257 EXPRESSION PROFILING OF GENES CRUCIAL FOR LINEAGE DETERMINATION IN IN VITRO-DERIVED EARLY BOVINE EMBRYOS

Abstract

In the early blastocyst, lineage segregation depends on the expression of several key specific transcription factors. In the mouse, commitment to inner cell mass (ICM), lineage is positively regulated by Oct-4, a repressor of trophectoderm (TE) cell fate, and Nanog, which inhibits the formation of extra-embryonic and primitive endoderm. Cdx2, a caudal-type homeodomain protein, is specifically expressed in the nascent TE. The mechanisms that drive Cdx2 segregation to the outside cells are still unclear. However, the expression of Fgf Receptor 2 (FgfR2), restricted to the outside cells, and the role for its ligand, Fgf4, in promoting TE development, suggest that this signalling pathway may act upstream or in parallel with Cdx2. Little information is available on these genes in bovine; therefore the aims of the present study were as follows: (a) to identify and characterize the expression profiles of Cdx2 and FgfR2 variants (IIIc and IIIb) in bovine oocytes and pre-implantation embryos; and (b) to compare their expression patterns in ICM and TE with that of Oct-4 and Nanog. Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. RNA was isolated from MII oocytes; 2-, 4-, 8-, and 16-cell embryos; morulae; blastocysts; ICMs; and TEs. Semi-quantitative analysis of Cdx2 and FgfR2 expression in oocytes and embryos was performed in the exponential phase of PCR amplification with rabbit globin as exogenous control. In order to exclude false negative results, PCR amplification in isolated TE and ICM was extended to the plateau phase for all genes considered. Fragment identity was confirmed by sequencing. Comparison of bovine Cdx2 cDNA sequence (EMBL AM293662) with databases revealed a 91% and 87% homology with human and mouse, respectively. Cdx2 expression was not detectable in MII oocytes, but increased in 2-cell embryos. Transcript levels decreased at the 4- and 8-cell stages and then increased again in the blastocyst. FgfR2 variants were present as both maternal and embryonic transcripts, because they were detectable throughout pre-implantation development. Cdx2 and FgfR2 IIIc and IIIb expression was restricted to TE cells. Nanog was detected only in ICM, whereas Oct-4 was expressed in both lineages, as previously described in bovine (van Eijk et al. 1999 Bio. Reprod. 60, 1093-1103). In conclusion, the expression profiles of Nanog, Cdx2, and FgfR2 in bovine pre-implantation embryos follow the pattern previously described in the mouse. Their differentially segregated expression is consistent with their role as selector factors of ICM vs. TE fates. The significance of Oct-4 ubiquitous distribution still remains to be elucidated. This work was supported by FIRB RBNE01HPMX_005, TECLA-MIUR, and EUROSTELLS-ESF.