Characterization of rDNA transcriptional activity in pre-implantation bovine embryos
Zusammenfassung der Projektergebnisse
Despite considerable progress made in the in vitro production of embryos, the overall efficiency, possibly caused by insufficient cytoplasmic maturation, remains low. Under our experimental conditions, FF-MAS can affect COC maturation, fertilization and early development in various ways. FF-MAS increased the degeneration rate of ova and the proportion of polyspermy regardless of the presence or absence of porcine follicular fluid in a dose dependent manner. In order to have a positive control for future experiments, we have developed technology which will support our experiments with sufficient numbers of embryos developed in vivo. The nucleolus is important for the development of oocytes and embryos and may serve as a marker for the completion of oocyte growth and regular activation of the embryonic genome. It appears that nucleolar formation in the embryo is less dependent on re-use of maternal proteins (or mRNA encoding them) inherited from the oocyte and to a greater extent on proteins synthesized from de novo transcribed mRNA from the embryonic genome. Moreover, maternally inherited rRNA target to the NORs where it is processed to ribosomal subunits independent of transcription of the rDNA. Activation of the rDNA transcription by RNA polymerase I is required for nucleolar formation and continued ribosome production. Information on the patterns of gene expression during initial embryonic development has become an important tool for understanding how embryo technological procedures may cause deviations in gene expression that can be detrimental for further embryonic development. The difference between embryos produced by different technical approaches regarding the activation of the embryonic genome indicates that cell cycle regulation is an important parameter for improving porcine fVP. It seems that under in vivo conditions the embryonic genome is activated like flipping a switch in all nuclei at the same time. The data from porcine embryos produced by SCNT suggest that it is possible to mimic this pattem. Although our IVP protocol has a success rate comparable that of other laboratories, activation of the embryonic transcription was asynchronous and delayed. Thus, the present data demonstrate that rRNA transcription is a useful marker for monitoring one of the crucial events of early embryonic development, the activation of the embryonic genome and, thus, for improving current FVP methods in the pig. Moreover, the similarity between parthenogenetic and SCNT embryos from 4 hpa during the first cell cycle underlines that maternal control to a high degree drives the development in the latter stages of the first cell cycle in SCNT embryos. Results of the project vrill be relevant as an alternative model to somatic cell nucleologenesis and will increase the number of checkpoints suitable for evaluation and improvement of the normality of embryos used in biomedical research or agricultural application. The research has resulted in eight full papers in refereed joumals, one book chapter and one book, one PhD thesis, several abstracts and oral presentations and thus provides a solid basis for future projects in this area.
Projektbezogene Publikationen (Auswahl)
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Centrosomal protein Centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos. Reproduction, 132, 423-434, 2006
Manandhar G., Feng D., Yi Y-J, Lai L., Ezashi T., Letko J., Laurincik J., Sutovsky M., Prather R.S., Schatten H., Sutovsky P.
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Nucleolar remodeling in nuclear transfer embryos. In: Somatic cell nuclear transfer. Ed.: Peter Sutovsky, Medical Intelligence Unit. Texas, USA, pp. 84-92, 2006
Laurincik, J. and Maddox-Hyttel, P.
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The effect of FF-MAS on porcine cumulus-oocyte complexes maturation, fertilization and pronucleus formation in vitro. Zygote, 14, 189-199, 2006
Faerge, I., Strejcek, F., Laurincik, J., Rath, D., Niemann, H., Schellander, K., Rosenkranz, Ch., Maddox-Hyttel, P., Grondahl, Ch.
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Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer. Mol. Reprod. Dev., 74, 35-41, 2007
Bjerregaard, B., Pedersen, H.G., Jakobsen, A.S., Rickords, L.F., Lai, L., Cheong, H.-T., Samuel, M., Prather, R.S., Strejcek, F., Rasmussen, Z., Laurincik, J., Niemann, H., Maddox-Hyttel, P., Dybdahl Thomsen, P.
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Early nuclear deprogramming in bovine somatic cell nuclear transfer embryo. International conference on farm animal reproduction. From egg to embryo. Program and Abstract Book, ICFAR, Rolduc, Netherlands, May 27-31, 2007, p. 90
Petrovicova, I., Svarcova, O., Strejcek, F., Lucas-Hahn, A., Lemme, E., Niemann, H., Laurincik, J., Maddox-Hyttel, P.
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Nuclear changes dutiny the first cell cycle in bovine somatic cell nuclear transfer embryos. Conference on reproductive and developmental biology, Prague, Czech Republic, June 21-22, 2007, p.35
Svarcova, O., Petrovicova, I., Strejcek, F., Lucas-Hahn, A., Lemme, E., Niemann, H., Laurincik, J., Maddox-Hyttel, P.
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Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos. Mol. Reprod. Dev., 74, 1428 - 35, 2007
Svarcova, O., Laurincik, J., Avery, B., Mlyncek, M., Niemann, H., Maddox-Hyttel, P.
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Nucleolar development requires transcriptrional activity during porcine embryonic genome activation. Proc. of the 33rd Annual Conference of the International Embryo Transfer Society, Kyoto, Japan, 7-9 January, 2007 Reprod. Fert. Dev., 19, 120-121, 2007
Svarcova, O., Maddox-Hyttel, P., Niemann, H., Hermann, D., Rasmussen, Z., Petrovicova, I., Strejcek, F., Laurincik, J.
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Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig. Theriogenology, 68 Suppl. 1, 63-70, 2007
Maddox-Hyttel, P., Svarcova, O., Laurincik, J.
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Early aspects of nuclear reprogramming following bovine somatic cell nuclear transfer. Proc. of the Annual Conference of the International Embryo Transfer Society, Denver, Colorado, USA, 5-9 January, Reprod., Fert. Dev., 20, 105, 2008
Petrovicova, I., Svarcova, O., Strejcek, F., Lucas-Hahn, A., Lemme, E., Niemann, H., Laurincik, J., Maddox-Hyttel, P.
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Endoscopic recovery of early preimplantation bovine embryos: Effect of hormonal stimulation, embryo kinetics and repeated collection. Reprod. Dom. Anim., 43, 566-572, 2008
Besenfelder, U., Havlicek, V., Moesslacher, G., Gilles, M., Tesfaye, D., Griese, J., Hoelker, M., Maddox-Hyttel, P., Laurincik, J., Brem, G., Schellander, K.
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The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos. Mol. Reprod. Dev., 75, 1095-1103, 2008
Svarcova, O., Strejcek, F., Petrovicova, I., Avery, B., Pedersen, H.G., Lucas-Hahn, A., Niemann, H., Laurincik, J., Maddox-Hyttel, P.
