Submitted to: Journal Of Reproduction, Fertility And Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 14, 2003
Publication Date: N/A
Interpretive Summary: The presence of the protein alkaline phosphatase (AP) has served as a biological marker for embryonic cells at the earliest stages of development and is considered a standard marker for the identification of embryonic stem (ES) cells in mouse and humans. This study was conducted to verify that AP staining could also serve as marker for ES cells in cattle and that embryonic treatments, nor culture conditions influenced the validity of the assay. However, after examining several hundred bovine embryos at various stages of development from the 1-cell stage to the blastocyst stage of development it was found that removing the protective coating around the embryo, the zona pellucida, dramatically changed the AP staining characteristics of bovine embryos. Furthermore, several of the most common bovine embryo culture mediums also change the AP staining pattern. It was concluded that AP staining may not serve as a reliable marker for early embryonic cells in bovine embryos.
Objectives were to 1) characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with zona pellucida (ZP) intact or removed, and 2) evaluate influence of culture medium constituents on AP activity. AP activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In greater than 90% of 2- to 16-cell embryos, AP activity was observed in perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. AP activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising culture medium altered AP activity. AP activity was reduced in 8-16 cell embryos and evacuated ZP cultured in CR1aa + 3 mg/mL BSA versus those cultured in CR1aa or CR1aa + 3 mg/mL BSA + 10% FBS. In summary, AP activity was present in bovine embryos as early as the 2-cell with greatest activity noted at blastomere contacts and ZP. AP activity may be altered by specific constituents of culture medium. Moreover, the presence of AP activity in trophectoderm and evacuated ZP limits its usefulness as a marker for differentiation of embryonic cells comprising the early embryo.