Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

Abstract

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.

Keywords: Antioxidant; Embryonic development; Free radicals; GSH; GSSG; Gcl; Gclc; Gclm; Gene expression; Glutathione; Gss; Oxidative stress; PCA; ROS; Redox; Zebrafish; glutamate–cysteine ligase; glutamate–cysteine ligase catalytic subunit; glutamate–cysteine ligase modifier subunit; glutathione disulfide; glutathione synthetase; perchloric acid; reactive oxygen species; reduced glutathione.

Figure 1. Diagram of the glutathione redox system

Glutathione is a tripeptide of cysteine, glutamate, and glycine, which undergoes oxidation and forms a homodimer GSSG. GSSG can participate in post-translation modification of proteins by s-thiylation. GSSG can also be recycled back to reduced glutathione by GSH reductase in a reaction which utilizes NADPH. GSH can also be shuttled to the extracellular space and utilized, after which its cysteine component can be recycled by gamma glutamyl transferase (GGT). The synthesis of glutathione draws from cysteine pools synthesized from cystathionine, which is made from homocysteine. Glutathione thus draws from the same source of homocysteine that is necessary to maintain levels of s-adenosylmethionine needed for DNA methylation and epigenetic gene control, which is especially relevant during embryonic development.

Figure 2

Total glutathione concentrations in zebrafish embryos double over the course of embryonic development. N = three pools of 30 embryos fixed in PCA buffer and analyzed by HPLC for total glutathione concentrations. This experiment is representative of at least 3 independent experiments. Data are presented as mean ± SEM. Please see Supplemental File 1 for statistically significant differences between embryo ages.

Figure 3

Glutathione concentrations and redox potential over the course of embryonic development in zebrafish embryos. A. Profiles of reduced GSH and GSSG over development. B. Redox potential, calculated from the reduced and oxidized glutathione components. N = three pools of 30. Data are representative of at least 3 independent experiments and are presented as mean ± SEM. Please see Supplemental File 1 for statistically significant differences between embryo ages.

Figure 4

Measurements of GSH, GSSG, and E_h in dissected body tissue compared to yolk in embryos at different ages. Since the proportion of body and yolk changes with development, the data are presented on a pmol per embryo basis. GSH, GSSG, and E_h were found to be in equilibrium during somitogenesis (24 hpf), pre-hatch (48 hpf) and post-hatch (72 hpf). Data are representative of three independent experiments; mean and SEM, N = 3 pools of 20 embryos.

Figure 5

Hatching status influences glutathione and E_h. Embryos at 48 hpf that hatched early were compared to age-matched manually dechorionated embryos that had not hatched. There were significant differences between the two groups of embryos with respect to A) GSH and GSSG, B) concentration of GSH_T, and C) E_h. Data are presented as the mean + SEM, and are representative of three independent experiments. N = 3 pools of 30 embryos. An asterisk (*) indicates a statistically significant difference (ANOVA, p < 0.01) between unhatched and hatched embryos.

Figure 6

Expression profiles of genes involved in glutathione synthesis, recycling, and utilization over the first 48 hpf of embryonic development. A) Genes involved in glutathione synthesis; B) Genes involved in recycling of glutathione, C) genes involved in the transport of cysteine. Data are the mean and SEM of three biological replicate pools of 100 embryos, measured by microarray.

Figure 7

Expression of genes involved in the utilization of glutathione. A) Highly expressed glutathione-S-transferase genes; B) glutathione S-transferase genes; C) membrane-bound glutathione-S-transferase genes; D) glutathione peroxidase genes; E) glutathione peroxidase 4 genes.

Figure 8

Concentrations of GSH_T and E_h during the first 120 hours of zebrafish development. Four “windows” of dynamic glutathione conditions during embryonic development are observed. A) reduced E_h and low GSH_T, observed in mature oocytes; B) oxidized E_h and low GSH_T, observed in embryos from the mid-blastula transition through somitogenesis (3–24 hpf); C) oxidized E_h and high GSH_T, observed in embryos undergoing organ differentiation (30–48 hpf), and D) reduced E_h and high GSH_T, observed in post-hatch eleutheroembryos.