Alberta Pork Research Centre: Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5

We have isolated and characterized a porcine trophoblast cell line designated JAG-1 which permits a simplified in vitro model in which to analyze the contributions of the trophoblast to the complex interactions which exist in vivo at the Òmaternal -fetalÓ interface during pregnancy in pigs. JAG-1 secretes growth factor(s) which cause cell proliferation in a porcine macrophage bioassay and which are believed to act in a paracrine and/or autocrine fashion in vivo . In order to determine the identity of this factor(s) a variety of approaches have been used. Northern blot analysis of total RNA from this cell line was carried out with probes for gene expression of a variety of growth factors and cytokines known to be important in regulating reproductive processes. The probes included those for CSF-1, GM-CSF, IFN-gamma, TGF-beta, IL-2, and IGF-1 which have been previously identified as being important in other mammalian species. In addition, protein purification strategies to isolate the unknown factor from conditioned media of JAG-1 cells were developed. Characterization of these proteins by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis under reducing conditions indicated the presence of an unique protein species of approximately 40 kDa in fractions which had activity in the bioassay. In conjunction with the Northern blot data which indicated a strong hybridization signal with a probe for CSF-1 (which encodes a protein whose monomeric molecular weight size is 36-45 kDa ), CSF-1 became a candidate for the JAG-1 growth factor. In order to isolate a gene for CSF-1 with which to compare our emerging protein microsequence information, we have cloned a full-length porcine CSF-1 cDNA ( expressed form of the gene) using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) techniques and screening of a JAG-1 cDNA gene library. We have also examined embryo and uterine-derived tissues obtained from animals at peri-implantation stages of pregnancy. Both tissues expressed CSF-1 mRNA, although the levels in the uterus were much higher. Future experiments will analyze tissues from earlier time points in pregnancy and will also examine the levels of CSF-1 receptor which may determine the responsiveness of the tissue to the CSF-1 signal. Using this combination of research approaches, we hope to confirm the identity of this potentially important embryo (trophoblast ) derived growth factor.

Implication: The identification of growth factors which control cell proliferation and growth of pig embryos will help us towards our goal of maximizing embryonic development and survival in pigs.