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Differential Inhibition of Trophoblast Outgrowth and Inner Cell Mass Growth by Actinomycin D in Cultured Mouse Embryos R

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Differential Inhibition of Trophoblast Outgrowth and Inner Cell Mass Growth by Actinomycin D in Cultured Mouse Embryos R Differential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured mouse embryos R. H. Glass, A. I. Spindle and R. A. Pedersen Department of Obstetrics and Gynecology and *Laboratory of Radiobiology, University of California, San Francisco, California 94143, U.S.A. Summary. Treatment of preimplantation mouse embryos in vitro with 10\p=n-\3 to 10\p=n-\1\g=m\g actinomycin D/ml for 2 hr showed that (i) postimplantation development in vitro was inhibited most when embryos were treated at the morula stage and (ii) after the morula stage actinomycin D inhibited trophoblast outgrowth less than inner cell mass de- velopment. Treatment of preimplantation mouse embryos with actinomycin D inhibits their preimplantation development in vitro (Skalko & Morse, 1969) and can cause developmental anomalies (Tuchmann- Duplessis & Mercier-Parot, 1960). It also impairs the capacity of the embryos for postimplantation development. When blastocysts are incubated briefly with 10_3-10_1 µg actinomycin D/ml and then transferred to pseudopregnant mice, fewer implant and develop into live fetuses than controls (Bell & Glass, 1975). When blastocysts are incubated continuously or for 24 hr with 5 IO-3 µg actinomycin D/ml and their postimplantation development in vitro is assessed, trophoblast outgrowths are formed but inner cell masses are not (Rowinski, Solter & Koprowski, 1975). To determine at what stage actinomycin D treatment begins to cause differential inhibition of trophoblast outgrowth and inner cell mass growth, we incubated embryos briefly with actinomycin D at the morula, early blastocyst, or late blastocyst stages and then allowed them to grow in vitro to early postimplantation stages for direct observation of inhibitory effects. Randomly bred 6- to 10-week-old Dub: (ICR) mice (Flow Research Animals, Inc., Dublin, Virginia) were induced to superovulate and were mated as described previously (Goldstein, Spindle & Pedersen, 1975). The day the plug was found was designated as Day 1 of pregnancy. The embryos were flushed from the oviducts and uteri on Day 3 (morulae) and from the uteri on Day 4 (early blastocysts) or Day 5 (late blastocysts, i.e. only those without a zona pellucida) with modified Hanks' balanced salt solution (BSS) and were cultured in modified Eagle's basal medium (BME) (Goldstein et al, 1975). A stock solution of actinomycin D (Calbiochem, La Jolla, California) was made with distilled water at a concentration of 10 µg/ml, kept at 4°C in the dark, and diluted to appropriate concentrations in modified BME before use. Embryos were incubated for 2 hr in medium containing actinomycin D, washed 5 times in modified Hanks' BSS, and transferred to 8-chamber culture slides (Lab Tek, Naperville, Illinois) containing modified BME. They were allowed to grow for 6 days (morulae), 5 days (early blastocysts) or 4 days (late blastocysts). The response of the embryos to actinomycin D treatment was evaluated at the end of the culture period by counting the number of embryos that developed to the following postimplantation end- points: (1) trophoblast outgrowth, (2) growth of a well defined inner cell mass on the trophoblast outgrowth, and (3) development of this inner cell mass into 2 germ layers (endoderm and ectoderm) recognizable under the dissecting microscope. Each treatment was repeated 4-12 times and the combined results were analysed with the 2 test for significant differences (1 d.f., <0-05 or <0-01). Downloaded from Bioscientifica.com at 09/25/2021 11:24:14AM via free access Table 1. Postimplantation development (as % of embryos treated) of mouse embryos treated at various preimplanta¬ tion stages with actinomycin D for 2 hr Dose of No. of Embryo stage actinomycin D embryos Trophoblast Growth of Development (culture period) ^g/ml) treated outgrowth inner cell mass of 2 layers Morula 0 188 73 52 29 (6 days) io-3 111 72 54 36 10"2 107 62* 39** 18**t IO"1 73 4** 0** 0** Early 0 280 94 80 52 blastocyst io-3 85 95 93 59 (5 days) io-2 66 89 77 44 io-1 145 71* 34**î