- 著者
- Kento MIURA Shogo MATOBA Narumi OGONUKI Takafumi NAMIKI Junya ITO Naomi KASHIWAZAKI Atsuo OGURA
- 出版者
- THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
- 雑誌
- Journal of Reproduction and Development (ISSN:09168818)
- 巻号頁・発行日
- pp.2018-053, (Released:2018-05-05)
- 被引用文献数
- 9
In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ–AID–EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ–AID–EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ–AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.
- 著者
- Naoki Shimizu Hannah Bersabe Junya Ito Shunji Kato Ryo Towada Takahiro Eitsuka Shigefumi Kuwahara Teruo Miyazawa Kiyotaka Nakagawa
- 出版者
- 公益社団法人 日本油化学会
- 雑誌
- Journal of Oleo Science (ISSN:13458957)
- 巻号頁・発行日
- pp.ess16159, (Released:2017-02-10)
- 被引用文献数
- 19
Squalene (SQ), a main component of human sebum, is readily photooxidized by exposure to sunlight, producing six squalene monohydroperoxide (SQ-OOH) isomers. Despite its known connection to various skin conditions, few studies have sought to analyze SQ-OOH at the isomeric level. In this study, we aimed to develop a method to discriminate each SQ-OOH isomer with the use of tandem mass spectrometry (MS/MS). The six standard SQ-OOH isomers were prepared by photooxidizing SQ in the presence of rose bengal, a photosensitizer, and isolated by semipreparative high-performance liquid chromatography (HPLC). To purify each isomer, 2-methoxypropene, which reversibly reacts with the hydroperoxide group of SQ-OOH, was utilized. Product ion scanning was then performed on the standard SQ-OOH isomers in the absence and presence of the sodium ion. In the absence of the sodium ion, the fragmentation patterns produced by atmospheric pressure chemical ionization were similar between the isomers, whereas in the presence of the sodium ion by electrospray ionization, unique fragmentation patterns were achieved. Based on these fragment ions, HPLC-MS/MS multiple reaction monitoring analysis was conducted on a mixture of the standard SQ-OOH isomers. We achieved discrimination of SQ-OOH isomers with high selectivity and detected SQ-OOH isomers at nanogram levels. These results may improve our understanding of the effect of SQ-OOH on skin conditions as well as the mechanism behind SQ peroxidation.