Biomedical subjects

Yasuo Sakuma

Publications and source records attributed to Yasuo Sakuma.

30 records · Page 2Linked to original sources

PubMed2004

Induction of Fos immunoreactivity in oxytocin neurons in the paraventricular nucleus after female odor exposure in male rats: effects of sexual experience.

1. We examined whether oxytocin (OT) neurons in the paraventricular nucleus of the hypothalamus (PVN) were activated by estrus female odor and sexual contact in sexually naïve and experienced Long-Evans rats. 2. Male rats were not presented to anesthetized estrus females (control) or presented to the females without (exposure to the female odor without sexual contact) or with direct contact (exposure to the female odor with sexual contact). 3. Exposure to the female odor with sexual contact significantly increased OT neurons with Fos-ir in both males. Exposure to the female odor without contact increased OT neurons with Fos-immunoreactive cells (Fos-ir) in sexually experienced males but not in naïve males, suggesting that the female odor without sexual contact activated the oxytocinergic neuronal system in the PVN in the experienced males. 4. Therefore, exposure to the estrus female odor itself may exert different effects on sexually naïve and experienced males.

Animals↗

PubMed2004

Expressional regulation of neuronal and cancer-related genes by estrogen in adult female rats.

Adult female rats were ovariectomized and treated with or without estrogen for two weeks. mRNA was obtained from the hypothalamus, uterus, liver, kidney and skeletal muscle and analyzed by Northern blotting and/or RT-PCR. We examined two types of estrogen-responsive genes from rats, neuronal system-related genes (Amphiregulin, AR; Neuropeptide Y-Y1 receptor, NPY-Y1R; Bassoon, BSN; N-Cadherin, N-CADH) and estrogen-susceptible cancer-related genes (C-terminal binding protein interacting protein, CtIP), based on the results of a cDNA microarray analysis which was carried out to profile estrogen-responsive genes in the human breast cancer cell line MCF-7. The N-CADH gene showed identical response to that in MCF-7 cells. In the hypothalamus, all except the AR gene were down-regulated in their expression. In other tissues, the expression showed marked differences: expression of the BSN gene was not detected by either method, and the NPY-Y1R gene showed down-regulation in most tissues except for skeletal muscle. We then analyzed the time course of the estrogen-responsiveness of these genes in several tissues, finding changes in expression patterns especially in skeletal muscle but not in the hypothalamus. Our results show that the estrogen-responsive genes, which were demonstrated simply as either up- or down-regulated in their expression by estrogen in a human cell line using cDNA microarrays, exhibit tissue and temporal-specific expression patterns in adult female rats.

Animals↗

PubMed2004

Using a customized DNA microarray for expression profiling of the estrogen-responsive genes to evaluate estrogen activity among natural estrogens and industrial chemicals.

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.

Biological Assay↗

PubMed2003

Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats.

Functional analysis of GnRH neurons is limited, although these neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiological analysis of GnRH neurons. To identify GnRH neurons, we tagged the neurons with green fluorescence protein by a transgenic technique. A dispersed culture of GnRH neurons was prepared from the transgenic rats. After overnight culture, a perforated patch clamp was applied to the identified GnRH neurons to analyze the Ca2+ currents. In neonatal GnRH neurons, high voltage-activated Ca2+ currents were clearly observed, but low voltage-activated Ca2+ current was negligible. Nimodipine (L-type channel blocker) and omega-conotoxin GVIA (N-type channel blocker) each attenuated the current by approximately 20%. The R-type channel blocker SNX-482 attenuated the current by approximately 55%. Inhibition by the P/Q-type channel blocker omega-agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca2+ currents were observed. Inhibitions by nifedipine, omega-conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by omega-agatoxin IVA was clearly seen in 40-61% of the GnRH neurons examined. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R-, and T-type channels. Expressions of P/Q- and T-type channels are developmentally regulated.

Animals↗

PubMed2003

Ghrelin gene in cichlid fish is modulated by sex and development.

We have identified the complementary and genomic DNA sequences of the novel growth hormone-releasing peptide, ghrelin, in the cichlid fish (tilapia Oreochromis niloticus). The tilapia ghrelin precursor cDNA was 855 bp long, consisting of 119 bp of 5(')-untranslated region, 324 bp open reading frame, and 412 bp of 3(')-untranslated region. The serine residues at positions 2 and 3 of the "active core" (GSSF) of the ghrelin mature peptide are conserved between tilapia and the mammalian species. The tilapia ghrelin gene has four exons and three introns and resembles the structure of other known ghrelin genes. RT-PCR analysis revealed ghrelin mRNA predominantly expressed in the stomach but absent in the brain, pituitary, heart, kidney, ovary, and testis. Real-time PCR analysis showed an age/body size dependent increase in gastric ghrelin, which stagnated at 7 cm body size (onset of maturation). Ghrelin mRNA levels were unchanged in food-deprived sexually mature animals but were significantly higher in females compared to males. The present study shows that the structure of ghrelin peptide is highly conserved, and the reported differences in somatic and gonadal growth in tilapia could be a consequence of age- and sex-related synthesis of gastric ghrelin.

