255 EXPRESSION OF TRANSCRIPTION FACTORS PRIOR TO THE MATERNAL-TO-ZYGOTIC TRANSITION IN BOVINE EMBRYOS

Abstract

During the first stages of bovine embryonic development, until the 8- to 16-cell stage, the zygote is maintained by the mRNA and proteins stored in the oocyte. New embryonic transcription is reported to begin only at the 8- to 16-cell stage even if some minor transcription is detected from the 2-cell stage. In order for this to occur, several factors are required to remodel the chromatin and activate the transcription machinery. Some regulating transcription factors are possibly present in the oocyte in their mRNA form, and their translation could enhance the maternal-to-zygotic transition (MZT). In our study, we observed the expression patterns of five transcription factors (ATF2, HMGN2, HMGB2, HUEL and MSY2) in bovine in vitro-produced embryos. Embryos were produced in vitro using selected cumulus-oocyte complexes from 3-5-mm follicles of slaughterhouse ovaries. Pooled GV or MII oocytes, and 2-, 4-, 8-cell and blastocyst-stage embryos (n = 40/stage) were washed in PBS and frozen at −80°C. Each pool was spiked with 1 pg of GFP RNA containing a poly(A) tail. The RNA was extracted using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA), co-precipitated with linear acrylamide (Ambion, Austin, TX, USA) and reverse-transcribed with Omniscript (Quiagen). The quantitative amplification of the transcription factors was performed in triplicate using the equivalent of 1 oocyte or embryo per reaction on a Lightcycler (Roche, Indianapolis, IN, USA). Data were normalized with the GFP levels found in each pool and a Least-Significant-Difference method was used for statistical analysis. Immunocytochemistry studies were performed on oocytes and embryos fixed and permeabilized in a solution of paraformaldehyde and Triton X-100, and results were observed on a confocal microscope. Our results show that the transcripts of the transcription factors studied are found at higher levels in pre-MZT embryos and at lower levels in subsequent stages. For HMGN2 and MSY2, there is a decrease in mRNA during oocyte maturation. For both genes, the residual mRNA remains constant up to the 4-cell stage before another loss in transcript levels in the 8-cell stage. In the case of ATF2, HMGB2 and HUEL, the maternal transcript levels are maintained until the 4-cell stage, suggesting that the mRNA is protected from degradation until its possible translation at the MZT. These results, combined to immunolocalization of the proteins, suggest a possible implication of some of these factors in the bovine MZT.