Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

被引:19
作者
Bjerregaard, B
Wrenzycki, C
Strejcek, F
Laurincik, J
Holm, P
Ochs, RL
Rosenkranz, C
Callesen, H
Rath, D
Niemann, H
Maddox-Hyttel, P
机构
[1] Royal Vet & Agr Univ, Dept Anat & Physiol, DK-1870 Frederiksberg C, Denmark
[2] Inst Anim Sci, Dept Biotechnol, D-31535 Neustadt, Germany
[3] Constantin Philosopher Univ, SK-94974 Nitra, Slovakia
[4] Res Inst Anim Prod, SK-94974 Nitra, Slovakia
[5] Danish Inst Agr Sci, Dept Anim Breeding & Genet, Sect Reprod Biol, DK-8830 Tjele, Denmark
[6] Precis Therapeut, Pittsburgh, PA 15213 USA
[7] Vet Univ Vienna, A-1210 Vienna, Austria
关键词
developmental biology; early development; embryo; gene regulation;
D O I
10.1095/biolreprod.103.021683
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase 1, RNA polymerase 1 [RNA Pol 1], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laserscanning microscopy, and mRNA expression was determined by semi quantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [H-3]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitintreated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with alpha-amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol 1, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with a-amanitin, indicating that RNA Pol 1, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBE Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first. time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitroproduced embryos.
引用
收藏
页码:867 / 876
页数:10
相关论文
共 81 条
[51]   AUTOANTIBODY TO RNA POLYMERASE-I IN SCLERODERMA SERA [J].
REIMER, G ;
ROSE, KM ;
SCHEER, U ;
TAN, EM .
JOURNAL OF CLINICAL INVESTIGATION, 1987, 79 (01) :65-72
[52]   CHARACTERIZATION OF DEOXYRIBONUCLEIC-ACID SYNTHESIS AND THE TRANSITION FROM MATERNAL TO EMBRYONIC CONTROL IN THE 4-CELL PORCINE EMBRYO [J].
SCHOENBECK, RA ;
PETERS, MS ;
RICKORDS, LF ;
STUMPF, TT ;
PRATHER, RS .
BIOLOGY OF REPRODUCTION, 1992, 47 (06) :1118-1125
[53]   Onset of nucleolar and extranucleolar transcription and expression of fibrillarin in macaque embryos developing in vitro [J].
Schramm, RD ;
Bavister, BD .
BIOLOGY OF REPRODUCTION, 1999, 60 (03) :721-728
[54]   REGULATION OF ZYGOTIC GENE ACTIVATION IN THE MOUSE [J].
SCHULTZ, RM .
BIOESSAYS, 1993, 15 (08) :531-538
[55]   Cancer cell cycles [J].
Sherr, CJ .
SCIENCE, 1996, 274 (5293) :1672-1677
[56]  
Shim C, 1996, MOL REPROD DEV, V44, P44, DOI 10.1002/(SICI)1098-2795(199605)44:1&lt
[57]  
44::AID-MRD5&gt
[58]  
3.0.CO
[59]  
2-T
[60]  
SMITH RKW, 1985, J EMBRYOL EXP MORPH, V89, P133