Development of 1-Cell Embryos from Different Strains of Mice in CZB Medium

OF REPRODUCTION BIOLOGY 42, 432-440 (1990) Development of 1-Cell Embryos from Difterent Strains of Mice in CZB Medium1 C. L. CHATOT,2 I. TORRES, Worcester J. L. LEWIS, and C. A. ZIOMEK Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB CZB medium can be used to culture CFI x B6SJLFI/J 1-cell embryos to the blastocyst stage provided is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range ofSi to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65 85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CFJ and DBAJ2J (both x B6SJLFJ/J) mice, which exhibit a “2-cell block” to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2FJ/J and CDI female mice (both x B6SJLFI/J males), which do not exhibit a “2-cell block” to in vitro development, optimum development to rnorula and blastocyst stages (95% and 50%) was in CZB medium containing both glutwnine and glucose from the start of culture. medium. glucose - INTRODUCTION B6D2F1) and an inbred strain (C3H) were capable of 30- 60% development to the blastocyst stage in a defined bicarbonate-buffered medium, while other strains (C3H x DBA, C57, DBA, and Swiss mice) were incapable of development beyond the 2-cell stage (0-8% blastocysts) in this medium. A variety of improvements in embryo culture medium have resulted in increased success in the culture of embryos from some strains whose development in vitro is arrested at the 2-cell stage, i.e. the “2-cell block.” However, many of these improvements have not worked equally well in other laboratories. It is unclear whether the differences are environmental (H20, gas, etC.) or are due to different strains (and/or their suppliers) in use in these laboratories. Cross and Brinster (1973) demonstrated an optimum lactate/pyruvate ratio of 120 for the development of 1-cell Swiss mouse embryos beyond the 2-cell stage. Later, Abramczuk et al. (1977) reported that the addition of ethylenediaminetetraacetic acid (EDTA) to Whitten’s medium was beneficial in promoting the development of 1-cell embryos from ICR, C57B1/6, and, to a lesser extent, Balb/c mice to the blastocyst stage. More recently, Loutradis et al. (1987) have shown that hypoxanthine can cause developmental arrest at the 2-cell stage in vitro in CD1 1-cell embryos, although hybrid embryos B6D2F1 do not appear to be affected. Success with culture of 1-cell mouse embryos to the blastocyst stage has been shown to be a function of both strain of mouse and culture conditions. In early studies, investigators succeeded in culturing embryos from Fl hybrid animals but not from most random-bred strains and some inbred strains (Whitten and Biggers, 1968; Kaufman and Sachs, 1976; Goddard and Pratt, 1983). Whitten and Biggers (1968) cultured Fl hybrid embryos (from a C57B1/1OJ x SJL/J cross) with 75% of embryos developing to the blastocyst stage in a bicarbonate-buffered medium containing lactate, pyruvate, and glucose. In contrast, that study (Whitten and Biggers, 1968) also showed that the inbred strains SJL/ J, C57B1/1OJ, 129/Rr, and a random-bred strain developed to the blastocyst stage at rates of only 3, 18, 17, and 0%, respectively. Biggers (1971) subsequently confirmed that several hybrid strains (B6AF1, C57 x SJL, Accepted Received tmThis work November August was 17, 1989. 8, 1989. done as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development. NIH, through cooperative agreement HD21942 (C.A.Z.) and by the National Cancer Institute, Nih, grant P030CA12708 (C.AZ.). 2Reprint requests: Dr. Clare L. Qiatot, Worcester Foundation for Experimental Biology. 222 Maple Ave.. Shrewsbury, MA 01545. 432 ABSTRACT CULTURE OF MOUSE EMBRYOS MATERIALS METHODS Collection Embryo Female strains AND CF1 mice used (Harlan in this study Sprague-Dawley, were of the blocking 202, mdi- Colony DIFFERENT STRAINS 433 anapolis, IN) and DBA/2J (Jackson Laboratories, Bar Harbor, ME) and the nonblocking strains B6D2FI /J (an F! hybrid from C57B1/6J x DBA/2J, Jackson Laboratories) and CD1 (Charles River Breeding Laboratories, Wilmington, MA). Mice were superovulated with i.p. injections of 10 IU pregnant mare’s serum gonadotropin (PMSG, Calbiochem, La Jolla, CA,) followed 48 h later by 5 IU human chorionic gonadotropin (hCG, Organon, W. Orange, NJ). CF1, DBA/2J, B6D2F1/J, and CD1 females were mated overnight with B6SJLFI /J males (an Fl hybrid from C57B1/6J x SJL/J, Jackson Laboratories). Embryos were flushed from excised oviducts at 25 -27 h after hCG (Day I of culture) into Hanks’ balanced salt solution with 4 mg bovine serum albumin! ml (HBSS+BSA), treated briefly if necessary with hyaluronidase (300 units/mi in phosphate-buffered saline [PBS] containing 1% polyvinylpyrrolidone, Sigma Chemical Co., St. Louis, MO) to remove cumulus cells, washed 3 times in HBSS+BSA, and placed in holding drops of CZB medium as previously described (Chatot et at., 1989). Average yields of 1-cell embryos per animal were 55.1 (n=28 mice) for CFI x B6SJLFI/J, 18.3 (n=41) for DBA x B6SJLF1/J, 26.1 (n=33) for B6D2F1!J x B6SJLF1/J, and 12.6 (n=50) for CD1 x B6SJLF1/J. Embryos from each mouse were kept in separate 50-iJ holding drops of CZB medium until adequate numbers of embryos were collected and then were randomly distributed across all samples in each experiment. All manipulations were performed in a darkened room (Schumacher and Fischer, 1988) and all solutions, dishes, and instruments were maintained at 3TC prior to use. Culture Media Embryos were cultured in CZB medium (for formula, see Chatot et a!., 1989), which contains 1 mM glutamine, 0.1 mM EDTA, and 5 mg/mI BSA and lacks glucose unless otherwise indicated. CZB medium was prepared on the basis of weight using sterile endotoxinfree tissue culture grade water (Cat. #W3500, Sigma Chemical Co.) as described (Chatot et al., 1989). When glutamine was present in the medium, it was added immediately prior to use from a freshly prepared 100 mM stock to give a final concentration of 1 mM. When glucose was present in the medium from the start of culture, it was added to the medium to give a final concentration of 5.56 mM (1 mg/mi). Medium was stored gassed with 5% CO2:5% 02:90% N2 at 4#{176}C and was freshly prepared every 2 wk. One-cell embryos from CF1 mice, which do not develop beyond the 2-cell stage (...truncated)