208 COMPARISON OF THE EXPRESSION OF SOX2 AND Stat3 IN BOVINE BLASTOCYST AND HATCHED BLASTOCYST

The investigation of the transcription factors involved in the regulation of pluripotency in pre-implantation embryos of cattle provides important information regarding the early embryonic development and derivation of embryonic stem cells. The present experiment aimed to compare the level of expression of SOX 2 and Stat3, pluripotency markers, in bovine blastocysts (D7) and hatched blastocysts (D10), because it is known that around hatching, the inner cell mass (ICM) differentiates into hypoblast and epiblast. Cumulus–oocyte complexes were matured in TCM 199 for 24 h and fertilized with frozen–thawed sperm. Presumptive zygotes were cultured in SOFaaci for 7 days (group 1) and for 10 days (group 2). All embryos were washed 3 times in PBS, pooled, and frozen at –80°C until RNA extraction. For quantification of SOX2 and Stat3 mRNA levels, total RNA was isolated from pools of 7 embryos per replicate (n = 3) and for each examined developmental stage using RNeasy Micro Kit (Qiagen). The reverse transcriptase Superscript III (Invitrogen) was used for the synthesis of cDNA (cDNA), and the qPCR was performed with the Gotaq qPCR Master Mix (Promega, Madison, WI, USA). As negative control, cDNA was replaced by nuclease-free water in the qPCR reaction. Quantification of expression was determined by the relative standard curve method and normalized to the housekeeping gene YWHAZ. Standard curves for SOX2, Stat 3, and YWHAZ were derived from 10-fold serial dilutions of bovine DNA and gave correlation coefficients greater than 0.99 and efficiencies greater than 94%. The data from 3 replicates were analysed by ANOVA, and it was found that the level of SOX2 abundance is 9.8-fold higher in D7 blastocyst cells compared with D10 blastocyst cells, and the level of Stat 3 transcription is 1.5-fold higher in blastocyst cells than in hatched blastocysts. The expression of pluripotency markers SOX2 and Stat3 was significantly higher in embryos with 7 days of development because at this stage the MCI had not yet begun the process of differentiation. Support of FAPESP is acknowledged.