103 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IS DISPENSIBLE DURING REGULATION OF TIGHT JUNCTION MEMBRANE ASSEMBLY BY CELL CONTACT PATTERN AND PKC SIGNALING

Contact symmetries are involved in regulating cell lineage segregation during blastocyst biogenesis when tight junction (TJ) membrane assembly is restricted to the epithelial trophectoderm (TE). Manipulation of cell contact patterns by immunosurgical isolation of inner cell masses (ICMs) providing a contact-free cell surface serves as a switch to induce TE differentiation upon in vitro culture. In this model, protein kinase C (PKC)-mediated signaling up-regulates TJ membrane assembly. Whether signaling via gap junctional intercellular communication (GJIC) affects these processes is controversial. The current study investigates the interrelationship between changes in cell contact pattern, PKC signaling, and GJIC on TE differentiation and TJ assembly. Eight-cell embryos flushed from MF1 mice were cultured in T6/BSA to time development to early blastocyst stage (<2 h of cavitation). Laser confocal microscopy (BioRad MRC 600, BioRad Laboratories, Inc., Hertfordshire, UK) after immunostaining with antibodies against PKCδ, θ, λ, or ζ isoforms (Transduction Labs, Oxford, UK or Sigma) or junctional proteins (E-cadherin, ZO-2, Occludin, ZO-1α+, Desmoplakin) combined with ALEXA 488 conjugated secondary antibodies (Cambridge Bioscience, Oxford, UK) was used to determine the distribution of PKCs and junctional proteins in intact blastocysts and fully and partially isolated ICMs after immunosurgery and in vitro culture in DMEM + 10% FCS (Eckert et al. 2004 Reproduction 127, 653). While broad PKC activators (1 μM 12-O-tetradecanoylphorbol-13-acetate or Indolactam; Calbiochem, Nottingham, UK) accelerate membrane assembly of the TJ proteins ZO-2 and ZO-1α+ in fully isolated ICMs this up-regulation was suppressed in intact blastocysts (n = 32–47 per treatment and antibody) and in partially isolated ICMs (remnants of lysed TE remaining surrounding the ICM; n = 17–21 per treatment and antibody) for up to 24 h with no TJ protein detectable within the ICM, even after two consecutive rounds of TE lysis (n = 13–22 per treatment and antibody). When GJIC was inhibited during blastocyst formation in vitro and in cultured fully isolatd ICMs by 18 α-glycyrrhetinic acid (AGA, 65 μM; Sigma), cavitation rate and distribution of PKCs or junction assembly were not affected compared to controls (70–80% cavitated with characteristic distribution of junctional proteins and PKCs; P > 0.05, ANOVA; n = 15–20 per treatment, antibody, and cell contact pattern). When GJIC inhibition by AGA was confirmed by Lucifer yellow (Sigma) injection (no dye transfer in 82–100%, n = 14–17 per contact pattern), GJIC was also absent in 50% of fully isolated ICMs without AGA treatment, suggesting that cell contact modulation may affect GJIC. Taken together, our data suggest that cell contact pattern regulates TJ assembly via PKC signaling pathways and may also affect GJIC. GJIC appeared dispensable during cavitation, TJ assembly, and PKC signaling. A better understanding of the interrelationships between different signaling mechanisms may help to improve embryo culture methods and viability. Funding by the Wellcome Trust and MRC is gratefully acknowledged.