Age Factors↗

PubMed2003

Activation of accessory olfactory bulb neurons during copulatory behavior after deprivation of vomeronasal inputs in male rats.

We examined whether the vomeronasal organ (VNO) is the sole receptor for pheromonal cues for male sexual behavior. Males carrying surgical removal of the VNO (VNOx) mated with stimulus females as sham-operated males, with a comparable number of mounts but a prolonged latency for ejaculation. In sham-operated males, mating increased cFos immunoreactivity in the granule and mitral cell layers of the accessory olfactory bulb and in the medial amygdala. VNOx diminished baseline as well as mating-induced cFos in the granule cell layer and in the medial amygdala; VNOx had no effect on either basal or induced cFos immunoreactivity in the mitral cell layer. Thus, during mating encounter, cFos expression in the mitral cell layer did not depend on VNO inputs. The medial amygdala may be modulated by impulses other than of mitral cell origin.

Animals↗

PubMed2003

FSH and LH-beta subunits in the preoptic nucleus: ontogenic expression in teleost.

In the present study we cloned, sequenced, and confirmed the presence of mRNAs of gonadotropins (FSH-beta, LH-beta subunits) from the brain and pituitary of tilapia, Oreochromis niloticus. Further, we examined the spatio-temporal expression pattern of FSH-beta and LH-beta in the brain and pituitary of two species of teleost (tilapia, O. niloticus; sockeye salmon, Oncorhynchus nerka), using in situ hybridization and immunological methods. The expression of FSH and LH immunoreactivity appeared simultaneously in the brain and pituitary (tilapia, 14 days; sockeye, 51 days after fertilization). In the pituitary, FSH mRNA and peptide expressing cells were distinct from LH expressing cells located in the ventral proximal pars distalis. In the brain, FSH and LH immunoreactivity was co-localized in cells of the preoptic nucleus parvocellularis, magnocellularis, and gigantocellularis. Fibers immunoreactive to FSH and LH antisera were seen along the forebrain-hypothalamus and in the neurohypophysis of the pituitary. Double-label immunofluorescence revealed FSH and LH immunoreactivity co-localized in arginine vasotocin synthesizing preoptic neurons. Our results show that FSH and LH-producing cells have developmental origins in the brain as well as in the pituitary. In addition, we propose that the brain-derived gonadotropins may function as hypophysiotropic hormones that regulate pituitary cells and along with arginine vasotocin could act as neuromodulators of reproductive behaviors.

Age Factors↗

PubMed2003

Single-cell real-time quantitative polymerase chain reaction of immunofluorescently identified neurons of gonadotropin-releasing hormone subtypes in cichlid fish.

The regulatory mechanisms controlling gene expression of GnRH subtypes, particularly of the evolutionarily conserved GnRH2, remain speculative. To address this issue, we have successfully coupled the anatomic specificity of immunofluorescently defined "cell picking" with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of GnRH mRNAs in individual neurons. Here, using RT-Q-RT-PCR, we report change in the levels of transcripts of GnRH subtypes in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. The levels of GnRH1 mRNA per cell and the percentage of neurons expressing GnRH1 transcripts exceeding 0.05 x 10(2) fg/cell were significantly higher in mature males (44.2%) compared with immature males (4.7%). In contrast, there was no difference in mRNA levels and the percentage of cells expressing GnRH2 and GnRH3 between the two reproductive states. Thus, using a novel approach that enables immunofluorescently labeled single-cell RT-Q-RT-PCR analysis of GnRH neurons, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Furthermore, we speculate that the use of this method will allow the identification and quantification of known and unknown genes in single GnRH neurons, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells of GnRH variants in vertebrate species.

Aging↗

PubMed2002

Ligand-dependent transcriptional enhancement by DNA curvature between two half motifs of the estrogen response element in the human estrogen receptor alpha gene.

We previously reported five DNA bend sites (ERB-4 to -1, and ERB+1) in the promoter region of the human estrogen receptor alpha (ERalpha) gene [FEBS Lett. 444 (1999) 117]. One of these sites, ERB-2, was accompanied by two half motifs of the estrogen response element (ERE) and several short poly(dA)(.)poly(dT) tracts including an A(4) tract located next to a half ERE motif. This A(4) tract and the 20 bp immediate flanking sequence containing a half ERE motif (T3B) exhibited DNA curvature. Transcription assays using luciferase as a reporter gene indicated that T3B sequence conferred positive estrogen responsiveness. Mutations introduced in this sequence indicated that both bendability and estrogen responsiveness were synergistically associated with the A(4) tract located next to the half ERE motif. This motif and a mutant sequence, GGTTA, had affinity for ERalpha protein, which seems to account for ERalpha protein binding to the region without an ERE motif. These findings suggest that some DNA curvature acts as a transcriptional modulator by modifying the state of ligand effects.

