Activation of Mouse Metallothionein-I Promoter in Mouse Preimplantation Embryos After Pronuclear Microinjection

Abstract

To analyze expression of developmental genes and their regulatory mechanisms in mouse preimplantation embryos, pronuclei of in vitro-fertilized eggs were microinjected 8-10 h after insemination (hpi) with a fusion gene composed of the mouse metallothionein-I promoter (MT-I) and the Escherichia coli β-galactosidase gene (lacZ). The eggs were cultured up to the blastocyst stage in Whitten's medium supplemented with 100 μM EDTA. Expression of the lacZ gene was examined by staining the embryos with X-gal as a substrate. Expression was first detected at the 2-cell stage, 27 hpi; the proportion of embryos expressing the lacZ gene reached a maximum of 80% at 33-48 hpi. The proportion declined from the 4-cell stage onward, but some embryos still showed positive staining at the blastocyst stage. α-Amanitin blocked the expression of lacZ gene only when added before 12 hpi, suggesting that transcription had started during the pronuclear stage rather than in cleavage stages. The presence of EDTA in the culture medium reduced the expression markedly from the morula stage on, but 25 μM ZnCl₂ enhanced expression after the 4-cell stage in the presence of EDTA. These results suggest that transcription is active at the pronuclear stage and that the MT-I promoter becomes inducible by ZnCl₂ after activation of the embryonic genome.