INTRAUTERINE INFUSION OF RECOMBINANT TRANSFORMING GROWTH FACTOR BETA LATENCY-ASSOCIATED PEPTIDE DISRUPTS PORCINE CONCEPTUS DEVELOPMENT

Abstract

The beta transforming growth factors (TGFbeta) and their receptors (TGFbeta-R) are present at the conceptus-maternal interface in pigs and other mammals. TGFbeta is released as latent precursors which cannot interact with TGFbeta-R prior to activation. In pigs, amounts of active TGFbeta increase during conceptus elongation in preparation for attachment to the endometrium; active TGFbeta may play roles in conceptus-uterine interactions at this time. The latency-associated peptide of TGFbeta (LAP) is part of a complex that comprises latent TGFbeta and contains an arg-gly-asp (RGD) amino acid sequence which can bind to and activate integrins. We hypothesize that both active TGFbeta and LAP contribute to normal conceptus development and implantation. Objectives of these studies are to: 1) determine temporal and spatial distribution of LAP at the conceptus-maternal interface of pigs during the implantation process, and 2) determine effects of intrauterine infusion of LAP on conceptus development during the peri-implantation phase of pregnancy. In study one, cross-bred gilts were ovariohysterectomized on Days 10, 12, 16, 20, and 24 of gestation (n=3/day). LAP distribution at implantation and nonimplantation sites was determined using immunofluorescence microscopy. On Days 10 and 12 of gestation, LAP was present at the apical and basal aspects of uterine luminal epithelia, apical surfaces of glandular epithelia and within the lumens of uterine glands. Epithelial expression decreased between Days 16 and 20, suggesting either decreased TGFbeta production or increased TGFbeta activation. On Day 24, when conceptus attachment is nearly complete, LAP was nearly undetectable at non-implantation sites, however aggregrates of LAP were present at sites of adhesion between conceptus and uterine tissues. Thus, collective temporal and spatial localization suggests a role for LAP as an extracellular matrix protein available for interaction with conceptus and/or maternal integrins that maintains conceptus attachment. In study 2, surgically-implanted mini-osmotic pumps were used to continually infuse uterine horns with molar excesses of recombinant LAP in a porcine serum albumin (PSA) vehicle or PSA alone beginning on Day 9 of gestation; others received no infusion. Gilts were ovariohysterectomized on Day 13 of gestation and conceptuses examined. Conceptuses from LAP-infused gilts (n=3 gilts) grossly appeared retarded in their development and some appeared to be degenerating compared to those recovered from gilts which received PSA or no infusion. In two litters, conceptuses were spherical or oval in contrast to the filamentous morphology of conceptuses in litters receiving PSA and no infusion. Histological evaluation confirmed degeneration of the trophoblast in LAP-infused gilts, while integrity of the uterine epithelium was maintained. Effects most likely resulted from inactivation of TGFbeta by the infused LAP, but effects of integrin activation by the RGD sequence of LAP are also possible and will be further investigated through infusion of an LAP mutant protein containing an RGE sequence in place of the RGD. This is the first report of targeted in vivo disruption of pre-implantation conceptus development by direct infusion of a recombinant protein into the uterus of pregnant pigs. Results suggest critical roles for TGFbeta in conceptus survival and elongation. Supported by NRICG #2005-35203-15798 from USDA CSREES. (platform)