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Study on the pathogenesis of oocyte pick-up dysfunction of oviduct in autoimmune disease-prone model mouse

齋藤, 実里奈 北海道大学

2022.09.26

概要

動物の繁殖障害及びヒトの雌性不妊症は、安定した畜産物の供給及び我が国の少子化克服を阻む重大な課題である。本課題を解決するため、排卵から着床までの生殖プロセスを妨げる雌性生殖器病態の解析が急務である。本研究では特に、卵管のピックアップ機能の病態に着目した。卵管の卵巣側末端・卵管ロートは、排卵卵子を卵管内へ誘導するピックアップ機能を担い、その機能異常・ピックアップ障害は繁殖障害及び不妊症の一因となる。しかし、ピックアップ機能の生理的制御機序のみならず、ピックアップ障害の詳細な発症機序は不明で、現在医学・獣医学領域の臨床現場における診断・治療法はない。

注目すべきことに、免疫異常は雌性不妊発症と深く関連する。家畜動物では、感染症や生殖器の炎症が繁殖率の低下を招く。さらに、女性の自己免疫疾患患者では不妊発症リスクが高いこと、及び自己免疫疾患モデルマウスでは卵巣内閉鎖卵胞の増加がみられることから、自己免疫異常は雌性生殖器の形態機能に重大な影響を及ぼすと考えられる。そこで本研究では、アポトーシス誘導受容体Fasのlpr変異に起因する全身性自己免疫疾患を発症するモデルマウスMRL/MpJ-Faslpr/lpr (MRL/lpr)の卵管の形態機能解析を通し、自己免疫異常とピックアップ機能の関連性を考察した。

まず第1章では、マウスにおけるピックアップ機能の定量法を検討するとともに、自己免疫異常がピックアップ機能に影響を与えるか調査した。過剰排卵処置を施した雌マウスの卵巣・卵管を摘出し、卵管内の卵子卵丘細胞複合体の数を実体顕微鏡下で計測しピックアップ卵子数とした。卵巣の連続切片を作製し、破裂卵胞と出血体の数を計測し排卵卵子数とした。排卵卵子数に対するピックアップ卵子数の割合をピックアップ効率とした。健常なC57BL/6N(B6)においてピックアップ効率(PUR)は約100%であり、本法はピックアップ機能の定量法として有用だった。一方、重篤な自己免疫疾患を発症する6ヶ月齢のMRL/lprにおいてPURは約63%であり、疾患症状の軽い3ヶ月齢時の約94%と比較して顕著に低下した。さらに、卵管ロートの超微形態観察の結果、6ヶ月齢のMRL/lprでは、同齢B6や3ヶ月齢のMRL/lprと比較し卵管ロートが腫脹し、上皮を覆う線毛が減少していた。以上より、MRL/lprにおいて、自己免疫疾患の進行が卵管ロートの形態的変化を伴ってピックアップ障害を招くことが示された。

続いて第2章では、卵巣囊の形態を解剖学的に精査した。マウスの卵巣及び卵管ロートは、卵巣間膜及び卵管間膜によって包まれた袋状の構造“卵巣囊”に位置するが、卵管峡部と子宮端を結ぶ卵管間膜上に0.04~0.12cm2の卵巣囊孔(FOB)が存在したため、腹腔との連続性を保っていた。FOBは平滑筋に富む卵巣囊孔間膜(LOB)に囲まれ、その筋収縮によってFOBの大きさが変動する可能性が示された。つまり、マウスの排卵卵子はFOBを通して腹腔内に脱落すると考えられた。一方、6ヶ月齢MRL/lprでは、LOBが線維化し、3ヶ月齢時と比較してFOBの面積が大きくなる傾向があったことから、FOBの形態変化がMRL/lprのピックアップ障害に関与することが示唆された。

第3章では、第1章において示唆されたMRL系統の卵巣機能における特異的形質の詳細を解析した。第1章において、3ヶ月齢のMRL/lprおよびその野生型であるMRL/MpJ(MRL/+)は、B6よりも過剰排卵処置後の排卵数が多かった。そこで、エクソーム解析によって、MRL/+における性ホルモン及びその受容体をコードする遺伝子配列をB6と比較した結果、これら蛋白質の構造や機能に影響を及ぼすと予測される変異はみられなかった。一方で、過剰排卵処置で得られた卵子の受精率はB6と比較してMRL/+で有意に低かった。また、第1章ではMRL/+のPURが100%を超えていた。そこで、過剰排卵処置後の黄体形成過程を組織学的に追跡した結果、MRL/+ではB6に比べて黄体形成が早期に進行し、これには排卵後卵胞における細胞外マトリックスのリモデリングの亢進が関与することが示唆された。

最後に第4章では、卵管ロートの線毛上皮の形態機能を精査した。規則的な線毛運動は健常なピックアップ機能における必須要素であるが、6ヶ月齢MRL/lprの卵管ロートでは、3ヶ月齢時と比較して線毛上皮細胞数の減少、線毛運動速度の低下、及び線毛運動方向のばらつきの増大がみられた。このことから、全身性の自己免疫異常が卵管ロート線毛上皮の形態機能に影響を与えることが示された。また、6ヶ月齢MRL/lprの卵管ロートの粘膜固有層には顕著な炎症細胞浸潤がみられ、これら細胞由来の液性免疫因子が線毛上皮の病態に関与すると考えられた。

結論として本研究は、マウスにおいて全身性自己免疫疾患が卵管の線毛上皮及びFOB・LOBを含む雌性生殖器の形態機能変化を通してピックアップ障害の発症に関与することを示した。今後、本知見を基に各種動物及びヒトの卵管病態を精査することで、原因不明の繁殖障害及び雌性不妊症の病態発生を説明できる可能性が示唆された。以上、本研究は免疫異常と卵管の機能障害の関連性を明らかにし、動物の繁殖障害及びヒトの雌性不妊症の病態発生機序の全貌を解明する上で必要不可欠な知見を提供した。

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