Gap junction formation and connexin distribution in pig trophoblast before implantation

This study describes the gap junctions in extraembryonic cell layers of the preimplantation pig embryo (trophectoderm and endoderm constituting the trophoblast). Using specific antibodies against connexins 31, 32 and 43, we found these connexins in embryos by immunodetection using Western blot and immunofluorescence analysis. By immunofluorescence, the first foci of connexin 31 were detected in the four-cell stage blastomeres, and the first diffuse gap junctions appeared at the eight-cell stage. Intercellular communication was observed with Lucifer yellow transfer to start also at the eight-cell stage around the onset of compaction. Typical gap junctions developed in the trophectoderm of blastocysts, as observed by transmission electron microscopy of thin sections and freeze-fracture replicas. Connexin proteins were differently expressed in time and space: connexin 31 was continuously present in trophectoderm, connexin 32 was essentially found in endoderm during elongation; connexin 43 was distributed in both trophectoderm and endoderm during blastulation and expansion. Connexin 43 was also found in two isoforms, phosphorylated or not, at day 14. Such developmentally regulated connexin expression may be essentially useful to control the exponential growth of trophoblast in preimplantation pig blastocysts.