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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.

SUBMITTER: Takeo T

PROVIDER: S-EPMC6366478 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.

Takeo Toru T Horikoshi Yuka Y Nakao Satohiro S Sakoh Kazuhito K Ishizuka Yuta Y Tsutsumi Aki A Fukumoto Kiyoko K Kondo Tomoko T Haruguchi Yukie Y Takeshita Yumi Y Nakamuta Yuko Y Tsuchiyama Shuuji S Nakagata Naomi N

Biology of reproduction 20150225 4

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 det ...[more]

PMID: 25715791

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Project description:Study questionHow and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization?Study findingOvastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization.What is known alreadyThe metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH.Study design, samples/materials, methodsWe isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test.Main results and the role of chanceMetaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage.Limitations, reasons for cautionFor this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH.Wider implications of the findingsThis study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans.Large scale dataNone.Study funding and competing interest(s)This work was supported by the Center of Natural Sciences and Medicine and by a start-up grant of the Johannes Gutenberg University Mainz to W.S., and by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University to J.F. and W.J.D. There are no competing interests to declare.

Project description:Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

Dataset Information

Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.

Publications

Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.

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