Synthesis of putative microtubule-associated proteins by mouse blastocysts during early outgrowth in vitro

Abstract

The outgrowth of mouse trophoblast in culture provides a simplified model system analogous in certain ways to blastocyst implantation in vivo. Day-four blastocysts cultured for 3 days in vitro undergo extensive changes in cell shape and motility which are likely to involve the complex cytoskeletal system of the trophoblast cells. To explore the biochemical basis of these changes, one set of cytoskeletal proteins, the microtubule-associated proteins (MAPs), was studied. Day 4 blastocysts were labeled with [35S]methionine and blastocyst outgrowths, after 3 days in culture from the blastocyst stage, were labeled with [3H] methionine. Labeled embryos were disrupted and the soluble supernatants were pooled, and newly synthesized proteins from the two stages were coassembled with taxol-stabilized brain microtubule polymer enriched for MAP-binding sites. Double-labeled coassembly proteins (putative MAPS) were then released from the microtubule polymer by treatment with 0.35 M NaCl and analyzed by one-dimensional polyacrylamide gel electrophoresis. 3H/35S dpm ratios were determined for individual protein bands to compare the relative synthesis rates for day 4 blastocyst and day 3 outgrowth MAPs. In spite of the extensive changes in cell shape and motility associated with blastocyst outgrowth, a common set of putative MAPs characterizes the two stages investigated, including several in the size range of tau factors. No synthesis of high molecular weight MAPs comparable with MAP 1 or MAP 2 from brain was detected. The synthesis rates of individual MAPs relative to each other remain constant over this period and are likely coordinated with total protein and tubulin synthesis.