Mayumi Tanaka

Expression of estrogen receptors (α, β) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei ; sex and species differences

Abstract Sex steroids have been inferred to be involved in the regulation of affective status at least partly through the serotonergic (5-HT) system, particularly in the dorsal raphe nucleus (DRN), which innervates enormous projections to the cerebral cortex and limbic system. In the present study, the expression of estrogen receptors-α and -β (ERα, ERβ), androgen receptor (AR) and 5-HT was examined immunohistochemically in the rat and mouse DRN in both sexes. The results showed that large numbers of ERα- and/or ERβ-immunoreactive (ERα-I, ERβ-I) cells were found in the DRN of both male and female mice, whereas only small numbers of ERα-I cells and no ERβ-I cells were seen in the rat DRN of each sex. With respect to AR-immunoreactive (AR-I) cells, moderate numbers of such cells were present only in male rats and mice, and no or very few could be observed in female ones. The ERα-I, ERβ-I, and AR-I cells were mainly distributed in the rostral DRN. In double-immunostaining, many 5-HT-I neurons were found to show ERα and/or ERβ expression specifically in the rostral DRN (particularly dorsal, ventral and interfascicular parts) of mice of both sexes, but not in that of rats. In contrast, only a few 5-HT neurons were observed to show AR expression in the DRN of both rodents. The current results strongly suggest that sex steroids can modulate the affective regulation of the serotonergic system through ERα and/or ERβ in 5-HT neurons of the mouse rostral DRN (but not so much through AR), and that such effects might be different depending on the sex and species, as shown by the prominent sex differences in AR expression and prominent species differences in ERα and ERβ expression.

Region-specific expression and sex-steroidal regulation on aromatase and its mRNA in the male rat brain: immunohistochemical and in situ hybridization analyses.

The brain has an estrogen‐biosynthetic potential resulting from the presence of neuronal aromatase, which controls the intraneural sex‐steroidal milieu and is involved in brain sexual differentiation, psychobehavioral regulation, and neuroprotection. In the rat brain, three distinct aromatase‐P450‐immunoreactive (AromP450‐I) neural groups have been categorized in terms of their peak expression time (fetal, fetoneonatal, and young‐to‐adult groups), suggesting the presence of region‐specific regulation on brain AromP450. In the present study, we compared the expressions between AromP450 protein and mRNA by using immunohistochemistry and in situ hybridization with an ovary‐derived cRNA probe in serial sections of fetal, fetoneonatal, and adult male rat brains and then performed steroidal manipulations to evaluate the sex‐steroidal effects on AromP450 in adult orchiectomized and adrenalectomized (OCX + ADX) male rats. As a result, prominent mRNA signals were detected in the fetal (i.e., the anterior medial preoptic nucleus) and fetoneonatal (i.e., the medial preopticoamygdaloid neuronal arc) groups, although no detectable signal was found in the “young‐to‐adult” group (i.e., the central amygdaloid nucleus). In addition, the “fetoneonatal” AromP450‐I neurons were prominently reduced in number and intensity after OCX + ADX and then were reinstated by the administration of dihydrotestosterone, testosterone, or 17β‐estradiol. In contrast, none of the sex steroids had any significant effects on the young‐to‐adult group. Several possible explanations were explored for why the young‐to‐adult group may differ in aromatase expression and regulation, including the possibility that distinct splicing variants or isozymes for aromatase exist in the rat brain. J. Comp. Neurol. 500:557–573, 2007.

