The objective of this proposal is to investigate the functional role of the lipid mediators generated by the 12/15-lipoxygenase (12/15-LOX) enzymes in the control of uterine function during implantation. The 12/15-LOX catalyzes the stereo-specific oxygenation of the 20-carbon polyunsaturated fatty acids, arachidonic acid and linoleic acid, into a complex series of derivatives, HETEs and HODES. Recent studies, using high-density oligonucleotide microarrays, revealed that progesterone markedly induces the synthesis of mRNAs encoding the 12/15-LOX family members, leukocyte-12/15-LOX and epidermal-12/15-LOX, in the surface epithelium of pregnant uterus precisely at the time of implantation. Administration of a LOX-specific inhibitor markedly inhibited steroid hormone-regulated vascular permeability during delayed implantation in mice, indicating an important role of these enzymes in creating a receptive uterus. The major goals of this proposal are to: 1. Identify the gene networks that mediate the physiological effects of these lipid metabolites in the uterus during implantation. The 12/15-LOX-regulated gene pathways will be identified by DNA microarray. The spatio-temporal expression of these genes in the peri-implantation uterus will be analyzed and their function will be determined by a newly developed antisense oligonucleotide strategy. 2. Investigate the role of PPARgamma in the 12/15-LOX signaling pathway during implantation. The 12/15-LOX-derived metabolites of arachidonic or linoleic acid activate gene transcription by the peroxisome proliferator-activated receptor gamma (PPARgamma) in cell-based assays. PPARgamma expression is also induced in the uterus in the peri-implantation period. We will test whether PPARgamma is indeed the endogenous receptor for the 12/15-LOX-derived metabolites in the peri-implantation uterus and identify its target genes in this tissue. The role of PPARgamma will be further investigated by generating a conditional knockout of its gene in the uterus and by analyzing the functional consequences of the loss-of-function mutation of this receptor during implantation. Collectively, the proposed experiments will test the hypothesis that a novel signaling pathway, involving 12/15-LOX-derived lipid mediators, PPARgamma, and its downstream target genes, regulates critical events during implantation.
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