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Unraveling the Physiology of Sperm Maintenance in the Vas Deferens of Japanese Quail (Coturnix japonica)

Prodip Kumar Sarkar 岐阜大学

2020.09.18

概要

鳥類では、精子は精巣内で産生され、雄性生殖道を通過する間に運動能力を獲得する。精子は射精前に男性生殖管に貯蔵されるが、受精能力が維持される分子機構は解明されていない。精子が男性生殖管内でどのようにして生存しているのかを理解するためには、精子の貯蔵・維持のメカニズムを理解することが重要である。また、雄性生殖道で添加される液性成分が、雄性生殖道での精子の維持に重要な役割を果たしている可能性がある。本研究の目的は、ウズラの雄性生殖道で精子がどのように生存・維持されているかを知ることである。目的を達成するために、鳥類の雄性生殖管に存在する様々な生理化学的条件で精子をインビトロインキュベートした。さらに、雄性生殖管における精子の維持を知るために、精漿タンパク質(アルブミン、トランスフェリン)とジペプチド(アンセリン、カルノシン)の影響を調査し、精子の生存率と運動性に及ぼす影響を評価した。

第二章では精子の生存率を調べるために、精子をハンクス平衡塩類溶液で希釈し、様々な温度、 pH、精子濃度、浸透圧で 24 時間インキュベートした。結果,精子の生存率は、21℃と 41℃に比べて 4℃で有意に高くなることが分かった。また、pH と精子の生存率の間には有意な関係が認められた。また、精子濃度が高くなるほど、精子の生存率が有意に高かった。精子の生存率は、浸透圧の影響を受けなかった。精子の生存率に対する精漿およびクロアカ腺分泌物の効果を調査した。しかし、精子の生存率はいずれの上清を添加しても改善されなかった。第三章では精漿の主要タンパク成分であるアルブミン(ALB)とトランスフェリン(TF)の精子の生存率への影響を調べた。精漿、クロアカ腺分泌物及び輸精管内腔液を、抗 ALB 又は抗 TF 抗血清を用いたウエスタンブロット分析を行った。ALB とTF は、射出精液、クロアカ腺分泌物及び輸精管内腔液に存在していた。免疫組織化学的解析の結果、ウズラの雄性生殖管に ALB と TF が発現していることが明らかになった。精子の生存率に及ぼす ALB とTF の影響を調べたところ、ALB とTF は精子の生存率を有意に改善した。第四章では精子の生存性に及ぼすアンセリン及びカルノシンの影響を調べた。アンセリンは精巣液中に存在し、カルノシンは精漿中に存在した。ヒスチジン、β-アラニン、1-メチルヒスチジンは精巣液、射出精液、クロアカ腺分泌物に存在した。精子をアンセリンまたはカルノシンと 15℃でインキュベートし、運動性を時間依存的に観察した。結果、アンセリンまたはカルノシンを添加して 6 時間培養した精子の運動性パラメータ(直線速度、曲線速度、平均経路速度、直線性、直線性、直進性、頭部振幅、頭部振動数、運動率)が有意に高いことが明らかになった。

精液中の ALB と TF の役割については、これまで限られた研究しか行われていない。ALB と TF は活性酸素の発生を最小限に抑え、精子の生存率を向上させているのではないかと考えられる。また、アンセリンやカルノシンも酸化ストレスを防ぎ、精子のダメージを最小限に抑えていると考えられる。今後、これらのタンパク質を組み合わせて、精子の生存力、運動性、受精能を研究することが考えられる。

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