166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

Abstract

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon^® Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.