Gonadotropin-releasing hormone (GnRH) II has been implicated in the interaction between female energy balance and reproductive function, in addition to an anti-proliferative/apoptotic role in cancer cell lines. Specifically, administration of GnRH II to underfed female mice or musk shrews rescued mating behavior. Moreover, endometrial and ovarian cancer cell proliferation is reduced by GnRH II, likely via interaction with the GnRH I receptor. Although the GnRH I receptor has been well characterized, the molecular mechanisms underlying regulation of the GnRH II receptor (GnRHRII) remain to be elucidated. To determine the importance of the GnRH II ligand-receptor system, the pig represents an ideal model since it produces GnRH II and 2 isoforms of GnRHRII. Our laboratory isolated 3029 bp of 5' flanking sequence for the porcine GnRHR II gene. Transient transfections with vectors containing the GnRHR II promoter fused to the cDNA encoding luciferase (3029pGL3) indicated that this promoter is active in cell lines derived from both reproductive and non-reproductive tissues. The objective of this study was to determine the transcription factors and corresponding binding sites that confer cell-specific activity of the porcine GnRHRII gene promoter in the swine testes (ST) cell line. Progressive 5' deletions of the GnRHR II promoter indicated that the region from -707/-488 bp upstream of the translational start site contained elements critical to promoter activity in ST cells. Electrophoretic mobility shift assays (EMSAs) were performed by incubating radiolabeled oligonucleotide probes spanning the -707/-488 bp promoter region with ST nuclear extracts. Specificity of binding was determined by addition of 50-fold molar excess of either homologous or heterologous competitors. Analysis of the region between -591 and -570 bp of proximal promoter indicated a specific binding complex, which was abrogated upon the addition of an unlabeled competitor containing consensus binding sites for members of the Sp family of transcription factors. Further, addition of antibodies directed against the Sp1 and Sp3 transcription factors, but not Sp2 or Sp4, resulted in a supershift of the specific DNA:protein complex. Transient transfection of ST cells with vectors containing a mutation of the Sp1/Sp3 site located at -581/-576 bp within the context of the 3029 bp promoter resulted in a 61% reduction in promoter activity relative to the unaltered promoter (P < 0.05). To determine the specific contribution of the Sp1 and Sp3 transcription factors to GnRHR II promoter activity in ST cells, Sp1 or Sp3 overexpression vectors were co-transfected with the 3029pGL3 reporter vector. Overexpression of Sp1 increased promoter activity 2-fold compared to control (P < 0.05), whereas increasing levels of Sp3 was unable to activate the promoter. Furthermore, analysis of the region between -606/-580 bp displayed specific binding complexes that were competed by either Sp1 or Egr1 consensus oligonucleotides. Additional EMSAs revealed that the specific complex binding to the -606/-580 radiolabeled probe shifted upon the addition of Egr1 or Sp1 antibodies. Mutation of the overlapping Sp1/Egr1 site residing between -597/-587 bp within 3029pGL3 and subsequent transfection of the ST cell line resulted in a 56% decrease in promoter activity compared to promoterless control (P < 0.05). Thus, Sp1 and Sp1/Egr1 binding sites are critical to expression of the porcine GnRHR II gene in ST cells. (poster)