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Improves Development of One-Cell Random-Bred Mouse Embryos in Vitro J

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Improves Development of One-Cell Random-Bred Mouse Embryos in Vitro J The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro J. J. G. Brown and D. G. Whittingham Medical Research Council, Experimental Embryology and Teratology Unit, St George's Hospital Medical School, London SW17 ORE, UK Summary. Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation develop- ment of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyru- vate and glucose, lactate pyruvate and glucose for the first 24 h, and medium contain- ing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Sub- sequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development. Keywords: preimplantation; embryo; blastocyst; two-cell block; culture; mouse Introduction Mouse embryos of all strains can develop into blastocysts when cultured from the late two-cell stage in simple chemically defined physiological salt solutions containing the energy substrates lactate, pyruvate and glucose (Brinster, 1965a, b; Biggers et al., 1967, 1971). However, the embryos of most strains arrest at the two- to four-cell stage when cultured from the one-cell stage, a phenomenon commonly referred to as the -vitro two-cell block'. Only the embryos of certain inbred strains, and their Fl hybrids, develop consistently from the one-cell to blastocyst stage under these conditions (Whitten & Biggers, 1968; Biggers, 1971; 1987). Previously, it has been shown that the block may be obviated when arrested embryos are transferred to explanted oviducts in organ culture (Whittingham & Biggers, 1967) or when the cytoplasm from embryos of a non- blocking strain is injected into arrested two-cell stage embryos (Muggleton-Harris et al., 1982; Muggleton-Harris & Brown, 1988). F2 hybrid embryos of inbred origin, which do not arrest at the two-cell stage in vitro, require glucose for the transition from morula to blastocyst in vitro (Brown & Whittingham, 1990b, 1991). Preliminary studies indicated that most one-cell embryos from random-bred MF1 mice avoid 'Present address: Department of Pathology, Tufts University, 136 Harrison Avenue, Boston MA 02111, USA. tCorrespondence and reprint requests. Downloaded from Bioscientifica.com at 09/24/2021 10:07:20PM via free access cleavage arrest at the two-cell stage, and develop into morulae, when cultured in modified M16 mouse embryo culture medium (Whittingham, 1971) containing lactate and pyruvate but lacking glucose (Brown & Whittingham, 1990a). This observation is consistent with the findings of Chatot et al. (1989), who developed an improved medium for the culture of one-cell random-bred mouse embryos. The formulation of the medium (CZB) is based on the observation that glucose inhibits development of cultured two-cell (Schini & Bavister, 1988) and eight-cell (Seshagiri & Bavister, 1989a) hamster embryos, and glutamine improves the development of cultured eight-cell hamster embryos (Carney & Bavister, 1987). It is a modification of BMOC-2 (Ebert & Brinster, 1983) with an increased lactate:pyruvate ratio of 116 (Cross & Brinster, 1973), 0-lmmol EDTA 1 (Abramczuk et al., 1977), 1 mmol glutamine l-1 (Carney & Bavister, 1987) and no glucose. This medium improves development from the one-cell to morula stage, but the addition of glucose after the first 48 h of culture is necessary for consistent development to the blastocyst stage (Chatot et al., 1989, 1990a, b). The interactions between substrates are important in meeting the energetic requirements of cultured mouse embryos (Brinster, 1965b). Improved development of one-cell embryos from random-bred mice has been achieved by changing the combination and concentration of energy substrates at various times during culture (Cross & Brinster, 1973). This study examines the specific roles of lactate, pyruvate and glucose (and their interactions), and the possible role of glutamine, at various times during culture of one-cell mouse embryos of random-bred origin. Materials and Methods Embryo collection and culture Fertilized one-cell embryos were obtained from random-bred albino MFl/Lac females mated to MFl/Lac males. The mice were superovulated with intraperitoneal injections of 5 iu pregnant mares' serum gonadotrophin (PMSG; Folligon: Intervet, UK) followed 