT). In both nuclei, no clear signals of ER-IR were encountered. On the other hand, ARO-IR were found in the PMv but only very few. In the PMv, although DHT did not increase nNOS-IR significantly in castrated males, all the nNOS-IR cells contained AR-IR at least in intact and castrated-DHT injected males. Thus, involvement of nNOS-nitric oxide system in the PMv in the androgenic action on male behaviors was suggested.

The transient expression of estrogen receptor (ER) in the ventromedial subnucleus of the facial nucleus was previously detected in the newborn rat, and the expression of ER molecules was down-regulated by daily injections of estradiol. Here we examined possible involvement of aromatization in this process. ER molecules were measured by immunohistochemistry and in situ hybridization histochemistry after daily injections of testosterone propionate (TP;100 mu g/0.02 ml) and estradiol benzoate (EB; 10 mu g/0.02 ml) in the male pups castrated within 24 h of birth. Daily injections of TP for 5 consecutive days did not suppress ER and ER mRNA in the facial nucleus, while they were both suppressed by daily injections of EB. Moreover, aromatase immunoreactivity was not detected in the facial nucleus of both castrated, TP injected and intact control males at 6 days of age. The present findings therefore suggest that ER molecules expressed transiently in the facial nucleus are not directly involved in masculine sexual differentiation of the brain in newborn rat.

Delayed-type hypersensitivity (DTH) responses to rat hepatocytes (HCs) in mice were investigated by flow cytometric analysis and immunohistochemistry with monoclonal antibodies directed against murine class II, CD4, and CD8 antigens. Mice were immunized subcutaneously (s.c.) with 10(6) rat HCs (referred to as s.c.-immunized mice), and control mice were injected s.c. with sterile Hanks' solution (non-immunized mice). Four days later, 10(5) rat HCs were injected into the footpad of s.c.-immunized mice and non-immunized mice. The DTH response in s.c.-immunized mice significantly increased after challenge when compared to that in non-immunized mice. The numbers of class II+, CD4(+), and CD8(+) cells in the footpad, and CD4(+) and CD8(+) cells in the inguinal lymph node of s.c.-immunized mice significantly increased during the DTH response. An increase in the number of CD4(+) cells in the footpad of s.c.-immunized mice after challenge was more significant than that of non-immunized mice. The number of CD4(+) cells increased more markedly in the footpad of s.c.-immunized mice as compared to that of CD8(+) cells. Furthermore, immunohistochemical studies of the footpad of s.c.-immunized mice showed more severe infiltration of CD4(+) cells rather than CD8(+) cells at the injection site of rat HCs. These results suggest that the DTH response in the footpad of mice immunized with rat HCs is associated with severe infiltration of CD4(+) cells.

Occurrence of estrogen receptor-immunoreactivity (ER-IR) in the cerebral cortex was examined in neonatal and adult rats. In newborn rats of postnatal day 1 (= day of birth) and postnatal day 5 (PD1 and PD5, respectively), ER-IR was not evident in the neocortex. On postnatal days 7, 10 and 13 (PD7, PD10 and PD13 respectively), a group of cells with distinct ER-IR appeared in the layer V of the auditory cortex. At the PD10, weak but specific ER-IR were also appeared in the somatosensory and the visual cortices. Among these areas, the ER-IR positive neurons occurred most frequently in the auditory cortex at PD10 rats. By examination of adjacent sections, one stained with Cresyl violet and the another stained with acethylcholinesterase (AChE) histochemistry, it was revealed that the region with ER-IR at PD7 to PD13 was limited to layer V of the neocortex. These signals, however, disappeared at PD15. In layer II of the neocortex, on the other hand, weak ER-IR signals were detected throughout the area sporadically at PD21 and in adults. The ER-IR detected transiently in the auditory cortex by the antiserum might contribute to maturation and establishment of the neurons of the rat auditory circuit.