Prevalence and risk factors of urinary incontinence and its influence on the quality of life of Japanese women

Urinary incontinence is a common problem as women age. The aims of this study were to assess the prevalence and risk factors of urinary incontinence and their impact on the quality of life of working Japanese women. In this cross-sectional study, 975 women completed the Urogenital Distress Inventory-6, the Short Form (SF)-36 Health Survey, and the Kings Health Questionnaire. Their mean age was 47.6 years and the reported prevalence of stress, urge, and mixed urinary incontinence symptoms during the past month was 19.3%, 4.2%, and 7.8%, respectively. The prevalence rate of all the three types of incontinence increased with age and Body Mass Index. There was no significant difference in the questionnaire subscale scores of the SF-36 Health Survey and the Kings Health Questionnaire between these types and those without urinary incontinence. However, in relation to the SF-36 scores, we found that urge urinary incontinence had more effect on women than stress urinary incontinence. There was a high prevalence of urinary incontinence in the participants. The results emphasize the need for women to be provided with education programs about incontinence and to perform preventive exercises.

Neuroanatomical distribution of huntingtin-associated protein 1-mRNA in the male mouse brain

Huntingtin‐associated protein 1 (HAP1) was identified as an interactor of the gene product (Huntingtin) responsible for Huntingtons disease and found to be a core component of the stigmoid body. Even though HAP1 is highly expressed in the brain, detailed information on HAP1 distribution has not been fully described. Focusing on the neuroanatomical analysis of HAP1‐mRNA expression using in situ hybridization histochemistry, the present study clarified its detailed regional distribution in the entire mouse brain. Mouse HAP1 (Hap1)‐mRNAs were abundantly expressed in the limbic‐related forebrain regions and midline/periventricular brainstem regions including the olfactory bulb, limbic‐associated cortices, hippocampus, septum, amygdala, bed nucleus of the stria terminalis, preoptico‐hypothalamic regions, central gray, raphe nuclei, locus coeruleus, parabrachial nuclei, nucleus of the solitary tract, and area postrema. In contrast, little expression was detected in the striatum and thalamus, implying that Hap1 is associated with neurodegeneration‐sparing regions rather than target lesions in Huntingtons disease. The distribution pattern, resembling that of the stigmoid body, suggests that HAP1 and the stigmoid body are implicated in protection from neuronal death rather than induction of neurodegeneration in Huntingtons disease, and that they play an important role in integrating instinct behaviors and underlying autonomic, visceral, arousal, drive, memory, and neuroendocrinergic functions, particularly during extensive homeostatic or emotional processes. These data will provide an important morphological base for a future understanding of functions of HAP1 and the stigmoid body in the brain. J. Comp. Neurol. 478:88–109, 2004.

Gonadal and adrenal effects on the glucocorticoid receptor in the rat hippocampus, with special reference to regulation by estrogen from an immunohistochemical view-point

Focusing on the hippocampal CA1 region, effects of peripheral gonadal and adrenal steroids on the glucocorticoid receptor (GR) were immunohistochemically evaluated in male and female adult rat brains after adrenalectomy (ADX), gonadectomy (GDX), and administration of estradiol (E2) and/or corticosterone (CS). In ADXed male rats, the hippocampal nuclear GR decreased and turned back to the cytoplasm, whereas in females, nuclear localization persisted even after ADX. In GDX+ADXed female rats, the GR was dispersedly translocated from the nucleus to the cytoplasm as well as in GDX+ADXed males. The dispersed cytoplasmic GR was again translocated into the nucleus by administration of CS. In addition, administration of a small dose of E2 for 4-13 days was found to sufficiently recover the nuclear location of GR in GDX+ADXed rat brains, whereas medium-to-large doses could not do this. Also, a longer administration more strongly enhances the nuclear GR location and expression. The present study provided strong immunohistochemical evidence that the sexually dimorphic effects of ADX on hippocampal GR are attributable to gonadal hormones, and that E2 is implicated in the effects in inversely-dose- and directly-duration-dependent manner. Taken together, intriguing gonadal and adrenal crosstalk is considered to play some important role in regulating hippocampal GR morphology and to have a possibly crucial influence on stress-related disorders such as depression.

