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Characterization of blastomeres derived from different stages of in vitro produced buffalo (Bubalus bubalis) embryos to determine the stemness/pluripotential ability

Author(s): Srinivasa Prasad Chigurupati, | Palanisamy, A, | Satheshkumar, S, | Gomathy, V.S., | Dhinakar Raj, | Thangavel, A

Journal: Research Opinions in Animal & Veterinary Sciences
ISSN 2221-1896

Volume: 2;
Issue: 2;
Start page: 72;
Date: 2012;
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Keywords: Embryonic stem cells | Oct-4 | Sox-2 | Gene expression-Buffalo

The present study was carried out to study the expression of genes for pluripotency Oct-4 and Sox-2 in blastomeres, derived from various stages of in vitro produced buffalo embryos, for determination of the stem cell characteristics or pluripotential ability. 0.5 per cent pronase was used for zona lysis and subsequent isolation of embryonic stem (ES) cells. RT-PCR was performed by using reverse specific primer (3’ primer) for the first strand synthesis. Strong Oct-4 and Sox-2 expression were consistently observed in cells, obtained from 16-cell stage embryos, morula and blastocyst, whereas in cells from 2, 4 and 8-cell embryos (pre ZGA) Oct-4 and Sox-2 expression was low and variable. Out of eight trials, in three trials the blastomeres of 2-cell, 4-cell and 8-cell stage embryos (Pre ZGA) did not express Oct-4 gene and in five trials a faint band was observed. With regard to Sox-2 gene expression, in four of six trials the blastomeres of 2-cell, 4-cell and 8-cell stage embryos (Pre ZGA) a faint band was observed, but in two trials Sox-2 gene was not expressed, eventhough they were believed to be totipotent. Low and variable expression of Oct-4 and Sox-2 genes in early stage embryos (pre-ZGA) might be related to the exhaustion of maternally-generated Oct-4 and Sox-2 transcripts and then their recovery appears to be via expression of zygotic transcripts, which takes place in buffalo embryos from 16 cell stages onwards. Epigenetic mechanisms might be the cause of the low levels of Oct -4 and Sox-2 expression after fertilization. The sequence results for Oct-4 (NCBI Ac. No: EU661360) and Sox-2 (NCBI Ac. No: EU661361) were analyzed by BLAST and it was found that Oct-4 had 88% - 97% homology and Sox-2 - 93% to 99% homology across the phylogeny with mouse being distant relative.
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