Ouabain Stimulates a Na+/K+-ATPase-Mediated SFK-Activated Signalling Pathway That Regulates Tight Junction Function in the Mouse Blastocyst

Abstract

The Na+/K+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

p38 MAPK Regulates Cavitation and Tight Junction Function in the Mouse Blastocyst

“…The TE mediates implantation to the uterine wall, contributes to the embryonic portion of the placenta and is characterized by the expression of transcription factors (TF), such as caudal homeobox two ( Cdx2 ) [3]. Functional adherens junctions (AJ) and tight junctions (TJ) form a seal between the cells of the TE, which is essential for fluid accumulation and formation of the blastocyst cavity [4]–[12].…”

Cell Signalling During Blastocyst Morphogenesis

“…A further elaboration of the polarised epithelial phenotype necessary for blastocyst formation is the accumulation of functional basolateral membrane Na Cell signalling regulating TE epithelial differentiation and blastocyst formation appears upstream of both tight junction biogenesis and Na + K + -ATPase activity. Thus, SRC family kinases are required for development and maintenance of tight junction integrity and specific SRK inhibitors induce tight junction permeability and blastocyst collapse (Giannatselis et al 2011). p38 MAPK regulates filamentous actin dynamics during the morula stage (Paliga et al 2005) and controls tight junction permeability and blastocyst expansion (Bell and Watson 2013).…”

“…To date, direct evidence for involvement of AMPK signalling triggering early embryonic responses to maternal diet is lacking. However, as discussed above, essential features of the maturing TE in blastocyst morphogenesis which are sensitive to environmental cues include tight junction formation and barrier function (Eckert and Fleming 2008) in concert with Na + /K + -ATPase activity (Armitage et al 2008), collectively establishing cavitation as an interdependent process (Madan et al 2007;Eckert and Fleming 2008;Giannatselis et al 2011). Evidence from other systems suggests that AMPK signalling contributes to regulation of tight junction formation, barrier function and Na + /K + -ATPase activity directly or indirectly, possibly in response to short-chain fatty acids (Zhang et al 2011;Benziane et al 2012;Elamin et al 2013) thus being a plausible target for transmitting responses to maternal diet in early embryos, too.…”

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

“…4,5 The Na-K ATPase is also a critical mediator of blastocyst formation as it establishes a transtrophectoderm ionic gradient that directs fluid movement across the epithelium of the TE. 4,6,7 Aquaporins (AQP3 and AQP9) in the TE membrane facilitate the movement of fluids across the TE from the 'outside' to the 'inside' of the blastocyst cavity, along the ionic gradient established by the Na-K ATPase localized on the baso-lateral membrane. 4,8 After segregation of the early lineages is complete, the blastocyst emerges from the zona pellucida to invade the maternal uterine endometrium, where it implants.…”

“…27,36 The ion channels (including Na-K ATPase and ENaC), tight junctions (including E-cadherin and ZO-1) and AQPs (including AQP3 and AQP9) contribute directly to the mechanism that enables the TEs to regulate cavitation and blastocyst formation. 6,37 Functional adheren junctions and tight junctions form a seal between the cells of the TE, which is essential for fluid accumulation and the formation of the blastocyst cavity during the process of expanded blastocyst formation. 5,38 In this study, we found that the knockdown of either FGF2 or FGFR2 reduced the expression of E-cadherin and ZO-1 and perturbed their distribution within the TE as determined by quantitative real-time PCR and immunofluorescence staining.…”