INITIATION OF TRANSCRIPTION AND NUCLEOLOGENESIS IN EQUINE EMBRYOS

The time of activation of the embryonic genome (maternal-embryonic tra nsition) in equine embryos was investigated by assessing incorporation of H-3-uridine and nucleolar development. In Experiment 1, embryos we re recovered from the oviduct (n = 15) and the uterus (n = 3). Recover ed embryos were assessed for morphologic development and quality score . Recovered embryos with less than 8 cells (two cells, n = 4; four cel ls, n = 5; five cells, n = 2) were incubated with H-3-uridine (560 mu Ci/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), a nd blastocysts (n = 3) were incubated with 280 mu Ci/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FB S and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embry os were spread onto glass slides, dipped into emulsion, and exposed fo r 8 d, then developed and counterstained with Giemsa and propidium iod ide. Embryos at the blastocyst, morula, eight-cell, and five-cell stag es incorporated H-3-uridine into their cell nuclei as detected by auto radiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleol ar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (n pb) consisting of homogeneous fibrillar structures. Two five- to six-c ell embryos also possessed reticulated nucleoli with both fibrillar an d granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granu lar components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of H-3-uridine and the forma tion of functional nucleoli with typical fibrillar and granular compon ents occurs between the four- to eight-cell stage in equine embryos. ( C) 1995 Wiley-Liss, Inc.