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メダカの視索前野においてステロイドホルモン感受性に性差を示すニューロン群

菊池, 結貴子 東京大学 DOI:10.15083/0002002282

2021.10.13

概要

生殖腺に精巣と卵巣があるように、脳にもオス型とメス型があり、その違いが種々の行動や内分泌のパターンに雌雄の違いをもたらしている。脳の性分化について、真骨魚類は脳が性分化した後でもその性を逆転させうる、つまり生涯にわたって性的な可逆性をもつという、他の脊椎動物とは異なる大きな特徴をもつ。真骨魚類のもつ脳の性的可逆性のメカニズムを解明することは、動物全体における脳の性、またその多様性を理解する上で非常に重要である。しかし現在、真骨魚類のもつ脳の性的可逆性のメカニズムはまったく未解明であり、その基盤となるはずの真骨魚類の脳の性差についても知見が不足している。
 こうした状況の中、近年、真骨魚類の脳内で発現に性差を示す遺伝子の探索が行われ、性行動の中枢として知られる終脳と間脳の境界領域、視索前野において多数の遺伝子の発現に性差が見出された。そこで本研究では、モデル動物として確立され性差研究において数々の利点をもつメダカを用い、視索前野に存在する性差をさらに深く探求する目的で、視索前野においてステロイドホルモン感受性に性差を示すニューロン群の探索やその性状解析を行った。

第1章 視索前野の神経ペプチド産生ニューロンにおけるグルココルチコイド受容体発現の性差
 本章では、ストレスホルモンとして知られるグルココルチコイドの受容体(GR)の全脳における発現パターンを解析し、雌雄で比較した。多くの脊椎動物において、ストレスに対する脳の応答には性差があることが知られており、これらには脳におけるグルココルチコイド感受性の性差が関与すると考えられた。さらに、グルココルチコイドが真骨魚類の性決定や性転換のプロセスに大きく影響することを示唆する現象も複数報告されており、脳におけるグルココルチコイドシグナル伝達の性差が真骨魚類の性転換メカニズムの一部を担う可能性も考えられた。また、これまで主要なステロイドホルモンのうち、エストロゲンとアンドロゲンの受容体遺伝子の脳における発現パターンとその性差は詳細に調べられていたにもかかわらず、グルココルチコイド受容体の発現パターンと性差に関する知見は真骨魚類で一切得られておらず、ステロイドホルモン感受性の性差に関する知見は不十分であった。そこで本研究では、メダカの脳におけるグルココルチコイド感受性の性差を解析した。
 解析の結果、視索前野の神経核PPa、Pblと、間脳の神経核NGpにおいて、GRがメスに偏って発現していることが明らかになった。PPaとPblでは攻撃行動や性行動、性成熟への関与が示されている神経ペプチドバソトシン(Vt)、イソトシン(It)、ゴナドトロピン放出ホルモン(GnRH)1が産生されていることから、次にこれらの神経ペプチド産生ニューロンにおけるGRの発現を検証した。その結果、GRがこれらの神経ペプチド産生ニューロンで発現すること、またPPaのVtニューロンとGnRH1ニューロンに占めるGR発現ニューロンの割合はメスの方が高いことがわかった。PPaのVtニューロンは脳下垂体を介したストレス応答、あるいは社会性行動に参加すると考えられる一方、PPaのGnRH1ニューロンの機能は未知であるが、本研究により、これらのこれらの神経ペプチドニューロンを介した生理作用にストレスが与える影響はオスよりもメスでより大きいことが示唆された。

