TY - JOUR

T1 - Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos.

AU - Viuff, Dorthe

AU - Greve, Torben

AU - Holm, Peter

AU - Callesen, Henrik

AU - Hyttel, Poul

AU - Thomsen, Preben D

N1 - Ukendte organisationer '\'Klinisk Institut\', \'90\', \'90\''

PY - 2002/3

Y1 - 2002/3

N2 - In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.

AB - In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.

U2 - 10.1095/biolreprod66.3.629

DO - 10.1095/biolreprod66.3.629

M3 - Journal article

C2 - 11870068

VL - 66

SP - 629

EP - 634

JO - Biology of Reproduction

JF - Biology of Reproduction

SN - 0006-3363

IS - 3

ER -