Oct4 expression in in-vitro-produced sheep blastocysts and embryonic-stem-like cells.
Transcription factor Oct4 (octamer-binding transcription factor-4) is important in early embryonic development and differentiation. It is also required for maintenance of pluripotency of the inner cell mass, and is used as a staminality marker of embryonic stem cells. Changes in Oct4 expression during the different stages of early embryo development have been reported, and therefore we have conducted a quantitative study of Oct4 gene expression of sheep blastocysts in vitro, and of embryonic-stem-like cells at the undifferentiated stage and in the course of differentiation. To characterize embryonic-stem-like cells, alkaline phosphatase activity, stage-specific embryonic surface antigens SSEA-1, SSEA-3, SSEA-4 and three specific gene markers Nanog, Sox2 and Stat3 were assayed. cDNA produced by RT (reverse transcriptase)-PCR was synthesized and amplified by PCR; sequencing gave 98, 95 and 98% homology with the bovine sequences of Oct4, Nanog and Stat3 respectively. Using the ovine sequence of 290 bp, quantitative expression of Oct4 in the inner cell mass, trophoblast and embryonic-stem-like cells was performed by qRT-PCR (quantitative real-time PCR). Oct4 was expressed in the inner cell mass, trophoblast and embryonic-stem-like cells. Expression in the inner cell mass was significantly higher than in the trophoblast. This could be useful in defining the quality of embryos produced and makes it possible to use Oct4 to detect pluripotency. In addition, the different levels of Oct4 expression between undifferentiated and differentiating embryonic-stem-like cell cultures could be used to detect this gene as a staminality marker.
Sanna
Daniela
D
Department of Research in Animal Production, Laboratory of Animal Reproduction and Biotechnology, 07040 Olmedo, Sardinia, Italy. danysanna@tiscali.it
Sanna
Angela
A
Mara
Laura
L
Pilichi
Susanna
S
Mastinu
Andrea
A
Chessa
Fabrizio
F
Pani
Luca
L
Dattena
Maria
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002454:Cell Differentiation; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism
D015295:Antigens, Tumor-Associated, Carbohydrate; D018398:Homeodomain Proteins; D016256:Lewis X Antigen; D050814:Octamer Transcription Factor-3; D050796:STAT3 Transcription Factor; D055746:Stage-Specific Embryonic Antigens; C052924:stage-specific embryonic antigen-3; C066584:stage-specific embryonic antigen-4; D000469:Alkaline Phosphatase