Studies on molecular mechanisms for functional evolution of oviduct to establish internal fertilization

2017 Fiscal Year Final Research Report

• Project/Area Number: 15K07165
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Evolutionary biology
• Research Institution: Yamagata University
• Principal Investigator: WATANABE ERIKO 山形大学, 学士課程基盤教育機構, 准教授 (20337405)
• Co-Investigator(Kenkyū-buntansha): 渡邉 明彦 山形大学, 理学部, 教授 (30250913) ; 越智 陽城 山形大学, 医学部, 准教授 (00505787)
• Project Period (FY): 2015-10-21 – 2018-03-31
• Keywords: 体内受精 / ゲノム編集 / 機能進化 / 輸卵管 / 精子運動 / 両生類

Outline of Final Research Achievements

Sperm motility-initiating substance (SMIS) is a key protein for the internal fertilization of Cynops pyrrhogaster. The deduced amino acid sequence of SMIS cDNA included 14 cysteine residues, and 6 of them construct the cysteine knot (CK) motif. CK motif is thought to mediate protein polymerization and stabilize protein conformation. 1) We performed genome editing using CRISPR/Cas9 system for targeting to 4th cysteine residues of the CK motif. Western blotting using the anti-SMIS antibodies revealed that the lack of the 4th cysteine resulted in the change of polymerization and/or conformation status of SMIS in correlation with the physiological activity. 2) SMIS genes of Cynops ensicauda and Hynobius lichenatus were identified by RNAseq and found the presence of the CK motif in the deduced amino acid sequences. 3) By the Western blotting, differences in the molecular sizes and the possible polymerization status were suggested among species that undergo distinct mode of reproduction.

Free Research Field

発生生物学