研究者詳細 - 井上　直子

Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats.

Sugimoto A, Tsuchida H, Nagae M, Inoue N, Uenoyama Y, Tsukamura H

The Journal of reproduction and development 2022年3月

Oikawa Mami, Nagae Mayuko, Mizuno Naoaki, Iwatsuki Kenyu, Yoshida Fumika, Inoue Naoko, Uenoyama Yoshihisa, Tsukamura Hiroko, Nakauchi Hiromitsu, Hirabayashi Masumi, Kobayashi Toshihiro

MOLECULAR REPRODUCTION AND DEVELOPMENT 89 巻 ( 3 ) 頁： 129 - 132 2022年3月

記述言語：日本語出版者・発行元：Molecular Reproduction and Development

Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.

DOI： 10.1002/mrd.23562

Cellular and molecular mechanisms regulating the KNDy neuronal activities to generate and modulate GnRH pulse in mammals

Ikegami Kana, Watanabe Youki, Nakamura Sho, Goto Teppei, Inoue Naoko, Uenoyama Yoshihisa, Tsukamura Hiroko

FRONTIERS IN NEUROENDOCRINOLOGY 64 巻頁： 100968 2022年1月

記述言語：日本語出版者・発行元：Frontiers in Neuroendocrinology

Accumulating findings during the past decades have demonstrated that the hypothalamic arcuate kisspeptin neurons are supposed to be responsible for pulsatile release of gonadotropin-releasing hormone (GnRH) to regulate gametogenesis and steroidogenesis in mammals. The arcuate kisspeptin neurons express neurokinin B (NKB) and dynorphin A (Dyn), thus, the neurons are also referred to as KNDy neurons. In the present article, we mainly focus on the cellular and molecular mechanisms underlying GnRH pulse generation, that is focused on the action of NKB and Dyn and an interaction between KNDy neurons and astrocytes to control GnRH pulse generation. Then, we also discuss the factors that modulate the activity of KNDy neurons and consequent pulsatile GnRH/LH release in mammals.

DOI： 10.1016/j.yfrne.2021.100968

Kisspeptin neurons as a key player bridging the endocrine system and sexual behavior in mammals

Nakamura Sho, Watanabe Youki, Goto Teppei, Ikegami Kana, Inoue Naoko, Uenoyama Yoshihisa, Tsukamura Hiroko

FRONTIERS IN NEUROENDOCRINOLOGY 64 巻頁： 100952 2022年1月

記述言語：日本語出版者・発行元：Frontiers in Neuroendocrinology

Reproductive behaviors are sexually differentiated: for example, male rodents show mounting behavior, while females in estrus show lordosis behavior as sex-specific sexual behaviors. Kisspeptin neurons govern reproductive function via direct stimulation of gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release for gonadal steroidogenesis in mammals. First, we discuss the role of hypothalamic kisspeptin neurons as an indispensable regulator of sexual behavior by stimulating the synthesis of gonadal steroids, which exert “activational effects” on the behavior in adulthood. Second, we discuss the central role of kisspeptin neurons that are directly involved in neural circuits controlling sexual behavior in adulthood. We then focused on the role of perinatal hypothalamic kisspeptin neurons in the induction of perinatal testosterone secretion for its “organizational effects” on masculinization/defeminization of the male brain in rodents during a critical period. We subsequently concluded that kisspeptin neurons are key players in bridging the endocrine system and sexual behavior in mammals.

DOI： 10.1016/j.yfrne.2021.100952

Central mu-Opioid Receptor Antagonism Blocks Glucoprivic LH Pulse Suppression and Gluconeogenesis/Feeding in Female Rats

Tsuchida Hitomi, Kawai Narumi, Yamada Koki, Takizawa Marina, Inoue Naoko, Uenoyama Yoshihisa, Tsukamura Hiroko

ENDOCRINOLOGY 162 巻 ( 10 ) 2021年10月

記述言語：日本語出版者・発行元：Endocrinology (United States)

Energetic status often affects reproductive function, glucose homeostasis, and feeding in mammals. Malnutrition suppresses pulsatile release of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) and increases gluconeogenesis and feeding. The present study aims to examine whether β-endorphin-μ-opioid receptor (MOR) signaling mediates the suppression of pulsatile GnRH/LH release and an increase in gluconeogenesis/feeding induced by malnutrition. Ovariectomized female rats treated with a negative feedback level of estradiol-17β (OVX+low E2) receiving 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, intravenously (iv) were used as a malnutrition model. An administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective MOR antagonist, into the third ventricle blocked the suppression of the LH pulse and increase in gluconeogenesis/feeding induced by iv 2DG administration. Histological analysis revealed that arcuate Kiss1 (kisspeptin gene)-expressing cells and preoptic Gnrh1 (GnRH gene)-expressing cells co-expressed little Oprm1 (MOR gene), while around 10% of arcuate Slc17a6 (glutamatergic marker gene)-expressing cells co-expressed Oprm1. Further, the CTOP treatment decreased the number of fos-positive cells in the paraventricular nucleus (PVN) in OVX+low E2 rats treated with iv 2DG but failed to affect the number of arcuate fos-expressing Slc17a6-positive cells. Taken together, these results suggest that the central β-endorphin-MOR signaling mediates the suppression of pulsatile LH release and that the β-endorphin may indirectly suppress the arcuate kisspeptin neurons, a master regulator for GnRH/LH pulses during malnutrition. Furthermore, the current study suggests that central β-endorphin-MOR signaling is also involved in gluconeogenesis and an increase in food intake by directly or indirectly acting on the PVN neurons during malnutrition in female rats.

