Introduction: The pre-implantation embryo has two distinct cell lineages at the blastocyst stage. An outer epithelial monolayer called trophectoderm and the non-polar inner cell mass. Tight junctions (TJ) form continuous intercellular contacts between neighbouring trophectoderm cells and are crucial in establishing a blastocoele cavity. TJs are composed of several transmembrane proteins including Claudins, a large family of proteins that play a central role in the formation of the epithelial barrier. Claudins (Cl) show distinct distribution patterns among epithelial tissues and several isotypes can exist in the same TJ.
Methods: mRNA extracted from different stages of mouse embryos was reverse-transcribed before amplification of cDNA for particular Claudin isotypes. Embryos at 2-cell, 8-cell, morula and blastocyst stage were fixed for immunofluorescence and confocal microscopy. Zygotes were microinjected with siRNA and development to blastocyst observed.
Results: RT-PCR showed that Cl-4 and 7 were transcribed from compaction to blastocyst stage, while mRNA for Cl-3 and 6 were present from late morula stage. mRNA for Cl-12 was only present in blastocysts and mRNA for Cl-2 and 15 was absent throughout. Immunofluorescence showed Cl-4 and 7 proteins assembling at cellcell contacts after compaction in a dis-continuous pattern. By late blastocyst stage, Cl-3, 4, and 7 all displayed continuous staining at trophectoderm TJs, whilst Cl-12 and 15 were absent at TJs. siRNA inhibition of Cl-4 and 6 reduced percentage of blastocysts formed, whilst cavitation was not affected by Cl-7 siRNA. Our study shows that five different claudins are expressed in the early embryo and two play a crucial role in blastocyst formation. Funding by BBSRC to TPF.
02 - 04 Sep 2014
World Congress of Reproductive Biology