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Transcriptional level of developmentally important genes in bovine preimplantation embryos generated in vitro
Theriogenology
191
TRANSCRIPTIONAL LEVEL OF DEVELOPMENTALLY IMPORTANT GENES IN BOVINE PREIMPLANTATION EMBRYOS GENERATED IN VITRO C. Wrenzycki, D. Herrmann, E. Lemme, K. Korsawe, J.W. Carnwath and H. Niemann Institut Rlr Tierzucht und Tierverhalten (FAL), Mariensee, 3 1535 Neustadt, Germany During preimplantation development the embryo undergoes cleavage divisions and distinct alterations in cellular morphology, finally forming a blastocyst with two distinct cell lineages, the trophectoderm and inner cell mass. These events require the expression of specific genes in a stagespecific manner. The purpose of our present study was to investigate the level of transcription of genes characterizing several physiological compartments involved in compaction and cavitation [gap junction protein connexin43 (Cx43), desmosomal protein plakophilin (Plako), desmosomal glycoproteins desmoglein 1 (Dg l), desmocollins I, II, III (DC I, DC II, DC III)], metabolism [glucosetransporter-l (Glut-l)], RNA processing [poly(A) polymerase (PolyA)], stress [heat shock protein 70.1 (Hsp)], and trophoblastic function [trophoblast protein (TP)]. Embryos were produced from slaughterhouse ovaries employing standard protocols for in vitro maturation (TCM199 + 10% oestrus cow serum + hormones), in vitro fertilization (semen prepared by “swim-up“ procedure, TALP) and in vitro culture (TCM199 + 10% oestrus cow serum). Immature (i.ooc.) and matured oocytes (m.ooc.), zygotes (zyg.), 2- to 4cell (2-4), 8- to 16-cell (8-16) stages, morulae (mo.), blastocysts (bla.) and hatched blastocysts (h.bla.) were used. For semi-quantitative RTPCR analysis, rabbit globin mRNA was added just prior to RNA isolation as an external standard. Poly(A)‘RNA was prepared using a Dynabead oligo-dT mRNA purification kit (Dynal, Oslo, Norway). After electrophoresis and ethidium bromide staining each gene-specific fragment was quantified by digital imaging. The identity of the tiagments was confirmed by sequencing. Assays were repeated at least three times with different embryo batches and gave similar results in all samples. The results are shown in the table. No transcripts were detected for the genes DC I and Dg 1. Table: Relative amount of specific gene transcripts in preimplantation bovine embryos 8-16 mo. bla. i.ooc. m.ooc. 2-4 h.bla. zyg. CO.01 < 0.01 0 0.16iO.01 0.37hO.l 1 0.45iO.05 0.23+0.03 0.36iO.14 cx43 0.07*0.02 0.55*0.18 1.21+0.56 Plako 0.1410.03 0.12iO.02 0.24iO.05 0.12iO.02 0 0 0 0.66*0.12 0.77*0.30 0.51+0.12 5.87hO.64 10.19+2.51 0 DC II 0 0 0.04ztO.03 0.18*0.05 0.36*0.07 0.06*0.02 0.22*0.18 0 DC III Glut-l 0.99iO.31 0.93iO.20 0.66kO.25 0.68kO.26 0.2tiO.02 2.21*0.73 4.64kl.37 3.88i1.27 PolyA 4.92i0.94 8.16+0.76 7.39kl.60 9.02i2.76 4.68iO.05 3.96kO.92 3.83+0.23 6.183tl.53 3.26hO.77 1.36*0.17 2.51*0.07 2.83h4.11 4.llAO.46 5.40-11.68 6.5311.42 7.35+3.61 Hsp 0 1.77ztO.44 1.72hO.71 0 0 0 0 0 TP Values are shown as mean f SEM (ratio of densities of each specific versus the globin fragment) Our results suggest that bovine preimplantation embryos transcribe different developmentallyrelated genes in a stage-specific manner and show a characteristic course of up and down regulation of transcription. These findings will help characterize the transcriptional activity of bovine embryos generated in vivo or in vitro using different culture systems. This work was supported by grant Ni 256/12-l from the Deutsche Forschungsgemeinsch (DFG).
