FGF2 Promotes Primitive Endoderm Development in Bovine Blastocysts.

Abstract

Cell fate specification begins in preimplantation embryo with the emergence of trophectoderm (TE). Primitive endoderm (PE) is the second extraembryonic lineage to form from cells within the inner cell mass (ICM) during embryogenesis. Ruminant conceptuses elongate dramatically prior to uterine attachment and extensive migration and proliferation of TE and PE is crucial for normal conceptus development and elongation. Work described here provides evidence that FGF2 and potentially other FGFs impact PE development during bovine embryonic development. Bovine blastocyst outgrowths were generated from in vitro-produced embryos using a feeder-layer free culture system containing a Matrigel-coated surface. Individually cultured bovine blastocysts were supplemented with FGF2 at the beginning of culture (day 8 post-IVF) and identification of PE-like cells was evaluated microscopically on days 13 and 15. In controls, only a single blastocyst outgrowth contained what appeared morphologically as PE cells (1.3% of all outgrowths). By contrast, 10.5±6.7%, 19.7±0.28% and 22.4±0.26% of outgrowths contained PE colonies on day 15 when embryos were exposed to 0.5, 5 and 50ng/ml FGF2, respectively. A subset of outgrowths (n=6 TE only and 6 PE-containing) were selected and cultured to day 20, then lineage-specific markers were evaluated using qRT-PCR. Transcripts for IFNT and CDX2 (TE markers) were present in TE outgrowths. Trace amounts of IFNT and CDX2 mRNA were detected in PE colonies (P<0.05) likely because residual TE cells still existed in some of these outgrowths. PE-containing outgrowths contained ample quantities of GATA4 and GATA6 mRNA (putative PE markers) whereas TE outgrowths contained no GATA4 and low amounts of GATA6 mRNA. None of the outgrowths contained NANOG mRNA whereas all TE and PE outgrowths contained OCT4 mRNA. Western blot analysis determined PE-containing outgrowths produced transferrin, a PE product. Moreover, immunoreactive GATA4 localized to nuclei of PE cells but was not detected in TE cells. By contrast, CDX2 localized to TE nuclei but was not detected in PE cells. Continued cultivation and selection for TE and PE yielded pure cell populations (n=6 of each). Differences in relative abundance of FGF receptors (FGFRs) and selective alternatively spliced receptor isoforms were evident between the cell types. Specifically, transcripts for FGFR2b and FGFR3 predominated in TE cells while FGFR1b represented the primary transcript in PE cells. TE and PE cells responded differently to FGF2 treatment. FGF2 supplementation did not affect the mitotic index of TE cells whereas the mitotic index of PE cells increased (P<0.05) following exposure to 5 or 50ng/ml FGF2 (35.1±0.09% and 34.7±0.11%, respectively versus 19.9±0.06% for controls). These observations indicate that the non-TE cells formed with blastocyst exposure to FGF2 are PE, and these cells respond differently to FGF2 than TE. Collectively, this work provides new insight into another vital activity for FGF2 and potentially other FGFs during early embryonic development in bovine. This project was supported by NRICGP number 2008-35203-19106 from the USDA-CSREES. (platform)