Interleukins affect equine endometrial cell function: modulatory action of ovarian steroids

Abstract

The aim of the present study was to investigate the interaction between ovarian steroids, interleukins and prostaglandins (PG) in equine epithelial and stromal cells in vitro. In Experiment 1, cells were exposed to IL-1α (10 ng/mL), IL-1β (10 ng/mL) or IL-6 (10 ng/mL) for 24 h and cell proliferation was determined using MTT. In Experiment 2, cells were exposed to progesterone (P4; 10(-7) M); 17-β estradiol (E2; 10(-9) M) or P4+E2 for 24 h and later medium was replaced with a fresh one treated with IL-1α, IL-1β or IL-6 (10 ng/mL, each) for 24 h. The oxytocin (OT; 10(-7) M) was used as a positive control. In Experiment 3, cells were exposed to P4 (10(-7) M), E2 (10(-9) M) or P4+E2 for 24 h and the IL receptor mRNAs transcription was determined using Real-time PCR. Prostaglandins concentration was determined using the direct enzyme immunoassay (EIA) method. Our findings reveal a functional linking between ovarian steroids and IL-stimulated PG secretion by equine endometrial cells. This interaction could be one of the mechanisms responsible for endometrial local orchestrating events during the estrous cycle and early pregnancy.

Figure 1

Representative morphology of cultured equine endometrial: (a) epithelial cells and (b) stromal cells. (c) Epithelial cells identification by immunofluorescent staining for cytokeratin; (d) stromal cells identification by immunofluorescent staining for vimentin. The scale bar = 50 μm (magnification: ×40).

Figure 2

The effect of IL-1α (10 ng/mL), IL-1β (10 ng/mL), or IL-6 (10 ng/mL) on proliferation of (a) epithelial cells (mean ± SEM; n = 5) and (b) stromal cells (mean ± SEM; n = 5) after 24 h incubation. All values are expressed as n-fold change from control. Asterisks indicate significant differences (*P < 0.05; **P < 0.01) from the respective control, as determined by parametric one-way ANOVA followed by Newmann-Keuls comparison test.

Figure 3

The effect of ovarian steroids on IL-1α stimulated PGE₂ and PGF_2α production by epithelial ((a), (c)) and stromal ((b), (d)) cells. The cells were treated with P₄ (10⁻⁷ M), E₂ (10⁻⁹ M), or P₄ + E₂ (10⁻⁷/10⁻⁹ M) for 24 h. Then, the medium was replaced with a fresh medium and the epithelial cells were stimulated with IL-1α (10 ng/mL) for 24 h. Oxytocin (OT; 10⁻⁷ M) was used as a positive control. All values are expressed as n-fold change from control. letters “a,” “b,” and “c” indicate significant differences (P < 0.05) between groups, as determined by parametric one-way ANOVA followed by Newmann-Keuls comparison test.

Figure 4

The effect of ovarian steroids on IL-1β stimulated PGE₂ and PGF_2α production by epithelial ((a), (c)) and stromal ((b), (d)) cells. The cells were treated with P₄ (10⁻⁷ M), E₂ (10⁻⁹ M), or P₄ + E₂ (10⁻⁷/10⁻⁹ M) for 24 h. Then, the medium was replaced with a fresh medium and the epithelial cells were stimulated with IL-1β (10 ng/mL) for 24 h. Oxytocin (OT; 10⁻⁷ M) was used as a positive control. All values are expressed as n-fold change from control. letters “a,” “b,” “c” indicate significant differences (P < 0.05) between groups, as determined by parametric one-way ANOVA followed by Newmann-Keuls comparison test.

Figure 5

The effect of ovarian steroids on IL-6 stimulated PGE₂ and PGF_2α production by epithelial ((a), (c)) and stromal ((b), (d)) cells. The cells were treated with P₄ (10⁻⁷ M), E₂ (10⁻⁹ M), or P₄ + E₂ (10⁻⁷/10⁻⁹ M) for 24 h. Then, the medium was replaced with a fresh medium and the epithelial cells were stimulated with IL-6 (10 ng/mL) for 24 h. Oxytocin (OT; 10⁻⁷ M) was used as a positive control. All values are expressed as n-fold change from control. letters “a,” “b,” and “c” indicate significant differences (P < 0.05) between groups, as determined by parametric one-way ANOVA followed by Newmann-Keuls comparison test.

Figure 6

The effect of P₄ (10⁻⁷ M), E₂ (10⁻⁹ M), and P₄ + E₂ (10⁻⁷/10⁻⁹ M) on IL-1RI ((a), (b)), IL-1RII ((c), (d)), and IL-6Rβ ((e), (f)) mRNA transcription in epithelial cells (n = 5) and stromal cells (n = 5) after 24 h incubation. Results are normalized against ACTB. Data are presented as fold induction relative to control. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) from the respective control, as determined by nonparametric one-way ANOVA Kruskala-Wallisa followed by Dunns'a test.

Figure 7

The effect of P₄ (10⁻⁷ M), E₂ (10⁻⁹ M), and P₄ + E₂ (10⁻⁷/10⁻⁹ M) on IL-1RI:IL-1RII mRNA transcription ratio in epithelial cells ((a); n = 5) and stromal cells ((b); n = 5) after 24 h incubation. Results are normalized against ACTB. Asterisks indicate significant differences (*P < 0.05; **P < 0.01) from the respective control, as determined by nonparametric one-way ANOVA Kruskala-Wallisa followed by Dunns'a test.