第2章 視索前野のメス特異的Npbaニューロンのエストロゲン応答性
 第2章からは対象をGR発現ニューロンから変更し、数年前に発見された、メダカの脳内でほぼメス特異的にエストロゲン受容体とアンドロゲン受容体を発現する視索前野の神経核PMm/PMgに含まれるペプチド産生ニューロンに着目して研究を行った。これらはNeuropeptide B(Npba)を発現する大細胞性ニューロンで、メスにはみられるがオスには全くみられないという顕著な性差を示す。さらに、これらのニューロンは性ステロイド環境に応答してnpbaの発現量を可逆的に変化させていることも知られていた。これらのメス特異的なNpbaニューロンはこのように性ステロイドの影響を強く受けることから、性的なゆらぎをもつと考えられた。しかし、これまでの研究はこれらのニューロンにおけるnpbaや性ステロイド受容体遺伝子の発現のみに着目して行われており、ニューロンの他の形質や他の遺伝子発現の面からの性状解析は行われてこなかった。そこで、本章ではこれらのニューロンの制御機構と機能の解明を目標として、ニューロン自体に着目した多角的な解析を行うこととし、まずはその詳細な形態解析を行った。
 その結果、これらのニューロンは大型の細胞体と核、発達した小胞体とゴルジ体をもち、核内にはユークロマチン領域を多く有するといった活発なペプチドニューロンに特徴的な形態をもつことがわかった。また、これらの核ではRNAPⅡ-Ser2P、RNAPⅡ-Ser5P、H3K36me、H3K36me2といった、転写の活性化との関連が知られている化学修飾が亢進していた。さらに、これらの特徴は卵巣除去によって失われ、卵巣を除去した個体にエストロゲンを投与すると回復することも明らかになった。このことから、これらの特徴はエストロゲン依存的に保たれており、可逆的に変化することが示唆された。
 また、本来オスのメダカはPMm/PMgにメスのNpbaニューロンに相当する大細胞性ニューロンをもたないが、エストロゲンを投与するとNpbaニューロンが神経新生を経ずに誘導されることも示された。この結果は、オスのメダカにエストロゲンを投与するとそれまでオス型の不活性な形態をとっていたニューロンが活性化されてメス型の大細胞性ニューロンになるとともにNpbaを発現するようになること、つまりこれらのニューロンがオス型からメス型へ性転換することを示唆している。このことから、PMm/PMgのNpbaニューロンに相当するニューロンは雌雄どちらにも存在し、それらがエストロゲンの有無によって活性化、あるいは不活性化することで可逆的な性的二型を形成していると考えられた。

第3章 視索前野のメス特異的Npbaニューロンの遺伝子発現と機能
 第2章で見出されたNpbaニューロンの形態的な特徴から、これらのニューロンはエストロゲンシグナルに依存してnpba以外にも多数の遺伝子を高発現していると考えられた。そこで、第3章ではNpbaニューロンでエストロゲン依存的に高発現する遺伝子を未知のものも含めて広く把握する目的でトランスクリプトーム解析を行った。これらのニューロンのトランスクリプトームをIntact群、Sham群とOVX群で比較したところ、OVX群において発現量が低下している転写産物が87個見出された。この結果から、予想通りこれらのニューロンは多数の遺伝子をエストロゲン依存的に発現していることが示された。
 次に、OVX群において発現量が低下していた転写産物の中から6つの遺伝子を選出し、これらに着目してさらなる解析を行うことにした。はじめに発現量の変動を追試したところ、5遺伝子で実際に卵巣除去による発現量の低下が確認できた。そこで、この5遺伝子のノックアウトメダカをCRISPR/Cas9システムを用いて作出し、それらの表現型解析を通してPMm/PMgのメス特異的なNpbaニューロンの機能解析を行った。その結果、そのうちの1つであるXLOC_004257のノックアウトメスでは、オスから求愛されてからそれを受け入れ産卵に至るまでの時間が野生型メスよりも短くなっていることがわかった。また、卵巣の観察により、これらのノックアウトメスの卵巣の発達と排卵は正常に行われていることが確かめられた。このことから、ノックアウトメスの性行動にみられた異常は脳内の変化に起因するものと考えられた。この結果から、エストロゲン存在下のPMm/PMgのNpbaニューロンが、オスの求愛を受け入れるか否かの判別に寄与することが示唆された。

 以上、本研究ではメダカ視索前野PPaのVtニューロン、GnRH1ニューロンにおいてGRの発現に性差があることを示した。また、視索前野PMm/PMgのメス特異的なNpbaニューロンは形態や化学修飾、遺伝子発現といった多数の形質についてエストロゲン依存的であることを示し、またこれらが求愛の受け入れに寄与することを示唆した。これらの成果により、本研究の最終目標であった「真骨魚類に特徴的な脳の性的可逆性のメカニズム解明」の基盤となる、脳の性差と性的可逆性についての知見を新たに提供した。特に本研究で強く示唆されたPMm/PMgのNpbaニューロンの性的可逆性は脳の性的可逆性のメカニズムに直結すると考えられる。今後、これらのニューロンにおける活発な遺伝子発現の機構、オスの脳内におけるこれらに相当するニューロンの探索と性状解析によって、今回示唆された性的可逆性の全容が解明され、真骨魚類の脳の性的可逆性のメカニズムが明らかになることが期待される。

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