Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

Abstract

In vitro culture during assisted reproduction technologies (ART) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2h in medium with osmolarity raised by 400mOsm induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation, and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

Protein O-GlcNAcylation Promotes Trophoblast Differentiation at Implantation

“…O-GlcNAcylation regulates the function of 1000+ proteins in line with metabolic and environmental status, contributing to the appropriate adaptation of cells to stressors [ 41 ]. Stress-induced differentiation has been implicated as a mechanism driving trophoblast invasion during implantation and placentation [ 9 , 10 , 11 ]; the present study evidences O-GlcNAcylation as a novel mediator of invasive trophoblast differentiation from TE that may play a role in the embryonic response to metabolic and environmental stress. Peri-implantation effects of ART procedures and maternal metabolic syndromes could, therefore, promote initial implantation, but this may be at the expense of long term placental function, which requires a balance of proliferative and invasive trophoblast populations that could be compromised by peri-implantation stress signalling [ 3 ].…”

“…To increase O-GlcNAcylated protein levels, mouse blastocysts were treated with the OGA inhibitor thiamet g (TMG) at 5 µM [ 33 , 34 , 35 ] during the E4.5–5.5 period when embryos are sensitive to signals that activate invasive implantation [ 10 , 36 , 37 ]. Untreated mouse blastocysts exhibited O-GlcNAcylated protein staining in the nucleus and especially the nuclear membrane ( Figure 1 A).…”

“…Our in vitro model of implantation suggests that increased O-GlcNAcylation does not affect TE function with regard to attachment to endometrial epithelial cells. Activation of embryos through initial contact with endometrial epithelial cells between E4.5 and 5.5, prior to stable attachment, is required for blastocysts to go on to breach the epithelial layer [ 36 ], and we have previously reported that hyperosmotic stress-induced signalling through JNK can activate embryos in the absence of epithelial contact [ 10 ]. Here, TMG treatment during E4.5–5.5 also activated the embryos, suggesting that TMG-induced O-GlcNAcylation mimicked a stress response to promote breaching of the epithelium.…”

“…Embryos use a variety of signalling pathways to sense environmental stressors and elicit responses enabling stress resolution or adaptation [ 9 ]. We have previously shown that in response to osmotic stress, the c-Jun N-terminal kinase (JNK) pathway triggers embryonic invasion of endometrial epithelium in vitro, implicating stress signalling in TE differentiation to trophoblast at implantation [ 10 ]. Moreover, JNK regulates trophoblast functions during placentation [ 11 ].…”

Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

“…The potential for OPN to alter trophoblast lineage allocation implies a regulatory role which could be important to balance populations of proliferating and invasive trophoblasts during preimplantation embryo transition to maternally recognised conceptus. External stressors disrupt this balance and induce excessive TGC allocation, which is associated with pregnancy failure [46,47].…”