One-step versus two-step culture of mouse preimplantation embryos: is there a difference?

Human Reproduction Vol.20, No.12 pp. 3376–3384, 2005 doi:10.1093/humrep/dei228 Advance Access publication August 25, 2005. One-step versus two-step culture of mouse preimplantation embryos: is there a difference? J.D.Biggers1,3, L.K.McGinnis1 and J.A.Lawitts2 1 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115 and 2Beth Israel Deaconess Hospital, Boston, MA 02115, USA 3 To whom correspondence should be addressed. E-mail: Introduction Preimplantation mammalian embryos have been cultured from the zygote to the blastocyst either by renewing the medium approximately midway in the culture period (two-step protocol) or not renewing the medium (one-step protocol). One-step and two-step protocols for the culture from the mouse zygote to the expanded blastocyst stage were first described by Whitten and Biggers (1968) and Chatot et al. (1989) respectively. The strong conviction that two-step protocols are absolutely necessary for the successful cultivation of human zygotes to the blastocyst stage has become widespread since the recommendations made by Gardner et al. (1998) (see also Gardner and Lane, 2003). There are several commercially available media sold for this purpose despite the fact that experimental support for this virtual dogma is relatively sparse (Summers and Biggers, 2003). In this paper we present results that compare the development of mouse zygotes to blastocysts using one- and two-step protocols. There are several variants of two-step protocols for the culture of preimplantation embryos. Gardner (1994) first advocated a two-step protocol to remove ammonium and pyrrolidone-5-carboxylic acid, the breakdown products of glutamine (Gln), which arise spontaneously in all media containing Gln (review: Biggers et al., 2004a). Renewing the medium, however, has potentially favourable and unfavourable effects. It may re-supply beneficial compounds that have been significantly depleted during the initial culture period. Conversely, it may remove any beneficial compounds secreted by the cells during the initial period, such as growth factors. Two-step culture protocols can also involve media in which the second medium differs in composition from the first medium. This can involve the mere addition of a substance, e.g. the addition of glucose to medium CZB, which contains no glucose (Chatot et al., 1989). Alternatively it can involve the omission of a single substance from the first medium, as was done by Lane and Gardner (1995) in which the second medium (DM1) is the first medium (DM2) less EDTA (Table I). The EDTA was considered toxic in the later stages of preimplantation development. More complex two-step procedures can also involve several additions and subtractions of compounds from the first medium to give the second medium, e.g. as is done using medium G1.1 and medium G1.2 (Gardner et al., 1998) (Table I). In these protocols the embryos may be subjected to stress by being transferred to a new chemical environment. The results summarized in this paper describe the effects of renewing potassium-enriched simplex optimized medium supplemented with glucose and amino acids (KSOMgAA), the effects of removing EDTA from KSOMgAA and the effects of replacing medium G1.2 with medium G2.2 and DM2 with DM1. Materials and methods Nomenclature and preparation of culture media The compositions of the media used in this work are shown in Table I. When two media are used sequentially they will be 3376 © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: BACKGROUND: A comparison has been made of the development of mouse zygotes in either one-step or two-step culture systems. METHODS: Embryo culture, blastocyst cell counts and embryo transfer were done. RESULTS: No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the inner cell mass (ICM) and trophectoderm (TE) that developed in protocols: one-step culture in potassium-enriched simplex optimized medium supplemented with glucose and amino acids (KSOMgAA), two-step culture in KSOMgAA/KSOMgAA, and two-step culture in G1.2/G2.2. No gross abnormalities were observed in the fetuses that developed from zygotes in the one-step protocol using KSOMgAA and a two-step protocol using G1.2/G2.2. The body weights of these two groups of fetuses were not significantly different and no developmental abnormalities were observed. No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the ICM and TE that developed in protocols: one-step culture in KSOMgAA, two-step culture in KSOMgAA/KSOMgAA, and two-step culture in DM2/DM1. EDTA is not toxic to the initial cleavage stages of development at a concentration of 0.01 mmol/ l in KSOMgAA. CONCLUSIONS: Two-step culture protocols are sufficient for the support of preimplantation mouse development in vitro but they are not necessary. One- and two-step embryo culture protocols Table I. Compositions of media KSOMgAA, G1, G2, DM1 and DM2 (Mmol/l) Component KSOMgAA a G1b DM2c DM1c 90.08 90.08 5.5 5.5 – – 0.25 0.25 1.8 1.8 1.0 1.0 25 25 0.32 0.1 10.5 5.87 0.5 3.15 – – 0.5 1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 – 0.6 – 0.1 – – 0.2 – 0.4 – 0.4 – 0.4 – 0.1 – 0.2 0.1 0.1 0.1 – – 0.4 – 0.5 – 0.2 – 0.4 0.01 – – – – – – – – – 98.4 98.4 4.78 4.78 1.19 1.19 – – 1.7 1.7 1.19 1.19 25 25 0.37 0.37 4.79 4.79 3.4 3.4 1.0 1.0 – – 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.1 4 4 5 5 100 100 0.0072 – – 0.0023 – – 0.01 – – 0.0082 – – 0.0042 – – 0.0049 – – 0.00027 – – 0.00296 – – a Biggers et al. (2000). Gardner and Lane (2002). Media G1 and G2 are often denoted G1.2 and G2.2, being later versions of earlier formulations. c Gardner and Lane (1996). b denoted generically as X/Y. Two two-step protocols were used: DM2/DM1 (Lane and Gardner, 1995; Gardner and Lane, 1996) was prepared in our laboratory, and G1.2/G2.2 was purchased from Vitro Life (Göteborg, Sweden). At the time our work was done the exact chemical compositions of media G1.2 and G2.2 were a trade secret, but recently their compositions have been published (Lane and Gardner, 2003). KSOMgAA is KSOM (Lawitts and Biggers, 1993) supplemented with amino acids (Biggers et al., 2000), and glucose at 5.56 mmol/l (Summers et al., 2000; Biggers and McGinnis, 2001). A modification of KSOMgAA was also used in which EDTA was omitted and is denoted EDTA-free KSOMgAA. Zygotes were collected in medium FHM (Lawitts and Biggers, 1993) which contained 1 mg/ml polyvinylalcohol (PVA) in place of bovine serum albumin (Biggers et al., 1997). All chemicals and reagents, unless otherwise stated, were from Sigma Corp. (St Louis, MO, USA). Culture Embryos were cultured in sets of 12 per micro-drop in a tri-gas a (...truncated)