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Fertility decreases during aging in human and bovine females, but the exact pathophysiological mechanisms in the oviducts and uteri are not clarified yet. Anti- Müllerian hormone (AMH) is a glycoprotein that belongs to the transforming growth factor -β superfamily. Plasma AMH concentration can predict the fertility of adult female goats, ewes, cows, and women via unknown physiological mechanisms. This thesis study attempted to clarify whether AMH, and the main receptor for AMH, AMH receptor type 2 (AMHR2) have important roles for the age-related infertility. In first, I investigated whether the primary receptor for AMH, AMHR2, is expressed in bovine oviducts and endometria. Reverse transcription polymerase chain reaction (RTPCR) detected expression of AMHR2 mRNA in oviductal and endometrial specimens. Western blotting and immunohistochemistry were performed to analyse AMHR2 protein expression using anti-bovine AMHR2 antibody. Immunohistochemistry revealed robust AMHR2 expression in the tunica mucosa of the ampulla and isthmus, as well as in the glandular and luminal epithelium of the endometrium. The number of AMHR2-positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. AMHR2 mRNA (measured RT-qPCR) and AMHR2 protein expression in these layers did not significantly differ among oestrous phases in adult Japanese Black (JB) cows (P>0.1). In addition, AMHR2 mRNA and protein expression in these layers did not differ Among old Holsteins (mean (±SEM age 91.9±6.4 months ) and young (26.6±0.8 months) and old (98.8±10.2 months) JB cows. Therefore, AMHR2 is expressed in bovine oviducts and endometria. Other important hormones for endocrinological regulation have paracrine and autocrine roles. Therefore, in the next study, I investigated whether bovine oviducts and endometria produce AMH. RT-PCR and western blotting detected AMH expression in oviductal and endometrial specimens. Immunohistochemistry revealed robust AMH expression in the ampulla and isthmus epithelia, and the glandular and luminal endometrial epithelia (caruncular endometria). The number of AMH-positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. AMH mRNA and protein expression in these layers did not significantly differ among estrous phases in adult JB heifers (p > .1). Furthermore, the expression in these layers also did not differ among Holstein cows (93.8 ± 5.8 months old), JB heifers (25.5 ± 0.4 months old), and JB cows (97.9 ± 7.9 months old). We also compared AMH concentrations in the oviduct and uterine horn fluids among the three groups (measured by immunoassays). Interestingly, the AMH concentration in the oviduct fluid, but not in the uterine horn fluid, of Holstein cows was lower than those in JB heifers and cows (p < .05). Therefore, bovine oviducts and endometria express AMH and likely secrete it into the oviduct and uterine fluids. Collagen, the most abundant extra-cellular matrix in oviducts and uteri, performs critical roles in pregnancies. I hypothesised that the locations and amounts of both denatured collagen and the collagen-specific molecular chaperone 47-kDa heat shock protein (HSP47) in the oviducts and uteri of old cows are different compared with those of young heifers because of repeated pregnancies. Since detecting damaged collagen in tissues is challenging, we developed a new method that uses a denatured collagen detection reagent. Then, we compared damaged collagen in the oviducts and uteri between postpubertal growing nulliparous heifers (22.1 ± 1.0 months old) and old multiparous cows (143.1 ± 15.6 months old). Further, I evaluated the relationship between denatured collagen and HSP47 by combining this method with fluorescence immunohistochemistry. Picro sirius red staining showed collagen in almost all parts of the oviducts and uteri. Expectedly, damaged collagen was increased in the oviducts and uteri of old cows. However, damaged collagen and HSP47 were not located in the same area in old cows. The number of HSP47- positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. These organs of old cows showed higher HSP47 protein amounts than those of heifers. However, the uteri, but not oviducts, of old cows had lower HSP47 mRNA amounts than those of heifers. These findings revealed the specific location and amounts of denatured collagen and HSP47 in the oviducts and uteri of old cows compared with those of heifers. Therefore, I discovered the AMH, AMHR2 expression in several important layers of oviducts and uteri, and I discovered the increased AMH, AMHR2, and HSP47 in the fibroblasts after aging. However, still role of AMH, AMHR2 in oviducts and uteri were not clarified yet. Therefore, in the next study, I hypothesized that AMH stimulate HSP47 expression in fibroblast and epithelium. I cultured uterine fibroblasts and epithelial cells obtained from heifers. Then, I treated the cells with recombinant with increasing concentrations (0, 1, 10, or 100 ng mL-1) of AMH. HSP47 expression was measured by western blotting. AMH stimulated (P<0.05) HSP47 expression in epithelial cells but not in fibroblasts. Therefore, these findings suggested the role of AMH to cause the abnormal high HSP47 expression in the oviducts and uteri of old cows. In conclusion, this thesis discovered the AMH and AMHR2 in bovine oviducts and uteri, which have important roles for collagen synthesis via HSP47.

