Mouse Zygote Development in Culture Medium without Protein in the Presence of Ethylenediaminetetraacetic Acid

BIOLOGY OF REPRODUCTION 41, 835-841 (1989) Mouse Zygote Development in Culture Medium Protein in the Presence of Ethylenediaminetetraacetic R. A. FISSORE, Department K. V. JACKSON, of Obstetrics, Harvard Boston, without Acid1 and A. A. KIESSLING2 Gynecology and Reproductive Medical Biology School Massachusetts 02115 Development of zygotes from a hybrid-inbred (B6D2FI) and two random-bred (CDJ and CFI) strains of mice were compared after culture in several modflcations of a simple, chemically defined medium based on Earle’s Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p<O.O5). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2FJ, 56% or 45% for CDI, and 19% or 28% for CFJ, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2FJ females developed to blas:ocysts better than those from CDI or CFJ females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20 p.M)partially substitutedfor EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino aciLc suggested, however, that the stimulatory effect of EDTA on embryo development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (20% vs. 83% blastocysts, p<O.0l), although better than the unsupplemented medium, were significantly less effective than FI)TA -supplemented medium (83% blastocysts, p<O.Ol). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role. The protein-free, chemically defined culture medium used in this report provides a basic milieu to study the embryonic requirements of those strains that do not achieve maximal development in the culture conditions routinely used. INTRODUCTION vitro (Whitten, 1957; Brinster, 1965b). Despite this finding, embryos from only a few inbred strains of mice developed to blastocysts when cultured from the pronuclear stage (Whitten and Biggers, 1968; Biggers, 1971; Shire and Whitten, 1980). Thus, whereas embryos explanted at the two-cell stage could develop to blastocysts, zygotes from the same strains did not. The difficulty in culturing zygotes past the initial cleavage has become known as the “two-cell block.” The nature of the block remains unresolved. It is thought to be due to deficiencies in oocyte cytoplasmic factors (McLaren, 1981; Muggleton-Harris et al., 1982; Goddard and Pratt, 1983; Pratt and Muggleton-Harris, 1988), although the importance of the nucleus in zygote development has recently been emphasized (Robi et al., 1988). The inability of blocking strains to develop to blastocysts in vitro is due either to unsatisfied environmental requirements or to uncompensated culture stresses. The protein requirements of mammalian embryos developing in vitro have been investigated for several years. Earlier research indicated the need for a fixed- The nutritional and environmental requirements for preimplantation development of mammalian embryos are not well understood. The earliest studies of in vitro culture of mouse embryos used saline/hen-egg extracts as the supportive medium for morula-to-blastocyst development (Hammond, 1949). Subsequent reports confirmed that mouse morulae develop into blastocysts under a variety of culture conditions (Whitten, 1956; McLaren and Biggers, 1958). However, it was not until the discovery that earlier cleavage stages require pyruvate or lactate as energy sources that development of two-cell embryos to blastocysts occurred routinely in Accepted JuLy 19, 1989. Received April 6. 1989. tThis work was funded in pan by a Multi-Center Cooperative Program on Non-Human In Vitro Fertilization and Preiinplantation Development, the National Institute of Child Health and Human Development, Nil, through cooerazive agreement HD21988, and in part by 141)21890. ‘Reprint requests: A. A. Kiessling. Harvard Medical School, Seeley 0. Mudd Building. 250 Longwood Ave., Boston, MA 02115. 835 ABSTRACT 836 FISSORE ment to blastocysts of hardy hybrid-inbred (B6D2F1) and more sensitive random-bred (CD1 and CF1) zygotes cultured in simple medium containing EDTA, and to begin to characterize the mechanism by which EDTA exerts its beneficial effects. The results of these studies support the notion that the ligand function of proteins may be more important to early embryo development than their nutritional effects. MATERIALS Animals and AND METHODS Breeding Five to 10-wk-old virgin female mice of three strains: B6D2F1 (an Fl hybrid of two inbred strains, C57B1/6 x DBAt2J, Jackson Labs, ME), CD-i (random-bred, Swiss), and CF-i (random-bred, albino, nonSwiss) (Charles River, Wilmington, MA) were superovulated with 5 IU of pregnant mare’s serum gonadotropin (PMSG; Sigma Chemical Co., St. Louis, MO), induced to ovulate 48 h later with 5 IU of human chorionic gonadotropin (hCG; Sigma Chemical Co.) and housed overnight with fertile B6D2F1 (5 to 50 wk old) males. The presence of a copulation plug the following morning (16-18 h post-hCG) was considered Day 1 of pregnancy. Embryo Collection and Culture Embryos were handled as described (John and Kiessling, 1988; Jackson and Kiessling, 1989). Briefly, zygotes were retrieved from oviducts 10-20 h post-hCG from 5-6 females and pooled into Dulbecco’s phosphate-buffered saline (DPBS, Grand Island Biological Co., Grand Island, NY) with 4 mg/ml BSA (Frac. V. Sigma Chemical Co.) and supplemented with 0.095 g/l penicillin (Calbiochem-Behring Corp., La Jolla, CA) and 0.05 g/l streptomycin (Calbiochem). Hyaluronidase, 67 lU/mI (Sigma Chemical Co.), was added for 5 mm to remove the cumulus cells. Embryos were rinsed twice in DPBS/BSA and randomly assigned to the respective treatments in groups of 15-30 per center well of organ culture dishes (Falcon 3037, Fisher Scientific, Boston, MA) containing 1 ml of medium under 1 ml silicone oil previously washed with Type I water. Three milliliters of medium were placed in the outer well for humidification purposes. Pronuclear stage embryos with one or two polar bodies and no signs of degeneration were selected for culture. Embryos were observed at 24 h for cleavage to two-cells and at 96 h for development to morulae or blastocysts. Embryos nitrogen source to support in vitro development from two-cells to blastocysts (Brinster, 1965a). Albumin of bovine origin (BSA) has been the most commonly used source of protein for embryo culture, even though the mechanism of its observed beneficial effects remains unc (...truncated)