The effects of estrogen, its antagonist ICI 182, 780, and interferon-tau on the expression of estrogen receptors and integrin alphaV beta 3 on cycle day 16 in bovine endometrium

Abstract

We have shown previously that downregulation of intercaruncular stromal integrin alphavbeta3 in bovine endometrium on day 16 of the estrous cycle coincided with the antibody recognition of estrogen receptors (ER) in the luminal epithelium. In pregnancy, these changes were not observed. Our hypothesis was that on day 16 of the estrous cycle, estrogen from the dominant follicle causes a reduction in integrin alphavbeta3 and affects ERalpha in the luminal epithelium. The pregnancy recognition protein, interferon-tau (IFN-tau), may prevent downregulation of integrin alphavbeta3 and suppress ERalpha expression in the luminal epithelium. On days 14 to 16, heifers received uterine infusions of the anti-estrogen ICI 182, 780, estradiol 17beta, IFN-tau or the saline control. On day 16, reproductive tracts were collected for analysis of integrin alphavbeta3 and ERalpha. Estrogen receptor alpha immunoreactivity was largely restricted to the luminal epithelium in control animals. Using anti-ERalpha recognizing the amino terminus, estrogen-treated animals showed reactivity in the stroma, shallow and deep glands and myometrium as is typical of estrus, whereas ICI 182, 870 treated heifers showed little or no reactivity. In contrast, carboxyl terminus-directed antibodies showed a widespread distribution of ERalpha with reactivity detected in the uterine epithelium, stroma and myometrium of both estrogen and ICI 182, 780 treated animals. Heifers treated with IFN-tau had low ERalpha reactivity overall. Control and IFN-tau treated heifers had lower intercaruncular stromal expression of integrin alphavbeta3 in comparison to estrogen and ICI 182, 780 treatments. Overall, the results suggest that on day 16 of the estrous cycle, estrogen effects on integrin alphavbeta3 are indirect and do not directly involve ERalpha in the luminal epithelium. During pregnancy, interferon-tau may block ERalpha in the luminal epithelium but likely does not rescue integrin alphavbeta3 expression.

Figure 1

Synchronization schedule, heat detection and treatment regimes for heifers used in the study. Heifers were injected with Estrumate^® 11 days apart and observed for heats after the second injection. All heifers were observed in standing heat within the same twelve hour period then observed for one cycle length of 18–20 days. Twelve days after non-induced estrus, heifers were injected a third time and observed for heats. At 6 pm, fourteen days following standing heat treatment began. Heifers received four uterine infusions 12 hr apart of either saline control, IFN-τ, estrogen or ICI 182,780. At 2 pm on day 16 of the estrous cycle, heifers were slaughtered and tissues were collected for analysis.

Figure 2

Immunofluorescent localization of the estrogen receptor using antibodies AER314 and AER311 in cyrostat cross-sections of endometrium obtained on day 16 of the estrous cycle after uterine infusion with a BSA-saline control solution (A-B), interferon-tau (C-D), ICI 182, 780 (E-F) or estrogen (G-H). A tissue section treated with mouse IgG in place of primary antibody is shown inset in A. Specific nuclear reactivity was present in some cells of the stroma (arrows) in control animals. LE – luminal epithelium, S – stroma, GE – glandular epithelium. Magnification for A-C was 400X, D,F,H was 200X and E,G 100X.

Figure 3

Immunofluorescent analysis of integrin α_vβ₃ in cryostat cross-sections of endometrium. (A) Immunoreactivity in sections from heifers treated with control solution, interferon-tau (IFN-τ), estrogen or ICI 182, 780. Diffuse staining was present in the dense stroma, low reactivity was observed in the glands (GE) and luminal epithelium (LE). Note the strong reactivity of intercaruncular subepithelial stromal cells (arrows) in sections from estrogen and ICI 182, 780 treated animals. CARS – caruncular stroma; ICS – intercaruncular stroma. Magnification was 100X. B) Least squares means ± standard error of signal intensity of integrin α_vβ₃ in the subepithelial stroma (SES) by treatment (3 animals × 4 replicate experiments per treatment). Intensity was scored on a six point scale (0-negative, 1-very weak, 2-weak but clearly positive, 3-intermediate, 4-high, and 5-very high) and the data were subjected to ANOVA and the Tukey-Kramer multiple comparison of means to determine effects of treatment in comparison to the control. Asterisks indicate means that are significantly different from the control (P < 0.05).

Figure 4

Effect of treatment onexpression of integrin subunit β₃ as detected by Northern blot analysis of intercaruncular endometrium. A) Three animals were used in each treatment group and received uterine infusions of either the saline control, interferon tau (IFN-τ), estrogen or ICI 182, 780. Thirty-five micrograms of total RNA from each animal were loaded per lane and biotin labeled riboprobes specific to integrin subunit β₃ were used for hybridization to the membrane. A single 2.4 kb transcript was detected. B) Least squares means ± standard error for integrin subunit β₃ mRNA band intensity by treatment (3 animals/treatment × 2 replicate blots) were calculated using 18S as a covariate. Asterisks indicate means that are significantly different from the Control and IFN-τ (P < 0.05).