A comparison of two autoradiographic methods for detecting radiolabeled nucleic acids in embryos

Plante, L.; Jw, Pollard; Wa, King

doi:10.1002/mrd.1080330205

Cited by 16 publications

(11 citation statements)

References 22 publications

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“…In no case was [ 3 H]uridine incorporation detected in pronucleate-stage embryos. Although the possibility of very low levels of mRNA synthesis by pronucleate-stage embryos cannot be ruled out, the whole embryo spreading technique used in this study for autoradiography is considerably more sensitive than autoradiography of embedded/sectioned embryos [31], which has commonly been used to assess the onset of embryonic transcription. Although it could be argued that [ 3 H]uridine might have been converted into DNA in 2-cell embryos during the 6-h culture period, treatment with ␣-amanitin, an inhibitor of RNA polymerase II, prevented incorporation of label into 2-cell embryos, and treatment with RNase reduced labeling to background levels, clearly demonstrating its incorporation into mRNA.…”

Section: Discussionmentioning

confidence: 99%

“…A total of 66 embryos from 11 rhesus macaques were incubated for either 6 h (pronucleate stage, n ϭ 14), 6-10 h (2-cell, n ϭ 18), 5 h (4-to 5-cell, n ϭ 12), 4 h (7-to 8- cell, n ϭ 14), 1 h (16-cell, n ϭ 4), or 10 min (blastocyst, n ϭ 4) in 25-l drops of CMRL-BCS medium containing 200 Ci/ml of [5,6-3 H]uridine (Amersham, Oakville, ON, Canada), as described by Plante et al [31]. Earlier-stage embryos were cultured for longer periods of time to insure detection of label in embryos demonstrating low levels of incorporation.…”

Section: Analysis Of Rna Synthesismentioning

confidence: 99%

“…Autoradiography was performed in total darkness as described by Plante et al [31]. Briefly, slides were dipped into NTB2 photographic emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 with water and dried at room temperature.…”

Section: Analysis Of Rna Synthesismentioning

confidence: 99%

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Onset of Nucleolar and Extranucleolar Transcription and Expression of Fibrillarin in Macaque Embryos Developing In Vitro1

Schramm

Bavister

1999

Biology of Reproduction

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Specific aims were to characterize the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during preimplantation development in vitro in macaque embryos using autoradiographic and immunocytochemical techniques. Autoradiography was performed on whole embryos cultured with [3H]uridine for assessment of nucleolar (rRNA) and extranucleolar (mRNA) transcription. Expression of fibrillarin was immunocytochemically assessed in whole embryos using a primary antibody against fibrillarin and a fluorescein isothiocyanate-conjugated secondary antibody. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos cultured 6-10 h with [3H]uridine. Culture with alpha-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with alpha-amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin.

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Section: Discussionmentioning

confidence: 99%

Section: Analysis Of Rna Synthesismentioning

confidence: 99%

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Onset of Nucleolar and Extranucleolar Transcription and Expression of Fibrillarin in Macaque Embryos Developing In Vitro1

Schramm

Bavister

1999

Biology of Reproduction

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“…After comparison between sectioning and spreading, Plante et al (1992) found that the spreading technique is more sensitive, simpler and faster for autoradiography. By autoradiography with [ 3 H]thymidine, Kopecny et al (1995) detected DNA synthesis in mouse zygotes and 4-cell embryos, and Laurincik et al (1998) studied DNA synthesis in bovine zygotes.…”

Section: Discussionmentioning

confidence: 98%

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Xia

et al. 2002

Zygote

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Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 μCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 μCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of ≤ 1 h, 22% had a G1-phase of ≤ 2 h, 22% had a G1-phase of ≤ 3 h and 13% had a G1-phase of ≤ 4 h. The G1-phase in 85% of the 4-cell embryos was ≤ 3 h, that in 8% of embryos was ≤ 4 h and that in 7% of embryos was ≤ 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 μM, and the appropriate concentrations which did not impair development were 0.05-0.5 μM.

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“…Recovered embryos (n = 18) were prepared for autoradiography as described by Plante et al (1992). Embryos were cultured in wells containing 5 ~1 of l o x…”

Section: Experiments L-incorporation Of 3h-uridinementioning

confidence: 99%

Initiation of transcription and nucleologenesis in equine embryos

Brinsko

Ball

Ignotz

et al. 1995

Molecular Reproduction Devel

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The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 microCi/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counterstained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated 3H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of 3H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos.

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A comparison of two autoradiographic methods for detecting radiolabeled nucleic acids in embryos

Cited by 16 publications

References 22 publications

Onset of Nucleolar and Extranucleolar Transcription and Expression of Fibrillarin in Macaque Embryos Developing In Vitro1

Onset of Nucleolar and Extranucleolar Transcription and Expression of Fibrillarin in Macaque Embryos Developing In Vitro1

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Initiation of transcription and nucleologenesis in equine embryos

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