ll**î Late 0 91 100 86 50 blastocyst IO"3 43 100 91 47 (4 days) -1 92 100 44* *î 10**î Significantly different ( 2) from respective control value, * <0 5; ** <0 1. Significantly different ( 2) from trophoblast outgrowth value, t <0-05; t <0·01. The results (Table 1) show that postimplantation development of mouse embryos was most in¬ hibited by actinomycin D when embryos were treated at the morula stage. At this stage inhibition of inner cell mass growth and trophoblast outgrowth was not significantly different at any dose, al¬ though at 10"2 µg/ml development to 2 layers was slightly more inhibited than trophoblast outgrowth. When early blastocysts were treated with 10"1 µg actinomycin D/ml, both growth of the inner cell mass and development to 2 layers were more inhibited than trophoblast outgrowth. When late ' blastocysts were treated with 10" µg actinomycin D/ml, only inner cell mass growth and develop¬ ment to 2 layers were inhibited. The primary effect of actinomycin D in mouse embryos is inhibition of RNA synthesis (Monesi, Molinaro, Spalleta & Davoli, 1970; Tasca & Hillman, 1970; Golbus, Calarco & Epstein, 1973), especially ribosomal RNA synthesis (Pikó, 1970), which in turn impairs protein synthesis. For example, a dose of 10" * µg/ml almost completely inhibits RNA synthesis and reduces protein synthesis by 20% (Monesi et al, 1970). The overall difference in sensitivity to actinomycin D that we found between morulae and blastocysts, as indicated by inhibition of postimplantation development, there¬ fore suggests that essential RNA or protein synthesis is not completed until after the morula stage. In addition, the differential inhibition of trophoblast outgrowth and inner cell mass growth, which appeared in embryos treated with actinomycin D at the early blastocyst stage and persisted in embryos treated at the late blastocyst stage, implies that trophoblast cells complete their essential gene expres¬ sion by the late blastocyst stage but that inner cell mass cells do not. Similarly, treatment of out¬ growing blastocysts for 24 hr with 5-bromodeoxyuridine, which also interferes with gene expression, indicates that the gene expression required for inner cell mass development continues until after the late blastocyst stage (Pedersen & Spindle, 1976). Work performed under the auspices of the U.S. Energy Research and Development Administra¬ tion. References Bell, P.S. & Glass, R.B. (1975) Development of the Goldstein, L.S., Spindle, A.I. & Pedersen, R.A. (1975) mouse blastocyst after actinomycin D treatment. X-ray sensitivity of the preimplantation mouse em¬ Fert. Steril. 26, 449^154. bryo in vitro. Radiât. Res. 62, 276-287. Golbus, M.S., Calarco, P.G. & Epstein, C.J. (1973) Monesi, V., Molinaro, M., Spalleta, E. & Davon, C The effects of inhibitors of RNA synthesis (a- (1970) Effect of metabolic inhibitors on macro- amanitin and actinomycin D) on preimplantation molecular synthesis and early development in the mouse embryogenesis. /. exp. Zool. 186, 207-216. mouse embryo. Expl Cell Res. 59, 197-206. Downloaded from Bioscientifica.com at 09/25/2021 11:24:14AM via free access Pedersen, R.A. & Spindle, A.I. (1976) Genetic effects Skalko, R.G. & Morse, J.M.D. (1969) The differential on mammalian development during and after im¬ response of the early mouse embryo to actinomycin plantation. In Embryogenesis in Mammals, Ciba D treatment in vitro. Teratology 2, 47-54. Fndn Symp. 40, 133-149. Elsevier, Amsterdam. Tasca, R.J. & Hillman, . (1970) Effects ofactinomycin , L. (1970) Synthesis of macromolecules in early D and cycloheximide on RNA and protein synthesis mouse embryos cultured in vitro: RNA, DNA, and a in cleavage stage mouse embryos. Nature, Lond. polysaccharide component. Devi Biol. 21, 257-279. 225, 1022-1025. Rowinski, J., Solter, D. & KopROWSKi, H. (1975) Tuchmann-Duplessis, H. & Mercier-Parot, L. (1960) Mouse embryo development in vitro: effects of The teratogenic action of the antibiotic actinomycin inhibitors of RNA and protein synthesis on blasto¬ D. In Congenital Malformations, Ciba Fndn Symp., cyst and post-blastocyst embryos. /. exp. Zool. 192, pp. 115-128. Eds. G.E.W. Wolstenholme & M. 133-142. O'Connor. J. & A. Churchill, London. Received 17 July 1976 Downloaded from Bioscientifica.com at 09/25/2021 11:24:14AM via free access.
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