Mechanisms underlying estrogen-induced sexual differentiation in the hypothalamus.

Estrogen plays critical roles in the sexual differentiation of the developing brain and gender-specific regulation of reproductive neuroendocrinology. Of the different regions of the brain, it is well known that hypothalamic areas contain key sexually differentiated neuronal circuits. Estrogen receptor (ER) proteins localized in the nucleus affect the expression of target genes when bound to their ligand estrogen. However, recent studies suggest that this may not be the only mechanism of estrogen action. Instead, estrogen can influence various cellular events through regulating different signaling pathways. Cross-talk between direct effects of estrogen on gene transcription and its effects on signaling pathways should be examined in future to elucidate mechanisms underlying sexual differentiation in the hypothalamus.

Circadian rhythm of melatonin release from the photoreceptive pineal organ of a teleost, ayu (Plecoglossus altivelis) in flow-thorough culture.

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow-through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free-running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six-hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.

Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats.

In an attempt to define the role of the pineal secretory melatonin and 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 GSH/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.

Developmental changes in the localization of activated C-JUN N-terminal kinase (JNK/SAPK) in the chick spinal cord.

To examine the role of c-Jun N-terminal kinase (JNK/SAPK) in the developing nervous system of vertebrates, the localization of an active form of JNK, phosphorylated JNK (p-JNK), was studied in the lumbosacral spinal cord of the chick embryo. We also examined the localization of phosphorylated neurofilaments (NFs, potential targets of p-JNK) and cyclin-dependent kinase 5 (Cdk5), which is known to phosphorylate cytoskeletal proteins, including NFs, and compared their expression with that of p-JNK. Additionally, the localization of phosphorylated forms of c-Jun and ATF-2 was compared with that of p-JNK. On embryonic day 3 (E3), the expression of p-JNK was observed in regions containing early-projecting axons. Axons in these regions also expressed phosphorylated NFs. Subsequently, on E5 and E8, the expression of both p-JNK and phosphorylated NFs increased concomitantly in the axonal tracts in the spinal white matter. Thus, white matter expressed both p-JNK and phosphorylated NFs, whereas there was only weak expression of Cdk5. By E13, the spinal cord expression pattern of p-JNK and phosphorylated NFs had changed compared to earlier ages. Although phosphorylated NFs were still expressed in the white matter, the expression of p-JNK was decreased in axons in the white matter, whereas strong p-JNK expression appeared in cell nuclei in the gray matter. In summary, the present study revealed that the localization of p-JNK in the spinal cord changes dramatically from axons to cell nuclei during development, suggesting multiple roles of p-JNK, depending on the developmental age.

Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors.

The ubiquitin-proteasome system has been regarded as being important in the progression of neurodegenerative diseases, although its exact role remains uncertain. This in vitro study using PC12h cell cultures examined whether interference with the ubiquitin-proteasome system by proteasome inhibitors induces the neuropathological features of neurodegenerative diseases. Perikaryal accumulation of phosphorylated neurofilaments and an increase in c-Jun as well as phosphorylated form of c-Jun and apoptosis-specific protein were induced by the proteasome inhibitors lactacystin and N-carbobenzoxy-leucyl-leucyl-leucinal. These changes were not observed when only calpain was inhibited. The present study therefore suggests the possibility that a perturbation of the ubiquitin-proteasome system may be one of the causes that result in the development of neuropathological features. Additionally, activity assays showed that the proteasome inhibitor caused an increase in the activity of c-Jun N-terminal kinase (JNK/SAPK), which can phosphorylate neurofilaments and c-Jun, suggesting the possible involvement of JNK in phosphorylation of these proteins.

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase).

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.

Female-soiled bedding induced fos immunoreactivity in the ventral part of the premammillary nucleus (PMv) of the male mouse.

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).

Proteasome inhibitors which induce neurite outgrowth from PC12h cells cause different subcellular accumulations of multi-ubiquitin chains.

