Effect of the conceptus on uterine natural killer cell numbers and function in the mouse uterus during decidualization

Abstract

Uterine natural killer (uNK) cells are the most abundant lymphocytes in the uterus during early pregnancy and play a role in spiral arteriole modifications. In the present study, we investigated whether uNK cell populations differed between mouse decidua and deciduoma. Histochemical staining using the Dolichos biflorus agglutinin (DBA) lectin was used to identify uNK cells and classify their stages of maturation. We found differences in the pattern of localization and density of uNK cells between the decidua and deciduoma at Days 2-4 after the onset of decidualization. The cells were more distributed and the densities were significantly greater in the mesometrial region of the decidua than in the deciduoma. Using double-labeling for DBA lectin binding and bromodeoxyuridine incorporation, we found that the higher number of uNK cells in the decidua was not due to an increase in uNK cell proliferation. Western blot analyses revealed that the increase in uNK cell number was accompanied by significant increases in the levels of interferon gamma (IFNG) and prointerleukin 18 when a conceptus was present. Vascular morphometry revealed that modifications of the spiral arterioles occurred in the mesometrial decidua but not in the deciduoma, which could be attributed to the differences observed in uNK cell number and IFNG production. The present study demonstrates that differences exist in uNK cell populations between the decidua and deciduoma, providing evidence that the conceptus generates signals that regulate uNK cell number and function in the uterus during implantation.

Figure 1

Time-lines for uteri collected on days after the onset decidualization. A. Pregnant uteri were collected on Days -1 to 5 corresponding to days of pregnancy (DOP) 3.5–9.5. B. Uteri undergoing artificially-induced decidualization in ovariectomized mice injected with estradiol-17β (E₂) and progesterone (P₄) to sensitize the uterus for a deciduogenic stimulus. Pseudopregnant females received a transfer of C. Concavalin A coated agarose beads or D. injection of sesame oil into the uterine lumen to artificially induce decidualization. Uteri were collected at Days 1–4 after the onset of artificially-induced decidualization (B–D).

Figure 2

Uterine NK cell maturation in the mouse. A. Uterine NK cells found in the mesometrial region of the decidua and deciduoma by DBA lectin histochemistry were classified as subtypes I–IV (scale bar = 10 μm). B. Day 4 after the onset of decidualization, uterine cross section illustrating subregions 1–3 (central axis toward conceptus from the myometrium) and 4 (lateral region). AM, antimesometrial region; C, conceptus; M, mesometrial region; MLAp, mesometrial lymphoid aggregate of pregnancy.

Figure 3

Uterine NK cell distribution and density. A. Photomicrographs of DBA lectin-stained cross sections of the mouse decidua and deciduomas on Day 3 after the onset of decidualization. Brown color indicates DBA lectin binding to uNK cells, Scale bar = 500 μm. .B. Graph showing density of uNK cells normalized to total area of mesometrial subregion in the mouse decidua and deciduomas at various days after the onset of decidualization. Bars represent the mean (±SEM; N= 6) and those with different letters on a given day are significantly different (P <0.01). An asterisk (*) denotes only determined for decidua.

Figure 4

Graphs summarizing regional uNK cell subtype (I–IV) density per test area in subregions 1–4 of the mouse Ovx-OID deciduoma and decidua on Days 2, 3 and 4 after the onset of decidualiztion. An asterisk (*) indicates a significant difference (P <0.05) between Ovx-OID deciduoma and decidua for a given subtype. Bars represent the mean and error bars denote standard error of the mean (N=6).

Figure 5

Graphs summarizing the percentage of uNK cells subtype (I–IV) in mesometrial subregions 1–4 of the mouse Ovx-OID deciduoma and decidua on Days 2, 3 and 4 after the onset of decidualization. Bars represent the mean and error bars denote standard error of the mean (N=6).

Figure 6

Graph showing the mean (± SEM, N=5–6) uNK cell proliferation index (proportion of uNK cells staining positive for BrdU) in the mouse Ovx-OID plus Pseudo-BID deciduomas and decidua on Days 2, 3 and 4 after the onset of decidualization. Bars with different are significantly different (P <0.01).

Figure 7

Western Blot Analysis of IL15, IFNG and IL18 in the mouse Ovx-OID plus Pseudo-BID deciduomas and decidua on Days 1–4 after the onset of decidualization. A. Representative Western blot analysis. ACTB was used as a control for equal protein loading. Graph bars represent (mean ± SEM, N=4) relative fluorescence normalized to ACTB for B. IL15, C. IFNG and D. IL18. An asterisk (*) indicates a significant difference (P <0.05) between deciduomas and decidua. N indicates no fluorescence signal detected above background. E. Rabbit IgG (2 μg/ml) negative control. F. Goat IgG (0.4 μg/ml) negative control.

Figure 8

Mouse spiral arteriole morphometry on Day 4 after the onset of decidualization. A. Photomicrographs of representative spiral arterioles in the Ovx-OID plus Pseudo-BID deciduomas and decidua. Scale bar equals 25 μm. B. Mean (± SEM, N=4–6) spiral arteriole thickness and C. lumen diameter in non-stimulated (control) plus stimulated (implantation stimulus) areas and non-implantation (control) plus implantation (implantation stimulus) sites of the deciduomas and decidua, respectively. V, vessel thickness; L, lumen diameter. Star denotes a significant (P<0.050 difference between the control and areas undergoing decidualization in response to an implantation stimulus.