title: Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.
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.
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takeo
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Horikoshi
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Satohiro
authors.initials: S
authors.lastname: Nakao
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Sakoh
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Ishizuka
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Tsutsumi
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Fukumoto
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Haruguchi
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Takeshita
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nakamuta
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan.
authors.forename: Shuuji
authors.initials: S
authors.lastname: Tsuchiyama
authors.affiliation: Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Chuo-ku, Kumamoto, Japan nakagata@kumamoto-u.ac.jp.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakagata
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D004624:Embryo Transfer; D005307:Fertilization in Vitro / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D011506:Proteins / Q000737:chemistry; D013094:Spermatozoa / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D015044:Zona Pellucida / Q000737:chemistry / Q000187:drug effects*
chemical_list: D000596:Amino Acids; D004220:Disulfides; D011506:Proteins; D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine
keywords: assisted reproductive technology; fertilization; in vitro fertilization (IVF); oocyte; zona pellucida