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Dynamics of sperm-uterine interaction that initiates a maternal pro-inflammatory response in the cow

MOHAMMADU AKUTHAR Muhammed Ihshan 帯広畜産大学

2020.06.23

概要

哺乳動物では、交配後に膨大な数の精子が子宮に侵入する。しかし多くの種では、ほんの数千の精子だけが卵管にたどり着く。精子が雌性生殖器を通過する際には、それらの上皮と同時に免疫細胞群とも相互に作用し合う。子宮に侵入した精子は上皮に作用 し、それに続く一連の免疫反応は余分な精子、欠陥のある精子、死んだ精子などを子宮内から迅速に排除して、その後に起こる胚の着床に向けた準備を始めるために重要である。ウシにおいて、精子が子宮内膜とどのようにして相互に作用しあって免疫反応が誘導されるかについては、ほとんどわかっていない。私たちの研究グループは、これまでに精子がウシ子宮上皮細胞に結合すると、Toll 様受容体(TLRs)に関係するシグナル伝達経路が主に TLR2 を介して誘導されることを示した。しかしながら、上皮細胞の単層培養系は生体内の子宮組織とは大きく異なる。そこで、生体の子宮内で起こる精子–子宮の相互作用と免疫反応を観察するために、本研究では、ex-vivo での子宮組織培養系モデルを確立して、詳細について検証した。

第1章では、新鮮なウシの子宮上皮組織培養系を用いた ex-vivo モデルを開発した。健常な発情後の子宮から、排卵直前の卵胞側に接する子宮角の子宮小丘の間の子宮上皮組織を直径 8 mm のバイオプシーパンチで円型に切り取り、2 mm の厚さに調整した。この子宮小片を、106/ml の射出精液から洗浄して得た精子と共培養した。精子ミトコンドリアを JC1 で蛍光ラベルして、精子の子宮小片上での動態をビデオ観察した。同時に、走査型電子顕微鏡(SEM)で微細な観察も行なった。その結果、精子は子宮小片の表面を泳いですぐに子宮腺に侵入した。SEM 観察では、精子は子宮腺に頭部を入れ、尾部の多くは子宮腺開口部の外に出していた。以上から、ウシ子宮小片の ex-vivo モデルは、精子の子宮内で示す動態を詳細に観察することができ、ウシ精子が子宮内で示す動態とその後の子宮の免疫応答の検証に極めて有効なアプローチであることがわかった。

第2章では、このウシ子宮小片の ex-vivo モデルを用いて、精子が子宮腺に侵入して誘導する子宮の免疫応答について詳細を検証した。精子は 30 min 以内に好中球の強力なケモカインである IL8 遺伝子発現を増加させ、他の主要な炎症性サイトカインである TNFAや IL1B 遺伝子発現も PGES と TLR2 遺伝子発現と共に 2 h 以内に刺激した。さらに免疫組織化学による観察で、精子が侵入した子宮腺の内腔側の上皮細胞は、炎症性サイトカイン TNFA タンパクが発現していた。驚くことに、SEM 観察では精子が侵入した子宮腺に好中球が出現しており、精子を攻撃していた。これは、授精後に起きる精子貪食による子宮の清浄化の最初の現象かもしれない。熱で非動化した精子や、黄体期の子宮では、精子が子宮腺に侵入することはなく、これは発情–排卵期特有の現象であると考えられた。

第3章では、精子の子宮腺への侵入に始まる炎症反応が、TLR2 を介しているかを検証した。TLR2 アンタゴニストは、子宮腺に侵入する精子数を減少させ、炎症性サイトカインの TNFA 発現を抑制した。この事実から、精子–子宮の相互作用と免疫反応は、少なくとも一部は TLR2 を介することが示唆された。関係するシグナル経路については、さらなる詳細な検証が必要である。

以上の一連の研究結果から、ウシ子宮小片の ex-vivo モデルは精子–子宮の相互作用に始まる免疫反応(炎症作用)の起点となる現象を検証するのに有効であった。発見した子宮腺への精子の侵入と、そこから始まる子宮の炎症反応は、授精直後に始まる子宮の精子排除と清浄化カスケードの起点であり、その後の初期胚の子宮への受け入れに重要な現象であることが強く示唆された。

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