The effects of two proteasome inhibitors on neurite outgrowth from PC12h cells were investigated in terms of the mean length of the neurites and the frequency of occurrence of cells with long neurites. Benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and benzyloxycarbonyl-isoleucyl-t-butyl-glutamyl-leucinal (PSI) caused a significant elongation of PC12h cell neurites. Since ZLLLal is known to inhibit both calpain and proteasome activity, we examined the effects ofbenzyloxycarbonyl-leucyl-leucinal (ZLLal) which inhibits calpain activity to the same degree as ZLLLal, but which inhibits proteasome activity only weakly. ZLLal did not induce the significant elongation of neurites at any of the concentrations we studied. These results show that the inhibition of proteasome activity causes neurite elongation. We also quantified subcellular levels of multi-ubiquitin chains and free ubiquitin after treatments with PSI, ZLLLal and ZLLal. Treatment with ZLLal had no effects on levels of water- and urea-soluble multi-ubiquitin chains or of free ubiquitin either in the nucleus or in the cytoplasm. PSI and ZLLLal induced a large accumulation of water- and urea-soluble multi-ubiquitin chains and free ubiquitin in the nucleus. Similarly, PSI and ZLLLal increased cytoplasmic levels of urea-soluble multi-ubiquitin chains. On the contrary, PSI and ZLLLal had no effect on levels of water-soluble multi-ubiquitin chains or free ubiquitin in the cytoplasm. This is the first study to demonstrate subcellular differences in the accumulation of multi-ubiquitin chains and free ubiquitin during the neurite elongation induced by proteasome inhibitors.

Effects of a low-protein diet on prolactin- and growth hormone-producing cells in the rat pituitary gland.

BACKGROUND: It is well known that an unbalanced diet induces various changes in the pituitary gland. However, little attention has been paid to the molecular aspects of this perturbation. We studied the influence of a low-protein diet (LPD) on the prolactin (PRL) and growth hormone (GH) cells in the rat pituitary gland using immunohistochemical staining and in situ hybridization. MATERIALS: Rats aged 20 days were fed a diet containing 27% protein or one with 8% protein (LPD) for 30 days. Pituitary glands were obtained and subjected to either immunohistochemistry or in situ hybridization. Quantitative morphological analysis was then conducted to determine cell number and area as well as the percentage of cells stained by the respective antisera and/or cDNA probe in each experimental group. RESULTS: The average sectional areas of both PRL- and GH-producing cells in the LPD group were smaller in size than those in the controls. The cell numbers per unit area (mm2) of PRL-positive cells and PRL mRNA-positive cells were 3,596.5 and 3,948.6, respectively, in the LPD group, and 3,179.6 and 4,888.5, respectively, in the controls. The numbers per unit area of GH-positive cells and GH mRNA-positive cells in the LPD group were similar (2,252.3 and 2,224.4), as compared to 2,161.3 and 1,684.2, respectively, in the well-fed rats. Whereas PRL-positive cells comprised about 27% of the total number of cells in both animal groups, those given the LPD contained a lower percentage (29%) of PRL mRNA-positive cells as compared to the controls (44%). On the other hand, GH mRNA-positive cells numbered about 15% of the total cell population both animal groups; however, the malnourished rats contained a lower percentage (16%) of GH-positive cells than did their well-fed counterparts (20%). CONCLUSIONS: Taken together, these results indicate that in the rat pituitary gland, administration of an LPD reduced the size of PRL- and GH-positive cells as well as differentially affecting a subpopulation of the PRL mRNA-positive cells and the GH-positive cells.

Characterization, guanosine 5'-O-(3-thiotriphosphate) modulation, daily variation, and localization of melatonin-binding sites in the catfish (Silurus asotus) brain.

Characteristics, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, daily variation, and localization of melatonin-binding sites in the brain of a nocturnal teleost, the catfish Silurus asotus, were studied by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The specific binding was rapid, stable, saturable, and reversible. The radioligand binds to a single class of receptor site with an affinity (Kd) of 30.7 +/- 7.3 pM and total binding capacity (Bmax) of 9.76 +/- 0.79 fmol/mg protein (mean +/- SE, n = 5). The binding sites were highly specific for 2-iodomelatonin and melatonin. The specificity was almost identical to that of functional melatonin receptors in the dermal and epidermal melanophores in this species and that of ML-1 subtype melatonin receptors in vertebrates, including melatonin-binding sites in the goldfish brain. GTPgammaS treatment altered both the Kd and Bmax values, indicating that melatonin-binding sites in the catfish brain are coupled to G protein. The Bmax values exhibited no daily variation under light-dark cycles of 12 hr light:12 hr dark whereas plasma melatonin levels and Kd fluctuated in a rhythmic fashion. The density of melatonin-binding sites in discrete brain areas was determined to be highest in optic tectum-thalamus and hypothalamus, intermediate in telencephalon, cerebellum, and medulla oblongata, and lowest in olfactory bulbs. These results suggest that melatonin secreted from the pineal organ and/or retina plays neuromodulatory roles in the catfish brain via G protein-coupled melatonin receptors. Characteristics of melatonin receptors seem to be highly conserved during evolution, although the density of melatonin receptors is not regulated by melatonin itself in this species.