転写因子CCAAT/enhancer-bringing protein beta（C/EBPβ）は、IGF-binding protein-1（IGFBP-1）やprolactin（ERL）遺伝子のプロモーターおよびエンハンサー領域において、転写活性マーカーであるHistone-H3 lysine-27 アセチル化(H3K27ac)を誘導するパイオニア因子であり、ヒト子宮内膜間質細胞（ESC）の脱落膜化に貢献することを我々はこれまでに報告している。パイオニア因子の一部はヒストンアセチルトランスフェラーゼ（HAT）活性を有するコファクターと複合体を形成することで機能する。我々は、C/EBPβと共役するHATタンパクとしてp300を同定しているが、それ以外のコファクターについては不明である。Peroxisome proliferator-activated receptor gamma comma coactivator 1-α（PGC-1α）はH3K27acを制御することが知られている転写共役因子である。PGC-1αはESCでも発現しているが、脱落膜化におけるPGC-1αの機能は不明である。そこで、PGC-1αがC/EBPβの転写共役因子として働き、脱落膜化過程においてH3K27ac誘導に関与しているのではないかと考え検討した。脱落膜化を誘導するためにESCをcAMP存在上で培養した。cAMPによるIGFBP-1およびPRLの発現上昇はPGC-1αのノックダウンにより抑制された。また、cAMPはIGFBP-1およびPRLのプロモーターとエンハンサー領域に存在するC/EBPβ結合部位へのPGC-1αとp300のリクルートを増加させた。さらに、PGC-1αをノックダウンするとC/EBPβとp300に結合、およびH3K27acレベルが低下したことから、PGC-1αはこれらの領域でC/EBPβおよびp300とヒストン修飾複合体を形成することでH3K27ac誘導に関与していることが示された。さらにPGC-1αの発現制御機構を調べるために、C/EBPβを上流因子として着目した。PGC-1αのエンハンサー領域へのC/EBPβの結合はcAMPで増加した。また、これらのエンハンサーをゲノム編集により欠失させた細胞ではPGC-1αの発現が減少したことから、C/EBPβは我々が見出したエンハンサー領域に結合することでPGC-1αの発現を上昇させると考えられた。以上より、PGC-1αはC/EBPβの新規エンハンサーへの結合によって発現誘導されること、また、C/EBPβとp300と共にヒストン修飾複合体を形成して、IGFBP-1およびPRLのプロモーターとエンハンサーにエピゲノム変化を引き起こすことで脱落膜化に貢献していることがわかった。

