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Abstract

Mechanisms of porcine trophectoderm binding to latency associated peptide of transforming growth factor beta and fibronectin

Aaron K. Spiegel, Laurie A. Jaeger*,

Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, 77843-4458.

Summary

The period of conceptus (embryo plus its extra-embryonic membranes) attachment and implantation is a critical time in pregnancy.  Early pregnancy losses remain a problem in human and domestic livestock, but the mechanisms of successful implantation are still not well-understood.  Growth factors, extracellular matrix proteins (ECMs), and integrins, which act as ECM receptors, appear to be important components of the implantation process.  In pigs, the implantation process is prolonged and the trophectoderm of the conceptus adheres to the endometrial epithelium which lines the lumen of the uterus.   The objective of this project is to determine the mechanisms of trophectoderm attachment to two transforming growth factor beta (TGFb)-related proteins that are present during early pregnancy - the TGFb latency-associated peptide (LAP) and fibronectin (FN).  The hypothesis is that trophectoderm attachment to LAP and FN is mediated through distinct integrins located in the cell membrane each with specific actions. 

A porcine trophectoderm cell line (pTr2), derived from conceptuses collected on day 12 of gestation, was maintained in culture (DMEM/F12, 5% charcoal-stripped fetal bovine serum) and used for all assays.  The pTr2 cells adhered, in a dose-dependent manner, to wells coated with graded doses of LAP or FN, and bound to a greater degree to LAP than to FN.   An LAP mutant protein, in which the arginine-glycine-aspartic acid (RGD amino acid sequence), an integrin binding site, was changed to arginine-glycine-glutamic acid (RGE) did not support pTr2 adhesion.   Adhesion to FN was decreased by addition of soluble RGD peptide, but not arginine-alanine-aspartic acid (RAD).   In competition experiments, soluble LAP added to the cells in a preincubation showed a decrease in cell adhesion to wells coated both with FN or LAP.  Soluble FN pretreatment of cells interestingly did not show a decrease in adhesion and at lower concentrations showed a mild up regulation.  These unexpected results raise questions which will be addressed by using cycloheximide and monensin to inhibit protein production and trafficking, respectively, to assess possible up-regulation of integrin expression, and by altering pre-incubation and incubation conditions, to assess possible effects of soluble and immobilized ECM interactions on cell binding.