Photic and circadian regulations of melatonin rhythms in fishes.

Photic and circadian regulations of melatonin rhythms in the pineal organ and the retina of several teleosts were studied to investigate the regulatory mechanisms of melatonin rhythms in fishes. In the eyecup preparations of the goldfish, Carassius auratus, both time of day and lighting conditions affected melatonin production, with high melatonin production observed only in the dark-treated group incubated during the 'subjective' night. Thus, in the goldfish retina, local photoreceptors and an ocular circadian clock seem to regulate melatonin production, as in the zebrafish retina and in the pineal organ of a number of teleosts, including the goldfish. However, this circadian regulation of melatonin rhythms is not universal among fishes. Although the superfused pineal organ of the masu salmon Oncorhynchus masou secreted melatonin in a rhythmic fashion under light-dark (LD) cycles, the rhythm disappeared under constant darkness (DD), as in the rainbow trout, with a large amount of melatonin released both during the subjective day and the subjective night. These results suggest that all salmonids lack circadian regulation of melatonin rhythms. Furthermore, when ocular melatonin rhythms were compared in two cyprinids, the ugui Tribolodon hakonensis and the oikawa Zacco platypus occupying different ecological niches, ocular melatonin contents exhibited daily variations, with higher values during the dark phase of LD cycles in both species. The rhythmic changes persisted in the ugui under DD, with higher levels at subjective midnight than at subjective midday; however, ocular melatonin levels in the oikawa were consistently high under DD. Thus, the circadian regulation of melatonin rhythms in fishes is influenced not only by phylogeny, but also by the ecological niches of the animals. These results suggest that the physiological functions of melatonin in the circadian and photoperiodic systems differ among fishes.

Ocular melatonin rhythms in the goldfish, Carassius auratus.

Ocular melatonin rhythms in the goldfish were studied and compared to those in the pineal organ and plasma. Under light:dark (LD) of 12 h light:12 h dark, melatonin contents in the eye as well as the pineal organ and plasma exhibited clear day-night changes with higher levels at mid-dark than at mid-light. However, melatonin contents in the eye at mid-light and mid-dark were approximately 100 and 9 times greater than those in the pineal organ, respectively. Day-night changes of ocular melatonin persisted after pinealectomy, which abolished those in plasma melatonin under LD 12:12. Ocular melatonin contents in the pinealectomized fish at mid-light were significantly higher than those in the sham-operated control. Under constant darkness (DD), circadian melatonin rhythms were observed in the eye but damped on the 3rd day, whereas plasma melatonin rhythms generated by the pineal organ persisted for at least 3 days. Under constant light, ocular melatonin contents exhibited a significant fluctuation with a smaller amplitude than that under DD, whereas plasma melatonin remained at low levels. These results indicate the involvement of LD cycles, a circadian clock, and the pineal organ in the regulation of ocular melatonin rhythms in the goldfish.

Administration of melatonin and related indoles prevents exercise-induced cellular oxidative changes in rats.

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.

Regulation by guanine nucleotides and cations of melatonin binding sites in the goldfish brain.

Effects of nucleotides and cations on 2-[125I]iodomelatonin binding sites in the goldfish brain were examined. Nucleotides (10(-6)-10(-3) M) dose-dependently inhibited the specific binding with the following order of potency: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) > GTP = GDP > GMP = ATP > cyclic GMP. Cyclic AMP was ineffective. The treatment of membranes with GTP gamma S induced rapid dissociation of 2-[125I]iodomelatonin from membranes when added at the steady state, increased the Kd and decreased the Bmax values as revealed by saturation analysis, and increased the IC50 value of melatonin to inhibit the specific binding. The treatment decreased the specific binding to membrane preparations obtained from six brain regions as well. Inorganic salts (5-200 mM) dose-dependently inhibited the specific binding with the following order of potency: CaCl2 > MgCl2 > LiCl > NaCl > choline chloride > KCl, except for 5 mM MgCl2, which enhanced the specific binding. Saturation experiments demonstrated that 75 mM CaCl2, 100 mM MgCl2 and 200 mM NaCl increased the Kd and decreased the Bmax while 5 mM MgCl2 increased the Bmax value. These results imply that G protein and physiological concentrations of cations are involved in the regulation of melatonin binding sites in the goldfish brain.