【目的】代謝状態の朝晩の変化を理解することは、代謝障害を管理するためには重要である。我々は、インスリン分泌とインスリン感受性の観点からこの朝晩の変化を解析し、さらに概日リズムとの関連性を含めて検討することを目的とした。 【方法】非糖尿病成人男性14人と10人に対し、それぞれ75ｇ経口ブドウ糖負荷試験(OGTT)と高インスリン正常血糖（HE）クランプ実験を行った。各被験者に対し、午前8時と午後8時にOGTTまたはHEクランプを1回ずつ行った。ただし試験時間の順序は無作為とした。各試験直前に毛根を採取し、毛包における時計遺伝子発現量をリアルタイムPCR法で解析した。また、マウスの肝臓と筋肉でもAKTリン酸化をウエスタンブロット法により解析した。 【結果】OGTTの結果から、耐糖能は午前8時の方が良好であることがわかった。これは、OGTTにおける負荷後60分までのインスリン早期分泌とHEクランプで説明される骨格筋インスリン感受性の違いに起因すると考えられた。一方、OGTTにおける肝臓のインスリン抵抗性指数によって推定される肝インスリン感受性は、午後8時の方が良好であった。60分までのインスリン分泌量と肝臓のインスリン抵抗性指数の朝晩の差は、Per2のｍRNAの相対的発現量と有意に相関していた。ΔGIR（20時の値－8時の値）は、Δ非エステル化脂肪酸（NEFA）と有意に相関したが、時計遺伝子発現との相関は認められなかった。ΔNEFAは、E4bp4のmRNA発現量およびΔコルチゾールと有意に相関していた。マウスでは、ヒトの研究から予想されるように、AKTリン酸化は活動時間の初めに肝臓で減少し、筋肉で増加していた。 【結語】各組織の糖代謝は朝と晩で大きく異なっており、脂質代謝、時計遺伝子およびコルチゾールの影響を受けていることがわかった。この関連性についてのより深い知見が代謝障害の新たな改善法の開発に寄与する可能性がある。

右心室 (RV) の機能障害とそれに関連する不整脈は、肺動脈性肺高血圧症 (PAH) の予後の重要な決定要因として認識されている。今回心臓リアノジン受容体 (RyR2) の安定剤であるダントロレン (DAN) による右室心筋への直接的な薬理学的介入が、モノクロタリン (MCT) 誘発性PAHラットモデルにおいてRV機能障害および不整脈に対する保護効果を有するかどうかを調査することを目的とした。方法として雄の8週齢のSprague-Dawleyラットに、PAHの誘発のためにMCTを腹腔内投与した。カテコールアミンによる心室頻拍 (VT) の誘発も、単離された心筋細胞におけるRyR2を介したCa^{2+}放出特性に関連して評価された。RVの形態と機能に対する慢性的な圧力過負荷の独立した影響を評価するためには、肺動脈縮窄モデルも確立した。結果、MCT誘発PHAラットモデルでは、RV肥大、拡張、および機能低下が観察され、MCT誘発2か月後の生存率は0%であった。対照的に、慢性DAN治療はこれらすべてのRVパラメータを改善し、生存率を80%に増加させた。慢性的なDAN療法はまた、RyR2からのカルモジュリンの解離を防ぎ、それによってMCTによって誘発された肥大したRV心筋細胞におけるCa^{2+}スパークと自発的なCa^{2+}トランジェストを抑制した。エピネフリンは、MCT誘発性PAHのラットの50%以上でVTを誘発したが、慢性DAN治療によってVTの完全に抑制した。以上よりDANによるRyR2の安定化は、PAHに関連するRV機能障害および致命的な不整脈の発症に対する新しい治療薬としての可能性を秘めている。

We previously discovered that SPC/Fyn/Rho-kinase (ROK) pathway mediates the Ca^{2+}-sensitization of coronary arterial smooth muscle (CASM) contraction leading to vasospasm, a major cause of sudden death. Lately, we have been trying to find and develop more natural edible compounds which can treat and/or prevent the SPC-induced abnormal CASM contraction, and finally the first to discover that tangeretin (5,6,7,8,4'-prentamethoxyflavone), a natural compound extracted from citrus plants, can inhibit the SPC-induced CASM contraction both in the pretreatment and posttreatment. In porcine CASM tissues, tangeretin showed remarkable inhibitory effects on the SPC-induced contraction with modest inhibitory effects on the high K^+-depolarization-induced Ca^{2+}-dependent contraction, both in pretreatment and posttreatment at the optimal concentrations; Regarding the mechanisms, tangeretin markedly abolished the SPC-induced cell contraction through inhibiting the SPC-induced activation and translocation of Fyn and ROK from the cytoplasm to the cell membrane in cultured CASM cells, resulting in the reduction of phosphorylation of myosin light chain. Taken together, these findings indicate that tangeretin, upon pre- or post- treatment, inhibits the SPC-induced CASM contraction through suppressing the Fyn/ROK signaling pathway, thereby suggesting that tangeretin can be a potential candidate for the treatment and/or prevention of vasospasm.

Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor and being considered as a core molecule of stigmoid body (STB). Brain or spinal cord regions with abundant STB/HAP1 expression are usually spared from neurodegeneration, whereas the regions with little STB/HAP1 expression are always neurodegenerative targets. The enteric nervous system (ENS) can act as a potential portal for pathogenesis of neurodegenerative disorders. To date, the expression of HAP1 and its neurochemical characterization have never been examined there. In the current study, we determined the expression and immunohistochemical phenotypes of HAP1 in ENS of adult rodents using Western blotting and light/fluorescence microscopy. HAP1 immunoreactivity was strongly expressed in both myenteric and submucosal plexuses of ENS. STBs were observed in the cytoplasm of most of the HAP1-immunoractive (ir) cells in ENS. In myenteric plexus, a large number of calretinin, calbindin, NOS, VIP, ChAT, SP, somatostatin, and TH-ir neurons showed HAP1 immunoreactivity. In contrast, most of the CGRP-ir neurons were devoid of HAP1-immunoreactivity. In submucosal plexus, almost all the cholinergic secretomotor neurons containing ChAT/ CGRP/ somatostatin/ calretinin, non-cholinergic secretomotor neurons containing VIP/TH/calretinin and vasodilator neurons containing VIP/calretinin express HAP1. Our current study is the first to clarify that HAP1 is highly expressed in excitatory motor neurons, inhibitory motor neurons, and interneurons but almost absent in sensory neurons in myenteric plexus. While, HAP1 is expressed in all neuronal subgroups of Meissner’s plexuses. These suggest that due to lack of putative STB/HAP1 protectivity, the sensory neurons (Dogiel type II) might be more vulnerable to neurodegeneration than STB/HAP1-expressing Dogiel type I neurons in myenteric plexus and secretomotor/vasodilator in Meissner’s plexuses. Our current results may reflect the involvement of HAP1 in modulation of excitatory and inhibitory motor neuron functions in myenteric plexus and the secretomotor and vasodilator functions of submucosal neurons. It will be of great interest to elucidate the physiological or pathological roles of HAP1 in ENS. Our current results might lay a basic foundation for future studies that seek to clarify the physiological/pathological effects of STB/HAP1 in the ENS.

Zero-determinant strategies are memory-one strategies in repeated　games which unilaterally enforce linear relations between expected payoffs of　players. Recently, the concept of zero-determinant strategies was extended　to　the class of memory-n strategies with n _ 1, which enables more compli-cated control of payoffs by one player. However, what we can do by　memory-n　zero-determinant strategies is still not clear. Here, we show that memory-n　zero-determinant strategies in repeated games can be used to control condi-tional expectations of payoffs. Equivalently, they can be used to control ex-pected payoffs in biased ensembles, where a history of action pro_les with large　value of bias function is more weighted. Controlling conditional expectations　of payoffs is useful for strengthening zero-determinant strategies, because play-ers can choose conditions in such a way that only unfavorable action pro_les　to one player are contained in the conditions. We provide several examples　of memory-n zero-determinant strategies in the repeated prisoner's dilemma　game. We also explain that a deformed version of zero-determinant strategies　is easily extended to the memory-n case.

To date, the process of implementing the global Sustainable Development Goals （SDGs） and global climate change mitigation and adaptation obligations has been largely decoupling in most countries. This creates administrative duplication, costs, and hinders the development of effective problem solving. Against this background, we show the present state of research on policy integration and integrated policymaking. Following the research results of Bauer et al. （2021） and Teebken et al. （2021）, this note briefly summarizes different understandings of policy integration and detailed criteria for evaluating policy integration from different　academic perspectives. Implementing an interactive agenda requires political　attention and institutional competence to address the interactions and complexity　of each, understand policies at a national level, and translation. In this note, we will　introduce the basic concept of what integration practice looks like, and focusing on　the interaction between the 2030 Agenda and the Paris Agreement.