Regulation of protein and prostaglandin secretion in polarized primary cultures of caprine uterine epithelial cells

127

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.

Section: Animalsmentioning

confidence: 99%

Expression of the Interferon Tau Inducible Ubiquitin Cross-Reactive Protein in the Ovine Uterus1

Johnson

Spencer

Hansen

et al. 1999

127

“…Endometrial LE, GE, and ST cells were isolated as previously described [26] with modifications. Immediately after hysterectomy, the uterus was separated into segments.…”

Section: Establishment Of Primary Cell Linesmentioning

confidence: 99%

Development and Characterization of Immortalized Ovine Endometrial Cell Lines1

Johnson

Burghardt

Newton

et al. 1999

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6-0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle alpha-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFNtau) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFNtau. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium.

“…Endometrial epithelial cells were enzymatically isolated as previously described [26]. Briefly, the uterine horn received enough sterile Ca 2ϩ /Mg 2ϩ -free Hanks' Balanced Salt Solution (HBSSϪ) containing pancreatin (2.5 mg/ml) and dispase (4.8 mg/ml) to cause mild distension (approximately 20 ml) and was incubated at 4ЊC for 1 h, warmed to 20ЊC for 20 min, and then warmed to 37ЊC for 10 min.…”

Section: Immunocytochemical Analysis Of the H-type 1 Antigen On Primamentioning

confidence: 99%

“…These include transepithelial resistance, tight junctions, apical microvilli, and domainspecific trafficking of proteins and prostaglandins. In addition, polarized ULE cells respond to IFN-and oxytocin, two major regulators of prostaglandin secretion in utero [26]. Polarized caprine (c) ULE cells (i.e., transmembrane resistance of 600 Ohm cm 2 or greater) were then cultured in DMEM:F12 containing 5% (v/v) charcoal-stripped (s) FBS for 24 h. Medium was replaced daily and was supplemented with either estradiol-17␤ (E 2 ; 10 Ϫ9 M), progesterone (P 4 ; 10 Ϫ7 M), E 2 ϩP 4 , or no additional steroids for an additional 5 days (n ϭ 8 filters per treatment using ULE cells from four goats).…”

Section: Immunocytochemical Analysis Of the H-type 1 Antigen On Primamentioning

confidence: 99%

“…Concentration of DNA in the supernatant was determined [28], and biotinylated proteins were precipitated using streptavidin-coupled beads. The precipitate was boiled in buffer containing 5% (w/v) SDS and 5% (v/v) 2-mercaptoethanol, separated by SDS-PAGE as previously described [26], and transferred electrophoretically to nitrocellulose. Blots were incubated sequentially with blocking solution, anti-H-type 1 antigen or NMS, washing solution, and rabbit anti-mouse gamma globulin coupled to horseradish peroxidase.…”

Section: Biotinylation Of Apical Membrane Proteins and Westernmentioning

confidence: 99%

See 1 more Smart Citation

Expression of Carbohydrate Antigens in the Goat Uterus During Early Pregnancy and on Steroid-Treated Polarized Uterine Epithelial Cells In Vitro1

Powell

Glasser

Woldesenbet

et al. 2000

Self Cite

Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P(4)) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P(4) on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.