191
TRANSCRIPTIONAL LEVEL OF DEVELOPMENTALLY IMPORTANT GENES IN BOVINE PREIMPLANTATION EMBRYOS GENERATED IN VITRO C. Wrenzycki, D. Herrmann, E. Lemme, K. Korsawe, J.W. Carnwath and H. Niemann Institut Rlr Tierzucht und Tierverhalten (FAL), Mariensee, 3 1535 Neustadt, Germany During preimplantation development the embryo undergoes cleavage divisions and distinct alterations in cellular morphology, finally forming a blastocyst with two distinct cell lineages, the trophectoderm and inner cell mass. These events require the expression of specific genes in a stagespecific manner. The purpose of our present study was to investigate the level of transcription of genes characterizing several physiological compartments involved in compaction and cavitation [gap junction protein connexin43 (Cx43), desmosomal protein plakophilin (Plako), desmosomal glycoproteins desmoglein 1 (Dg l), desmocollins I, II, III (DC I, DC II, DC III)], metabolism [glucosetransporter-l (Glut-l)], RNA processing [poly(A) polymerase (PolyA)], stress [heat shock protein 70.1 (Hsp)], and trophoblastic function [trophoblast protein (TP)]. Embryos were produced from slaughterhouse ovaries employing standard protocols for in vitro maturation (TCM199 + 10% oestrus cow serum + hormones), in vitro fertilization (semen prepared by “swim-up“ procedure, TALP) and in vitro culture (TCM199 + 10% oestrus cow serum). Immature (i.ooc.) and matured oocytes (m.ooc.), zygotes (zyg.), 2- to 4cell (2-4), 8- to 16-cell (8-16) stages, morulae (mo.), blastocysts (bla.) and hatched blastocysts (h.bla.) were used. For semi-quantitative RTPCR analysis, rabbit globin mRNA was added just prior to RNA isolation as an external standard. Poly(A)‘RNA was prepared using a Dynabead oligo-dT mRNA purification kit (Dynal, Oslo, Norway). After electrophoresis and ethidium bromide staining each gene-specific fragment was quantified by digital imaging. The identity of the tiagments was confirmed by sequencing. Assays were repeated at least three times with different embryo batches and gave similar results in all samples. The results are shown in the table. No transcripts were detected for the genes DC I and Dg 1. Table: Relative amount of specific gene transcripts in preimplantation bovine embryos 8-16 mo. bla. i.ooc. m.ooc. 2-4 h.bla. zyg. CO.01 < 0.01 0 0.16iO.01 0.37hO.l 1 0.45iO.05 0.23+0.03 0.36iO.14 cx43 0.07*0.02 0.55*0.18 1.21+0.56 Plako 0.1410.03 0.12iO.02 0.24iO.05 0.12iO.02 0 0 0 0.66*0.12 0.77*0.30 0.51+0.12 5.87hO.64 10.19+2.51 0 DC II 0 0 0.04ztO.03 0.18*0.05 0.36*0.07 0.06*0.02 0.22*0.18 0 DC III Glut-l 0.99iO.31 0.93iO.20 0.66kO.25 0.68kO.26 0.2tiO.02 2.21*0.73 4.64kl.37 3.88i1.27 PolyA 4.92i0.94 8.16+0.76 7.39kl.60 9.02i2.76 4.68iO.05 3.96kO.92 3.83+0.23 6.183tl.53 3.26hO.77 1.36*0.17 2.51*0.07 2.83h4.11 4.llAO.46 5.40-11.68 6.5311.42 7.35+3.61 Hsp 0 1.77ztO.44 1.72hO.71 0 0 0 0 0 TP Values are shown as mean f SEM (ratio of densities of each specific versus the globin fragment) Our results suggest that bovine preimplantation embryos transcribe different developmentallyrelated genes in a stage-specific manner and show a characteristic course of up and down regulation of transcription. These findings will help characterize the transcriptional activity of bovine embryos generated in vivo or in vitro using different culture systems. This work was supported by grant Ni 256/12-l from the Deutsche Forschungsgemeinsch (DFG).