Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA.
This study investigated the effects of a semi-defined culture system on the temporal pattern of expression of RNA from genes involved in compaction and cavitation: gap junction protein connexin43 (Cx43), desmosomal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (PolyA); heat shock protein 70.1 (HSP); and trophoblastic function trophoblast protein (TP) in bovine oocytes and embryos generated in vitro using TCM199 supplemented with BSA as the culture medium. Morulae and blastocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)+ RNA was extracted from pools of 20-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing. Assays were repeated at least three times for each developmental stage and provided consistent results in all replicates. In bovine embryos produced in vitro, mRNA encoding Cx43 was detectable up to the morula stage, whereas blastocysts and hatched blastocysts did not express this gene. No transcripts were found for Dg 1 and Dc I throughout the tested preimplantation stages. Dc II and Dc III transcripts were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encoding Plako was detected in immature and mature oocytes and zygotes, while no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for Glut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, with one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine embryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. These data contribute to the molecular characterization of this widely used in vitro culture system for bovine embryos and provide a major advance towards production of 'physiologically normal' embryos.
Wrenzycki
C
C
Department of Biotechnology, Institut für Tierzucht und Tierverhalten (FAL), Mariensee, Neustadt, Germany.
Herrmann
D
D
Carnwath
J W
JW
Niemann
H
H
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle / Q000502:physiology*; D018031:Connexin 43 / Q000235:genetics; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000235:genetics; D051187:Desmocollins; D051183:Desmoglein 1; D051182:Desmogleins; D051180:Desmoplakins; D005796:Genes; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D007370:Interferon Type I / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D009028:Morula / Q000502:physiology; D051191:Plakophilins; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics; D011257:Pregnancy Proteins / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D012710:Serum Albumin, Bovine; D014158:Transcription, Genetic
D018031:Connexin 43; D003470:Culture Media; D003598:Cytoskeletal Proteins; D051187:Desmocollins; D051183:Desmoglein 1; D051182:Desmogleins; D051180:Desmoplakins; D018840:HSP70 Heat-Shock Proteins; D007370:Interferon Type I; D009004:Monosaccharide Transport Proteins; D051191:Plakophilins; D011257:Pregnancy Proteins; D011506:Proteins; D012333:RNA, Messenger; C020947:interferon tau; D012710:Serum Albumin, Bovine; D011062:Polynucleotide Adenylyltransferase