Nitric oxide and estrogen have been shown to play a critical role in the control of female reproductive function. In order to determine an anatomical relationship between nitric oxide generating neurons and estrogen target neurons, NADPH-diaphorase histochemistry was combined with estrogen receptor immunohistochemistry in the female medial preoptic area. While only a few weakly stained neurons for NADPH-diaphorase were found in ovariectomized control rats, a drastic increase in NADPH-diaphorase activity was observed in the medial preoptic nucleus of estradiol-treated ovariectomized animals. The total number of NADPH-diaphorase neurons in the estradiol-treated group increased three-fold relative to controls, and more than 80% of those neurons contained estrogen receptor-immunoreactivity in their nuclei. Since neuronal NADPH-diaphorase is nitric oxide synthase, the present result suggests that nitric oxide synthase activity can be positively regulated by estradiol in neurons containing estrogen receptor in the female medial preoptic nucleus.

Effects of gonadal steroids on numbers of neurons containing estrogen receptor (ER) and/or substance P (SP) were examined in the anteroventral periventricular nucleus (AVPV) of female and male rats by double-labeling immunohistochemistry employing antibodies specific for ER and SP. Animals were gonadectomized and received subcutaneously either oil alone (Control group), sequential injections of estradiol benzoate and oil (EB + Oil group), or those of EB and progesterone (EB + P group). In the female control rat, a large population of ER-immunoreactive (in) cells were found clustered throughout the AVPV. They were counted more than 2,000 in total of 4 sections in this nucleus. On the contrary, SP-IR neurons were scarcely observed in the same area of this group. Administration of estrogen to female animals decreased the total number of ER-IR cells to 67% of the control group. In contrast to the suppressive effect of estrogen to its own receptor, it induced SP-IR neurons in the AVPV of the female. Approximately 50-80 SP-IR neurons were counted in the 4 sections, and 59% of these neurons expressed ER-IR material in their nuclei. In the female EB + P group, the number of ER-IR neurons also decreased to 79% of the control group. Although the number of SP-IR neurons in this group decreased to 32% of that in the EB + Oil group, a ratio of coexistence of ER-IR material in these neurons increased to 75%. The male control group contained a smaller population of ER-IR cells relative to the female control (1497 vs 2143). SP-IR neurons were rarely observed as were in the female control. Administration of estrogen to the male also decreased the number of ER-IR cells in a manner similar to that in the female. However, unlike the female, the steroid failed to induce the SP-IR neurons in the male. These results demonstrate sexual dimorphism in the AVPV not only in the number of ER-IR neurons but also in the responsiveness of SP neurons to estrogen. They further provide anatomical evidence that a subset of SP neurons are regulated by estradiol in estrogen sensitive neurons in the female rat, The data also suggest that this peptide is involved in mechanisms of luteinizing hormone surge by mediating actions of gonadal steroids in the AVPV.

NADPH-diaphorase (NADPH-d) histochemistry was combined with estrogen receptor (ER) immunohistochemistry in order to study effect of estrogen on NADPH-d activity and establish an anatomical relationship between NADPH-d and ER-immunoreactivity (ir) containing neurons in the ventromedial hypothalamic nucleus (VMN). Gonadectomized female and male rats received either oil (control group) or 10 mu g of estradiol benzoate (EB) for two successive days and were sacrificed on day 4. While NADPH-d histochemistry stained a specific subpopulation of neurons in the ventrolateral portion of the VMN of all groups, there was a marked sex difference in the effect of EB treatment. EB elevated the number of NADPH-d positive neurons in females but had no significant effect in males. Double-labeling histochemistry revealed that more than 70% of the NADPH-d positive neurons contained ER-ir in the VMN of all groups, with EB-treated females having a significantly higher frequency than all other groups. Since neuronal NADPH-d is nitric oxide synthase (NOS), these results provide anatomical evidence for an association of estrogen with NOS in the rat VMN and suggest that NOS activity can be modulated by estradiol in estrogen sensitive neurons.