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25 February 2015 Cysteine Analogs with a Free Thiol Group Promote Fertilization by Reducing Disulfide Bonds in the Zona Pellucida of Mice
Toru Takeo, Yuka Horikoshi, Satohiro Nakao, Kazuhito Sakoh, Yuta Ishizuka, Aki Tsutsumi, Kiyoko Fukumoto, Tomoko Kondo, Yukie Haruguchi, Yumi Takeshita, Yuko Nakamuta, Shuuji Tsuchiyama, Naomi Nakagata
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Abstract

Archives of cryopreserved sperm harvested from genetically engineered mice, in mouse resource centers, are a readily accessible genetic resource for the scientific community. We previously reported that exposure of oocytes to reduced glutathione (GSH) greatly improves the fertilization rate of frozen-thawed mouse sperm. Application of GSH to in vitro fertilization techniques is widely accepted as a standard protocol to produce sufficient numbers of mice from cryopreserved sperm. However, the detailed mechanism of the enhancement of fertilization mediated by GSH in vitro is not fully understood. Here we focused on the chemical by determining the effects of its amino acid constituents and cysteine analogs on the fertilization of oocytes by frozen-thawed sperm. Furthermore, we determined the stability of these compounds in aqueous solution. We show here that l-cysteine (l-Cys), d-cysteine (d-Cys), or N-acetyl-l-cysteine (NAC) increased the rate of fertilization when added to the medium but did not adversely affect embryo development in vitro or in vivo. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by l-Cys, d-Cys, and NAC. These effects were abrogated by the methylation of the thiol group of l-Cys. NAC was the most stable of these compounds in the fertilization medium at 4°C. These results suggest that the thiol groups of cysteine analogs markedly enhance the fertilization rate of mouse oocytes.

Toru Takeo, Yuka Horikoshi, Satohiro Nakao, Kazuhito Sakoh, Yuta Ishizuka, Aki Tsutsumi, Kiyoko Fukumoto, Tomoko Kondo, Yukie Haruguchi, Yumi Takeshita, Yuko Nakamuta, Shuuji Tsuchiyama, and Naomi Nakagata "Cysteine Analogs with a Free Thiol Group Promote Fertilization by Reducing Disulfide Bonds in the Zona Pellucida of Mice," Biology of Reproduction 92(4), (25 February 2015). https://doi.org/10.1095/biolreprod.114.125443
Received: 24 September 2014; Accepted: 1 February 2015; Published: 25 February 2015
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KEYWORDS
assisted reproductive technology
fertilization
in vitro fertilization (IVF)
oocyte
zona pellucida
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