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Downloaded from Bioscientifica.Com at 09/27/2021 05:05:07PM Via Free Access 66 T Reproduction (2002) 124, 65–71 Research Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators T. Matsukawa, S. Ikeda, H. Imai and M. Yamada* Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan The present study was undertaken to examine the with that (< 25%) in medium without polyamino- effects of various transition metal ion chelators, both poly- carboxylates. Although EDDA, EDTA and DTPA at aminocarboxylates (including nitrilotriacetate (NTA), 10 µmol l–1 induced the development of most one-cell ethylenediaminediacetate (EDDA), ethyleneglycolbistetra- embryos to the four-cell stage and beyond, a higher acetate (EGTA), ethylenediaminetetraacetate (EDTA) and concentration (100 µmol l–1) of NTA and EGTA was diethylenetriaminepentaacetate (DTPA)) and non- required to obtain a similar result. Therefore, the ability of polyaminocarboxylates (dipicolinic acid and deferoxamine), polyaminocarboxylates to overcome the two-cell block is on the development in vitro of one-cell ICR strain mouse not correlated with their potency to chelate transition embryos to the four-cell and blastocyst stages. The order of metal ions. In contrast, the non-polyaminocarboxylates stability constants of polyaminocarboxylates for transition dipicolinic acid and deferoxamine, at 10 and 100 µmol l–1, metal ions such as zinc, copper and iron is as follows: did not have the same effect. Taken together, the results NTA р EDDA < EGTA < EDTA < DTPA. Addition of 10 indicate that the ability of polyaminocarboxylates to or 100 µmol polyaminocarboxylates l–1 to the medium overcome the two-cell block in embryo development is significantly enhanced the development of most one-cell due to some common feature or features other than the embryos (66–88%) beyond the two-cell stage compared ability to chelate transition metal ions. a chelator of transition metal ions to prevent extracellular Introduction generation of oxygen radicals. However, although poly- In vitro culture of one-cell embryos from most strains of aminocarboxylate metal chelators, including EDTA, mouse is known to result in developmental arrest during the nitrilotriacetate (NTA), ethylenediaminediacetate (EDDA), G2 phase of the second cell cycle, when the major zygotic ethyleneglycolbistetraacetate (EGTA), and diethylenetri- gene activation occurs. This developmental arrest is referred aminepentaacetate (DTPA), are commonly used to prevent to widely as the ‘two-cell block’. In some strains, such as the metal-catalysed oxidation because they sequester metals, a ICR strain, the developmental block is alleviated by adding number of studies have indicated that iron– or copper– ethylenediaminetetraacetate (EDTA) to the culture medium, polyaminocarboxylate complexes may be catalytically resulting in an increased proportion of one-cell embryos active as a result of their specific co-ordination geometry that develop to the blastocyst stage (Abramczuk et al., 1977; (Yamada et al., 1987; Stadtman, 1993; Asaumi et al., 1996; Hoshi and Toyoda, 1985; Chatot et al., 1989; Lawitts and Okada, 1996; Zhao et al., 1996). Production of oxygen Biggers, 1992; Gardner and Lane, 1996). As EDTA is a cell radicals, such as hydroxyl radicals, in the presence of membrane-impermeable metal ion chelator, the effects of polyaminocarboxylates tends to increase with decreased EDTA may be exerted by chelating extracellular transition complex stability: NTA у EDDA ӷ EDTA > DTPA (Asaumi metal ions such as zinc, copper and iron. For example, et al., 1996; Okada, 1996; Zhao et al., 1996). Furthermore, chelation of transition metal ions by EDTA generally it has been reported that iron-chelated EDTA (Fe–EDTA) prevents the ions from participating in chemical reactions plays a catalytic role and promotes the oxidation of amino that generate harmful oxygen radicals (Halliwell and acids by the classical Fenton system (Asaumi et al., 1996; Gutteridge, 1984). Inclusion of superoxide dismutase (SOD) Zhao et al., 1996). Moreover, although the metal ion in culture medium without EDTA partially alleviates the complexes formed with EDTA and EGTA have similar two-cell block, although the effects of SOD are disputed stability constants, Hoshi and Toyoda (1985) reported that and variable (Legge and Sellens, 1991; Noda et al., 1991; EGTA did not alleviate the two-cell block in embryo devel- Payne et al., 1992; Johnson and Nasr-Esfahani, 1994). Thus, opment. Thus, it remains unclear whether the alleviation one possible role of EDTA in the culture medium is to act as effect of EDTA is mediated by its ability to chelate transition metal ions, resulting in either sequestration or generation of *Correspondence reactive oxygen radicals. Email: [email protected] The aim of the present study was to examine the effects © 2002 Society for Reproduction and Fertility 1470-1626/2002 Downloaded from Bioscientifica.com at 09/27/2021 05:05:07PM via free access 66 T. Matsukawa et al. Table 1. Dose effects of