Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings

Thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs) have been characterized as electron donors, guards of the intracellular redox state, and “antioxidants”. Today, these redox catalysts are increasingly recognized for their specific role in redox signaling. The number of publications published on the functions of these proteins continues to increase exponentially. The field is experiencing an exciting transformation, from looking at a general redox homeostasis and the pathological oxidative stress model to realizing redox changes as a part of localized, rapid, specific, and reversible redox-regulated signaling events. This review summarizes the almost 50 years of research on these proteins, focusing primarily on data from vertebrates and mammals. The role of Trx fold proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners. On the basis of this analysis, th...

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Thioredoxins, glutaredoxins, and peroxiredoxins--molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling.

Transcript profiling during preimplantation mouse development.

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development: effect of β-mercaptoethanol, cysteine and cystine.

Function of reactive oxygen species during animal development: passive or active?

Experiments were conducted to elucidate the status of glutathione present in the oxidized (GSSG), reduced (GSH), and protein-mixed disulfide (GSSprotein) forms in preimplantation mouse embryos during development and after treatment with tertiary-butyl hydroperoxide (tBH) to cause oxidative stress. Glutathione was measured at picomolar levels by fluorimetric HPLC after derivatization of extracted embryonic samples with dansyl chloride. GSH content decreased approximately 10-fold from that in the unfertilized oocyte to 0.12 pmol/blastocyst, representing an estimated change in concentration from 7 to 0.7 mM. GSH levels were lower in embryos cultured in vitro than in embryos that developed in vivo. Addition of GSH to the culture medium improved in vitro development of mouse embryos, but surprisingly the addition of glutathione monoethyl ester did not. Addition of low levels of the oxidant tBH (13.2 microM) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 microM tBH, GSH levels were markedly decreased in the two-cell stage embryo (75%), but only slightly decreased (25%) in the blastocyst. The loss of GSH was accounted for by increases in GSSG and GSSprotein, indicating that the embryo was undergoing oxidative stress. These data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.

Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo.

Changes in sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content during preimplantation mouse embryo development were determined. Western blotting, using polyclonal antiserum against guinea pig Na+/K+ ATPase, was used to detect changes in Na+/K+ ATPase alpha- and beta-subunit content during mouse embryo development. Total RNA from mouse embryos was analyzed using Northern and slot blots hybridized with random-primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kb) in sheep and mouse kidneys and mouse embryos. Although Na+/K+ ATPase alpha-subunit mRNA content of mouse embryos increased 45-fold between Day 1 and Day 4 of development, Na+/K+ ATPase alpha-subunit content remained constant, and beta-subunit content increased 9-fold. The Na+/K+ ATPase alpha-subunit and alpha-subunit mRNA content did not increase in a similar manner. The results suggest that, in mouse embryos, blastocoel formation is not triggered by an increase in Na+/K+ ATPase alpha-subunit content. Changes in beta-subunit content may be important in regulating Na+/K+ ATPase activity and blastocoel formation.

Sodium/potassium adenosine triphosphatase alpha- and beta-subunit and alpha-subunit mRNA levels during mouse embryo development in vitro.

Plasminogen activator production by ovine embryos and the effects of plasminogen on ovine embryo development and zona pellucida integrity were evaluated. Eight-cell to sixteen-cell embryos were cultured in Whitten's medium containing 0, 60, or 120 micrograms/ml plasminogen. Plasmin and plasminogen activator concentrations in the medium were determined by a caseinolytic assay. More blastocysts hatched in medium containing 60 and 120 micrograms/ml plasminogen (33 and 21%, respectively) than 0 microgram/ml plasminogen (0%; p less than 0.05). Zona pellucida dissolution time in acidified phosphate-buffered saline was less after incubation in medium with 60 and 120 micrograms/ml plasminogen (7.2 and 5.9 min, respectively) than 0 microgram/ml plasminogen (9.4 min; p less than 0.05). Plasminogen activator production was low until the morula stage, increased during morula-blastocyst transition, and remained elevated through blastocoelic expansion and hatching. Zona pellucida solubility, plasminogen activator production, and plasminogen conversion to plasmin increased as embryonic stage advanced; however, plasminogen activator production and plasmin conversion to plasmin were poorly correlated with zona pellucida solubility. The results indicate that ovine embryos produce plasminogen activator, and plasmin can increase zona pellucida solubility; however, other factors may also be involved in altering zona pellucida integrity prior to hatching.

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The effects of plasminogen on in vitro ovine embryo development.

Sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content of rabbit embryos during preimplantation development were evaluated. Changes in Na+/K+ ATPase alpha-subunit content were detected with Western blotting using polyclonal antiserum against guinea pig Na+/K+ ATPase. Total RNA samples from rabbit embryos were analyzed by using Northern blots hybridized with random primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kilobases) in sheep kidneys and rabbit embryos. Between Day 4 and Day 6 of development, Na+/K+ ATPase alpha-subunit mRNA content increased 35-fold whereas Na+/K+ ATPase alpha-subunit content increased 22-fold. The similar increase in Na+/K+ ATPase alpha-subunit mRNA and alpha-subunit content in rabbit embryos suggests that Na+/K+ ATPase is partly regulated at the mRNA level during blastocyst expansion.

Sodium/potassium adenosine triphosphatase alpha-subunit and alpha-subunit mRNA levels in early rabbit embryos.

The fertilized mouse egg undergoes a series of cleavage divisions that result in an embryo composed of 8 similar totipotent cells. These cells subsequently develop cytoplasmic and plasma membrane polarity with distinct apical and basolateral regions. Polarization of cells is the first step in embryonic differentiation. Cleavage divisions of these polarized cells produce two distinct cell types: polar cells on the outside of the embryo and apolar cells on the inside of the embryo. Polar cells develop into trophectoderm—the first transporting epithelium of the embryo—and subsequently will form extraembryonic membranes in the conceptus. The apolar cells develop into inner cell mass that later forms the embryo proper.

C.S. Gardiner

Papers

Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo.

Sodium/potassium adenosine triphosphatase alpha- and beta-subunit and alpha-subunit mRNA levels during mouse embryo development in vitro.

The effects of plasminogen on in vitro ovine embryo development.

Sodium/potassium adenosine triphosphatase alpha-subunit and alpha-subunit mRNA levels in early rabbit embryos.

Development of Na/K ATPase Activity and Blastocoel Formation