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

Abstract

The interferon-stimulated gene ISG15, a ubiquitin homolog, becomes conjugated to and regulates uterine proteins in response to conceptus-derived interferon-τ 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.

Interferon Stimulated Genes (Isgs): Novel Pregnancy Specific Biomarker In Buffaloes (Bubalus bubalis

“…ISG15 gene expression was up-regulated from day 18 to 23 and down-regulated from day 23 to 45 and did not register any change on day 50 of pregnancy indicating higher expression of ISG15 in pregnant as compared to non-pregnant cows 24,14 . In contrast, Shirasuna et al (2012) did not observe any change in expression pattern of ISG15 in pregnant and non-pregnant cows 25 .…”

Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity

“…A couple of genes related to the so-called ISG15ylation process were found. The ubiquitin-like protein, ISG15, is suggested to play an important role in IFNT signalling (Austin et al 2004). ISG15 is conjugated to intracellular proteins and thereby regulates their function.…”

2 Identification of Genes Induced by the Conceptus in the Bovine Endometrium During the Pre-Implantation Period

“…Of particular interest among the interferon stimulated genes is ISG15, and recently several studies were dedicated to clarify the role of ISG15 at the embryo-maternal interface Austin et al, 2004;Joyce et al, 2005). ISG15, one of the most markedly upregulated genes in the present study, encodes an ubiquitin-like protein that is conjugated to intracellular proteins (Narasimhan et al, 1996).…”

“…Some of these genes have already been described in the context of early pregnancy in ruminants, e.g. ISG15 (Austin et al, 1996;Johnson et al, 1998;Austin et al, 2004) OAS1 (Schmitt et al, 1993), IRF1 , IFITM1 and IFITM3 (Pru et al, 2001), MX protein Hicks et al, 2003), IFIT1 , ISG12 , MHC class I genes and B2M , but only little information exists concerning the putative roles of these ISGs in the context of early pregnancy.…”