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ブタ成熟精巣におけるインスリン関連因子3(リラキシン関連因子)の構造と機能に関する研究

皆川, 至 岐阜大学

2020.03.13

概要

妊孕性のある精子の産生は、精子が形成される一連の生殖細胞や形成段階で特異的に発現する遺伝子産物の組織だった作用によって制御され、内分泌および傍分泌因子を含むいろいろな分子の複雑な相互調節により最適に維持されている。その中の 1 つに、インスリン関連因子 3(INSL3)(以前はリラキシン関連因子と呼ばれていた)がある。INSL3 は胎子や成体の精巣ライディッヒ細胞で産生され、胎子期の精巣下降に必須であるが、成体での機能はよくわかっていない。さらに、INSL3 の構造に関しても、cDNA 配列から推定されているものの、一部の動物を除き、実際にどのような構造で精巣より産生・分泌されるのかよくわかっていない。本研究では、ブタを用いて INSL3 の構造と機能の解明を目的とした。なお、本論文は 5 章よりなり、第 1 章は緒論、第 5 章は総括である。

第 2 章では、成熟ブタ精巣より INSL3 を単離・精製し、構造解析を行うと共に、その生物活性を調べた。INSL3 はゲルロカ、陽イオン交換 FPLC および逆相 HPLC を組み合わせて精製し、シングルピークとして単離した。本精製物をトリプシン消化しMALDI-MS/MS解析に供した結果、66%シークエンスカバレッジでブタ INSL3 は同定され、cDNA から推定された B-、C-および A-ドメインからなるモノマー構造と判明した。精製物の MALDI- MS 解析では、INSL3 の質量(m/z 値)が 12031 と特定され、さらに PMF 解析から予測される 3 つのジスルフィド結合が部位特異的に架橋されていることもわかった。加えて、単離した INSL3 が十分な生物活性を有していることを、本受容体 RXFP2 を強制発現させたヒト胎児腎細胞(HEK-293 細胞)で示した。これらの結果はブタ INSL3 が十分な生物活性を持ったB–C–A モノマー構造として精巣から分泌されることを示唆した。

第 3 章では、ブタ INSL3 の時間分解蛍光免疫測定法(TR-FIA)を開発し、性成熟に伴う INSL3 発現の動態と、精巣における INSL3 ホルモン–受容体システムの発現を調べた。
全ての試料は Duroc 種の雄ブタから採集した。確立した TR-FIA は感度が 8.2 pg/well(164 pg/ml)で、それを用いて血中 INSL3 は性成熟に伴い漸次増加すること、さらに、ライディッヒ細胞から分泌されたINSL3 は血中のみならず精巣間隙および精細管内に十分な濃度で放出されることを示した。一方、精巣分画法を用いた解析では、本受容体 RXFP2 遺伝子が精巣内の生殖細胞で発現していること、加えて、レセプターアッセイより INSL3 は生殖細胞膜画分に高い親和性で結合することを示した。これらの結果から、ライディッヒ細胞より精巣間隙に分泌された INSL3 は精細管内に輸送され、そこで受容体 RXFP2 を発現する生殖細胞と出会い、INSL3 を結合することがわかり、INSL3 は精細管内の生殖細胞における傍分泌因子として機能することを示唆した。

第 4 章では、INSL3 の成体における役割を解明するために、INSL3 抗体を用いた受動免疫により内因性 INSL3 の中和化を施した成熟雄ブタを作製し、精巣機能と精子産生に及ぼすその影響を調べた。実験には Duroc 種の雄ブタを用い、受動免疫区と対照区(各区 n = 3)に分け、 受動免疫区には INSL3 のB ドメインに対して作製した抗 INSL3 抗体 IgG 画分を 21 週齢(春機発動開始期)から 40 週齢(春機発動後期)まで 2 週間隔で静脈内注射した。対照区は正常 IgG を同様に投与した。その結果、抗体投与は精巣重量を減少させ、アポトーシス陽性の生殖細胞の出現頻度において4倍の増加を引き起こした。それはアポトーシス促進性 CASP3 および BAX の発現上昇、ならびにアポトーシス抑制性 XIAP および BCL2 の発現低下と関連していた。これらの結果から、受動免疫による INSL3 の中和化は生殖細胞のアポトーシスを誘発することで精巣重量と精子濃度を減少させ、INSL3 が精子生産の維持において生殖細胞の生存/抗アポトーシス因子として作用することを示唆した。

以上、本研究はブタ精巣において INSL3 は十分な生物活性を有した B–C–A モノマー構造として産生・分泌され、精子形成の場である精細管内に十分な濃度で輸送され、そこで受容体 RXFP2 を発現する生殖細胞と出会い、INSL3 を結合すること、さらに、INSL3 抗体を受動免疫法で成体に投与して INSL3 リガンドー受容体の結合を阻害すると、生殖細胞のアポトーシス増加とそれに伴う精巣重量および精子濃度の減少をもたらすことを明らかにした。これらのことから、INSL3 は精子生産の維持において生殖細胞の生存/抗アポトーシス因子として発現していることが示唆された。

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参考文献

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