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Localization of ISG15 and Conjugated Proteins in Bovine Endometrium Using Immunohistochemistry and Electron Microscopy

Reproductive Biology Program, Department of Animal Science (K.J.A., A.L.C., J.K.P., E.L.G.), and Department of Veterinary Science and Veterinary Diagnostic Laboratory (E.L.B., C.E.H.), University of Wyoming, Laramie, Wyoming 82071

Address all correspondence and requests for reprints to: Thomas R. Hansen, Reproductive Biology Program, Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071-3684. E-mail: thansen{at}uwyo.edu.

The interferon-stimulated gene ISG15, a ubiquitin homolog, becomes conjugated to and regulates uterine proteins in response to conceptus-derived interferon- on d 18 of pregnancy. It was hypothesized here that cellular localization of ISG15 within endometrial cells might provide insight regarding function. Uteri were collected from cows (21-d estrous cycle) on d 17–21/0 of the estrous cycle and pregnancy and d 23, 45, and 50 of pregnancy. Intracellular ISG15 and its conjugates were present on d 17 of pregnancy, peaked to highest levels from d 18 to 23 and then declined to low but detectable levels by d 45 (P < 0.05) based on Western blotting. ISG15 and its conjugates were not detected on d 50 of pregnancy or during the estrous cycle. Immunohistochemistry revealed that ISG15 was localized throughout the endometrium on d 18–23, with heaviest staining in the sublumenal stratum compactum and the glandular epithelium throughout the stratum spongiosum. By d 45 and 50, ISG15 was lightly stained only in the stratum compactum immediately beneath the lumenal epithelium. Using transmission electron microscopy and immunogold labeling, ISG15 was specifically localized to organelles and compartments of endometrial epithelial cells and stromal cells: nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum, and cell membrane. This specific localization in epithelial and stromal cells led to the conclusion that ISG15 has diverse intracellular functions. The sustained presence of conjugated ISG15 through d 50 of pregnancy might reflect stabilization of conjugated proteins in response to implantation and the development of the placenta.

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