Nerve growth factor (NGF) induces increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin in nuclei of PC12h.

Changes in Ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PC12h cells. Ubiquitin-immunoreactivity was increased in the nucleus of NGF-treated cells. The quantitative analysis revealed that, after 7 days of NGF treatment, almost 20% of cells had ubiquitin-immunoreactive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions: urea- and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Levels of multi-ubiquitin chains did not change in urea-soluble cytoplasmic extracts as well as nuclear urea-soluble ones after NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC12h cells.

Effects of pinealectomy and constant light exposure on day-night changes of melatonin binding sites in the goldfish brain.

Effects of pinealectomy and constant light exposure on day-night changes of melatonin binding sites in the goldfish brain were examined. The density and affinity of binding sites were higher at mid-day than at mid-night in sham-pinealectomized goldfish under light-dark cycles. The rhythms disappeared after pinealectomy, or constant light exposure both of which abolish plasma melatonin rhythms. The effects of pinealectomy and constant light exposure were not additive. These results indicate that diel changes of melatonin binding sites in the goldfish brain are regulated by endogenous melatonin of pineal origin.

Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates.

Twenty-four edible plants were investigated for the presence of melatonin, heretofore considered to be a molecule found only in the animal kingdom. The amount of melatonin in different plants varied greatly with highest melatonin being present in plants of the rice family. Melatonin was identified by radioimmunoassay and verified by high performance liquid chromatography with fluorescence detection. Feeding a diet containing plant products rich in melatonin to chicks increased radioimmunoassayable levels of melatonin in their blood. Likewise, melatonin extracted from plants inhibited binding of [125I]iodomelatonin to rabbit brain. Thus, melatonin ingested in foodstuffs enters the blood and is capable of binding to melatonin binding sites in the brain of mammals.

Characteristics, day-night changes, subcellular distribution and localization of melatonin binding sites in the goldfish brain.

Melatonin binding sites in the goldfish brain were characterized by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. Specific binding of 2-[125I]iodomelatonin was rapid, stable, saturable and reversible. Saturation experiments demonstrated that 2-[125I]iodomelatonin binds to a single class of receptor site with an affinity constant (Kd) of 29.8 +/- 0.7 pM and a total binding capacity (Bmax) of 11.47 +/- 0.33 fmol/mg protein at mid-light. At mid-dark, the Bmax value decreased significantly to 7.90 +/- 0.23 fmol/mg protein (P < 0.01) with no significant variation in the Kd value (33.8 +/- 1.5 pM). Competition experiments revealed the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine > 5-methoxytryptophol > 5-methoxyindole-3-acetic acid. 5-Hydroxytryptamine, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, norepinephrine and acetylcholine exhibited no inhibition. Subcellular distribution of melatonin binding sites was demonstrated to be greatest in the P2 and P3 fractions as compared with the P1 fraction. Localization of melatonin binding sites in discrete brain areas was determined to be highest in the optic tectum-thalamus and hypothalamus, intermediate in the telencephalon, cerebellum and medulla oblongata, and lowest in the olfactory bulbs and pituitary gland. These results suggest that characteristics of melatonin receptors are highly conserved during evolution and that in this species melatonin plays neuromodulatory roles in the central nervous system through specific receptors.

Increase in ubiquitin-immunoreactive nuclei in rat pituitary luteinizing hormone cells after castration.

Immunocytochemical detection of ubiquitin in the nucleus of rat LH cells and the effects of castration and testosterone replacement on the occurrence of immunoreactive ubiquitin in the nucleus were investigated. Immunoreactive ubiquitin occurred in certain nuclei, mostly belonging to identified LH cells. The concentration of testosterone in blood was altered by castration and implantation of testosterone into castrated rats, and the occurrence of ubiquitin was examined weekly for the following 4 weeks. In castrated rats, the proportion of LH cells with ubiquitin-immunoreactive nuclei was high throughout the experiment. In castrated rats implanted with testosterone, on the contrary, the proportion remained significantly lower. Ubiquitin may be involved in the cellular activity of LH cells in the rat pituitary.