DOI： 10.1210/endocr/bqab140

Kisspeptin Neurons and Estrogen-Estrogen Receptor alpha Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction

Uenoyama Yoshihisa, Inoue Naoko, Nakamura Sho, Tsukamura Hiroko

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 17 ) 2021年9月

記述言語：日本語出版者・発行元：International Journal of Molecular Sciences

Estrogen produced by ovarian follicles plays a key role in the central mechanisms control-ling reproduction via regulation of gonadotropin‐releasing hormone (GnRH) release by its negative and positive feedback actions in female mammals. It has been well accepted that estrogen receptor α (ERα) mediates both estrogen feedback actions, but precise targets had remained as a mystery for decades. Ever since the discovery of kisspeptin neurons as afferent ERα‐expressing neurons to gov-ern GnRH neurons, the mechanisms mediating estrogen feedback are gradually being unraveled. The present article overviews the role of kisspeptin neurons in the arcuate nucleus (ARC), which are considered to drive pulsatile GnRH/gonadotropin release and folliculogenesis, in mediating the estrogen negative feedback action, and the role of kisspeptin neurons located in the anteroventral periventricular nucleus‐periventricular nucleus (AVPV‐PeN), which are thought to drive GnRH/lu-teinizing hormone (LH) surge and consequent ovulation, in mediating the estrogen positive feedback action. This implication has been confirmed by the studies showing that estrogen‐bound ERα down‐ and up‐regulates kisspeptin gene (Kiss1) expression in the ARC and AVPV‐PeN kisspeptin neurons, respectively. The article also provides the molecular and epigenetic mechanisms regulating Kiss1 expression in kisspeptin neurons by estrogen. Further, afferent ERα‐expressing neurons that may regulate kisspeptin release are discussed.

DOI： 10.3390/ijms22179229

Morphological Analysis of the Hindbrain Glucose Sensor-Hypothalamic Neural Pathway Activated by Hindbrain Glucoprivation

Sato Marimo, Minabe Shiori, Sakono Takahiro, Magata Fumie, Nakamura Sho, Watanabe Youki, Inoue Naoko, Uenoyama Yoshihisa, Tsukamura Hiroko, Matsuda Fuko

ENDOCRINOLOGY 162 巻 ( 9 ) 2021年9月

記述言語：日本語出版者・発行元：Endocrinology (United States)

Lowered glucose availability, sensed by the hindbrain, has been suggested to enhance gluconeogenesis and food intake as well as suppress reproductive function. In fact, our previous histological and in vitro studies suggest that hindbrain ependymal cells function as a glucose sensor. The present study aimed to clarify the hindbrain glucose sensor-hypothalamic neural pathway activated in response to hindbrain glucoprivation to mediate counterregulatory physiological responses. Administration of 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, into the fourth ventricle (4V) of male rats for 0.5 hour induced messenger RNA (mRNA) expression of c-fos, a marker for cellular activation, in ependymal cells in the 4V, but not in the lateral ventricle, the third ventricle or the central canal without a significant change in blood glucose and testosterone levels. Administration of 2DG into the 4V for 1 hour significantly increased blood glucose levels, food intake, and decreased blood testosterone levels. Simultaneously, the expression of c-Fos protein was detected in the 4V ependymal cells; dopamine β-hydroxylase-immunoreactive cells in the C1, C2, and A6 regions; neuropeptideY (NPY) mRNA-positive cells in the C2; corticotropin-releasing hormone (CRH) mRNA-positive cells in the hypothalamic paraventricular nucleus (PVN); and NPY mRNA-positive cells in the arcuate nucleus (ARC).Taken together, these results suggest that lowered glucose availability, sensed by 4V ependymal cells, activates hindbrain catecholaminergic and/or NPY neurons followed by CRH neurons in the PVN and NPY neurons in the ARC, thereby leading to counterregulatory responses, such as an enhancement of gluconeogenesis, increased food intake, and suppression of sex steroid secretion.

DOI： 10.1210/endocr/bqab125