Hematopoietic progenitor cell count, but not immature platelet fraction value, predicts successful harvest of autologous peripheral blood stem cells

Mobilized stem cells in the peripheral blood (PB) must be efficiently harvested at the appropriate time before autologous PB stem cell (PBSC) transplantation. Enumeration of CD34+ cells in the PB before apheresis predicts the number of PBSCs that can be collected, but the cytometric techniques used are complex and expensive. Therefore, it is necessary to identify an alternative to the CD34+ cell count in PBSC harvest‐time monitoring. Fully automated flow cytometry using blood cell counters now allows reliable quantification of immature myeloid cells in the PB, referred to as hematopoietic progenitor cells (HPC), and reticulated platelets, expressed as the immature platelet fraction (IPF). Immature or reticulated platelets are thought to correlate with thrombopoietic activity of the marrow. Following a chemotherapy nadir, the recovery of white blood cell and platelet counts has been used to determine the right time for apheresis. Therefore, we examined whether the HPC count and IPF value could be used to predict PBSC mobilization in 20 patients with hematological malignancies. The HPC count was found to be correlated with the CD34+ cell count (r = 0.84, P < 0.01), whereas the IPF value was not (r = 0.37, P = 0.44). Therefore, the HPC count, but not the IPF value, is a possible predictor of the timing of autologous stem cell transplantation. J. Clin. Apheresis, 2011.

Alteration of adipokines during peripheral blood stem cell mobilization induced by granulocyte colony-stimulating factor.

Adipokines, soluble mediators produced by adipocytes, have been shown to play a role in various physiological and pathological conditions. We investigated the involvement of adipokines in granulocyte colony‐stimulating factor (G‐CSF)‐induced mobilization of hematopoietic stem cells in 21 healthy donors. We found that serum visfatin and resistin levels, but not leptin and adiponectin levels, were significantly elevated by G‐CSF treatment. G‐CSF treatment activated signaling proteins like extracellular signal‐regulated kinase and stimulated secretion of visfatin from 3T3‐L1 adipocytes. These findings suggest that some adipokines may play a role in G‐CSF‐induced mobilization of stem cells from the bone marrow into systemic circulation. J. Clin. Apheresis 2009.

Activation of the unfolded protein response in primary acute myeloid leukemia cells.

The unfolded protein response (UPR), which plays an important role in maintaining homeostasis of the endoplasmic reticulum (ER), is known to be activated in various solid tumors [1]. The role of the UPR in different forms of cancer or metastasis remains poorly characterized, and it is unclear whether UPR activation in cancer is due solely to microenvironmental stress, or to other mechanisms. The influence of the UPR on leukemogenesis remains largely uninvestigated. We previously reported that the UPR is activated in Philadelphia (Ph)-positive leukemia cells using cell lines and primary cells from Ph-positive acute lymphoid leukemia (ALL) patients [2]. In the present study, we investigated whether the UPR is activated in another type of acute leukemia, primary acute myeloid leukemia (AML).

Fluctuations in thrombopoietin, immature platelet fraction, and glycocalicin levels in a patient with cyclic thrombocytopenia