Base Sequence↗

PubMed2002

Sexually dimorphic expression of estrogen receptor beta in the anteroventral periventricular nucleus of the rat preoptic area: implication in luteinizing hormone surge.

Striking sex difference was detected in the expression of estrogen receptor (ER) beta mRNA and protein by nonisotopic in situ hybridization and immunohistochemistry in the anteroventral periventricular nucleus (AVPV) of the rat preoptic area. In females more than in males, a significantly larger number of ERbeta mRNA-positive cells were visualized in the medial-most portion of the AVPV within 50 microm from the ependymal lining of the third ventricle. Rats of 7, 14, 21, 35, and 60 days of age (d 1 = day of birth) showed the sex difference. Orchidectomy of male neonates or estrogen treatment of female pups reversed the brain phenotype when examined on d 14. In the AVPV of adult females, ERalpha immunoreactivity colocalized in 83% of ERbeta mRNA-positive cells. Tyrosine hydroxylase immunoreactivity colocalized in 18% of ERbeta immunoreactive cells in d 21 females. Infusion of an ERbeta antisense oligonucleotide into the third ventricle in the vicinity of the AVPV resulted in significantly longer days of successive estrus and a 50% reduction in the number of ERbeta-immunoreactive cells in the AVPV. These findings provide support for the hypothesis that activation of ERbeta in the AVPV is an important regulatory event in the female-typical induction of luteinizing hormone surge by estrogen.

Animals↗

PubMed2002

Sex difference in the expression and regulation of nitric oxide synthase gene in the rat preoptic area.

Neuronal nitric oxide synthase (nNOS) mRNA-positive cells were visualized by non-isotopic in situ hybridization histochemistry in the organum vasculosum of the lamina terminalis (OVLT) and the preoptic area (POA) in gonadectomized juvenile female and male rats. In the rostral POA (rPOA) at the level of the anteroventral periventricular nucleus, nNOS mRNA-positive cells were distributed in an inverted V-shaped area over the third ventricle and were in close proximity to cell bodies of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. In the caudal POA (cPOA) at the level of the medial preoptic nucleus, no topological association existed between GnRH and nNOS. Throughout the rPOA, both the number and the area of nNOS mRNA positive cells were significantly larger in the gonadectomized females than in the gonadectomized males. Treatment with estradiol for 2 days, followed by progesterone in the next morning, which caused an increase in serum luteinizing hormone 6 h later, induced a significant reduction of the nNOS mRNA expression in the rPOA in the female but not in the male rat at the time of sacrifice. In the OVLT and the cPOA, ovarian steroids had no effect on nNOS mRNA expression of both sexes. The results indicate that nNOS mRNA expression in the rPOA is sexually dimorphic and regulated by ovarian steroids in a sex specific manner.

Animals↗

PubMed2002

3',5'-cyclic adenosine monophosphate augments intracellular Ca2+ concentration and gonadotropin-releasing hormone (GnRH) release in immortalized GnRH neurons in an Na+ -dependent manner.

In GT1-7 cells, cAMP increases the intracellular Ca2+ concentration ([Ca2+](i)) through activation of the voltage-gated Ca2+ channels, thereby facilitating GnRH release. To activate these channels, the membrane potential must be depolarized. In the present study we hypothesize that cAMP depolarizes the cells by increasing the membrane Na+ permeability, as in the case of somatotrophs and pancreatic beta-cells. To examine this, we analyzed [Ca2+](i) and [Na+](i) in GT1-7 cells by an intracellular ion-imaging technique along with cAMP assay by RIA. Forskolin, a direct activator of adenylyl cyclase, increased [Ca2+](i) and [Na+](i) via cAMP formation. The forskolin-induced increase in [Ca2+](i) depended on the presence of Ca2+ and Na+ in the extracellular solution. This response was blocked by the voltage-gated Ca2+ channel blocker, nifedipine; the nonselective cation channel blocker, gadolinium (Gd3+); and the cyclic nucleotide-gated channel blocker, l-cis-diltiazem. In contrast, the forskolin-induced increase in [Na+](i) depended only on extracellular Na+, not on Ca2+. Gd3+ and l-cis-diltiazem also blocked the increase in [Na+](i). Furthermore, the forskolin-induced increase in GnRH release was blunted in both low Ca2+ and low Na+ media. The results indicate that cAMP increases the membrane Na+ permeability, probably through nonselective cation channels on GT1-7 cells, thereby promoting GnRH release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