The establishment of pregnancy in ruminants is dependent on the production of interferon-tau (IFNT) by the conceptus which stimulates the production of interferon stimulated gene 15 (ISG15) in maternal tissues. ISG15 covalently binds to (ISGylation) and regulates intracellular proteins processes in a manner like ubiquitin. The aim of this study was to identify ISGylated proteins and infer their specific functions and biochemical pathways within endometrium. Endometrium was collected from pregnant ewes on day 17 (P17, n=4), synchronized non-inseminated ewes on Day 10 (EC10, n=4) and Day 17 (EC17, n=4) after estrus (Day 0). In addition, bovine endometrial (BEND) cells were cultured with 10 ng/ml of recombinant bovine (rb)IFNT for 24 h. ISGylated proteins in protein extracts were immunoprecipitated with anti-bovine ISG15 antibody. The ISGylated proteins in eluates were identified using Liquid Chromatography-Tandem Mass Spectrometry and confirmed by reciprocal immunoprecipitation of selected ISGylated proteins. Seventy-five ISGylated proteins were differentially expressed in the endometrium of EC10, EC17 and P17 ewes. Ingenuity pathway analysis indicated that interferon α/β signaling, ISG15 antiviral mechanism, Glycolysis I, and Gluconeogenesis I pathways were the most highly significant pathways induced in the pregnant endometrium. Enrichment of the ISGylated proteins STAT1, MX1, and ENO1 was achieved by immunoprecipitation with specific antibodies and their conjugation to ISG15 was confirmed. The ISGylated endometrial proteins identified in pregnant ovine endometrium may function in IFN signaling, antiviral mechanisms and glucose metabolism with implications for these pathways in the preparation of endometrium for successful implantation and maintenance of pregnancy.
Guzeloglu et al. (Tue,) studied this question.
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