Cyclic thrombocytopenia (CT) is an uncommon disorder characterized by periodic fluctuations in platelet count, resulting in alternating episodes of thrombocytopenia and normal platelet counts. Its pathogenesis remains unclear. Here, we report the case of a 65-year-old man with CT who was initially diagnosed with idiopathic thrombocytopenic purpura (ITP) concomitant with chronic thyroiditis. ITP was diagnosed 12 years ago in the absence of periodic fluctuations in platelet counts. He had been treated with corticosteroids; underwent splenectomy; and subsequently been treated with azathioprine, vincristine, high doses of dexamethasone, and herbal medicine. He had not received antibiotic eradication therapy for Helicobacter pylori because there was no evidence of infection with this organism. Since his symptoms did not improve, the treatments were discontinued, and he was placed under observation. He complained of mild bleeding symptoms, such as petechiae and purpura, which occurred intermittently for several months before he presented to us to undergo detailed investigations. His platelet count varied between 3 9 10/ll and 30 9 10/ll approximately every 3–4 weeks. His white blood cell count and hemoglobin concentration remained unchanged. To elucidate the mechanism underlying this disease, we evaluated the serum thrombopoietin (TPO) level, immature platelet fraction (IPF), plasma glycocalicin index normalized for the individual platelet count (GCI), and T helper (Th)1/Th2 ratio. The serum TPO level was measured with a human TPO ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). The serum levels of TPO, a principal regulator of megakaryogenesis and thrombopoiesis, are thought to be regulated via receptor-mediated clearance by platelets and megakaryocytes. The median serum TPO concentration in healthy individuals is 51 pg/ml. The plasma glycocalicin level was measured with a Glycocalicin EIA kit (Takara Bio Inc., Ohtsu, Japan). The GCI was calculated as proposed by Steinberg et al. [1]: GCI = glycocalicin level 9 250 9 10/individual platelet count (/ll). Glycocalicin is a proteolytic fragment of the a-chain of glycoprotein Ib, a platelet membrane. The GCI reflects the rate of platelet destruction [2]. The mean GCI in healthy individuals is 1.27 (range 0.87–1.77). The IPF was determined to reliably quantify reticulated platelet counts by using a fully automated hematology analyzer XE-2100 equipped with special software IPF Master (Sysmex, Kobe, Japan). Reticulated platelets are RNA-rich immature platelets and are thought to reflect the ability of the bone marrow to produce platelets. Therefore, the IPF, as a surrogate marker of the reticulated platelet count, reflects the rate of thrombopoiesis [3]. The mean IPF in healthy individuals is 2.0% (range 0.5–5.7%). The Th1/Th2 ratio was determined by using flow cytometry analysis to measure the ratio of CD4? cells containing intracellular interferon-c to those containing interleukin-4. The fluctuations in the TPO levels, percentage of IPF, and GCI were synchronous and inversely proportional to those in the platelet counts (Fig. 1). In the thrombocytopenic phase, elevated GCI indicated platelet destruction in this patient. Thrombopoiesis was simultaneously accelerated in response to TPO. Consequently, the IPF, which is as an indicator of thrombopoiesis, increased. In the present case, CT seemed to be closely associated with cyclic increases in platelet destruction. However, the mechanism of cyclic platelet destruction remains to be elucidated. Th1/Th2 balance is T. Yujiri (&) Y. Tanaka M. Tanaka Y. Tanizawa Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan e-mail: yujirit@yamaguchi-u.ac.jp

Altered expression of circadian clock genes during peripheral blood stem cell mobilization induced by granulocyte colony-stimulating factor

Circulating hematopoietic stem cells exhibit robust circadian fluctuations, which influence the mobilized cell yield, even during enforced stem cell mobilization. However, alterations in the expression of circadian clock genes during granulocyte colony-stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization are not fully elucidated. Therefore, we measured the expression of these genes in human peripheral blood leukocytes from 21 healthy donors. While CRY1 mRNA expression significantly increased by 3.9-fold (p < 0.01), the expression of PER3, CRY2 and BMAL1 mRNAs significantly decreased (by 0.2-fold, 0.2-fold, and 0.6-fold, respectively; p < 0.001) after G-CSF administration. Moreover, CRY1 mRNA expression was inversely correlated with the plasma level of noradrenaline (r = −0.36, p < 0.05), while PER3, CRY2, and BMAL1 mRNA expression directly correlated with the plasma level of noradrenaline (r = 0.55, r = 0.66, and r = 0.57, respectively; p < 0.001). Thus, significant correlations between the levels of circadian clock gene mRNAs and the plasma level of noradrenaline, a sympathetic nervous system neurotransmitter, were established. The modulation of sympathetic activation and of the circadian clock may be novel therapeutic targets for increasing stem cell yields in PBSC donors.