Cripto Pathway Regulation of Embryo-Maternal Recognition and Embryo Attachment

Many embryonic and maternal factors regulate embryo-maternal recognition and embryo implantation. One such factor is Cripto-1 (TDGF1), a GPI-anchored and secreted member of the EGF-CFC family. Cripto regulates multiple signaling pathways and stimulates cell proliferation, migration, angiogenesis and endothelial to mesenchymal transition in embryogenesis and tumorigenesis.

We aimed to explore the role Cripto in embryo survival and implantation.

We used western blot and immunohistochemistry analysis to analyze Cripto expression and localization in mouse uteri at different days of pregnancy. We also implanted control embryos or embryos with targeted knockdown of Cripto expression in trophoblast cells in uteri of pseudopregnant females in order to explore the role of Cripto in embryo implantation. Implantation sites were histologically examined on day 4.5 of implantation (E4.5) and tested by quantitive PCR and immunostaining to measure expression of genes involved in embryo attachment, proliferation and apoptosis.

We find that Cripto is expressed by the implant blastocyst and in the uterus at embryo implantation sites. Cripto knockdown in trophoblast cells inhibited its function as reflected by decreased levels of the Cripto signaling mediators phosphorylated SMAD2 (pSMAD2), pSRC and pERK. Knockdown of Cripto expression in the embryonic trophoblast cells also reduced the proliferation of these cells while increasing their apoptosis. Notably, loss of Cripto in embryonic trophoblasts resulted in abnormally enlarged implantation sites and defective implantation attributed to inappropriate attachment of the embryo to the uterine wall.

Our findings indicate that Cripto may play an important role in trophoblasts of the pre-implanted embryo and during embryo attachment and implantation.