ethylenediaminetetraacetate (EDTA) on development of one-cell ICR mouse embryos in vitro Number of Percentage of embryos developed to EDTA embryos (µmol l–1) examined Two-cell stage Four-cell stage Blastocyst 0.0 47 100a 24a 3a 0.25 50 100a 46b 21ab 0.5 55 100a 75c 37b 1.0 55 100a 84c 59b 10 56 100a 92c 82c 100 38 100a 95c 85c 1000 53 100a 64c 3a abcValues in the same column with different superscripts are significantly different. of polyaminocarboxylates and other chelators (non- EDTA, supplemented with 0.1% (w/v) polyvinylpyrrolidone polyaminocarboxylates) with different metal ion stability instead of BSA and designated here as modified KSOM constants on the two-cell block in cultured ICR mouse (mKSOM). embryos. Statistical analysis Materials and Methods Each experiment was repeated three times. Subclass Chemicals means were analysed by ANOVA and Scheffe’s test. Percent- age data were subjected to arcsine transformation before The chemicals used were obtained as follows: NTA, statistical analysis. A value of P < 0.05 was considered to be 2+ 2+ EDDA, EDTA, EGTA, DTPA, and Ca - or Mg -saturated an indication of significance. EDTA (Ca–EDTA and Mg–EDTA, respectively) from Dojindo Laboratories (Kumamoto); dipicolinic acid from Molecular Probes (Eugene, OR); and deferoxamine from Sigma (St Results Louis, MO). The first experiment examined the effects of EDTA at various concentrations on development in vitro of one-cell Embryo collection and culture embryos at the pronuclear stage to the four-cell and Female ICR mice, aged 3–6 weeks, were superovulated blastocyst stages. When concentrations of EDTA > 0.25 µmol by injection of 5 iu equine chorionic gonadotrophin (eCG) l–1 were added to mKSOM, the proportions of embryos followed 48 h later by 5 iu human chorionic gonadotropin reaching the four-cell and blastocyst stages increased (hCG). The females were subsequently mated with male significantly in a dose-dependent manner (Table 1). The mice of the same strain, and vaginal plug formation was prominent effects of EDTA on development to the four-cell confirmed on the next morning (day 1). The female mice and blastocyst stages were observed at concentrations of were anaesthetized with ether and killed by cervical dis- 0.5–100.0 µmol l–1 and 10–100 µmol l–1, respectively (the location. All procedures involving animals were approved rates of development to the four-cell and blastocyst stages by the Kyoto University Animal Care and Use Committee. were 75–92% and 80–82%, respectively). However, ad- Fertilized one-cell embryos were collected 20 h after dition of 1 mmol EDTA l–1 to the medium significantly administration of hCG from the ampullae of oviducts of reduced the proportion of embryos that developed to the superovulated females by tearing the ampullae with a hypo- four-cell stage, and the resulting four-cell embryos did not dermic needle. After removal of cumulus cells by digestion develop to the blastocyst stage. In the control group, in with 0.1% (w/v) hyaluronidase (Sigma) for approximately which the embryos were cultured in mKSOM without 5 min, the embryos were placed in 50 µl drops of culture EDTA, the developmental rates to the four-cell and blasto- medium, covered with mineral oil and cultured for 5 days cyst stages were 24% and 3%, respectively. at 37ЊC under 5% CO2 in air. The embryos were observed Lane and Gardner (2001) suggested that the alleviation every 24 h under a Nikon inverted microscope. The culture effect of EDTA is due to the chelation of divalent cations efficiency was evaluated by determining the proportion of such as magnesium. Therefore, the next experiment embryos reaching the two-cell (day 2), four-cell (day 3) and examined the effects of Mg–EDTA and Ca–EDTA on blastocyst (day 5) stages. development of one-cell embryos in vitro. Mg–EDTA and The basal culture medium used was potassium simplex Ca–EDTA are non-magnesium and non-calcium chelators optimized medium (KSOM; Erbach et al., 1994) without that are able to bind to metal ions with higher stability Downloaded from Bioscientifica.com at 09/27/2021 05:05:07PM via free access Effect of polyaminocarboxylates on the two-cell embryo block 67 Table 2. Effects of Ca– and Mg–ethylenediaminetetraacetate (EDTA) on development of one-cell ICR mouse embryos in vitro Number of Percentage of embryos developed to Concentration embryos Additive (µmol l–1) examined Two-cell stage Four-cell stage Blastocyst – – 65 100a 14a 0a Ca–EDTA 10 37 100a 91b 69b 100 40 100a 89b 73b Mg–EDTA 10 38 100a 97b 79b 100 38 100a 98b 74b abcValues in the same column with different superscripts are significantly different. Table 3. Stability constants of chelators for transition metal ions Log stability constant for Chelator Zn2+ Cu2+ Fe3+ Ca2+ Mg2+ NTAa 10.7 13.0 15.9 6.4 5.5 EDDAa 11.2 16.2 17.0 na 3.9 EGTAa 14.5 17.8 20.5 11.0 5.2 EDTAa 16.5 18.8 25.1 11.0 8.7 DTPAa 18.8 21.5 28.6 10.7 9.3 Dipicolinic acidb 6.4 9.1 na 4.4 2.3 Deferoxaminec 11.1 14.1 30.6 2.6 na aData from Dojindo Laboratories, Kumamoto, Japan.
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