126 MOUSE BLASTOCYST FORMATION IS REGULATED BY SPECIFIC PKC ISOFORMS THAT AFFECT LOCALIZATION OF NA/K-ATPASE

Abstract

During blastocyst biogenesis, establishment of a trans-trophectodermal ion gradient by the Na/K-ATPase ion transporter family is essential for fluid accumulation within the blastocoelic cavity. In the mouse, the α1β1 isozyme combination confined to the basolateral membrane within the trophectoderm is considered mainly responsible. Enzyme subunits, e.g. α1, can become inactive by internalization upon phosphorylation potentially mediated by protein kinase C (PKC). At least 11 PKC isotypes with variable biological functions dependent upon cellular contexts have been identified so far. The present study examined the role of specific PKC isoforms during cavitation in relation to potential influence on Na/K-ATPase localization using cell-permeable PKC isoform-specific activating or inhibiting peptides. At 20 h post-compaction, in vitro-generated (T6-BSA) late morulae (MF1) were incubated for up to 4 hrs in 500 μL DMEM with 10% FCS and 0, 0.1, 0.5 or 1 μM PKCδ agonist or antagonist or PKCζ antagonist alone or with 0.1 μM PKCδ and ζ antagonist combined. Either peptides (obtained from Dr. Daria Mochly-Rosen, Stanford University, Standford, CA, USA) were renewed after 2 h or embryos were washed and placed into peptide-free medium (release). Cavitation was recorded hourly before fixation in ice-cold methanol for 10 min. Embryos were double-labelled with antibodies against a-catenin for visualization of the cell membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the α1 subunit of the Na/K-ATPase (Upstate Biotechnology, Wolverton Mill South, UK) combined with ALEXA 488 or 568 conjugated respective secondary antibodies (Molecular Probes, Eugene, OR, USA). Protein localization was analyzed (Confocal Assistant software) after laser confocal microscopy (BioRad MRC 600, Hemel Hampstead, Hertfordshire, UK) at 60× magnification under oil. Cavitation was significantly (P < 0.05; ANOVA) delayed in a partially dose-dependent and reversible manner by PKCδ and/or ζ antagonists compared to controls (n = 60–73 per treatment; cavitation after e.g. 3 h: 7.4–21% in control, 1.2–2.9% in PKCδ or ζ antagonist alone or combined v. 12.5–18.5% in PKCδ agonist or 5.5–7.5% peptide release). Toxicity was excluded as carrier peptides alone had no effect on cavitation, and 70–90% formed blastocysts after overnight culture with peptides. In addition, inhibition of PKCζ or activation PKCδ caused internalization of the α1 subunit in 85–87.5% of embryos as compared to 22.2% within the control embryos (P < 0.05, ANOVA; n = 17–43 per treatment). This reversal of membrane insertion of one functional enzyme subunit indicative of decreased enzyme function occurred similarly within cavitated and non-cavitated embryos. Taken together, our data suggest that PKCδ and ζ participate in regulating cavitation by affecting different mechanisms. While decreased PKCζ activity delays cavitation and reduces Na/K-ATPase function, the various targets of PKCδ need further examination. A better understanding of underlying mechanisms involved in regulating the complex process of cavitation could help to improve embryo viability. Funding by the Wellcome Trust and MRC is gratefully acknowledged.