48 h later by 5 iu human chorionic gonadotrophin (hCG; Chorulon: Intervet, UK). In all experiments, fertilized one-cell embryos were recovered from the oviductal ampullae of mated females between ' 26 27 h after hCG The cumulus cells were with ( 150 iu ml in and injection. dispersed hyaluronidase " ) Hepes-buffered mouse embryo handling medium, M2 (Fulton & Whittingham, 1978; Quinn et al, 1982), and the embryos washed twice in M2 (2 2 ml). Embryos were cultured in bicarbonate-buffered mouse embryo culture medium, M16 (Whittingham, 1971) or modified M16 in which one or more of the energy substrates (lactate; pyruvate; glucose) was omitted. Media are referred to in the text according to the energy substrates they contain. Culture medium was freshly prepared each week from isotonic stock solutions of the various components so that changes in osmolarity, caused by the omission of energy substrate(s), could be adjusted simply by the addition of extra NaCl stock. The concentrations of lactate, pyruvate and glucose in all media were the same as those in unmodified M16 (23-28 mmol sodium lactate 1, 0-33 mmol sodium pyruvate I"1 (Sigma, UK), 5-56 mmol glucose l"1 (BDH, UK)). After washing thoroughly through 2 2 ml changes of the medium in which they were to be cultured, embryos were placed in small drops (10 µ ) of the same medium (10 embryos per drop) under light paraffin oil (BDH, UK) in 60 mm plastic tissue cul¬ ture dishes (Falcon, UK). Medium and oil were previously equilibrated with 5% C02 in air at 37°C. Embryos were cultured in a humidified atmosphere of 5% C02 in air at 37°C as described previously (Biggers et al, 1971), and developmental progress scored daily. Embryos in all treatments were taken through the washing procedures irrespective of whether they were to be transferred to the same, or different, media for the next culture period. Experimental design and statistical analysis Each experiment was repeated three times on three consecutive days, except for Expt 1 which was repeated three times in three separate weeks. Owing to the large number of treatments (36) involved in Expt 1, and thus the consider¬ able amount of washing involved in the transfer of embryos to different media each day, this was divided into three component experiments (12 treatments in each) carried out on different days. We ensured that experimental bias was not introduced by the use of embryos collected from different batches of mice on different days by randomly allocat¬ ing the treatments in each replicate to the three component experiments using a table of random numbers between 1 and 100 (Fisher & Yates, 1963). A randomized incomplete block design was used, which omitted the glucose-only medium, since preliminary studies showed this could not support first or second cleavage in random-bred MF1 embryos in vitro (J. Brown, unpublished observations). All embryos collected on each day were pooled in the final M 2 wash before being allocated to the different treatments. Since no marked differences in development were observed within treatments between replicates, the data Downloaded from Bioscientifica.com at 09/24/2021 10:07:20PM via free access Table 1. The effects of lactate, pyruvate and glucose on development of one-cell MF1 embryos in vitro Percentage of embryos developing to the various preimplantation Medium* stages during 96 h in culture % two-cell Treatment Day Days Number of after 24 h Four-to number 1 2-5 embryos0 in culture Two-cell eight-cell Morula Blastocyst L L 70 69 16 0 0 L 70 59 3 0 o L LP 70 53 61 20 0 o L LG 70 64 II 0 o L PG 70 76 11 0 o L LPG 60 67 17 0 o L 60 93 27 o o 60 93 37 o o 9 LP 70 81 S3 69 41 o 10 LG 70 SO 9 0 o 11 PG 70 90 II 0 o 12 LPG 70 VI) 59 10 6 13 LP L 70 93 34 1 o 14 LP 70 94 47 1 o 15 LP LP 60 89 90 75 33 o 16 LP LG 80 42 32 1 1 17 LP PG 80 79 20 3 3 18 LP LPG 60 97 73 22 13 19 LG L 60 45 7 0 0 20 LG 60 60 10 0 0 21 LG LP 70 55 70 21 4 0 22 LG LG 60 6S 17 0 o 23 LG PG 70 77 7 0 o 24 LG LPG 50 86 34 o o 25 PG L 80 95 32 10 3 26 PG 50 94 60 o 0 27 PG LP 70 88 93 S3 59 9 28 PG LG SO S9 30 0 0 29 PG PG 70 93 24 0 0 30 PG LPG 70 94 77 24 9 31 LPG L 60 93 23 2 0 32 LPG 60 87 32 0 0 33 LPG LP 70 92 97 SI 44 o 34 LPG LG 50 92 3 S 4 o 35 LPG PG 60 95 3 S 0 o 36 LPG LPG 70 94 76 17 3 'Embryos transferred to medium on day 1, 26-27 h after hCG; day 2, 46-48 h after hCG.
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